Ultrastructural and immunocytochemical studies of neuromuscular junctions in oviduct of Locusta migratoria

The ultrastructure of nerve endings in the oviduct visceral muscles of Locusta migratoria was studied by electron microscopy and by immunogold labeling for two kinds of neuromodulators, the pentapeptide proctolin and FMRFamide-related peptides. Nerve endings contained electron-lucent round vesicles and two kinds of granules (round and avoid), and formed two types of synapses or release sites with the muscle. The morphologically distinct nerve endings were classified into three different categories based on the composition of synaptic vesicles and granules. Type-I nerve endings were dominated by electron-lucent round vesicles and contained only a few round electron-dense granules. Type-II nerve endings contained mostly electron-dense round granules and electron-lucent round vesicles. A few electron-dense ovoid granules were also present. Electron-dense ovoid granules dominated the type-III nerve endings, which usually contained less electron-lucent vesicles than either type-I or II nerve endings. Both proctolin and FMRFamide-like immunoreactivity was associated with electron-dense round granules. However, FMRFamide-like immunoreactivity was only found in the type-II nerve endings, while proctolin immunoreactivity was found within type-I nerve endings as well as in some type-II nerve endings. Immunological results therefore allow us to further divide type-II nerve endings into type-IIa (immunonegative for proctolin) and type-IIb (immunopositive for proctolin). Type-III nerve endings show no immunolabeling to either proctolin or FMRFamide.     

Ultrastructural characteristics of rat neuromuscular junctions after physical load

It is interesting to ascertain the adaptive reaction of rat neuromuscular junctions (NMJ) of muscle fibers of different types to a chronic physical load. We examined ultrastructural changes in NMJ following both static load (pre- and postnatal ontogenesis of Wistar rats till a 2 month age took place under a constant rotation on the centrifuge at hypergravity conditions 2G), and after three kinds of dynamic loads (1/run on treadmill with a speed 35 m/min for 6 wks, 10-60 min/day; 2/swimmings, each 10 hrs/day for 10 days; 3/strength exercises on a vertical treadmill with load for 6 wks). Differences in NMJ reaction of muscle fibers of the same type to various loads were established. A low secretory activity of axonal terminals of type I muscle fibers of m. soleus was shown after the static load. The dynamic load (run) is accompanied with a high secretory activity of axonal terminals in m. soleus type I muscle fibers and of some axonal terminals of m. quadriceps femoris IIB type muscle fibers after strength exercises; the secretory activity of axonal terminals of m. quadriceps femoris IIA and IIB types muscle fibers is expressed in a lesser degree after swimming. The NMJ ultrastructure remodelling (terminal renewal) of type I muscle fibers of the 2 month old control rats increases after static and dynamic (run) loads. Some correlations between different kinds of physical load, muscle fiber type and the degree of NMJ ultrastructure transformation have been shown.     

Ultrastructural features of neuromuscular junctions in the stapedius muscle of Gallus gallus

The ultrastructure of neuromuscular junctions in the twitch fibers of the stapedius muscle of Gallus gallus (domesticus) was investigated as part of a series of neurophysiological studies. Among the morphological features observed were elongated end-plates with numerous large and clear synaptic vesicles mixed with larger dense core vesicles and irregular or aperiodic “active sites” in the presynaptic membrane where synaptic vesicles were focused. The most remarkable features of these junctions were large synaptic clefts (50-80 nm) and the absence of junctional folds in the sarcolemmal surface. Unlike the large periodic junctional folds seen in the neuromuscular junctions of frogs and in the fast twitch fibers of the mammalian stapedius, the preparations studied only show small aperiodic invaginations (primitive folds) in the postsynaptic membranes. This morphological feature remains essentially constant from newly hatched to adult chickens. While these smooth junctions are consistent with earlier findings of inconspicuous junctional folds in the twitch fibers of the chicken posterior latissimus dorsi they are unlike those seen in the fast twitch fibers of the mammalian stapedius muscle, or other twitch fibers in general. The morphological findings of the present study may also suggest that the simple, unmodified neuromuscular junctions in the stapedius of Gallus may be a useful preparation for studies of synaptic membrane structures that employ the freeze-fracture technique.     

Ultrastructural studies on neuromuscular contacts and the formation of junctions in the flight muscle of Antheraea polyphemus (Lep.)

In the moth Antheraea polyphemed at the onset of adult development. The subsequent breakdown of the isolated motor stulongated vesicles similar in structure to channels of smooth ER, appear in large numbers in the axoplasm. Their nature as well as the functional aspects of early axonal changes are discussed. From the 7th day onward two types of axonal breakdown become prominent. The first is characterized 0y swelling axon profiles, distorted vesicles and strongly shrunken mitochondria, uhile shrinking axon profiles containing tightly packed mitochondria and unaltered vesicles are typical of the second. Both types presumably take place independently of each other in different axon terminals. Axons and the contents of at least the first type are finally removed by transformation into lamellar bodies. Glial processes obviously behave independently of degenerating terminals; they loose any contact with them and never act as phagocytes for axon remnants. During the whole period of breakdown undifferentiated contacts between nerve fibers and muscle anlagen are present but synaptic structures as in normal developing dlm have never been observed. This fact, in comparison with earlier studies, suggests a lack of trophic nervous activity on the muscle anlagen tissue. A short time after removal of the isolated stumps new nerve tracts appear between dlm-fibers (which are, of course, strongly retarded in development). They are presumably sensory wing nerves which lack a guide structure to the central target, due to axotomy. Neuromuscular contacts or even junctions formed by axons of these nerves have occasionally been detected on the dlm. Their nature is discussed. Wallerian axon degeneration is compared to the normal, metamorphic breakdown of the innervation of the larval dlm-precursor. In contrast to the former, glial processes here remain in contact with the terminals. Glia and axons first swell. Then most glial processes are transformed into lamellar bodies whereas neurites shrink and become electron-dense. Axonal organelles remain intact for a long period.     

