Glial cell development and function in the Drosophila visual system

In the developing nervous system, building a functional neuronal network relies on coordinating the formation, specification and survival to diverse neuronal and glial cell subtypes. The establishment of neuronal connections further depends on sequential neuron-neuron and neuron-glia interactions that regulate cell-migration patterns and axon guidance. The visual system of Drosophila has a highly regular, retinotopic organization into reiterated interconnected synaptic circuits. It is therefore an excellent invertebrate model to investigate basic cellular strategies and molecular determinants regulating the different developmental processes that lead to network formation. Studies in the visual system have provided important insights into the mechanisms by which photoreceptor axons connect with their synaptic partners within the optic lobe. In this review, we highlight that this system is also well suited for uncovering general principles that underlie glial cell biology. We describe the glial cell subtypes in the visual system and discuss recent findings about their development and migration. Finally, we outline the pivotal roles of glial cells in mediating neural circuit assembly, boundary formation, neural proliferation and survival, as well as synaptic function.     

Assessing the Role of Cell-Surface Molecules in Central Synaptogenesis in the Drosophila Visual System

A hallmark of the central nervous system is its spatial and functional organization in synaptic layers. During neuronal development, axons form transient contacts with potential post-synaptic elements and establish synapses with appropriate partners at specific layers. These processes are regulated by synaptic cell-adhesion molecules. In the Drosophila visual system, R7 and R8 photoreceptor subtypes target distinct layers and form en passant pre-synaptic terminals at stereotypic loci of the axonal shaft. A leucine-rich repeat transmembrane protein, Capricious (Caps), is known to be selectively expressed in R8 axons and their recipient layer, which led to the attractive hypothesis that Caps mediates R8 synaptic specificity by homophilic adhesion. Contradicting this assumption, our results indicate that Caps does not have a prominent role in synaptic-layer targeting and synapse formation in Drosophila photoreceptors, and that the specific recognition of the R8 target layer does not involve Caps homophilic axon-target interactions. We generated flies that express a tagged synaptic marker to evaluate the presence and localization of synapses in R7 and R8 photoreceptors. These genetic tools were used to assess how the synaptic profile is affected when axons are forced to target abnormal layers by expressing axon guidance molecules. When R7 axons were mistargeted to the R8-recipient layer, R7s either maintained an R7-like synaptic profile or acquired a similar profile to r8s depending on the overexpressed protein. When R7 axons were redirected to a more superficial medulla layer, the number of presynaptic terminals was reduced. These results indicate that cell-surface molecules are able to dictate synapse loci by changing the axon terminal identity in a partially cell-autonomous manner, but that presynapse formation at specific sites also requires complex interactions between pre- and post-synaptic elements.     

The function of p120 catenin in filopodial growth and synaptic vesicle clustering in neurons

At the developing neuromuscular junction (NMJ), physical contact between motor axons and muscle cells initiates presynaptic and postsynaptic differentiation. Using Xenopus nerve-muscle cocultures, we previously showed that innervating axons induced muscle filopodia (myopodia), which facilitated interactions between the synaptic partners and promoted NMJ formation. The myopodia were generated by nerve-released signals through muscle p120 catenin (p120ctn), a protein of the cadherin complex that modulates the activity of Rho GTPases. Because axons also extend filopodia that mediate early nerve-muscle interactions, here we test p120ctn's function in the assembly of these presynaptic processes. Overexpression of wild-type p120ctn in Xenopus spinal neurons leads to an increase in filopodial growth and synaptic vesicle (SV) clustering along axons, whereas the development of these specializations is inhibited following the expression of a p120ctn mutant lacking sequences important for regulating Rho GTPases. The p120ctn mutant also inhibits the induction of axonal filopodia and SV clusters by basic fibroblast growth factor, a muscle-derived molecule that triggers presynaptic differentiation. Of importance, introduction of the p120ctn mutant into neurons hinders NMJ formation, which is observed as a reduction in the accumulation of acetylcholine receptors at innervation sites in muscle. Our results suggest that p120ctn signaling in motor neurons promotes nerve-muscle interaction and NMJ assembly.     

In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells

Targeting of axons and dendrites to particular synaptic laminae is an important mechanism by which precise patterns of neuronal connectivity are established. Although axons target specific laminae during development, dendritic lamination has been thought to occur largely by pruning of inappropriately placed arbors. We discovered by in vivo time-lapse imaging that retinal ganglion cell (RGC) dendrites in zebrafish show growth patterns implicating dendritic targeting as a mechanism for contacting appropriate synaptic partners. Populations of RGCs labeled in transgenic animals establish distinct dendritic strata sequentially, predominantly from the inner to outer retina. Imaging individual cells over successive days confirmed that multistratified RGCs generate strata sequentially, each arbor elaborating within a specific lamina. Simultaneous imaging of RGCs and subpopulations of presynaptic amacrine interneurons revealed that RGC dendrites appear to target amacrine plexuses that had already laminated. Dendritic targeting of prepatterned afferents may thus be a novel mechanism for establishing proper synaptic connectivity.     

