The ultrastructural interactions of identified pre- and postsynaptic cells during synaptic target recognition in Drosophila embryos

During the development of neural networks, what sets synaptogenic interactions apart from nonsynaptogenic interactions is not well understood at the subcellular level. Using a combination of intracellular dye injection and electron microscopy, we show that a specific motoneuron (RP3) and its synaptic partners (muscles 6 and 7), both often bearing microprocesses, develop intimate membrane contact sites characterized by junctional structures, prior to their initiating synaptogenesis in Drosophila embryos. Other motoneuron growth cones that extend alongside the RP3 growth cone to innervate surrounding muscles do not form such contacts with muscles 6 and 7. We also examined how specific target recognition molecules affect the development of these ultrastructural associations between synaptic partner cells. When Fasciclin III (Fas3), a "positive" target recognition molecule for RP3, is ectopically expressed in neighboring muscles, the RP3 growth cone ectopically develops membrane contact sites with Fas3-misexpressing muscles with which it would not normally associate. In contrast, when Toll, a "negative" target recognition molecule normally expressed by a subset of muscles that surrounds muscles 6 and 7, is misexpressed on muscles 6 and 7, the RP3 growth cone fails to exhibit its normal close contact with these muscles. We propose that the formation of close membrane associations and junctional structures can be regulated under the influence of synaptic target recognition molecules and signifies the beginning of subcellular events during synaptic target recognition.     

The Drosophila SH2-SH3 adapter protein Dock is expressed in embryonic axons and facilitates synapse formation by the RP3 motoneuron

The Dock SH2-SH3 domain adapter protein, a homolog of the mammalian Nck oncoprotein, is required for axon guidance and target recognition by photoreceptor axons in Drosophila larvae. Here we show that Dock is widely expressed in neurons and at muscle attachment sites in the embryo, and that this expression pattern has both maternal and zygotic components. In motoneurons, Dock is concentrated in growth cones. Loss of zygotic dock function causes a selective delay in synapse formation by the RP3 motoneuron at the cleft between muscles 7 and 6. These muscles often completely lack innervation in late stage 16 dock mutant embryos. RP3 does form a synapse later in development, however, because muscles 7 and 6 are normally innervated in third-instar mutant larvae. The absence of zygotically expressed Dock also results in subtle defects in a longitudinal axon pathway in the embryonic central nervous system. Concomitant loss of both maternally and zygotically derived Dock dramatically enhances these central nervous system defects, but does not increase the delay in RP3 synaptogenesis. These results indicate that Dock facilitates synapse formation by the RP3 motoneuron and is also required for guidance of some interneuronal axons The involvement of Dock in the conversion of the RP3 growth cone into a presynaptic terminal may reflect a role for Dock-mediated signaling in remodeling of the growth cone's cytoskeleton.     

Early ablation of target muscles modulates the arborisation pattern of an identified embryonic Drosophila motor axon.

The Drosophila RP3 motor axon establishes a stereotypic arborisation along the adjoining edges of muscles 6 and 7 by the end of embryogenesis. The present study has examined the role of the target muscles in determining this axonal arborisation pattern. Target muscles were surgically ablated prior to the arrival of the RP3 axon. Following further development of the embryo in culture medium, the morphology of target-deprived RP3 motor axons was assayed by intracellular injection with the dye Lucifer Yellow. Axonal arborisations were formed on a variety of non-target muscles when muscles 6 and 7 were removed and these contacts were maintained into stage 16. The pattern of axonal arborisations over non-target muscles varied between preparations in terms of the number of muscles contacted, and the distribution of arborisations on individual muscles. Following removal of muscle 6, the RP3 motor axon frequently contacted muscle 7, and axonal arborisations were present along the distal edge of the muscle. In the absence of muscle 7, the RP3 axon often did not contact muscle 6 and when muscle 6 was contacted, the arborisation of RP3 was poorly developed. Axonal processes were retained on non-target muscles when only one target muscle was present. Therefore, the establishment of a stereotypic arborisation by the RP3 motor axon is apparently dependent on growth cone contact with both target muscles.     

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.     

“Painting” the target: how local molecular cues define synaptic relationships

A majority of studies on neuronal growth cones focus on the features that particular groups of neurons share. In contrast, questions such as how specific growth cones respond very differently to the same extrinsic cues require cell-specific experimentation. The most succinct cell-specific growth cone responses occur during synaptic targeting. Recent studies have examined one specific growth cone, the Drosophila RP3 motoneuron growth cone, in variously altered microenvironments. In this review, we summarize how such studies are beginning to uncover the repertoire of extrinsic cues that influence the synaptic targeting of a single growth cone. BioEssays 20:941–948, 1998. © 1998 John Wiley & Sons, Inc.     

