Mary B. Rheuben

Ultrastructural Correlates of Neuromuscular Junction Development

Publisher Summary The larval and late embryonic neuromuscular junctions found on the abdominal muscles of Drosophila are a very useful experimental model system for examining the normal developmental processes underlying synapse formation and for examining the effects of lethal or semi-lethal mutations producing defects in proteins important to synaptic function. However, because some of the defects may be subtle, involving only quantitative changes in the sizes or number of pre- and postsynaptic organelles, it has been important to characterize the normal ultrastructure of the different types of nerve terminals that innervate these muscle fibers and to characterize the morphological responses that the muscle fiber makes to the presence of each of them in turn. Each of the abdominal muscle fibers is innervated by a unique combination of the subtypes of motor and neuromodulatory neurons. The secondary motor nerve branch containing their axons typically contacts each muscle fiber at a particular spot, and the type I terminals then give rise to a fairly consistent number and placement of branches that spread out from this point on each muscle fiber. Other terminal types diverge in a less consistent pattern. This region, referred to as the nerve entry point, is thus interesting from the point of view that it is the target for the ingrowing axons during development, as well as the point from which their terminals must disassociate from each other and form a specific branch pattern on the muscle fiber. The group of terminals from the various motor and neuromodulatory neurons that innervate each fiber is referred to as the junctional aggregate.

Disruption of mitochondrial DNA replication in Drosophila increases mitochondrial fast axonal transport in vivo

Mutations in mitochondrial DNA polymerase (pol γ) cause several progressive human diseases including Parkinsons disease, Alpers syndrome, and progressive external ophthalmoplegia. At the cellular level, disruption of pol γ leads to depletion of mtDNA, disrupts the mitochondrial respiratory chain, and increases susceptibility to oxidative stress. Although recent studies have intensified focus on the role of mtDNA in neuronal diseases, the changes that take place in mitochondrial biogenesis and mitochondrial axonal transport when mtDNA replication is disrupted are unknown. Using high-speed confocal microscopy, electron microscopy and biochemical approaches, we report that mutations in pol γ deplete mtDNA levels and lead to an increase in mitochondrial density in Drosophila proximal nerves and muscles, without a noticeable increase in mitochondrial fragmentation. Furthermore, there is a rise in flux of bidirectional mitochondrial axonal transport, albeit with slower kinesin-based anterograde transport. In contrast, flux of synaptic vesicle precursors was modestly decreased in pol γ−α mutants. Our data indicate that disruption of mtDNA replication does not hinder mitochondrial biogenesis, increases mitochondrial axonal transport, and raises the question of whether high levels of circulating mtDNA-deficient mitochondria are beneficial or deleterious in mtDNA diseases.

Neuromuscular Mechanisms of Insect Flight

The stringent requirements for successful flapping flight have resulted in remarkable adaptions of the skeleton, muscles and nervous system of animals capable of this means of locomotion. The wings have a large surface area but are lightweight to reduce the inertial costs of flapping. The flight muscles can contract and relax repeatedly and rapidly. Their high metabolic rate is adequately supported by sufficient fuel and oxygen that in many species flights lasting for hours are possible. The central nervous system produces the motor output that coordinates the contractions of the flight muscles and integrates information from a variety of receptors to adjust the motor pattern as required by environmental stimuli and the behavior of the insect. Some of these aspects of insect flight are discussed in other chapters of this volume. In this chapter we consider three topics: (1) flight muscles and their innervation, (2) motor patterns and their possible usefulness as an indicator of metabolic costs, and (3) putative roles of octopamine in flight.

Leech photoreceptors project their galectin‐containing processes into the optic neuropils where they contact AP cells