Myopodia (postsynaptic filopodia) participate in synaptic target recognition

Synaptic partner cells recognize one another by utilizing a variety of molecular cues. Prior to neuromuscular synapse formation, Drosophila embryonic muscles extend dynamic actin-based filopodia called "myopodia." In wild-type animals, myopodia are initially extended randomly from the muscle surface but become gradually restricted to the site of motoneuron innervation, a spatial redistribution we call "clustering." Previous experiments with prospero mutant embryos demonstrated that myopodia clustering does not occur in the absence of motoneuron outgrowth into the muscle field. However, whether myopodia clustering is due to a general signal from passing axons or is a result of the specific interactions between synaptic partners remained to be investigated. Here, we have examined the relationship of myopodia to the specific events of synaptic target recognition, the stable adhesion of synaptic partners. We manipulated the embryonic expression of alphaPS2 integrin and Toll, molecules known to affect synaptic development, to specifically alter synaptic targeting on identified muscles. Then, we used a vital single-cell labeling approach to visualize the behavior of myopodia in these animals. We demonstrate a strong positive correlation between myopodia activity and synaptic target recognition. The frequency of myopodia clustering is lowered in cases where synaptic targeting is disrupted. Myopodia clustering seems to result from the adherence of a subset of myopodia to the innervating growth cone while the rest are eliminated. The data suggest that postsynaptic cells play a dynamic role in the process of synaptic target recognition.     

A role for local calcium signaling in rapid synaptic partner selection by dendritic filopodia

Synapse elimination is an important process underlying the establishment of functional neuronal networks during development. Here, we tested the idea that neurons select among potential synaptic partners already during initial contact formation between dendritic filopodia and axons-well before mature synapses are established. We show that filopodia frequently make contact with axons, and while some contacts are selectively stabilized, many are short-lived. More specifically, we demonstrate that contacts with a certain population of GABAergic axons never get stabilized, indicating that filopodia already early on select between different types of axons. Local dendritic calcium transients that are independent of glutamate occur within seconds after contact formation, and their frequency is high where contacts become stabilized and low at short-lived contacts. Thus, filopodia are capable of choosing between potential synaptic partners well before a mature synapse is established.     

Close encounters: how cortical neurons find and connect to their correct synaptic partners depends on the cell type

In this issue of Neuron, Stepanyants et al. embark on quantitative analyses of the axonal and dendritic arbors of cortical neurons. They test whether the paths taken by axons actually bring them closer to their true synaptic partners than they would have gotten by chance encounters based on the same axon trajectories. The results depend on the type of presynaptic neuron. Inhibitory cells take paths that explicitly link them to their actual partners while excitatory axons are as likely to have close encounters with actual partners as with other neurons. These results suggest that the mechanisms for selectivity depend on the type of presynaptic neuron.     

Axon branch removal at developing synapses by axosome shedding

In many parts of the developing nervous system, the number of axonal inputs to each postsynaptic cell is dramatically reduced. This synapse elimination has been extensively studied at the neuromuscular junction, but how axons are lost is unknown. Here, we combine time-lapse imaging of fluorescently labeled axons and serial electron microscopy to show that axons at neuromuscular junctions are removed by an unusual cellular mechanism. As axons disappear, they shed numerous membrane bound remnants. These "axosomes" contain a high density of synaptic organelles and are formed by engulfment of axon tips by Schwann cells. After this engulfment, the axosome's contents mix with the cytoplasm of the glial cell. Axosome shedding might underlie other forms of axon loss and may provide a pathway for interactions between axons and glia.     

Binding partners for the myelin-associated glycoprotein of N2A neuroblastoma cells

The myelin-associated glycoprotein (MAG) has been proposed to be important for the integrity of myelinated axons. For a better understanding of the interactions involved in the binding of MAG to neuronal axons, we performed this study to identify the binding partners for MAG on neuronal cells. Experiments with glycosylation inhibitors revealed that sialylated N-glycans of glycoproteins represent the major binding sites for MAG on the neuroblastoma cell line N2A. From extracts of [3H]glucosamine-labelled N2A cells several glycoproteins with molecular weights between 20 and 230 kDa were affinity-precipitated using immobilised MAG. The interactions of these proteins with MAG were sialic acid-dependent and specific for MAG.     

Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase

Activity-dependent and -independent signals collaborate to regulate synaptogenesis, but their relative contributions are unclear. Here, we describe the formation of neuromuscular synapses at which neurotransmission is completely and specifically blocked by mutation of the neurotransmitter-synthesizing enzyme choline acetyltransferase. Nerve terminals differentiate extensively in the absence of neurotransmitter, but neurotransmission plays multiple roles in synaptic differentiation. These include influences on the numbers of pre- and postsynaptic partners, the distribution of synapses in the target field, the number of synaptic sites per target cell, and the number of axons per synaptic site. Neurotransmission also regulates the formation or stability of transient acetylcholine receptor-rich processes (myopodia) that may initiate nerve-muscle contact. At subsequent stages, neurotransmission delays some steps in synaptic maturation but accelerates others. Thus, neurotransmission affects synaptogenesis from early stages and coordinates rather than drives synaptic maturation.     

Glial cells mediate target layer selection of retinal axons in the developing visual system of Drosophila

In the fly visual system, each class of photoreceptor neurons (R cells) projects to a different synaptic layer in the brain. R1-R6 axons terminate in the lamina, while R7 and R8 axons pass through the lamina and stop in the medulla. As R cell axons enter the lamina, they encounter both glial cells and neurons. The cellular requirement for R1-R6 targeting was determined using loss-of-function mutations affecting different cell types in the lamina. nonstop (encoding a ubiquitin-specific protease) is required for glial cell development and hedgehog for neuronal development. Removal of glial cells but not neurons disrupts R1-R6 targeting. We propose that glial cells provide the initial stop signal promoting growth cone termination in the lamina. These findings uncover a novel function for neuron-glial interactions in regulating target specificity.     

Single-cell analysis of Drosophila larval neuromuscular synapses

The neuromuscular system of Drosophila has been widely used in studies on synaptic development. In the embryo, the cellular components of this model system are well established, with uniquely identified motoneurons displaying specific connectivity with distinct muscles. Such knowledge is essential to analyzing axon guidance and synaptic matching mechanisms with single-cell resolution. In contrast, to date the cellular identities of the larval neuromuscular synapses are hardly established. It is not known whether synaptic connections seen in the embryo persist, nor is it known how individual motor endings may differentiate through the larval stages. In this study, we combine single-cell dye labeling of individual synaptic boutons and counterstaining of the entire nervous system to characterize the synaptic partners and bouton differentiation of the 30 motoneuron axons from four nerve branches (ISN, SNa, SNb, and SNd). We also show the cell body locations of 4 larval motoneurons (RP3, RP5, V, and MN13-Ib) and the types of innervation they develop. Our observations support the following: (1) Only 1 motoneuron axon of a given bouton type innervates a single muscle, while up to 4 motoneuron axons of different bouton types can innervate the same muscle. (2) The type of boutons which each motoneuron axon forms is likely influenced by cell-autonomous factors. The data offer a basis for studying the properties of synaptic differentiation, maintenance, and plasticity with a high cellular resolution.     

Competitive interactions between retinal ganglion cells during prenatal development

In the mammalian visual system, retinal ganglion cell axons terminate within the LGN in a series of alternating eye-specific layers. These layers are not present initially during development. In the cat they emerge secondarily following a prenatal period in which originally intermixed inputs from the two eyes gradually segregate from each other to give rise to the characteristic set of layers by birth. Many lines of evidence suggest that activity-dependent competitive interactions between ganglion cell axons from the two eyes for LGN neurons play an important role in the final patterning of retinogeniculate connections. Studies of the branching patterns of individual ganglion cell axons suggest that during the period when inputs from the two eyes are intermixed, axons from one eye send side branches into territory later occupied exclusively by axons from the other eye. Ultrastructural studies indicate that these branches in fact are sites of synaptic contacts, which are later eliminated since the side branches disappear as axons form their mature terminal arbors in appropriate territory. In vitro microelectrode recordings from LGN neurons indicate that they can receive convergent synaptic excitation from electrical stimulation of the optic nerves before but not after the eye-specific layers form, suggesting that at least some of the synaptic contacts seen at the ultrastructural level are functonal. Finally, experiments in which tetrodotoxin was infused intracranially during the two week period during which the eye-specific layers normally form demonstrate that it is possible to prevent, or at least delay, the formation of the layers. Accordingly, individual axons fail to develop their restricted terminal arbor branching pattern and instead branch widely throughout the LGN. These results indicate that all of the machinery necessary for synaptic function and competition is present during fetal life. Moreover, it is highly likely that neuronal activity is required for the formation of the eye-specific layers. If so, then activity would have to be present in the form of spontaneously generated action potentials, since vision is not possible at these early ages. Thus, the functioning of the retinogeniculate system many weeks before it is put to the use for which it is ultimately designed may contribute to the final patterning of connections present in the adult.     

