How to build a central synapse: clues from cell culture

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Central neurons develop and maintain molecularly distinct synaptic specializations for excitatory and inhibitory transmitters, often only microns apart on their dendritic arbor. Progress towards understanding the molecular basis of synaptogenesis has come from several recent studies using a coculture system of non-neuronal cells expressing molecules that generate presynaptic or postsynaptic ‘hemi-synapses’ on contacting neurons. Together with molecular properties of these protein families, such studies have yielded interesting clues to how glutamatergic and GABAergic synapses are assembled. Other clues come from heterochronic cultures, manipulations of activity in subsets of neurons in a network, and of course many in vivo studies. Taking into account these data, we consider here how basic parameters of synapses – competence, placement, composition, size and longevity – might be determined.

Introduction

Synaptogenesis involves a complex series of events, spanning neuronal differentiation, cell–cell contact and localized induction of presynaptic and postsynaptic differentiation. Synaptic specificity is determined by the developmental status of both partner cells, by neuronal and glial cues that influence competence for synaptogenesis, by long-range and local axon and dendrite guidance cues, by cell-adhesion molecules that mediate contact, and by local presentation of differentiation-inducing molecules. Although activity is a major force in sculpting circuitry during development [1] and regulates synaptic composition and strength 2, 3, 4, it is not essential for the basic assembly of synapses. Synapses form normally when neurotransmitter release is chronically blocked using clostridial neurotoxins or genetic methods 5, 6, 7. We focus here on molecular cues involved in the later stages of synaptogenesis, once appropriate axons and dendrites are brought into proximity. Studies of several major synaptogenic molecules identified for glutamatergic and/or GABAergic synapses are summarized in Table 1, and partial molecular linkages are shown in Figure 1 8, 9, 10. We also focus on aspects of recent studies that particularly illuminate how basic parameters of synapses are shaped.

For this review, we consider a ‘synapse’ to mean a functional synapse (noting that other more limited definitions of synapses, based on structure or molecular composition, can be useful in many circumstances) (Box 1). We use ‘hemi-presynapse’ or ‘presynaptic differentiation’ to refer to clusters of release-competent synaptic vesicles, and ‘hemi-postsynapse’ or ‘postsynaptic differentiation’ to refer to clusters of surface neurotransmitter receptors and associated signaling and scaffolding molecules. These hemi-synaptic elements can be combined in a bona fide synapse or induced in isolated axons or dendrites by individual synaptogenic molecules (Figure 2).

Section snippets

Competence

Neurons acquire the ability to form synapses as part of a developmental maturation process. Intrinsic limitations in competence to form synapses have been demonstrated in cell culture studies, where a difference in experience of two days can be crucial. For example, hippocampal neurons from embryonic day (E)18 rats form functional synapses in culture but, under the same conditions, E16 neurons form morphological synapses that are largely presynaptically silent, regardless of how long they are

Matching cellular partners

For synapse assembly to occur, the plasma membranes of the appropriate presynaptic and postsynaptic cells must be brought into contact. Members of the cadherin and immunoglobulin (Ig) superfamilies are thought to mediate this function. Cadherin expression patterns and some function-blocking studies support the idea that cadherins have a key role in mediating selective adhesion leading to formation of synapses between the appropriate partners 24, 25. In Drosophila, single-cell mosaic analyses

Recruiting presynaptic components

Once the cell membranes are brought into contact, the next step is to recruit the molecular assemblies that mediate transmitter release and response. Several studies in the past few years have demonstrated the surprising ability of a handful of isolated molecules to induce focal aggregation of release-competent synaptic vesicles when presented to axons of cultured neurons. Neuroligins were the first of these molecules to be identified [55] and appear to be the most potent inducers of

Size

At a typical CNS synapse composed of a single active zone, the areas of the active zone and postsynaptic density, the numbers of synaptic and docked vesicles and the volumes of the axon varicosity and spine head are all highly correlated 91, 92, 93. This size correlation suggests a coordinated regulation of presynaptic active zone and bouton size with PSD and spine size via connecting transmembrane and cytoskeletal proteins. Active zone and PSD sizes range over about two orders of magnitude 92,

Longevity and plasticity

The majority of spine synapses in the mature brain are stable for months 114, 115, presumably through continual replenishment of the synaptogenic signals. However, as exemplified by studies of ephrins and Eph receptors [116] and cadherins [117], the same molecules that function centrally in synaptogenesis also contribute to activity-dependent synaptic plasticity in more mature systems. Such plasticity can occur through synapse assembly or disassembly and through altering the strength of

Concluding remarks

A key question is whether any single factor or even single family of proteins is essential for synaptogenesis. At present, mutant mice for individual synaptogenic proteins mentioned here are viable and form synapses. Moreover, directed screens in C. elegans and Drosophila have not revealed any proteins essential for basic synapse assembly – that is, mutants completely lacking synapses have not been found 98, 99. These results, or lack of them, suggest either that the key synaptogenic factors

Acknowledgements

We thank Huaiyang Wu for preparation of cultures used in Figure 2, and YunHee Kang and other members of the Craig laboratory for helpful comments. Supported by grants from NIH, CIHR and MSFHR, and by Canada Research Chair (AMC) and NSF Predoctoral Fellow (ERG) awards.

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