Identification of common excitatory motoneurons in Drosophila melanogaster larvae

In insects, four types of motoneurons have long been known, including fast motoneurons, slow motoneurons, common inhibitory motoneurons, and DUM neurons. They innervate the same muscle and control its contraction together. Recent studies in Drosophila have suggested the existence of another type of motoneuron, the common excitatory motoneuron. Here, we found that shakB-GAL4 produced by labels this type of motoneuron in Drosophila larvae. We found that Drosophila larvae have two common excitatory motoneurons in each abdominal segment, RP2 for dorsal muscles and MNSNb/d-Is for ventral muscles. They innervate most of the internal longitudinal or oblique muscles on the dorsal or ventral body wall with type-Is terminals and use glutamate as a transmitter. Electrophysiological recording indicated that stimulation of the RP2 axon evoked excitatory junctional potential in a dorsal muscle.     

Axon pathfinding proceeds normally despite disrupted growth cone decisions at CNS midline

Axons in the bilateral brain of Drosophila decide whether or not to cross the midline before following their specific subsequent pathways. In commissureless mutants, the RP3 and V motoneuron axons often fail to cross the midline but subsequently follow the mirror-image pathways and innervate corresponding muscle targets on the ipsilateral side. Conversely, in roundabout mutants, the RP2 and aCC motoneuron axons sometimes cross the midline abnormally but their subsequent pathways and synaptic targeting are the perfect mirror images of those seen in wild type. Furthermore, within a single segment of these mutants, bilateral pairs of motoneuron axons can make their midline decisions independently of each other. Thus, neither the growth cones' particular molecular experience nor the decision at the midline caused by these mutations affects their ability to respond normally to subsequently presented cues.     

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.     

Fine structural analysis of membrane changes during reaggregation culture of chick optic tectum

Thin section and freeze-fracture electron microscopy have been used to characterize the changes in membrane morphology of reaggregating cultures of chick optic tectum. The cells are rounded and freely dispersed at 0 hr after dissociation. Between 2 and 6 hr the cells become closely apposed on all sides by other cells and form small aggregates. At this time punta adhaerentia junctions and focal densities are seen along the membranes of neighboring cells. Between 1 and 5 days in vitro (DIV) neurites containing growth cone regions are present. At 5 DIV the first synaptic contacts are observed. Between 7 and 14 DIV, the number of synaptic contacts increase and fewer growth cone regions are observed. As early as 7 DIV profiles are observed which strongly resemble both astrocytic and oligodendroglial cell somata and processes. Freeze-fracture analysis of aggregates at 0–4 hr reveals a sparse particle distribution on the P and E faces of apposed cells. By 1 DIV small clusters of loosely packed, large sized particles are seen on the P face of apposed cell membranes which may represent junctional contacts. Apparent coated vesicle fusion sites are common on the P face at 1–2 DIV. By 7 DIV, E face particle arrays are seen on cell bodies and neurites which correspond to specializations characteristic of excitatory synaptic junctions. By 8–10 DIV particle arrays are seen on the P face of post-synaptic membrane which may represent inhibitory synaptic contacts. Other types of particle specializations seen in freeze-fracture replicas include: specializations characteristic of gap junctions between cells and orthogonal assemblies of particles thought to be characteristic of astrocytes.     

Intramembrane organization of specialized contacts in the outer plexiform layer of the retina. A freeze-fracture study in monkeys and rabbits

