Chapter 2 Synapse Formation in Developing Neural Circuits

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Abstract

The nervous system consists of hundreds of billions of neurons interconnected into the functional neural networks that underlie behaviors. The capacity of a neuron to innervate and function within a network is mediated via specialized cell junctions known as synapses. Synapses are macromolecular structures that regulate intercellular communication in the nervous system, and are the main gatekeepers of information flow within neural networks. Where and when synapses form determines the connectivity and functionality of neural networks. Therefore, our knowledge of how synapse formation is regulated is critical to our understanding of the nervous system and how it goes awry in neurological disorders.

Synapse formation involves pairing of the pre‐ and postsynaptic partners at a specific neurospatial coordinate. The specificity of synapse formation requires the precise execution of multiple developmental events, including cell fate specification, cell migration, axon guidance, dendritic growth, synaptic target selection, and synaptogenesis (Juttner and Rathjen in Cell. Mol. Life Sci. 62:2811, 2005; Salie et al., in Neuron 45:189, 2005; Waites et al., in Annu. Rev. Neurosci. 28:251, 2005). Remarkably, during the development of the vertebrate nervous system, these developmental processes occur almost simultaneously in billions of neurons, resulting in the formation of trillions of synapses. How this remarkable specificity is orchestrated during development is one of the outstanding questions in the field of neurobiology, and the focus of discussion of this chapter.

We center the discussion of this chapter on the early developmental events that orchestrate the process of synaptogenesis prior to activity‐dependent mechanisms. We have therefore limited the discussion of important activity‐dependent synaptogenic events, which are discussed in other chapters of this book. Moreover, our discussion is biased toward lessons we have learned from invertebrate systems, in particular from C. elegans and Drosophila. We did so to complement the discussions from other chapters in this book, which focus on the important findings that have recently emerged from the vertebrate literature.

The chapter begins with a brief history of the field of synaptic biology. This serves as a backdrop to introduce some of the historically outstanding questions of synaptic development that have eluded us during the past century, and which are the focus of this review. We then discuss some general features of synaptic structure as it relates to its function. In particular, we will highlight evolutionarily conserved traits shared by all synaptic structures, and how these features have helped optimize these ancient cellular junctions for interneural communication.

We then discuss the regulatory signals that orchestrate the precise assembly of these conserved macromolecular structures. This discussion will be framed in the context of the neurodevelopmental process. Specifically, much of our discussion will focus on how the seemingly disparate developmental processes are intimately linked at a molecular level, and how this relationship might be crucial in the developmental orchestration of circuit assembly. We hope that the discussion of the multifunctional cues that direct circuit development provides a conceptual framework into understanding how, with a limited set of signaling molecules, precise neural wiring can be coordinated between synaptic partners.

Introduction

The history of synapse biology starts at the end of the eighteenth century, with the studies of Luigi Galvani and his descriptions of “animal electricity.” In these classical studies, Galvani observed that he could induce the contraction of limb muscles when he inserted a metal hook into the medulla of the frog and attached the other end to an iron railing. These observations marked the first experimental demonstrations of synaptic transmission (Cowan and Kandel, 2001).

Most of the subsequent synaptic studies in the nineteenth century and earlier half of the twentieth century also focused around the functionality of synapses, or synaptic transmission. It is therefore befitting that the actual term “synapse” was not coined by a neuroanatomist, but by a physiologist named Charles Sherrington. Sherrington coined the term “synapse” to refer to the special connections from one nerve cell to another that facilitated the transmission of nervous impulses (Cowan and Kandel, 2001).

While physiologists and neuropharmacologists were functionally defining the concept of synapses, neuroanatomists tangled in a bitter debate on their existence. The main reason for this debate was that during the nineteenth century and earlier part of the twentieth century, nobody could visualize cell membranes and establish conclusively the existence of synapses. However, in spite of these technological limitations, some insightful neurobiologists garnered enough experimental evidence to propose the anatomical existence of synapses.

Most of these early observations came from a specialized synapse: the neuromuscular junction (NMJ). Because of its size, morphology and functional readouts, NMJs informed then, as they do now, most of our knowledge on synaptic biology. Taking advantage of this system, physiologist Willy Kühne and anatomist Wilhelm Krause independently hypothesized the existence of synapses at the site of contact between nerve cells and muscles (Cowan and Kandel, 2001).

