Effects of the ecdysoneless mutant on synaptic efficacy and structure at the neuromuscular junction in Drosophila larvae during normal and prolonged development

Neuronal axons are cellular extensions that can reach more than a meter in length. To maintain such a structure, macromolecules synthesized in cell bodies must be transported to the distal axons. Proteins associated with membranous organelles are generally transported in several fast transported groups, while cytoplasmic proteins, mostly composed of cytoskeletal proteins, are transported in slowly transported groups. Neurofilaments are a main component in the slowly transported group. Composed of three polypeptide subunits (NF-H, NF-M and NF-L), they are the most abundant cytoskeletal element in large myelinated axons. In various neurological or neurotoxic disorders, selective accumulation of neurofilaments was observed in different compartments of a neuron (cell bodies, proximal or distal axons). The underlying mechanism for this regional selectivity has been unclear. Using the classical pulse labeling method, we examined the changes in neurofilament transport velocity in transgenic mice that overexpress different neurofilament subunits. We present evidence that at least three velocities of neurofilament transport exist along peripheral motor axons. Each of these velocities was altered differently depending on which neurofilament subunit was overexpressed. We suggest that neurofilament transport in motor axons consists of multiple successive stages and that each of these stages is carried out by different transport mechanisms. These differences provide a basis for the regional deficiencies in axonal transport associated with several neurological disorders.     

Ultrastructural and physiological effects of the ionophore A23187 at identified frog neuromuscular junctions

The physiological and morphological effects of the calcium ionophore A23187 (calimycin) at the frog neuromuscular junction in vitro were examined. Miniature endplate potentials were recorded intracellularly during exposure to the ionophore. Preparations fixed 15 or 30 min after adding the drug to the incubating medium, which exhibited a greatly increased miniature endplate potential frequency, showed no obvious morphological differences when compared to controls with regard to synaptic vesicle number or distribution of vesicles within the terminal. However, after 45-60 min of exposure to the ionophore, when miniature endplate potential frequency had declined almost to zero, most of the nerve endings appeared devoid of synaptic vesicles and other organelles while the plasma membrane was intact. It is suggested that the apparent depletion of vesicles from the terminal induced by the calcium ionophore is a consequence of irreversible changes at the terminal.     

Structural alterations at the neuromuscular junctions of matrix metalloproteinase 3 null mutant mice

Matrix metalloproteinases are important regulators of extracellular matrix molecules and cell-cell signaling. Antibodies to matrix metalloproteinase 3 (MMP3) recognize molecules at the frog neuromuscular junction, and MMP3 can remove agrin from synaptic basal lamina (VanSaun & Werle, 2000). To gain insight into the possible roles of MMP3 at the neuromuscular junction, detailed observations were made on the structure and function of the neuromuscular junctions in MMP3 null mutant mice. Striking differences were found in the appearance of the postsynaptic apparatus of MMP3 null mutant mice. Endplates had an increased volume of AChR stained regions within the endplate structure, leaving only small regions devoid of AChRs. Individual postsynaptic gutters were wider, containing prominent lines that represent the AChRs concentrated at the tops of the junctional folds. Electron microscopy revealed a dramatic increase in the number and size of the junctional folds, in addition to ectopically located junctional folds. Electrophysiological recordings revealed no change in quantal content or MEPP frequency, but there was an increase in MEPP rise time in a subset of endplates. No differences were observed in the rate or extent of developmental synapse elimination. In vitro cleavage experiments revealed that MMP3 directly cleaves agrin. Increased agrin immunofluorescence was observed at the neuromuscular junctions of MMP3 null mutant mice. These results provide strong evidence that MMP3 is involved in the control of synaptic structure at the neuromuscular junction and they support the hypothesis that MMP3 is involved in the regulation of agrin at the neuromuscular junction.     

