Joanne Pearce

Motor nerve terminals on abdominal muscles in larval flesh flies, Sarcophaga bullata: Comparisons with Drosophila

Motor nerve terminals on abdominal body‐wall muscles 6A and 7A in larval flesh flies were investigated to establish their general structural features with confocal microscopy, transmission electron microscopy, and freeze‐fracture procedures. As in Drosophila and other dipterans, two motor axons supply these muscles, and two morphologically different terminals were discerned with confocal microscopy: thin terminals with relatively small varicosities (Type Is), and thicker terminals with larger varicosities (Type Ib). In serial electron micrographs, Type Ib terminals were distinguished from Type Is terminals by their larger cross‐sectional area, more extensive subsynaptic reticulum, more mitochondrial profiles, and more clear synaptic vesicles. Type Ib terminals possessed larger synapses and more synaptic contact area per unit terminal length. Although presynaptic dense bars of active zones were similar in mean length for the two terminal types, there were almost twice as many dense bars per synapse for Type Ib terminals. Freeze‐fractures through the presynaptic membrane showed particle‐free areas indicative of synapses on the P‐face, within which were localized aggregations of large intramembranous particles indicative of active zones. These particles were similar in number to those found at active zones of several other arthropod neuromuscular junctions. In general, synaptic structural parameters strongly paralleled those of the anatomically homologous muscles in Drosophila melanogaster. In live preparations, simultaneous focal recording from identified varicosities and intracellular recording indicated that the two terminals produced excitatory junction potentials of similar amplitude in a physiological solution similar to that used for Drosophila. J. Comp. Neurol. 402:197–209, 1998.

Differential reflex activity determines claw and closer muscle asymmetry in developing lobsters

The paired claws and closer muscles of the lobster, Homarus americanus, are identical in the early juvenile stages, but subsequently differentiate into a stout crusher claw with only slow fibers and a slender cutter with largely fast fibers. Rearing with different substrates or exercise of the claws revealed that claw laterality is determined in the central nervous system by differential reflex activity in the paired claws; the side with greater activity becomes the crusher, while the contralateral side becomes the cutter.

Intramembranous organization of lobster excitatory neuromuscular synapses.

SummaryThe fine structure of identified neuromuscular synapses of the single excitatory axon to the distal accessory flexor muscle in lobster limbs was examined with freeze-fracture and serial thin-section electron microscopy. The latter technique reveals presynaptic dense bars with synaptic vesicles aligned on either side of these bars and often fused to the membrane, suggesting exocytosis and confirming our previous contention that these bars are active zones of transmitter release. The intramembranous organization of these active zones, as revealed in freeze-etched tissue, is a ridge-like elevation of the P-face of the axolemma with a matching trough on the complementary E-face. The ridge on the P-face has rows of large scattered intramembranous particles along the apex and is often bordered by a series of small, circular depressions which are presumed to represent exocytotic vesicles attached to the presynaptic membrane. Complementing these depressions are a few volcano-like protuberances seen occasionally on the E-face membrane. Because such evidence for transmitter release occurred in both stimulated and non-stimulated preparations, it demonstrates that chemical fixatives employing aldehydes induce transmitter release. The postsynaptic receptor sites of these excitatory synapses are characterized by oval-shaped patches of densely packed particles on the E-face, arranged in a random pattern on the sarcolemma. The complementary P-face view exhibits a regular square array of particle imprints or pits.

Proliferation and relocation of developing lobster neuromuscular synapses

Abstract The development of multiterminal innervation from a single identifiable excitatory motoneuron to the lobster distal accessory flexor muscle (DAFM) was studied by serial section electron microscopy. The number, size, and location of neuromuscular synapses and presynaptic dense bars within the peripheral branching pattern of the axon was determined in cross sections of the DAFM in 1st (24-hr-old)-, 4th (2-week-old)-, and 12th (1-year-old)-stage lobsters. The mean size of synapses remains fairly constant in these three stages but synaptic density, i.e., the number of synapses per unit length of fiber, increased more than 20-fold between the 1st and 4th stages and more than 5-fold between the 4th and 12th stages. Synaptic surface area per fiber length showed a parallel increase. Consequently there is a proliferation of synapses along the length of individual muscle fibers during primary development. Furthermore from the 1st stage where only a few fibers are innervated, synapses proliferate to many more fibers in the 4th and to all fibers in the 12th stage. The neuromuscular synapses are distributed in different proportions within the axonal branching pattern in the three stages. Based on the number and size of synapses and presynaptic dense bars, the main axon and primary branches provide almost equal amounts of innervation in the 1st stage. With further branching in the 4th stage, the main axon accounts for only 20–25% of the innervation; the primary branches for 45% and other finer branches the remainder. By the 12th-stage synapses are found only on branches other than the main axon and its primary offshoots. There is therefore a shift in innervation from the main axon to the primary branches and then to the finer branches during primary development. This shift in innervation involves the formation of new synaptic terminals and the restructuring of existing ones into axonal areas. In this way the multiterminal innervation arising from an identifiable motoneuron is remodeled.