Freeze-fracture analysis of the neural connections in the outer plexiform layer of the retina of primates (Macaca mulatta and Macaca arctoides) demonstrates a remarkable diversity in the internal structure of the synaptic membranes. In the invaginating synapses of cone pedicles, the plasma membrane of the photoreceptor ending contains an aggregate of A-face particles, a hexagonal array of synaptic vesicle sites, and rows of coated vesicle sites, which are deployed in sequence from apex to base of the synaptic ridge. The horizontal cell dendrites lack vesicle sites and have two aggregates of intramembrane A-face particles, one at the interface with the apex of the synaptic ridge, the other opposite the tip of the invaginating midget bipolar dendrite. Furthermore, the horizontal cell dendrites are interconnected by a novel type of specialized junction, characterized by: (a) enlarged intercellular cleft, bisected by a dense plate and traversed by uniformly spaced crossbars; (b) symmetrical arrays of B-face particles arranged in parallel rows within the junctional membranes; and (c) a layer of dense material on the cytoplasmic surface of the membranes. The plasmalemma of the invaginating midget bipolar dendrite is unspecialized. At the contact region between the basal surface of cone pedicles and the dendrites of the flat midget and diffuse cone bipolar cells, the pedicle membrane has moderately clustered A-face particles, but no vesicle sites, whereas the adjoining membrane of the bipolar dendrites contains an aggregate of B-face particles. The invaginating synapse of rod spherules differs from that of cone pedicles, because the membrane of the axonal endings of the horizontal cells only has an A-face particle aggregate opposite the apex of the synaptic ridge. Specialized junctions between horizontal cell processes, characterized by symmetrical arrays of intramembrane B-face particles, are also present in the neuropil underlying the photoreceptor endings. Small gap junctions connect the processes of the horizontal cells; other gap junctions probably connect the bipolar cell dendrites which make contact with each cone pedicle. Most of the junctional specializations typical of the primate outer plexiform layer are also found in the rabbit retina. The fact that specialized contacts between different types of neurons interacting in the outer plexiform layer have specific arrangements of intramembrane particles strongly suggests that the internal structure of the synaptic membranes is intimately correlated with synaptic function.     

Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture

Our object was to characterize the morphological changes occurring in pre- and postsynaptic elements during their initial contact and subsequent maturation into typical synaptic profiles. Neurons from superior cervical ganglia (SCG) of perinatal rats were freed of their supporting cells and established as isolated cells in culture. To these were added explants of embryonic rat thoracic spinal cord to allow interaction between outgrowing cord neurites and the isolated autonomic neurons. Time of initial contact was assessed by light microscopy; at timed intervals thereafter, cultures were fixed for electron microscopy. Upon contact, growth cone filopodia became extensively applied to the SCG neuronal plasmalemma and manifested numerous punctate regions in which the apposing plasma membranes were separated by only 7-10 nm. The Golgi apparatus of the target neuron hypertrophied, and its production of coated vesicles increased. Similar vesicles were seen in continuity with the SCG plasmalemma near the close contact site; their apparent contribution of a region of postsynaptic membrane with undercoating was considered to be the first definitive sign of synapse formation. Tracer work with peroxidase and ferritin confirmed that the traffic of coated vesicles within the neuronal soma is largely from Golgi region to somal surface. Subsequent to the appearance of postsynaptic density, the form and content of the growth cone was altered by the loss of filopodia and the appearance of synaptic vesicles which gradually became clustered opposite the postsynaptic density. As the synapse matured, synaptic vesicles increased in number, cleft width and content increased, presynaptic density appeared, branched membranous reticulum became greatly diminished, and most lysosomal structures disappeared. Coated vesicles continued to be associated with the postsynaptic membrane at all stages of maturation. The incorporation of Golgi-derived vesicles into discrete regions of the cell membrane could provide the mechanism for confining specific characteristics of the neuronal membrane to the synaptic region.     

wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila

We conducted a large-scale screen for Drosophila mutants that have structural abnormalities of the larval neuromuscular junction (NMJ). We recovered mutations in wishful thinking (wit), a gene that positively regulates synaptic growth. wit encodes a BMP type II receptor. In wit mutant larvae, the size of the NMJs is greatly reduced relative to the size of the muscles. wit NMJs have reduced evoked excitatory junctional potentials, decreased levels of the synaptic cell adhesion molecule Fasciclin II, and synaptic membrane detachment at active zones. Wit is expressed by a subset of neurons, including motoneurons. The NMJ phenotype is specifically rescued by transgenic expression of Wit only in motoneurons. Thus, Wit appears to function as a presynaptic receptor that regulates synaptic size at the Drosophila NMJ.     