A comparison of miniature end-plate potentials at normal,denervated, and long-term botulinum toxin type A poisoned frog neuromuscular junctions

The effects of denervation and long-term botulinum toxin type A (BoTx) poisoning on miniature endplate potentials (m.e.p.p.s) in the frog were studied with intracellular microelectrode recording. BoTx reduced the frequency of m.e.p.p.s. to less than 1% of the level seen in untreated frogs, leaving a large percentage of tiny m.e.p.p.s and slow-rising m.e.p.p.s (slow m.e.p.p.s). Unlike what is observed in the rat, the frequency of slow m.e.p.p.s never increased above the low rate measured in the untreated controls, and in fact slightly but significantly decreased after BoTx. A comparison of the m.e.p.p.s seen after BoTx poisoning (BoTx m.e.p.p.s) and m.e.p.p.s seen after denervation (Schwann m.e.p.p.s) revealed many similarities between the two including amplitude and time-to-peak distributions, temperature Q₁₀ values and responses to several drugs and procedures. However, it was concluded that BoTx m.e.p.p.s do not originate from the Schwann cells because 1. denervation of BoTx-paralysed frogs abolishes all m.e.p.p.s and 2. the drug 4-aminoquinoline affects BoTx m.e.p.p.s and Schwann m.e.p.p.s in opposite ways, increasing the frequency of the former while almost eliminating the latter. BoTx m.e.p.p.s and Schwann m.e.p.p.s probably represent similar processes of secretion which are non-specific in nature, having a lower energy barrier than for normal release and not originating from specialized areas of transmitter release.     

Hypertonicity-induced transmitter release at Drosophila neuromuscular junctions is partly mediated by integrins and cAMP/protein kinase A

The frequency of quantal transmitter release increases upon application of hypertonic solutions. This effect bypasses the Ca²⁺ triggering step, but requires the presence of key molecules involved in vesicle fusion, and hence could be a useful tool for dissecting the molecular process of vesicle fusion. We have examined the hypertonicity response at neuromuscular junctions of Drosophila embryos in Ca²⁺-free saline. Relative to wild-type, the response induced by puff application of hypertonic solution was enhanced in a mutant, dunce , in which the cAMP level is elevated, or in wild-type embryos treated with forskolin, an activator of adenylyl cyclase, while protein kinase A (PKA) inhibitors decreased it. The response was also smaller in a mutant, DC0 , which lacks the major subunit of PKA. Thus the cAMP/PKA cascade is involved in the hypertonicity response. Peptides containing the sequence Arg-Gly-Asp (RGD), which inhibit binding of integrins to natural ligands, reduced the response, whereas a peptide containing the non-binding sequence Arg-Gly-Glu (RGE) did not. A reduced response persisted in a mutant, myospheroid , which expresses no integrins, and the response in DC0 was unaffected by RGD peptides. These data indicate that there are at lease two components in the hypertonicity response: one that is integrin mediated and involves the cAMP/PKA cascade, and another that is not integrin mediated and does not involve the cAMP/PKA cascade.     

Ultrastructural correlates of synapse withdrawal at axotomized neuromuscular junctions in mutant and transgenic mice expressing the Wld gene

We carried out an ultrastructural analysis of axotomized synaptic terminals in Wld(s) and Ube4b/Nmnat (Wld) transgenic mice, in which severed distal axons are protected from Wallerian degeneration. Previous studies have suggested that axotomy in juvenile (< 2 months) Wld mice induced a progressive nerve terminal withdrawal from motor endplates. In this study we confirm that axotomy-induced terminal withdrawal occurs in the absence of all major ultrastructural characteristics of Wallerian degeneration. Pre- and post-synaptic membranes showed no signs of disruption or fragmentation, synaptic vesicle densities remained at pre-axotomy levels, the numbers of synaptic vesicles clustered towards presynaptic active zones did not diminish, and mitochondria retained their membranes and cristae. However, motor nerve terminal ultrastructure was measurably different following axotomy in Wld transgenic 4836 line mice, which strongly express Wld protein: axotomized presynaptic terminals were retained, but many were significantly depleted of synaptic vesicles. These findings suggest that the Wld gene interacts with the mechanisms regulating transmitter release and vesicle recycling.     