Presynaptic dense bars at neuromuscular synapses of the lobster, Homarus americanus

SummaryThe threedimensional ultrastructure of presynaptic dense bars was examined by serial section electron microscopy in the excitatory neuromuscular synapses of the accessory flexor muscle in the limbs of larval, juvenile, and adult lobsters. The cross-sectional profile of the dense bar resembles an asymmetric hourglass, the part contacting the presynaptic membrane being larger than that projecting into the terminal. The bar has a height of 55–65 nm and varies in length from 75–600 nm. In its dimensions it resembles the dense projections in the synapses of the CNS of insects and vertebrates. The usual location of these dense bars is at well defined synapses, though a few are found at extrasynaptic sites either in the axon or terminal. In the latter case the bars are close to synapse-bearing regions, particularly in the larval terminals, suggesting that the extrasynaptic bars denote early events in synapse formation. In all cases the bars are intimately associated with electron lucent, synaptic vesicles located on either side, in the indentation of its hourglass-shaped cross sectional profile. The vesicles occur along the length of the bar and contact the presynaptic membrane. Consequently the dense bar may serve to align the vesicles at the presynaptic membrane prior to exocytosis. A similar role has been suggested for the presynaptic dense bodies at the neuromuscular junction of the frog, where synaptic vesicles form a row on either side of this structure.

Differentiation of identifiable lobster neuromuscular synapses during development

SummaryThe ultrastructure of physiologically identified low and high release synapses arising from a single axon on fibres of the distal accessory flexor muscle (DAFM) in a mature lobster was examined by serial section electron microscopy. Low release neuromuscular terminals located only on the proximal fibre were characterized by large synapses (mean area 2.084 μm2), small presynaptic dense bars (mean area 0.021 μm2) and hence a low (2.3%) ratio of dense bar area to synaptic area. In contrast high output terminals located only on the distal fibre had smaller synapses (mean area 0.625 μm2), larger dense bars (mean area 0.066 μm2) and a high (23.9%) ratio of bar area to synaptic area. A similar ratio was consistently found for each synaptic type in several other examples of mature lobsters. Hence it was used as a criterion for determining the point at which differentiation occurs during development. In the first larval stage (24 h old) the innervation was localized and undifferentiated. In the fourth (2 week old) and twelfth (1 y old) stage lobsters, the innervation had proliferated to small bundles of proximal and distal fibres. During development synapses increase in their mean surface area in the proximal fibre while remaining constant in the distal fibre. The mean surface area of the dense bars is similar in all stages except for the proximal fibres of the twelfth stage where it is smaller by 50%. Similarly the ratio of dense bar area to synaptic area is not significantly different for all stages except for the twelfth stage proximal fibres where it is half the value. Consequently differentiation of low and high release neuromuscular terminals occurs by the twelfth stage with an increase in the mean surface area of synapses and a decrease in the mean surface area of dense bars. This morphological differentiation is enhanced in the mature lobster.

Growth-related features of lobster neuromuscular terminals

Neuromuscular terminals of the low-output type formed by the single excitor axon to the limb distal accessory flexor muscle in the lobster Homarus americanus were studied with serial section electron microscopy. This type of innervation was compared between a small and a large lobster where a two-fold difference in mean quantal content of synaptic transmission was found. Several growth-related changes in the fine structure of these low-output synaptic terminals were seen. First, there was a proliferation of multiterminal innervation consisting of an increase in the number of nerve terminals, synapses and presynaptic dense bars between the small and large lobster. Also the mean surface area of the synapses increased significantly in the large compared to the small lobster. Second, synapses possessed distinct areas of non-specialized membrane or perforations which showed a growth-related increase in their number per synapse between small and large lobsters. Such perforations also occurred in the high-output synapses but only amongst the larger synapses of the older lobster. It is proposed that these perforations subdivided synapses into smaller functional units for membrane recycling as they provide a ready source of non-synaptic axolemma for nearby active sites (dense bars). Third, the branch point between subsidiary and principal terminals as well as the ending of a terminal is composed of synaptic membrane which is presumably involved respectively in the sprouting and elongation of nerve terminals during growth. Altogether these observations signify both qualitative and quantitative changes in identified neuromuscular terminals with growth.