Fluorescence microscopy of calcium and synaptic vesicle dynamics during synapse formation in tissue culture

The signal transduction process involved in the development of the nerve terminal is an intriguing question in developmental neurobiology. During the formation of the neuromuscular junction, presynaptic development is induced by growth cone's contact with the target muscle cell. Fluorescence microscopy with specific markers has made it possible to follow signalling events during this process. By using fluorescent calcium indicators, such as fura-2 and fluo-3, we found that a rise in intracellular calcium is elicited in the growth cone upon its contact with a target, and this calcium signal can also be elicited by local application of basic fibroblast growth factor. To monitor the clustering of synaptic vesicles in response to target contact, the fluorescent vesicular probe FMl-43 was used. With this probe, we observed that packets of synaptic vesicle are already present along the length of naive neurite, which has not encountered its synaptic target. The activity-dependent loading of FMl-43 indicates that these packets can undergo exocytosis and endocytosis upon depolarization. Time-lapse recording showed that these packets are quite mobile. Upon target contact, synaptic vesicles become clustered and immobilized at the contact site. The methodology and instrumentation used in these studies are described in this article. 1998 © Chapman & Hall     

Intercellular communication and tissue growth: VII. A cancer cell strain with retarded formation of permeable membrane junction and reduced exchange of a 330-dalton molecule

Summary A cancer (hepatoma) cell strain is described in which the formation of junctional membrane channels is abnormally slow. The development of electrical junctional coupling following the establishment of contact between these (reaggregated) cells is at least 15 times slower than that between their normal counterparts; and junctional transfer of fluorescein eventually develops, but only in about 5% of the contacts (as against 100% normally). This deviant membrane behavior is interpreted as a retardation in the process of accretion of junctional membrane channels. Its possible etiological role in defective growth regulation is discussed.     

Intercellular communication and tissue growth: VII. A cancer cell strain with retarded formation of permeable membrane junction and reduced exchange of a 330-dalton molecule.

A cancer (hepatoma) cell strain is described in which the formation of junctional membrane channels is abnormally slow. The development of electrical junctional coupling following the establishment of contact between these (reaggregated) cells is at least 15 times slower than that between their normal counterparts; and junctional transfer of fluorescein eventually develops, but only in about 5 per cent of the contacts (as against 100 per cent normally). This deviant membrane behavior is interpreted as a retardation in the process of accretion of junctional membrane channels. Its possible etiological role in defective growth regulation is discussed.     

Growth cones stall and collapse during axon outgrowth in Caenorhabditis elegans.

During nervous system development, neurons form synaptic contacts with distant target cells. These connections are formed by the extension of axonal processes along predetermined pathways. Axon outgrowth is directed by growth cones located at the tips of these neuronal processes. Although the behavior of growth cones has been well-characterized in vitro, it is difficult to observe growth cones in vivo. We have observed motor neuron growth cones migrating in living Caenorhabditis elegans larvae using time-lapse confocal microscopy. Specifically, we observed the VD motor neurons extend axons from the ventral to dorsal nerve cord during the L2 stage. The growth cones of these neurons are round and migrate rapidly across the epidermis if they are unobstructed. When they contact axons of the lateral nerve fascicles, growth cones stall and spread out along the fascicle to form anvil-shaped structures. After pausing for a few minutes, they extend lamellipodia beyond the fascicle and resume migration toward the dorsal nerve cord. Growth cones stall again when they contact the body wall muscles. These muscles are tightly attached to the epidermis by narrowly spaced circumferential attachment structures. Stalled growth cones extend fingers dorsally between these hypodermal attachment structures. When a single finger has projected through the body wall muscle quadrant, the growth cone located on the ventral side of the muscle collapses and a new growth cone forms at the dorsal tip of the predominating finger. Thus, we observe that complete growth cone collapse occurs in vivo and not just in culture assays. In contrast to studies indicating that collapse occurs upon contact with repulsive substrata, collapse of the VD growth cones may result from an intrinsic signal that serves to maintain growth cone primacy and conserve cellular material.     

Evidence for the involvement of KIF4 in the anterograde transport of L1-containing vesicles

In this study we present evidence about the cellular functions of KIF4. Using subcellular fractionation techniques and immunoisolation, we have now identified a type of vesicle that associates with KIF4, an NH(2)-terminal globular motor domain kinesin-like protein. This vesicle is highly concentrated in growth cones and contains L1, a cell adhesion molecule implicated in axonal elongation. It lacks synaptic vesicle markers, receptors for neurotrophins, and membrane proteins involved in growth cone guidance. In cultured neurons, KIF4 and L1 predominantly localize to the axonal shaft and its growth cone. Suppression of KIF4 with antisense oligonucleotides results in the accumulation of L1 within the cell body and in its complete disappearance from axonal tips. In addition, KIF4 suppression prevents L1-enhanced axonal elongation. Taken collectively, our results suggest an important role for KIF4 during neuronal development, a phenomenon which may be related to the anterograde transport of L1-containing vesicles.