Drosophila olfactory local interneurons and projection neurons derive from a common neuroblast lineage specified by the empty spiracles gene

Background

The skeletal neuromuscular junction is a useful model for elucidating mechanisms that regulate synaptogenesis. Developmentally important intercellular interactions at the neuromuscular junction are mediated by the synaptic portion of a basal lamina that completely ensheaths each muscle fiber. Basal laminas in general are composed of four main types of glycosylated proteins: laminins, collagens IV, heparan sulfate proteoglycans and nidogens (entactins). The portion of the muscle fiber basal lamina that passes between the motor nerve terminal and postsynaptic membrane has been shown to bear distinct isoforms of the first three of these. For laminins and collagens IV, the proteins are deposited by the muscle; a synaptic proteoglycan, z-agrin, is deposited by the nerve. In each case, the synaptic isoform plays key roles in organizing the neuromuscular junction. Here, we analyze the fourth family, composed of nidogen-1 and -2.

Results

In adult muscle, nidogen-1 is present throughout muscle fiber basal lamina, while nidogen-2 is concentrated at synapses. Nidogen-2 is initially present throughout muscle basal lamina, but is lost from extrasynaptic regions during the first three postnatal weeks. Neuromuscular junctions in mutant mice lacking nidogen-2 appear normal at birth, but become topologically abnormal as they mature. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting the phenotype is not secondary to a general defect in the integrity of synaptic basal lamina. Further genetic studies suggest that synaptic localization of each of the four families of synaptic basal lamina components is independent of the other three.

Conclusion

All four core components of the basal lamina have synaptically enriched isoforms. Together, they form a highly specialized synaptic cleft material. Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction.     

Dermatan sulfate and de-sulfated heparin solubilized collagen-tailed acetylcholinesterase from the rat neuromuscular junction

We are interested in the study of the interactions involved in the attachment of collagen-tailed acetylcholinesterase (AChE) to the synaptic basal lamina. The fact that AChE occupies less than 0.1% of the muscle basal lamina, suggests that there is a very high specificity in the interaction that defines its distribution. We have previously found that asymmetric AChE is bound to the neuromuscular junction via heparan sulfate proteoglycans. Sulfated glycosaminoglycans as heparan sulfate and heparin extracted the asymmetric AChE from the synaptic basal lamina. Here we show that dermatan sulfate as well as de-sulfated heparin, are also able to extract collagen-tailed AChE. Taking into account that the solubilization of the asymmetric AChE is concomitant with the liberation of a dermatan sulfate proteoglycan from the rat neuromuscular junction, the present results open the possibility that the collagen-tailed AChE is also anchored to dermatan sulfate proteoglycans at the synaptic basal lamina.     

Structure and physiology of developing neuromuscular synapses in culture

The structure and function of developing neuromuscular synapses in culture have been investigated. We used neuromuscular junctions formed by coculturing dissociated muscle cells and dissociated neurons obtained from Xenopus embryos. After recording nerve-evoked endplate potentials (e.p.p.s) and spontaneously occurring miniature endplate potentials (m.e.p.p.s) from a given junction, the same specimen was investigated for electron-microscopic histology. We surveyed almost the total area of the junctional region by making serial sections. Even in preparations cocultured for only a short time (4-11 hr), both e.p.p.s and m.e.p.p.s could be obtained. The junctional region of these early synapses revealed a simple structure. The presynaptic terminals contained smooth-surfaced clear vesicles, but there were no presynaptic specializations such as active zones. The width of the synaptic cleft was variable, with predominance of narrow regions (10-30 nm), and there was no basal lamina inside the cleft. When the coculture time was 1 d or longer, the junctional area started to show structural features resembling a mature neuromuscular synapse. In the presynaptic terminal there were active zones, consisting of the presynaptic density and an accumulation of vesicles near the density. In many junctions, the postsynaptic membrane showed densities and thickenings, with a widened synaptic cleft, that contained basal lamina. It is known that growth cones, prior to making neuromuscular junctions, can release the transmitter substance with a very long latency if stimulated repetitively. In contrast, e.p.p.s with short latencies can be evoked by single stimuli soon after the growth cones attach to muscle cells. However, our data did not reveal any structural changes to account for such functional changes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of postsynaptic-like specializations of the neuromuscular synapse in the absence of motor nerve