An ultrastructural comparison of neuromuscular junctions in normal and developmentally arrested Rana pipiens larvae: Limited maturation in the absence of metamorphosis

Neuromuscular junctions in the rectus abdominis muscles of normal and developmentally arrested Rana pipiens larvae were studied with freeze fracture and conventional electron microscopy to determine whether structural aspects of junctional maturation depend on metamorphosis. Comparison was made between junctions in premetamorphic larvae 1–3 months old and junctions in larvae that had remained in premetamorphosis for more than a year (more than four times as long as normal). In most respects, junctions from the two groups of larvae were similar. Unlike adult junctions, nerve-muscle contacts in both larval groups were pleomorphic and often involved more than one neuronal process; Schwann cell processes very rarely extended between nerve and muscle. Active zone structure ranged from total disorganization to an adult pattern of highly ordered double rows of particles aligned over junctional folds. Only quantitative analysis revealed differences between junctions in old and young larvae. The older larvae had fewer nerve-muscle contact sites involving multiple neuronal elements and a higher ratio of active zone length to presynaptic membrane area, although the mean active zone length was the same in the two groups. The results indicate that the maturation of junctional shape, the branching pattern of the axons, and the relationship of presynaptic axons to Schwann cells must be directly or indirectly dependent on the hormonal or behavioral changes associated with metamorphosis.     

A method to identify,dissect and stain equine neuromuscular junctions for morphological analysis

Morphological study of the neuromuscular junction (NMJ), a specialised peripheral synapse formed between a lower motor neuron and skeletal muscle fibre, has significantly contributed to the understanding of synaptic biology and neuromuscular disease pathogenesis. Rodent NMJs are readily accessible, and research into conditions such as amyotrophic lateral sclerosis (ALS), Charcot–Marie–Tooth disease (CMT), and spinal muscular atrophy (SMA) has relied heavily on experimental work in these small mammals. However, given that nerve length dependency is an important feature of many peripheral neuropathies, these rodent models have clear shortcomings; large animal models might be preferable, but their size presents novel anatomical challenges. Overcoming these constraints to study the NMJ morphology of large mammalian distal limb muscles is of prime importance to increase cross‐species translational neuromuscular research potential, particularly in the study of long motor units. In the past, NMJ phenotype analysis of large muscle bodies within the equine distal pelvic limb, such as the tibialis cranialis, or within muscles of high fibrous content, such as the soleus, has posed a distinct experimental hurdle. We optimised a technique for NMJ location and dissection from equine pelvic limb muscles. Using a quantification method validated in smaller species, we demonstrate their morphology and show that equine NMJs can be reliably dissected, stained and analysed. We reveal that the NMJs within the equine soleus have distinctly different morphologies when compared to the extensor digitorum longus and tibialis cranialis muscles. Overall, we demonstrate that equine distal pelvic limb muscles can be regionally dissected, with samples whole‐mounted and their innervation patterns visualised. These methods will allow the localisation and analysis of neuromuscular junctions within the muscle bodies of large mammals to identify neuroanatomical and neuropathological features.     

Ultrastructure of the ocellar visual system in normal and mutant Drosophila melanogaster

Between the two compound eyes on the vertex on the adult head are the three simple eyes, ocelli. Transmission and scanning electron microscopy were used to investigate the corneal lenses, ocellar photoreceptors, and axonal projections in normal and mutant Drosophila melanogaster. In wild type flies, the cornea consists of about 45 lamellae. It has corneal nipples distally and is underlaid with a monolayer of corneagenous cells. Retinula cells have open rhabdomeres of about 2 microns (diameter) x 7 microns (length). Rhabdomeres extend to the distal extent of the cell and do not have caps. Microvilli have a rodlet within. Retinula cells are joined by belt desmosomes on the lateral borders. Eye color pigment granules are housed within the retinula cells of normal flies, not in accessory cells. The granules do not migrate in response to light. No screening pigment granules exist in the white mutant. Each ocellus has about 80 retinula cells whose axons project to corresponding ganglia from which 4 giant afferent interneurons (per ganglion) project to the brain. receptor terminals are invested with capitate projections from glia. Receptors synapse onto dyads of follower cells, usually interneuron processes, at sites of T shaped presynaptic ribbons. These "T bars" are surrounded by indistinct flattened vesicles. Interneurons make feed back synapses onto receptor terminals at T bars clustered with distinct round vesicles. Three mutants with abnormal ocelli were investigated. The none mutant has unusual compound eye and ocellar corneas. The compound eye is devoid of differentiated photoreceptors but some axons from undifferentiated cells from synapses. No receptors were found in the ocelli of none. The oc mutant has no ocelli, although sometimes an ocellar cornea like that of none is seen; the compound eye is normal. The rdo mutant is also specific to ocelli with smaller ocelli having half the wild type allotment of receptor cells; the number of giant afferents is unaffected. Mutants best known for their compound eye defects were examined. The norpA mutant loses its ocellar rhabdomeres with age but has normal feed forward and feed back synapses. This normal synaptology prevails despite the electrophysiological defects in norpA ocelli reported earlier. The rdgABS12 mutant has poorly formed ocellar receptors which show some degeneration with age but synapses survive. The trp. rdgBKS222 and rgdAPC47 mutants are essentially normal with respect to structure and survival of ocellar receptors and synapses.     

