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.

The Drosophila neuromuscular junction has attracted widespread attention as an excellent model system for studying the cellular and molecular mechanisms of synaptic development and neurotransmission. In Drosophila the advantages of invertebrate small systems, where individual cells can be examined with single-cell resolution, are combined with the powerful techniques of patch-clamp analysis and molecular genetics. In this review we examine myogenesis and motoneuron development, the problems of axon outgrowth and target selection, the differentiation of the synapse, and the mechanisms of both synaptic function and plasticity in this model genetic system.

Visual information received from the three types of photoreceptor neurons(R1-R6, R7 and R8) in the fly compound eyes converges to the external part of the medulla neuropil (M1-M6 layers) in a layer-specific fashion: R7 and R8 axons terminate at the M6 and M3 layers, respectively, whereas lamina neurons(L1-L5) relay R1-R6 to multiple medulla layers (M1-M5). Here, we show that during development, R7 and R8 neurons establish layer-specific projections in two separate stages: during the first stage, R7 and R8 axons sequentially target to the R7- and R8-temporary layers, respectively; and at the second stage, R7 and R8 growth cones progress synchronously to their destined layers. Using a set of mutations that delete different afferent subsets or alter R7 connectivity, we defined the mechanism of layer selection. We observed that R8, R7 and L1-L5 afferents target to their temporary layers independently,suggesting that afferent-target, but not afferent-afferent, interactions dictate the targeting specificity. N-cadherin is required in the first stage for R7 growth cones to reach and remain in the R7-temporary layer. The Ncad gene contains three pairs of alternatively spliced exons and encodes 12 isoforms. However, expressing a single Ncad isoform in Ncad mutant R7s is sufficient to rescue mistargeting phenotypes. Furthermore, Ncad isoforms mediate promiscuous heterophilic interactions in an in vitro cell-aggregation assay. We propose that Ncad isoforms do not form an adhesion code; rather, they provide permissive adhesion between R7 growth cones and their temporary targets.

Immunocytochemical electron-microscopic observation indicated that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) and/or its degradation products are localized in small spherical bodies having a diameter of 0.4-1.2 micro m in naturally senescing leaves of wheat (Triticum aestivum L.). These Rubisco-containing bodies (RCBs) were found in the cytoplasm and in the vacuole. RCBs contained another stromal protein, chloroplastic glutamine synthetase, but not thylakoid proteins. Ultrastructural analysis suggested that RCBs had double membranes, which seemed to be derived from the chloroplast envelope, and that RCBs were further surrounded by the other membrane structures in the cytoplasm. The appearance of RCBs was the most remarkable when the amount of Rubisco started to decrease at the early phase of leaf senescence. These results suggest that RCBs might be involved in the degradation process of Rubisco outside of chloroplasts during leaf senescence.

Precise synaptogenesis is crucial to brain development, and depends on the ability of specific partner cells to locate and communicate with one another. Dynamic properties of axonal filopodia during synaptic targeting are well documented, but the cytomorphological dynamics of postsynaptic cells have received less attention. In Drosophila embryos, muscle cells bear numerous postsynaptic filopodia ('myopodia') during motoneuron targeting. Here we show that myopodia are actin-filled microprocesses, which progressively clustered at the site of motoneuron innervation while intermingling with presynaptic filopodia. In prospero mutants, which have severe delays in axon outgrowth from the CNS, myopodia were present initially but clustering behavior was not observed, demonstrating that clustering depends on innervating axons. Thus, postsynaptic filopodia are capable of intimate interaction with innervating presynaptic axons. We propose that, by contributing to direct long-distance cellular communication, they are dynamically involved in synaptic matchmaking.

Memory and learning in animals are mediated by neurotransmitters that are released from vesicles clustered at the synapse. As a synapse is used more frequently, its neurotransmission efficiency increases, partly because of increased vesicle clustering in the presynaptic neuron. Vesicle clustering has been believed to result primarily from biochemical signaling processes that require the connectivity of the presynaptic terminal with the cell body, the central nervous system, and the postsynaptic cell. Our in vivo experiments on the embryonic Drosophila nervous system show that vesicle clustering at the neuromuscular presynaptic terminal depends on mechanical tension within the axons. Vesicle clustering vanishes upon severing the axon from the cell body, but is restored when mechanical tension is applied to the severed end of the axon. Clustering increases when intact axons are stretched mechanically by pulling the postsynaptic muscle. Using micro mechanical force sensors, we find that embryonic axons that have formed neuromuscular junctions maintain a rest tension of Ϸ1 nanonewton. If the rest tension is perturbed mechanically, axons restore the rest tension either by relaxing or by contracting over a period of Ϸ15 min. Our results suggest that neuromuscular synapses employ mechanical tension as a signal to modulate vesicle accumulation and synaptic plasticity.T he accumulation of neurotransmitter containing vesicles at the presynaptic terminal is essential for neural communication. On the arrival of an action potential at the terminal, neurotransmitters are released through exocytosis of the vesicles. The transmitters excite the postsynaptic terminal in a millisecond time frame (1). The amount of neurotransmitter release for a given action potential depends on several factors including how frequently the synapse has been used. This usage dependent plasticity is believed to be the basis of memory and learning (2). Despite a wealth of knowledge on the molecular components of the synapse (3, 4) and the modulation of the postsynaptic machinery during long-term potentiation and depression (5-7), the mechanism of vesicle accumulation at the presynaptic terminal and regulation of neurotransmission in a usage-dependent manner remains unclear (8). There is increasing experimental evidence suggesting that the mechanical microenvironment has a significant influence on a variety of cell functions including gene expression, cell growth and morphology, cytoskeletal organization, and apoptosis (9-13). Our in vivo experiments reveal that mechanical tension in axons plays a key role in vesicle accumulation.We examine the neuromuscular synapse within live embryos of Drosophila melanogaster (14) (Fig. 1A). In Drosophila, an aCC (anterior corner cell) pioneer motoneuron extends a single axon and invariably innervates its target, muscle1. A pair of aCC motoneurons occurs in every segment of the embryo, and their development is highly stereotyped. The first contact between the aCC axon and muscle1 occurs at hour 14 of embryogenesis, ...

Each muscle fiber in the segmented body wall of Drosophila larvae is innervated by anatomically stereotyped neuromuscular junctions. These synapses arise through the selective choices of motoneuronal growth cones at their peripheral targets. Using digital optical microscopy of staged intracellular dye fills, we have singly identified embryonic motoneurons and have examined individual growth cones when they contact and differentiate at the target cells. There is a precise connectivity between motoneuron and muscle fiber, which is the direct consequence of growth cone behavior. We have also found that Drosophila muscle fibers possess molecularly heterogeneous cell surfaces that may be involved in growth cone recognition of appropriate targets. Fasciclin III, a homophilic adhesion molecule, is coexpressed by several of the efferent growth cones and in a site-specific fashion by the target muscle fiber's membrane. The fasciclin III expression is transient, corresponding to the period in embryogenesis when the first neuromuscular contacts are made. Upon encountering the target cell surface, the growth cones can sprout stereotypically arrayed filopodial processes, orient along the anterior-posterior axis, and turn in predictable directions. Subsequently, terminal branches are established in a nonrandom order. These phenomena were found to occur in two motoneurons that innervate adjacent muscle fiber targets, and may be general features of neuromuscular synaptogenesis in Drosophila.

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