It was previously reported that the acetylcholine receptor clusters and acetylcholinesterase appear on embryonic superior oblique muscle cells developing in vivo without motor nerve contacts. The objective of this study was to examine whether some other components of neuromuscular junction also form on muscle cells developing in vivo in the absence of motor neurons. In the present study, postsynaptic specializations such as junctional folds, postsynaptic density and basal lamina were studied in normal and aneural muscles. The superior oblique muscle of duck embryos was made aneural by permanent destruction of trochlear motor neurons by cauterizing midbrain on embryonic day 7; 3 days before the motor neurons normally project their axons into the muscle. Normal and aneural muscles from embryonic days 10 to 25 were processed for electron microscopy. The results indicate that morphological specializations such as junction-like folds, postsynaptic-like density, and basal lamina also develop in the absence of motor neuron contacts. Whether the differentiation of specialized synaptic basal lamina is dependent on the presence of motor neurons was examined by utilizing a monoclonal antibody against heparan sulfate proteoglycan. Immunohistochemical studies indicate that specialized synaptic basal lamina differentiates in the absence of motor neurons. Thus, the mechanism of development of postsynaptic components of neuromuscular junction in this muscle is not dependent on motor neuron contacts. These results also suggest that the postsynaptic cell plays a more active role in synapse formation than previously realized. The results are discussed in relation to the control of synapse numbers by the postsynaptic cell.     

Biochemical and Functional Characterization of Basal Lamina-bound Agrin in the Chick Central Nervous System

Agrin is a high-molecular weight extracellular matrix molecule, initially purified from the electric organ of the marine ray Torpedo californica , which induces on the surface of cultured myotubes the formation of postsynaptic specializations similar to those found at the neuromuscular junction. Agrin immunoreactivity is highly concentrated in the basal lamina of the synaptic cleft but is also found in a number of other tissues where its function is not known. We characterized agrin associated with two basal laminae from the central nervous system, the inner limiting membrane of the retina and the mesencephalic external limiting membrane. A major broad band with an apparent molecular weight of <300 kDa was identified in immunoblots of isolated basal laminae from retina, mesencephalon, kidney and muscle, showing that basal lamina-bound agrin from the central nervous system and that from non-neural tissues have similar molecular sizes. Agrin is stably but not covalently bound to the inner limiting membrane and could be completely removed only with strong detergents. Agrin could be partially extracted with buffers that are also able to partially release acetylcholine receptor aggregation activity from the neuromuscular junction or from the electric organ. Despite these immunological and biochemical similarities, agrin from both central nervous system-derived basal laminae was not able to induce acetylcholine receptor aggregation on cultured myotubes. This shows that functionally different agrin isoforms are associated with basal laminae in the central nervous system compared to the neuromuscular junction or the electric organ.     

NEURONAL UPTAKE OF IRON: SOMATOPETAL AXONAL TRANSPORT AND FATE OF CATIONIZED AND NATIVE FERRITIN, AND IRON-DEXTRAN AFTER INTRAMUSCULAR INJECTIONS

Mice were injected into the muscles of the vibrissae with native ferritin (NF), cationized ferritin (CF) and iron-dextran. CF adsorbed on to the surface of the axon terminal at the neuromuscular junction, while NF did not. Both CF and NF were incorporated into vesicles and vacuoles at the synapse, but CF uptake was detected after injections at much lower concentrations than NF. In contrast to NF, CF was also found histochemically in cell bodies of facial neurones after a single i.m. injection, showing that the electrical charge of a molecule is one factor of importance for its potential to be incorporated in axons and transported somatopetally. Repeated i.m. injection of iron-dextran into suckling mice resulted in a marked iron load of Schwann cells and nerve cell bodies. This produced no signs of toxicity and the nerve fibre developed normally. Iron had disappeared from the nerve cell bodies after 25 days, while in Schwann cells it still persisted after 223 days.     

