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It is estimated that there are 10¹¹ neurons in the human brain, connected by 10¹⁴ synapses. The connectivity of a neuron (its unique constellation of synaptic inputs and outputs) is essential for its function. A large body of anatomical and physiological evidence demonstrates that neuronal connections are made with exquisite accuracy between specific types of neurons, often within certain subcellular compartments (1). How each neuron finds its synaptic partners has been a central question in developmental neurobiology.

Neuronal connections are specified in stepwise fashion during a series of developmental stages. Neuronal cell fate determination, migration, and axon guidance establish the long-range axon projections of the nervous system and form a blueprint for the connectivity map (2). After reaching the target field, however, individual axons are surrounded by dendrites from both synaptic targets and nontarget neurons. Neurons are somehow capable of distinguishing between correct and incorrect targets and forming selective connections only with appropriate synaptic partners. It is not yet understood how this specificity is achieved during development; whether genetically encoded or experience-driven mechanisms are more important in the mutual recognition of pre- and postsynaptic partners. It has long been hypothesized, beginning with Roger Sperry’s famous “eye rotation experiment,” that there might be “chemoaffinity tags” present on the membranes of synaptic partners that determine connectivity (3). This implies that synaptic choice is a hard-wired decision made through direct interaction between pre- and postsynaptic cells. Unlike the well-established chemoaffinity gradients of ephrins and Eph receptors in the topographic mapping of retinal axons (4), the chemoaffinity tags on individual neurons that directly participate in synapse formation have not been definitively identified.

The nematode worm Caenorhabditis elegans, with its well-characterized nervous system and genetics, provides a potentially powerful system in which to identify chemoaffinity tags in synapse formation. Through labeling and studying a small set of worm synapses, we identified a pair of transmembrane immunoglobulin superfamily proteins, SYG-1 and SYG-2, which serve as a chemoaffinity receptor and ligand to specify synaptic connections through heterophilic interactions. Our results also indicate that synaptic specificity is not a hard-wired decision between synaptic partners but is rather a competition between a variety of targets that certain targets invariably win. SYG-1 and SYG-2 act to tip the balance of this competition and thus generate synaptic specificity. We have also discovered that cells other than pre- and postsynaptic neurons can play essential roles in determining the specificity of connections.

The two motor neurons involved in egg laying, HSNL and HSNR, form synapses onto vulval muscle cells and onto the motor neurons VC4 and VC5. Although HSN axons contact many other cells, they do not normally form synapses onto them (5, 6). By studying the development of the HSNL synapses, we discovered that a group of epithelial cells play an essential guidepost role for HSNL synaptogenesis. These guidepost cells contact the HSNL axon and induce the clustering of synaptic vesicles at the site of contact, shortly before the normal postsynaptic targets are innervated. In the absence of guidepost cells, clusters of HSNL synaptic vesicles accumulate at ectopic locations (see the figure).

Anatomy of the egg-laying circuit. The HSNL axon travels ventrally and then anteriorly around the vulval, making en passant synapses with VC4,5 and vulval muscle near the guidepost vulval epithelial cells. Anterior ectopic synaptic vesicle clusters form in syg-1 and syg-2 mutant animals.

In this sytem, SYG-1 and SYG-2 bind to each other and function as receptor and ligand, respectively, in specifying HSNL synaptic connections. SYG-2 is expressed transiently by the guidepost cells during the early stages of HSNL synaptogenesis. SYG-1 functions in the presynaptic HSNL neuron and localizes to synapses early during synapse formation. In loss-of-function syg-1 and syg-2 mutants, the HSNL axon fails to form synaptic connections with its normal targets and instead forms synapses to adjacent cells that do not normally receive synaptic input from HSNL (7, 8). When SYG-2 is expressed in the secondary vulval epithelial cells, which are located next to the guidepost cells and do not normally express SYG-2, both SYG-1 and synaptic vesicles localize to the segment of the HSNL axon that contacts the secondary vulval epithelial cells. This gain-of-function result supports the idea that interactions between SYG-1 and SYG-2 are sufficient to trigger synaptic vesicle clustering (8).