The question of the existence of interneuronal synapses was much harder to settle. Synapses in the central nervous system are much smaller than NMJs, in closed apposition to one another and packed at very high densities. This made their visualization with the methods used during nineteenth century downright impossible and triggered the postulation of the “reticular theory”: the idea that the nervous system lacked functional separation of nerve cells and was syncytial, rather than synaptic, in nature (Cowan and Kandel, 2001, Westfall, 1996).

The theory turned out, of course, to be wrong. Although this was not conclusively shown until the advent of electron microscopy in the 1950s, the first evidence that neurons were discrete units came from developmental, pathological, and anatomical observations in the nineteenth century. Most notable among these early studies are Santiago Ramón y Cajal's. By using a method derived by Golgi, which stains only 1% of the cells, Cajal was able to visualize the morphology of individual cells in the context of the nervous system. His detailed characterization of neurons not only provided critical evidence for the neuron doctrine, but also stated the “Principios de la Especificidad de la Conexión”: the idea that nerve cells connect to each other in a specific fashion to form precise networks (Cowan and Kandel, 2001).

Although it would take another half a century for cell biologists to visualize synapses, Cajal's observations and insights at the turn of the nineteenth century provided the conceptual basis that has driven most of the neurodevelopmental questions since then. Over a century after Cajal's initial descriptions, we are still untangling the complex morass that is the central nervous system and tackling the questions staged by his landmark observations: How are the numerous cell types in the nervous system specified? What directs neurites to connect to each other? What are the cellular and molecular factors that underlie the “Principles of connection specificity”?

During the last century however, and thanks in great part to technical advances in cell biology, the field has made great progress in its understanding of the synaptic structure as it relates to synaptic function. Most notably, electron microscopy allowed the visualization of synapses for the first time in the 1950s. This work, spearheaded by George Palade and Keith Porter, provided unequivocal evidence for the neuron doctrine and the existence of synapses, and identified the different types of synapses and their structural components (Cowan and Kandel, 2001, De Camilli et al., 2001).

There are two general categories of synapses: electrical synapses and chemical synapses. Physiologists and neuropharmacologists functionally defined these two categories of synapses well before they were visualized by cell biologists (Cowan and Kandel, 2001). But the cell biological work that proceeded from the physiological studies demonstrated that these two functional categories corresponded to completely different structures. Electrical synapses are gap junctions that allow bidirectional propagation of signals, including electrical stimuli. They allow the fastest mode of electrical propagation across cells, and are now known to be important in synchronizing neural activity across networks (De Camilli et al., 2001). These gap junctions will not be further discussed in this chapter.

Chemical synapses allow communication between discontinuous neurons via the highly regulated secretion of chemical intermediate signals. Unlike electrical synapses, chemical synapses are polarized junctions that allow the flow of information in just a single direction. Because of their highly regulated and directional transfer of information, chemical synapses have been the focus of most of the synaptic biology studies, and as such will remain the focus of our chapter.

Although there is great morphological and molecular variability among chemical synapses, all chemical synapses share common structural and functional features (De Camilli et al., 2001). They consist of two asymmetrically juxtaposed components linking two separate cells: a presynaptic specialization and a postsynaptic region. The presynaptic specializations are specialized regions in the presynaptic cell with an abundance of neurotransmitter‐filled synaptic vesicles. Presynaptic specializations also contain the active zone structures that facilitate vesicle fusion and the release of neurotransmitter content to the intersynaptic space, called the synaptic cleft. The postsynaptic region is an area of the postsynaptic cell with a high concentration of neurotransmitter receptors, channels, and downstream signaling molecules. The neurotransmitters released by the presynaptic specializations are sensed by the receptors at the postsynaptic site, activating downstream signaling molecules, opening channels, and propagating the nervous impulse to the postsynaptic partner. These general features of the presynaptic and postsynaptic specializations are shared by all classes of synaptic structures.