A lectin, peanut agglutinin, as a probe for the extracellular matrix in living neuromuscular junctions

Summary The extracellular matrix plays important roles in the differentiation of synapses. To identify molecules concentrated specifically in the synaptic extracellular matrix, fluorescently-labelled lectins were applied to neuromuscular junctions. A lectin, peanut agglutinin (PNA), stains the neuromuscular region selectively and irreversibly (up to at least 3 weeksin situ), outlining the periphery of the nerve terminal arborization in the frog. Snake neuromuscular junctions also stain intensely with fluorescent PNA, while mouse diaphragm staining is faint. At the electron microscopic level, the reaction products of horseradish peroxidase-conjugated PNA are found primarily in the extracellular matrix flanking Schwann cells in the frog endplate regions. Fluorescently labelled PNA does not affect synaptic potentials and can serve as a simple stain for correlating functional studies of living neuromuscular junctions. Moreover, it can be combined with a presynaptic dye to observe nerve terminals and synaptic extracellular matrix in the same junctionsin situ. This report reveals the existence of synapse-specific carbohydrates associated with Schwann cell extracellular matrix in the frog neuromuscular junction. The specific binding and its physiological compatibility make PNA a useful probe for further investigation of synaptic differentiation, plasticity and maintenance.     

Formation of the active zone at developing neuromuscular junctions in larval and adult bullfrogs

Summary Development of the presynaptic active zone was studied at neuromuscular junctions with freeze-fracture electron microscopy in larval and adult bullfrogs. In rudimentary larval neuromuscular junctions, clusters of active zone particles were scattered over the P-face of the presynaptic membrane. Vesicle openings were observed at these terminals even though active zone particles lacked the mature pattern of two double rows. Gradually, active zone particles became organized into rows, but they were still randomly located and oriented. Once junctional folds were observed in replicas, developing active zones were located opposite to the folds, as in mature terminals. Multiple terminals occupying the same junctional gutters were also observed. At the end of metamorphosis, most active zones were still immature in appearance and had only grown to one third of their mature length. After metamorphosis, the number of active zone segments aligned at the same junctional fold increased. These discontinuous short active zones then elongated, joined together, and finally formed the mature active zones. Signs suggesting synapse elimination such as disorganization of active zones, absence of intramembrane particles in varicosities, and exposed muscle membranes with patches of acetylcholine receptor aggregates were observed. In some multiply innervated junctions, one terminal had mature active zones with vesicle openings, the other in the same gutter displayed disorganized active zones without vesicle openings, although both terminals showed similar sizes and distributions of background particles. This study suggests that developing active zones, as is the case for regenerating active zones in the adult, are functional before the mature organization is formed. The sequence of development of active zones is also similar to that of regeneration except for the random location and orientation of early active zones in tadpoles. The comparison between regeneration and development further indicates that the process of active zone formation is related to junctional folds and/or associated structures. It is also suggested that synapse elimination may involve degenerative changes in presynaptic membranes, although direct evidence remains to be provided.     