We characterized a subset of leech sensory afferents, the photoreceptors, in terms of their molecular composition, anatomical distribution, and candidate postsynaptic partners. For reagents, we used an antiserum generated against purified LL35, a 35 kD leech lactose‐binding protein (galectin); monoclonal antibody (mAb). Lan3‐2, which is specific for a mannose‐containing epitope common to the full set of sensory afferents; and dye injections. Photoreceptors differ from other types of sensory afferents by their abundant expression of galectin. However, photoreceptors share in common with other sensory modalities the mannose‐containing epitope recognized by mAb Lan3‐2. Photoreceptors from a given segment project their axons directly into the CNS ganglion innervating the same segment. They assemble in a target region, the optic neuropil, which is separate from the target regions of other sensory modalities. They also extend their axons as an optic tract into the connective to innervate optic neuropils of other CNS ganglia, thereby providing extensive intersegmental innervation for the 33 CNS ganglia comprising the leech nerve cord. Because of its intimate contact with the optic neuropil, a central neuron, the AP effector cell, is a strong candidate second order visual neuron. In confocal images, the AP cell projects its primary axon for about 100 μm alongside the optic neuropil. In electron micrographs, spines emanating from the axon of the AP cell make contact with vesicle laden nerve terminals of photoreceptors. Leech photoreceptors and their second order visual neurons represent a simple visual system for studying the mechanisms of axonal targeting.

Neuromuscular junctions are pathological but not denervated in two mouse models of spinal bulbar muscular atrophy

Spinal bulbar muscular atrophy (SBMA) is a progressive, late onset neuromuscular disease causing motor dysfunction in men. While the morphology of the neuromuscular junction (NMJ) is typically affected by neuromuscular disease, whether NMJs in SBMA are similarly affected by disease is not known. Such information will shed light on whether defective NMJs might contribute to the loss of motor function and represent a potential therapeutic target for treating symptoms of SBMA. To address this gap in information, the morphology of NMJs was examined in two mouse models of SBMA, a myogenic model that overexpresses wildtype androgen receptor (AR) exclusively in muscle fibres and a knockin (KI) model expressing a humanized mutant AR gene. The tripartite motor synapse consisting of motor nerve terminal, terminal Schwann cells (tSCs) and postsynaptic specialization were visualized and analysed using confocal microscopy. Counter to expectation, we found no evidence of denervation in either model, but junctions in both models show pathological fragmentation and an abnormal synaptophysin distribution consistent with functionally weak synapses. Neurofilament accumulations were observed only in the myogenic model, even though axonal transport dysfunction is characteristic of both models. The ultrastructure of NMJs revealed additional pathology, including deficits in docked vesicles presynaptically, wider synaptic clefts, and simpler secondary folds postsynaptically. The observed pathology of NMJs in diseased SBMA mice is likely the morphological correlates of defects in synaptic function which may underlie motor impairments associated with SBMA.

Affiliation of large numbers of fibroblasts with larval muscle fibers

Muscle fibers from fourth and fifth instar caterpillars were examined with scanning and thin section electron microscopy. Scanning micrographs showed that early fifth instar specimens had a population of cells lying beneath the basal lamina over the surface of the muscle fiber and in conjunction with tracheoles and nerves. At least two cell types were present. One type could be categorized as tracheoblasts of their close association with the tracheoles and the presence of taenidia within the tracheoblast cytoplasm in sectioned material. A second cell type, characterized by long filamentous processes, contained extensive rough endoplasmic reticulum and cisternae swollen with an electron-dense substance similar in appearance to the basal lamina. This ultrastructural appearance is characteristic of vertebrate fibroblasts and certain types of insect hemocytes. Early and late fourth instar specimens had few cells on their muscle fiber surfaces. Measurements of the basal lamina thickness were taken from thin sections of nondigested muscle fibers of early fourth, late fourth, and early fifth instar animals. The results showed that the basal lamina underwent a large increase in thickness between the fourth and fifth instars. The proliferation of cells which appeared to be in an actively synthesizing state paralleled the increase in basal lamina thickness. This suggests the hypothesis that these cells are active in connective tissue formation, and contribute to the formation of the basal lamina that lies over both them and the muscle fiber.

The Use of Gold Chloride to Stain Developing and Adult Neuromuscular Junctions in the Insect

Individual insect muscle fibers, whose neuromuscular junctions have been stained with a modification of Ranviers gold chloride method, can be dissected free and mounted whole if the muscle is prefixed in aldehydes. The neuromuscular junctions along the length of the individual fibers are well delineated and can be measured and counted. Effective procedures include fixation with glutaraldehyde buffered to low pH with sodium citrate, or glutaraldehyde and paraformaldehyde combined in phosphate buffer at neutral pH, followed by exposure to citric acid and to gold chloride. The method is convenient, and could be useful for the study of arthropod neuromuscular junctions in general, since their nerve terminals do not release acetylcholine as a transmitter and cannot be stained by the more commonly used cholinesterase methods.