Molecular architecture of the neuromuscular junction

The neuromuscular junction (NMJ) is a complex structure that serves to efficiently communicate the electrical impulse from the motor neuron to the skeletal muscle to signal contraction. Over the last 200 years, technological advances in microscopy allowed visualization of the existence of a gap between the motor neuron and skeletal muscle that necessitated the existence of a messenger, which proved to be acetylcholine. Ultrastructural analysis identified vesicles in the presynaptic nerve terminal, which provided a beautiful structural correlate for the quantal nature of neuromuscular transmission, and the imaging of synaptic folds on the muscle surface demonstrated that specializations of the underlying protein scaffold were required. Molecular analysis in the last 20 years has confirmed the preferential expression of synaptic proteins, which is guided by a precise developmental program and maintained by signals from nerve. Although often overlooked, the Schwann cell that caps the NMJ and the basal lamina is proving to be critical in maintenance of the junction. Genetic and autoimmune disorders are known that compromise neuromuscular transmission and provide further insights into the complexities of NMJ function as well as the subtle differences that exist among NMJ that may underlie the differential susceptibility of muscle groups to neuromuscular transmission diseases. In this review we summarize the synaptic physiology, architecture, and variations in synaptic structure among muscle types. The important roles of specific signaling pathways involved in NMJ development and acetylcholine receptor (AChR) clustering are reviewed. Finally, genetic and autoimmune disorders and their effects on NMJ architecture and neuromuscular transmission are examined.     

Agrin and acetylcholine receptor distribution following electrical stimulation

Electrical stimulation is a therapeutic modality available for the preservation of muscle function following peripheral nerve injury. Agrin, a synaptic basal lamina protein, induces accumulation of acetylcholine receptors (AChRs) and other molecules at the neuromuscular junction. Electrical stimulation of denervated muscle does not alter agrin and AChR distribution at abandoned synaptic sites, supporting the hypothesis that the existing aggregation of synaptic molecules, which may be necessary for successful reinnervation, is unaltered by electrical stimulation of denervated muscle. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:407–409, 1998.     

Novel antigens at the neuromuscular junction

Three novel components of neuromuscular junctions have been identified by use of monoclonal antibodies (McAb) against glycoproteins obtained from a mouse neuroblastoma X human dorsal root ganglion cell hybrid line. Antigen distribution was assessed by fluorescent immunohistochemistry on frozen sections of human intercostal muscle counterstained with labeled alpha-bungarotoxin to identify neuromuscular junctions. Antigen SOS 6 stained exclusively in the neuromuscular junction, whereas antigens SOS 5 and SOS 13 were highly enriched in the junction but also stained extrasynaptic regions. These antigens can be distinguished from previously described components of the neuromuscular junction by their molecular weights, insensitivity to collagenase treatment, and solubility in 0.1% Triton X-100. Indirect evidence suggests that these species-specific antigens are located in the postsynaptic muscle membrane, but location in the junctional basal lamina or subsarcolemmal region cannot be excluded.     