Disruption of Synaptic Development and Ultrastructure by Drosophila NSF2 Alleles

First identified as the cytosolic component that restored intra‐Golgi vesicle trafficking following N‐ethylmaleimide poisoning, N‐ethylmaleimide‐sensitive factor (NSF) was later shown to be an ATPase that participates in many vesicular trafficking events. Current models hold that NSF disassembles postfusion SNARE protein complexes, allowing them to participate in further rounds of vesicle cycling. To further understand the role of NSF in neural function, we have embarked on genetic studies of Drosophila NSF2. In one approach, we employed transgenic flies that carry a dominant‐negative form of NSF2 (NSFE/Q). When expressed in neurons this construct suppresses synaptic transmission, increases activity‐dependent fatigue of transmitter release, and reduces the functional size of the pool of vesicles available for release. Unexpectedly, it also induced pronounced overgrowth of the neuromuscular junction. The aim of the present study was twofold. First, we sought to determine if the neuromuscular junction (NMJ) overgrowth phenotype is present throughout development. Second, we examined NSF2E/Q larval synapses by serial section electron microscopy in order to determine if there are ultrastructural correlates to the observed physiological and morphological phenotypes. We indeed found that the NMJ overgrowth phenotype is present at the embryonic neuromuscular synapse. Likewise, at the ultrastructural level, we found considerable alterations in the number and distribution of synapses and active zones, whereas the number of vesicles present was not changed. From these data we conclude that a primary phenotype of the NSF2E/Q transgene is a developmental one and that alteration in the number and distribution of active zones contributes to the NSF2E/Q physiological phenotype. J. Comp. Neurol. 487:101–111, 2005.

Enhanced reappearance of fast fibers in regenerating crayfish claw closer muscles

In the pristine claws of adult crayfish the muscle fibers of the closer are all of slow type as judged by sarcomere lengths of greater than 6 micron, and a uniform degree of myofibrillar ATPase activity. In regenerating claws of mature and immature crayfish, the muscle has a central band of fast type fibers as characterized by shorter sarcomeres (less than 6 micron) and a higher degree of ATPase activity than the surrounding slow fibers. During primary development, the closer muscle has a fiber composition similar to that of the regenerating muscle except for a smaller proportion of fast fibers. Thus the reappearance of fast fibers during regeneration recapitulates ontogeny while their enhanced proportions may reflect epigenetic influences such as restriction of nerve-mediated muscle activity in the limb bud.

Structure of allotransplanted ganglia and regenerated neuromuscular connections in crayfish.

In adult crayfish, Procambarus clarkii, motoneurons to a denervated abdominal superficial flexor muscle regenerate long-lasting and highly specific synaptic connections as seen from recordings of excitatory postsynaptic potentials, even when they arise from the ganglion of another crayfish. To confirm the morphological origins of these physiological connections we examined the fine structure of the allotransplanted tissue that consisted of the third abdominal ganglion and the nerve to the superficial flexor muscle (the fourth ganglion and the connecting ventral nerve cord were also included). Although there is considerable degeneration, the allotransplanted ganglia display intact areas of axon tracts, neuropil, and somata. Thus in both short (6-8 weeks) and long (24-30 weeks) term transplants approximately 20 healthy somata are present and this is more than the five axons regenerated to the host muscle. The principal neurite and dendrites of these somata receive both excitatory and inhibitory synaptic inputs, and these types of synaptic contacts also occur among the dendritic profiles of the neuropil. Axon tracts in the allotransplanted ganglia and ventral nerve cord consist largely of small diameter axons; most of the large axons including the medial and lateral giant axons are lost. The transplanted ganglia have many blood vessels and blood lacunae ensuring long-term survival. The transplanted superficial flexor nerve regenerates from the ventral to the dorsal surface of the muscle where it has five axons, each consisting of many profiles rather than a single profile. This indicates sprouting of the individual axons and accounts for the enlarged size of the regenerated nerve. The regenerated axons give rise to normal-looking synaptic terminals with well-defined synaptic contacts and presynaptic dense bars or active zones. Some of these synaptic terminals lie in close proximity to degenerating terminals, suggesting that they may inhabit old sites and in this way ensure target specificity. The presence of intact somata, neuropil, and axon tracts are factors that would contribute to the spontaneous firing of the transplanted motoneurons.