Reversing the outcome of synapse elimination at developing neuromuscular junctions in vivo: evidence for synaptic competition and its mechanism

During mammalian development, neuromuscular junctions and some other postsynaptic cells transition from multiple- to single-innervation as synaptic sites are exchanged between different axons. It is unclear whether one axon invades synaptic sites to drive off other inputs or alternatively axons expand their territory in response to sites vacated by other axons. Here we show that soon-to-be-eliminated axons rapidly reverse fate and grow to occupy vacant sites at a neuromuscular junction after laser removal of a stronger input. This reversal supports the idea that axons take over sites that were previously vacated. Indeed, during normal development we observed withdrawal followed by takeover. The stimulus for axon growth is not postsynaptic cell inactivity because axons grow into unoccupied sites even when target cells are functionally innervated. These results demonstrate competition at the synaptic level and enable us to provide a conceptual framework for understanding this form of synaptic plasticity.     

Temporal target restriction of olfactory receptor neurons by Semaphorin-1a/PlexinA-mediated axon-axon interactions

Axon-axon interactions have been implicated in neural circuit assembly, but the underlying mechanisms are poorly understood. Here, we show that in the Drosophila antennal lobe, early-arriving axons of olfactory receptor neurons (ORNs) from the antenna are required for the proper targeting of late-arriving ORN axons from the maxillary palp (MP). Semaphorin-1a is required for targeting of all MP but only half of the antennal ORN classes examined. Sema-1a acts nonautonomously to control ORN axon-axon interactions, in contrast to its cell-autonomous function in olfactory projection neurons. Phenotypic and genetic interaction analyses implicate PlexinA as the Sema-1a receptor in ORN targeting. Sema-1a on antennal ORN axons is required for correct targeting of MP axons within the antennal lobe, while interactions amongst MP axons facilitate their entry into the antennal lobe. We propose that Sema-1a/PlexinA-mediated repulsion provides a mechanism by which early-arriving ORN axons constrain the target choices of late-arriving axons.     

Neurexins and Neuroligins: Recent Insights from Invertebrates

During brain development, each neuron must find and synapse with the correct pre- and postsynaptic partners. The complexity of these connections and the relatively large distances some neurons must send their axons to find the correct partners makes studying brain development one of the most challenging, and yet fascinating disciplines in biology. Furthermore, once the initial connections have been made, the neurons constantly remodel their dendritic and axonal arbours in response to changing demands. Neurexin and neuroligin are two cell adhesion molecules identified as important regulators of this process. The importance of these genes in the development and modulation of synaptic connectivity is emphasised by the observation that mutations in these genes in humans have been associated with cognitive disorders such as Autism spectrum disorders, Tourette syndrome and Schizophrenia. The present review will discuss recent advances in our understanding of the role of these genes in synaptic development and modulation, and in particular, we will focus on recent work in invertebrate models, and how these results relate to studies in mammals.     

Axon navigation in the mammalian primary olfactory pathway: where to next?

The process of establishing long-range neuronal connections can be divided into at least three discrete steps. First, axons need to be stimulated to grow and this growth must be towards appropriate targets. Second, after arriving at their target, axons need to be directed to their topographically appropriate position and in some cases, such as in cortical structures, they must grow radially to reach the correct laminar layer. Third, axons then arborize and form synaptic connections with only a defined subpopulation of potential post-synaptic partners. Attempts to understand these mechanisms in the visual system have been ongoing since pioneer studies in the 1940s highlighted the specificity of neuronal connections in the retino-tectal pathway. These classical systems-based approaches culminated in the 1990s with the discovery that Eph-ephrin repulsive interactions were involved in topographical mapping. In marked contrast, it was the cloning of the odorant receptor family that quickly led to a better understanding of axon targeting in the olfactory system. The last 10 years have seen the olfactory pathway rise in prominence as a model system for axon guidance. Once considered to be experimentally intractable, it is now providing a wealth of information on all aspects of axon guidance and targeting with implications not only for our understanding of these mechanisms in the olfactory system but also in other regions of the nervous system.     

Development of cell type-specific connectivity patterns of converging excitatory axons in the retina

To integrate information from different presynaptic cell types, dendrites receive distinct patterns of synapses from converging axons. How different afferents in?vivo establish specific connectivity patterns with the same dendrite is poorly understood. Here, we examine the synaptic development of three glutamatergic bipolar cell types converging onto?a common postsynaptic retinal ganglion cell. We find that after axons and dendrites target appropriate synaptic layers, patterns of connections among these neurons?diverge through selective changes in the conversion of axo-dendritic appositions to synapses. This process is differentially regulated by neurotransmission, which is required for the shift from single to multisynaptic appositions of one bipolar cell type but not for maintenance and elimination, respectively, of connections from the other two types. Thus, synaptic specificity among converging excitatory inputs in the?retina emerges via differential synaptic maturation of axo-dendritic appositions and is shaped by neurotransmission in a cell type-dependent manner.