Accelerated Structural Maturation Induced by Synapsin I at Developing Neuromuscular Synapses of Xenopus laevis

The role of synapsin I, a synaptic vesicle-associated phosphoprotein, in the maturation of nerve–muscle synapses was investigated in nerve–muscle co-cultures prepared from Xenopus embryos loaded with the protein by the early blastomere injection method. The stage of maturation of the synapses was analysed by electron microscopy as well as by whole-cell patch-clamp recording. The acceleration in the functional maturation of neuromuscular synapses induced by synapsin I was accompanied by a profound rearrangement in the ultrastructure of the nerve terminal. Nerve terminals formed by synapsin I-loaded neurons were characterized by a higher number of small synaptic vesicles organized in clusters and predominantly localized close to the nerve terminal plasma membrane, a smaller number of large dense-core vesicles and no significant change in the number of coated vesicles. Precocious development of active zone-like structures as well as deposition of basal lamina into the synaptic cleft were also observed at these synapses. These results support a role for synapsin I in the architectural changes which occur during synaptogenesis and lead to the maturation of quantal neurotransmitter release mechanisms.     

Miller Fisher syndrome: immunofluorescence and immunoelectron microscopic localization of IgG at the mouse neuromuscular junction

In vitro electrophysiological experiments have demonstrated that IgG antibodies from patients with Miller Fisher syndrome (MFS) impair neuromuscular transmission by a fast and completely reversible combined pre- and postsynaptic blockade. In this study we investigated the cellular and subcellular binding sites of IgG from four MFS patients at the mouse hemidiaphragm by immunofluorescence and immunoelectron microscopy. IgG from all patients produced significant immunostaining at the neuromuscular junction, whereas sera from healthy volunteers or from patients with other neurological diseases did not stain neuromuscular junction. Immunoelectron microscopy revealed that, when living hemidiaphragms were incubated with IgG from MFS patients, labeling was found on both pre- and postsynaptic membranes of the neuromuscular junction, whereas terminal Schwann cells and the basal lamina covering the synaptic membranes were not labeled. These findings demonstrate that IgG from MFS patients binds to synaptic membranes of the neuromuscular junction where it might interfere with the function of both the pre- and postsynaptic activities.     

Thickness of the basal lamina at the frog neuromuscular junction

The Schwann cell basal lamina at the frog neuromuscular junction is much thicker than previously supposed. Its thickness has been demonstrated by modifying the fixation: the nerve muscle preparation is fixed in situ without prior exposure to Ringer solution and the osmolarity of the glutaraldehyde fixative is increased to 285 mOsm. With this procedure the basal lamina is seen to be up to 1 micron thick around the Schwann cells of the nerve terminal and the preterminal axon. The thickness of the basal lamina surrounding the Schwann cells of the myelinated portion of the axons within the perineural sheath (20 nm) is not changed by this modification of fixation. The present results indicate that in addition to the ability to form myelin, other properties of the Schwann cells differ inside and outside the perineurial sheath.     

A minigene of neural agrin encoding the laminin‐binding and acetylcholine receptor‐aggregating domains is sufficient to induce postsynaptic differentiation in muscle fibres

The extracellular matrix molecule agrin is both necessary and sufficient for inducing the formation of postsynaptic specializations at the neuromuscular junction (NMJ). At the mature NMJ, agrin is stably incorporated in synaptic basal lamina. The postsynapse‐inducing activity of chick agrin, as assayed by its capability of causing aggregation of acetylcholine receptors (AChRs) on cultured muscle cells, maps to a 21 kDa, C‐terminal domain. Binding of chick agrin to muscle basal lamina is mediated by the laminins and maps to a 25 kDa, N‐terminal fragment of agrin. Here we show that an expression construct encoding a 'mini'‐agrin, in which the laminin‐binding fragment was fused to the AChR‐clustering domain, is sufficient to induce postsynaptic differentiation in vivo when injected into non‐synaptic sites of rat soleus muscle. As shown for ectopic postsynaptic differentiation induced by full‐length neural agrin, myonuclei underneath the ectopic sites expressed the gene for the AChR ε‐subunit. Altogether, our data show that a 'mini'‐agrin construct encoding only a small fraction of the entire agrin protein is sufficient to induce postsynapse‐like structures that are reminiscent of those induced by full‐length neural agrin or innervation by motor neurons.     

Structure of autonomic neuromuscular junctions in the sinus venosus of the toad.