The synaptic structure as described above is also very well conserved across evolution. Sea anemones and hydra (Phylum Cnidaria) have the most primitive nervous system, which consists of a diffuse network of neurons. These nerve nets, however, are connected via chemical and electrical synapses that are fully capable of transmitting and regulating information flow (Anderson and Spencer, 1989, Peteya, 1973, Westfall, 1996). Close inspection of these synaptic structures reveal that Cnidarian synapses have similar structural components as those of higher organisms, with defined presynaptic and postsynaptic specializations in close juxtaposition (Anderson and Spencer, 1989, Peteya, 1973, Westfall, 1996).

The presence of a conserved synaptic structure in these primitive nervous systems reveals that synapses are as ancient as the nervous system itself. This evolutionary conservation of the synaptic structure also underscores the importance of these specialized cell junctions in interneuronal communication and the functioning of the neural networks (Anderson and Spencer, 1989, Peteya, 1973, Westfall, 1996).

Interestingly, a recent study suggests that the evolution of the synaptic molecular machine might even precede the evolution of the nervous system (Sakarya et al., 2007). Although sponges (Phylum Porifera) are the only metazoans without a nervous system, it was found that sponges express a nearly complete set of postsynaptic protein homologues that are hypothesized to assemble into synaptic‐like scaffolds. Although sponges do not have neurons, these postsynaptic‐like structures are hypothesized to act as chemosensory structures capable of responding to environmental cues (Sakarya et al., 2007).

Other molecular components of the presynaptic machine, such as the synaptic vesicle cycle regulators, also predate the existence of the nervous system and are very well conserved across evolution (Sudhof, 2004). It is provocative that these macromolecular machines, presumable “building blocks” of the synapse, might be found even in the absence of a nervous system itself, an observation that underscores the importance and conservation of these signaling complexes throughout evolution (Sakarya et al., 2007).

Molecular and genetic studies in model invertebrate and vertebrate animals have also supported the notion that the ultrastructural conservation of synapses corresponds to a conservation at the molecular level. For instance, in the simple nervous system of the nematode Caenorhabditis elegans, which consists of only 302 neurons, the number of neurotransmitters and receptors required for the proper functioning of its ∼5000 synapses approaches in complexity those used by the hundreds of trillions of synapses in the vertebrate nervous system (Rand and Nonet, 1997).

This suggests that throughout evolution, the increased capacity of information processing and storage observed in higher organisms is not the result of marked changes in the complexity of the synaptic structure. We speculate that this complexity results from an increasingly sophisticated neural framework in way of the abundance and organization of neural networks. Where, when, and how synapses form during development play critical roles on the wiring and function of neural networks. Although the neural network organization varies vastly across animals, the biological basis of synapses is shared from the simplest networks of Cnidaria to the complex neuropils of the human brain.

Section snippets

Synaptogenesis During Development

The organization of where, when, and how synapses are formed plays an instrumental role in directing the connectivity of circuits and organizing the neuroarchitecture that enables information processing, storage, and ultimately behaviors. As such, the developmental questions postulated by Cajal in his “Principles of connection specificity” are of great importance to our understanding of the assembly and function of the nervous system. What are the molecular and cellular factors that direct the

Building a Synapse

The process of guidance and target recognition is followed by synapse formation. How synapses are assembled is a formidable developmental question in its own right. As discussed previously, synapses need to form onto the right partner, at the right density, and at a specific subcellular location with respect to the dendrites. Moreover, the assembly of presynaptic sites also needs to match the postsynaptic densities in terms of localization and identity of the neurotransmitter and postsynaptic

Perspective

Correct circuit formation requires an intricate orchestration of multiple developmental events including cell migration, axon guidance, dendritic growth, target selection, and synaptogenesis (Juttner and Rathjen, 2005, Salie et al., 2005, Waites et al., 2005). These events are integrated to enable correct synapse formation between neuronal partners. The developmental innervation of synaptic partners results in hundreds of trillions of precisely wired synaptic connections. Since the human genome

Acknowledgments

I thank M. Hammarlund, S. Margolis, M. Margeta, and G. Maro for thoughtful comments concerning this chapter. I particularly thank K. Shen for helpful discussions, generous advice, and thoughtful comments on the chapter. I also thank J. Blagburn, V. Poon, and F. Ango for contributing images. I apologize to those whose work I did not cite here due to oversight or space constrains. During the preparation of this chapter, I was supported by NIH grant 4R00NS057931‐03.

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