How to build a central synapse: clues from cell culture

Abstract

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.

Figure 1

Molecular components of glutamatergic (a) and GABAergic (b) synapses. Only some of the components are shown, emphasizing cleft and transmembrane proteins and their interacting partners. Solid lines indicate reported protein–protein interactions; broken lines indicate presumed indirect interactions. For references, see main text.

Figure 2

Hemi-synapse induction by neuroligins or neurexins presented to isolated axons or dendrites on the surface of fibroblasts. (a) Fibroblasts (F) expressing neuroligins induce clusters of presynaptic components, including GAD, at contact sites with axons of cultured neurons (N). These induced clusters of GAD (arrowheads) lack the normal postsynaptic proteins such as gephyrin, in contrast to endogenous synapses (arrow). (b) Fibroblasts (F) expressing neurexins induce clusters of postsynaptic components,including the GABA_A receptor γ2 subunit (GABARγ2), at contact sites with dendrites of cultured neurons (N). These induced clusters of GABARγ2 (arrowheads) lack the normal presynaptic proteins such as synapsin, in contrast to endogenous synapses (arrow). (c) Hemi-presynapse (left) and hemi-postsynapse (right) formation in isolated axons and dendrites compared with bona fide synapses at axon–dendrite contacts (centre). Neuroligins, SynCAM, soluble FGF22 and soluble Wnt7a induce hemi-presynapses [18,21,29,55]; neurexins, NARP and ephrins induce full or partial hemi-postsynapses [60,67,71]. Scale bar, 10 μm.

Figure 3

Potential role for neurexins in matching postsynaptic and presynaptic composition. (a) It is possible that glutamatergic axons express neurexin isoforms (A) that bind most strongly to neuroligins-1, -3 and -4, whereas GABAergic axons express neurexins (B) that bind most strongly to neuroligin-2. Neurexins (A) versus (B) represent different forms that could result from differential gene usage, promoter usage, alternative splicing and/or glycosylation. Before synapse formation, all neuroligin isoforms might be diffusely distributed over the dendritic surface, whereas neurexins might be distributed ubiquitously over the axonal surface. (b) Synapse formation could be triggered when presynaptic neurexins interact with the appropriate postsynaptic neuroligins, stabilizing and aggregating both at nascent contact sites. (c) By specifically aggregating neuroligins-1, -3 and -4, glutamatergic neurexins could then cause the subsequent clustering of glutamate postsynaptic proteins. Likewise, by binding to and aggregating neuroligin-2, GABAergic neurexins could thereby influence the clustering of GABAergic postsynaptic proteins. In a complementary manner, the interaction of neuroligins with neurexins would also stabilize axon contacts and induce presynaptic specializations.

Figure 4

Comparison of predicted size differences between synaptic adhesion molecule pairs. The classical cadherin trans-dimer is modeled after an experimentally determined structure [108]. In this model, homophilic interaction between cadherin molecules located on opposing membrane surfaces is mediated by a strand exchange between the two extracellular cadherin 1 (EC1) domains. Cis-interactions (not shown) also contribute to defining the inter-membrane spacing of ~25 nm. The other structures are shown relative to a 20 nm inter-membrane spacing typical of a synaptic cleft [44]. The neurexin 1-β LNS domain structure is oriented so that the loops involved in interactions with neuroligins are oriented away from the membrane. Based on sequence homology, the crystal structure of acetylcholinesterase and the tandem fibronectin type III (FNIII) repeats of Drosophila neuroglian are used here to represent the size of the neuroligin ectodomain and the tandem EphB2 FNIII repeats, respectively. Ephrin-B2, EphB2, β-neurexins and neuroligins also have additional extracellular peptide sequence that cannot be modeled or predicted. Secondary structural elements are coded by color: red, α-helix; orange, 3–10 helix; green, β-sheet; yellow, turn; blue, coil. The structures were visualized using the Visual Molecular Dynamics (VMD) program [142] (http://www.ks.uiuc.edu/Research/vmd/). Protein Databank (PDB) files: C-cadherin (1L3W) [108], neurexin 1-β (1C4R) [143], mouse acetylcholinesterase (1MAA) [144], EphB2–Ephrin-B2 complex (1KGY) [145], Drosophila neuroglian (1CFB) [146].