The structure of cholinergic and adrenergic neuromuscular junctions in the sinus venosus of the toad, Bufo marinus, was determined by electron microscopy. From random sections of sinus venosus tissue it appeared that there were variable separations between cholinergic or adrenergic varicosities and the nearest sinus venosus muscle cell. However, when the structure of complete cholinergic and adrenergic varicosities was determined by examining serial electron micrographs, virtually all varicosities that lost their covering of Schwann cell were found to form an area of close apposition with an adjacent muscle cell. At the region of close apposition, the neuromuscular cleft was filled with a single layer of basal lamina to give a neuromuscular separation of about 70 nm. Synaptic vesicles within a varicosity were usually found to be concentrated towards the region of close apposition. These observations are discussed in relationship to the idea that when transmission occurs at these neuromuscular junctions the transmitters act on discrete pools of specialized subsynaptic receptors.     

Nitric oxide is a downstream mediator of agrin-induced acetylcholine receptor aggregation

The synaptic basal lamina protein, agrin, is required for the formation of the neuromuscular junction. Agrin signals through a muscle-specific receptor tyrosine kinase (MuSK) initiating a cascade of events that lead to the aggregation of acetylcholine receptors (AChR) at the postsynaptic site. Another important synaptic signalling molecule is nitric oxide (NO), which is produced by the enzyme, nitric oxide synthase (NOS). We investigated the interaction between the agrin signalling cascade and the NO signalling cascade by treating cultured myotubes with agrin, NOS inhibitors, and NO donors. NOS inhibitors prevented agrin induced AChR aggregation and phosphorylation of the AChR beta subunit. Furthermore, NO donors induced AChR aggregation in the absence of agrin, as well as phosphorylation of the AChR beta subunit. These results demonstrate a role for NO as a downstream mediator of agrin induced AChR aggregation and AChR beta subunit phosphorylation at the neuromuscular junction.     

Distinct localization of collagen Q and PRiMA forms of acetylcholinesterase at the neuromuscular junction

Acetylcholinesterase (AChE) terminates the action of acetylcholine at cholinergic synapses thereby preventing rebinding of acetylcholine to nicotinic postsynaptic receptors at the neuromuscular junction. Here we show that AChE is not localized close to these receptors on the postsynaptic surface, but is instead clustered along the presynaptic membrane and deep in the postsynaptic folds. Because AChE is anchored by ColQ in the basal lamina and is linked to the plasma membrane by a transmembrane subunit (PRiMA), we used a genetic approach to evaluate the respective contribution of each anchoring oligomer. By visualization and quantification of AChE in mouse strains devoid of ColQ, PRiMA or AChE, specifically in the muscle, we found that along the nerve terminus the vast majority of AChE is anchored by ColQ that is only produced by the muscle, whereas very minor amounts of AChE are anchored by PRiMA that is produced by motoneurons. In its synaptic location, AChE is therefore positioned to scavenge ACh that effluxes from the nerve by non-quantal release. AChE-PRiMA, produced by the muscle, is diffusely distributed along the muscle in extrajunctional regions.     

Nerve terminal growth remodels neuromuscular synapses in mice following regeneration of the postsynaptic muscle fiber

Muscle fibers degenerate and regenerate in response to contractile damage, during aging, and in various muscle diseases that weaken the fibers. It is known that degeneration and regeneration of the segment of the postsynaptic fiber produces dramatic alterations in the neuromuscular junction (NMJ) that forms on the regenerated fiber, but the mechanisms here are incompletely understood. We have used a laser microbeam to damage the postsynaptic fibers at individual NMJs in the sternomastoid muscle of living young adult mice and then followed the synapses vitally over time using fluorescent proteins expressed in motor neurons and glial cells and staining of postsynaptic acetylcholine receptors. We find, in contrast to previous reports, that the mouse nerve terminal retains contact with the synaptic basal lamina marked by cholinesterase staining even in the absence of the target, showing that this terminal does not require a continuous supply of target-derived molecules for its maintenance. Thus, remodeling of the nerve terminal during the period of target absence does not explain the subsequent changes in the new NMJ. Rather, we see that the synapse becomes altered as the new fiber segment regenerates. Mechanisms for remodeling the synapse include failure of the regenerating muscle fiber to contact the old basal lamina and nerve terminal, growth of the nerve terminal and its glia toward the regenerating fiber, and remodeling of the initial contact as the nerve terminal becomes varicose.     