Facilitation,augmentation and potentiation of transmitter release at frog neuromuscular junctions poisoned with botulinum toxin

Botulinum toxin type A (Botx) is a potent neurotoxin which inhibits specifically cholinergic synaptic transmission by an unknown mechanism. In order to gain further insight into the mode of action of this toxin, the effect of conditioning nerve stimuli on neuromuscular transmission was studied at endplates of Botx-poisoned and umpoisoned control cutaneous pectoris muscles in the frog. Effects of single conditioning stimuli (facilitation) and multiple high-frequency stimuli (augmentation and potentiation) on epp amplitude and mepp frequency were studied. The main results were that initial facilitation was significantly increased and its decay time constant significantly decreased in Botx-poisoned muscles, while augmentation was unchanged and potentiation was abolished. These changes could be detected before the muscle became completely paralysed, suggesting that they reflect a primary disturbance in the Ca²⁺-dependent release process.     

Strong immunoreactivity of beta-amyloid precursor protein, including the beta-amyloid protein sequence, at human neuromuscular junctions.

At the postsynaptic domain of the human neuromuscular junction (NMJ), we have demonstrated strong concentrations of the N-terminus 45-62, C-terminus 676-695 and beta-amyloid protein sequences of beta-amyloid precursor protein (beta APP). We used well-characterized monoclonal and polyclonal antibodies for co-localization with three other postsynaptic proteins, applying double and triple fluorescence labeling. Strong immunoreactivity of all three beta APP sequences was found at all NMJs identified by bound alpha-bungarotoxin (alpha BT), where they co-localized with alpha BT and with immunoreactive desmin and dystrophin, which are postsynaptic proteins of human NMJs. This appears to be the first demonstration of beta APP sequences concentrated postsynaptically at human NMJs. beta APP may have a role in normal junction biology and possibly in some diseases affecting NMJs.     

Two synaptic vesicle pools, vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction

Drosophila neuromuscular junctions ( D NMJs) are malleable and its synaptic strength changes with activities. Mobilization and recruitment of synaptic vesicles (SVs), and replenishment of SV pools in the presynaptic terminal are involved in control of synaptic efficacy. We have studied dynamics of SVs using a fluorescent styryl dye, FM1-43, which is loaded into SVs during endocytosis and released during exocytosis, and identified two SV pools. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low frequency nerve stimulation and releases FM1-43 during exocytosis induced by high K(+). The ECP locates close to release sites in the periphery of presynaptic boutons. The reserve pool (RP) is loaded and unloaded only during high frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the efficacy of synaptic transmission during low frequency neuronal firing. An increase of cAMP facilitates SV movement from RP to ECP. Post-tetanic potentiation (PTP) correlates well with recruitment of SVs from RP. Neither PTP nor post-tetanic recruitment of SVs from RP occurs in memory mutants that have defects in the cAMP/PKA cascade. Cyotochalasin D slows mobilization of SVs from RP, suggesting involvement of actin filaments in SV movement. During repetitive nerve stimulation the ECP is replenished, while RP replenishment occurs after tetanic stimulation in the absence of external Ca(2+). Mobilization of internal Ca(2+) stores underlies RP replenishment. SV dynamics is involved in synaptic plasticity and D NMJs are suitable for further studies.     

A synaptic balancing act: local and global signaling in the clustering of ACh receptors at vertebrate neuromuscular junctions

The clustering of acetylcholine receptors (AChRs) in muscle is a hallmark step in the development of vertebrate neuromuscular junctions (NMJs). It involves localized signaling, which is initiated by the activation of MuSK (muscle-specific kinase) and leads to the concentration and cytoskeletal anchoring of AChRs at the synapse, and global signaling, which traverses the muscle to disperse extra-synaptic AChR clusters. Here we review some of the recent findings that indicate important roles for the actin cytoskeleton and tyrosine phosphatases in mediating these processes.     