This study provides evidence that synaptic specificity can be encoded by molecular cues. Both SYG-1 and SYG-2 are conserved throughout evolution. Interestingly, their homologs in Drosophila, SNS/Hibris (for SYG-1) and DUF/Kirre (for SYG-2), play essential functions in cell-cell recognition between muscle founder cells and fusion-competent cells (9, 10). The human homologs, NEPH1 and Nephrin, are indispensable for the formation of the slit diaphragm, a cellular junction in the kidney that functions as a molecular filter (11-13). In all three cases, these immunoglobulin superfamily proteins mediate recognition between contacting cells during development. Nature has connected these recognition modules to different downstream molecular pathways to achieve diverse biological functions.

In addition, we found, unexpectedly, that cells other than the pre- and postsynaptic neurons can play instructive roles during synaptogenesis. The concept of guidepost cells is well established in axon guidance (14). However, examples of synaptic guidepost cells in vertebrates are rare and poorly characterized (15-17). SYG-2 is the first molecular cue associated with these transient targets to be identified. Temporal discrepancies between the differentiation of the pre- and postysynaptic cells are frequently encountered in neural development. Guidepost cells appear to be particularly important for synaptogenesis under these circumstances.

Finally, our studies reveal that the choice of a synaptic partner is not an absolutely hard-wired decision. Among the cells contacting a given axon in the target field, many cells are capable of forming synapses with the axon. Certain targets are favored during development, which is suggestive of a hierarchy among the potential targets. When preferred targets fail to form connections, synapses then form with alternative choices.

Many questions remain unanswered. SYG-1 and SYG-2 are obviously not the solution to establishing synaptic specificity throughout the entire central nervous system. Do similar membrane molecules specify other synapses? Synapses outnumber genes in our genome by 10¹⁰. How are such a large number of connections specified by a limited number of genes? How do molecules that underlie specificity build functional synapses? All of these questions must be answered before we have a complete understanding of how the brain is wired.

References

1. D. L. Benson, D. R. Colman, G. W. Huntley, Nature Rev. Neurosci. 2, 899 (2001).
2. T. M. Jessell, J. R. Sanes, Curr. Opin. Neurobiol. 10, 599 (2000).
3. R. W. Sperry, Proc. Natl. Acad. Sci. U.S.A. 50, 703 (1963).
4. J. G. Flanagan, P. Vanderhaeghen, Annu. Rev. Neurosci. 21, 309 (1998).
5. J. G. White, E. Southgate, J. N. Thomson, S. Brenner, Philos. Trans. R. Soc. London Ser. B 275, 327 (1976).
6. J. G. White, E. Southgate, J. N. Thomson, S. Brenner, Philos. Trans. R. Soc. London Ser. B 314, 1 (1986).
7. K. Shen, R. D. Fetter, C. I. Bargmann, Cell 116, 869 (2004).
8. K. Shen, C. I. Bargmann, Cell 112, 619 (2003).
9. M. Ruiz-Gomez, N. Coutts, A. Price, M. V. Taylor, M. Bate, Cell 102, 189 (2000).
10. M. Strunkelnberg et al., Development 128, 4229 (2001).
11. D. B. Donoviel et al., Mol. Cell Biol. 14, 4829 (2001).
12. H. Putaala, R. Soininen, P. Kilpelainen, J. Wartiovaara, K. Tryggvason, Hum. Mol. Genet. 10, 1 (2001).
13. J. Khoshnoodi, K. Tryggvason, Exp. Nephrol. 9, 355 (2001).
14. T. P. O'Connor, Cell. Mol. Life Sci 55, 1358 (1999).
15. K. L. Allendoerfer, C. J. Shatz, Annu. Rev. Neurosci. 17, 185 (1994).
16. J. A. Del Rio et al., Nature 385, 70 (1997).
17. H. Super, A. Martinez, J. A. Del Rio, E. Soriano, J. Neurosci. 18, 4616 (1998).

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