The membrane morphology of the neuromuscular junction, sarcolemma, sarcoplasmic reticulum and transverse tubule system in murine muscular dystrophy studied by freeze-fracture electron microscopy

‘Dystrophic’ mice of the 129/ReJ-dy strain have a genetic defect in Schwann cell proliferation and neuromuscular junction formation. The presynaptic membrane specialization associated with vesicle fusion and acetylcholine release, as well as the postsynaptic membrane specializations associated with acetylcholine receptivity, appear normal in these animals when visualized with freeze-fracture techniques. However, there is a reduction in the infolding of the postsynaptic membrane, which forms the secondary synaptic cleft at the motor endplate and is the site of acetylcholinesterase activity. The orthogonal arrays of the non-junctional sarcolemma are found on dystrophic muscles, but at lower than normal densities. These observations are made on muscle fibers in which the membrane molecular organization of the sarcoplasmic reticulum and transverse tubule systems appear normal. Several possible linkages between the deficit in myelination and the altered synaptic morphology are discussed in the context of neuromuscular interaction.     

Regenerated EDL muscle of rats requires innervation to maintain AChE molecular forms

Extensores digitorum longi of rats, infarcted and denervated by different surgical procedures, were used to analyze by biochemical and cytochemical methods the acetylcholinesterase (AChE) changes during muscle degeneration, regeneration, and early or delayed reinnervation. Biochemical tests showed that the regenerating muscle produces globular AChE forms (36% of controls) and small amounts of A12 (16S) asymmetric form (5% of controls); at the end of the regeneration, innervation and electromechanical function are required for the complete recovery of globular forms, and are absolutely critical to prevent A12 (16S) disappearance. Cytochemical observations showed that, unlike nicotinic receptor, AChE deposited at the neuromuscular junction before ischemic necrosis is protected from breakdown, as is the basal lamina of muscle fibers. Taken together, these observations contribute to the understanding of the factors that play a critical role in muscle repair and are, therefore, of clinical relevance.     

Immunogold and freeze etch electron microscopic studies of merosin localization in basal lamina of human skeletal muscle fibers

Merosin is a basement-membrane-associated protein found in striated muscle, peripheral nerve and placenta, the deficiency of which causes the muscle wasting condition in C57BL/6J-dy/dy, so-called dy/dy mouse. Moreover, merosin is the binding protein of 156 kDa α-dystroglycan which binds dystrophin by way of 43 kDa β-dystroglycan. Therefore, merosin is an important component of the basal lamina of normal skeletal myofibers. We investigated the ultrastructural localization of merosin antibody in normal human skeletal myofibers by using immunogold electron microscopy and freeze etch electron microscopy. The ultrastructure of the basal lamina showed the presence of the lamina lucida, lamina densa and lamina reticularis. The lamina lucida appeared electron translucent with the exception of fuzzy fibrils. The immunogold electron microscopy disclosed that the merosin was present at the innermost layer (lamina lucida) of the basal lamina of normal human skeletal myofibers. With freeze etch replica electron microscopy, short cross-bridge fine fibrils were noted in the lamina lucida, connecting the basal lamina to the outer leaflet of the muscle plasma membrane. They measured 3–13 nm in diameter, 20–90 nm in length and were distributed with a spacing of 30–40 nm. The immunogold particles showing the presence of the merosin epitope were associated with these connecting structures. Received: 3 April 1996 / Revised, accepted: 15 July 1996