Depression of synaptic efficacy in high- and low-output Drosophila neuromuscular junctions by the molting hormone (20-HE)

The molt-related steroid hormone, 20-hydroxyecdysone (20-HE), was applied to muscles 6 and 7 of third instar larval of Drosophila melanogaster neuromuscular junction preparations to examine if rapid, nongenomic responses could be observed as was shown recently to occur in crustacean neuromuscular junctions. At a dose of 10 microM, the excitatory junction potentials were reduced in amplitude within minutes. To elucidate the site of action of the hormone, focal-macropatch recordings of synaptic currents were obtained over the neuromuscular junctions. The results showed that the high-output (Is) and the low-output (Ib) motor nerve terminals, which innervate muscles 6 and 7, released fewer synaptic vesicles for each stimulation while exposed to 20-HE. Because the size and shape of synaptic currents from spontaneous releases did not change, the effects of the 20-HE are presynaptic. The rapid effects of this hormone may account in part for the quiescent behavior associated with molts among insects and crustaceans.     

Formation of hydroxyapatite in water,Hank's solution,and serum at physiological temperature

The influence of de-ionized water, Hank's saline solution, and bovine calf serum on formation of stoichiometric (Ca/P = 1.67) hydroxyapatite (SHAp) at physiological temperature was studied. SHAp formed in aqueous solution by acid-base reaction of particulate Ca(H(2)PO(4))(2).H(2)O and Ca(4)(PO(4))(2)O. Hydroxyapatite formation is accompanied by an initial period of surface hydration of the precursors, an induction period, and a period during which the bulk of the conversion to hydroxyapatites occurs. The formation of SHAp occurred more rapidly in Hank's solution and distilled water than in serum. The formation of SHAp from these precursors is strongly inhibited by serum. There were two primary exothermal events associated with SHAp formation: initial heat evolution peak, which was associated with reactant dissolution, and the major heat evolution peak, which was associated with SHAp formation. The presence of the constitutents in serum depresses both. This is a result of serum macromolecules adsorbing onto the available surfaces regardless of whether they are reactants or products. Variations in heat evolution behavior, pH, and the times of disappearance of the reactants and appearance of SHAp correlate with one another.     

Examining the timing of miniature endplate potential releases at the frog and mouse neuromuscular junctions for deviations from Poisson expectations

The intervals between miniature endplate potentials (MEPPs) were measured at frog and mouse neuromuscular junctions in several different solutions. Data sets with monotonic trends in MEPP frequency were discarded. The remaining data sets had between 283 and 5024 MEPPs. The fit to the exponential distribution, which describes a Poisson process, was tested with Sherman's statistic. If the intervals are not distributed exponentially, this statistic indicates whether they deviate because they are more concentrated or more diffuse. In 6 of 20 data sets from the frog and in 3 of 7 data sets from the mouse the intervals between MEPPs were not distributed exponentially. Some of these were clustered, while others were diffuse. In one frog data set release was periodic. In all data sets releases appeared to be independent, judging from the integrated power spectra. One data set may show fractal behavior. Calculations of the approximate entropy gave no indication of an underlying regularity, so there is no evidence for a chaotic process. The lack of fit to the exponential distribution due to either concentrated or diffuse interval distributions is mimicked by a model in which release is Poisson, but with a mean rate that randomly shifts to a different level.     

Specific and innervation-regulated expression of the intermediate filament protein nestin at neuromuscular and myotendinous junctions in skeletal muscle

The intermediate filament proteins nestin, vimentin, and desmin show a specific temporal expression pattern during the development of myofibers from myogenic precursor cells. Nestin and vimentin are actively expressed during early developmental stages to be later down-regulated, vimentin completely and nestin to minimal levels, whereas desmin expression begins later and is maintained in mature myofibers, in which desmin participates in maintaining structural integrity. In this study we have analyzed the expression levels and distribution pattern of nestin in intact and denervated muscle in rat and in human. Nestin immunoreactivity was specifically and focally localized in the sarcoplasm underneath neuromuscular junctions (NMJs) and in the vicinity of the myotendinous junctions (MTJs), ie, in regions associated with acetylcholine receptors (AChRs). This association prompted us to analyze nestin in neurogenically and myogenically denervated muscle. Immunoblot analysis disclosed a marked overall increase of accumulated nestin protein. Similar to the extrajunctional redistribution of AChRs in denervated myofibers, nestin immunoreactivity extended widely beyond the NMJ region. Re-innervation caused complete reversion of these changes. Our study demonstrates that the expression levels and distribution pattern of nestin are regulated by innervation, ie, signal transduction into myofibers.