Ultrastructural studies on neuromuscular contacts and the formation of junctions in the flight muscle of Antheraea polyphemus (Lep.)

The ultrastructure of neuromuscular connections on developing dorsolongitudinal flight muscles were studied in the moth Antheraea polyphemus. Undifferentiated membrane contacts between axon terminals and muscle-fiber anlagen are present in the diapause pupa. They persist during the period of nerve outgrowth, which probably provides a pathway of contact guidance. By the 4th day of adult development some of these contact areas have differentiated into structures similar to neuromuscular junctions although differentiation of muscle structure does not start earlier than the eighth day. Dense-cored vesicles are abundant in many axon terminals at the beginning of development. They later decrease in number quite rapidly. The significance of the above-mentioned early junctions, their possible mode of action and the role of the dense-cored vesicles are discussed. It is proposed that they exercise a stimulating (trophic) influence on the growth of the undifferentiated muscular tissue. The imaginal neuromuscular junctions are formed during the second half of adult development. Clusters of vesicles and electron-dense depositions along the inner face of the axo- and lemma seem to initiate junction formation. Glial processes then grow between the axo- and sarcolemma and divide the large contact area into several small segments. Mutual invaginations and protrusions of the sarcolemma and the glial cell membrane subsequently form an extensive "rete synapticum." Six days before eclosion the glial and sarcoplasmic parts of the rete synapticum are similar in size. Up to eclosion, all glial processes shrink and increase in electron density. Most of the observations are discussed also in relation to findings in vertebrates.     

Assembly,plasticity and selective vulnerability to disease of mouse neuromuscular junctions

Although physiological differences among neuromuscular junctions (NMJs) have long been known, NMJs have usually been considered as one type of synapse, restricting their potential value as model systems to investigate mechanisms controlling synapse assembly and plasticity. Here we discuss recent evidence that skeletal muscles in the mouse can be subdivided into two previously unrecognized subtypes, designated FaSyn and DeSyn muscles. These muscles differ in the pattern of neuromuscular synaptogenesis during embryonic development. Differences between classes are intrinsic to the muscles, and manifest in the absence of innervation or agrin. The distinct rates of synaptogenesis in the periphery may influence processes of circuit maturation through retrograde signals. While NMJs on FaSyn and DeSyn muscles exhibit a comparable anatomical organization in postnatal mice, treatments that challenge synaptic stability result in nerve sprouting, NMJ remodeling, and ectopic synaptogenesis selectively on DeSyn muscles. This anatomical plasticity of NMJs diminishes greatly between 2 and 6 months postnatally. NMJs lacking this plasticity are lost selectively and very early on in mouse models of motoneuron disease, suggesting that disease-associated motoneuron dysfunction may fail to initiate maintenance processes at “non-plastic” NMJs. Transgenic mice overexpressing growth-promoting proteins in motoneurons exhibit greatly enhanced stimulus-induced sprouting restricted to DeSyn muscles, supporting the notion that anatomical plasticity at the NMJ is primarily controlled by processes in the postsynaptic muscle. The discovery that entire muscles in the mouse differ substantially in the anatomical plasticity of their synapses establishes NMJs as a uniquely advantageous experimental system to investigate mechanisms controlling synaptic rearrangements at defined synapses in vivo.     

Physiological studies during formation and development of rat neuromuscular junctions in tissue culture

Developing neuromuscular junctions in tissue cultures of rat embryo spinal cord and muscle were studied with intracellular recording. Excitatory junctional potentials (ejps) were found during nerve or spinal cord stimulation, and also arising spontaneously. The time-course of the potentials tended to be shorter in cultures older than 9 days in vitro than in more recently innervated cultures. Evidence of multiple innervation was found in many cells. In order to test the hypothesis that transmitter was released in integral multiples of a quantal amount and according to a Poisson distribution, mean quantum content was calculated from the coefficient of variation of ejp amplitudes, the percentage of zero responses, and the ratio of mean amplitude to mean of the smallest class of amplitudes. The three independent measures were in fair or good mutual agreement, implying that the mechanism of transmitter release in newly developed junctions is the same as in the adult. In newly formed junctions, ejps were subthreshold for action potential generation, but afterwards mean quantum content increased and action potentials were generated by single ejps. In fibers developing both with and without innervation, the entire muscle cell surface was as sensitive to acetylcholine as the adult end plate region. Innervation was related to cross-striation: every cross-striated fiber tested was found to be innervated, and denervation in vitro led to loss of distinct cross-striations.     

Striking denervation of neuromuscular junctions without lumbar motoneuron loss in geriatric mouse muscle

Reasons for the progressive age-related loss of skeletal muscle mass and function, namely sarcopenia, are complex. Few studies describe sarcopenia in mice, although this species is the mammalian model of choice for genetic intervention and development of pharmaceutical interventions for muscle degeneration. One factor, important to sarcopenia-associated neuromuscular change, is myofibre denervation. Here we describe the morphology of the neuromuscular compartment in young (3 month) compared to geriatric (29 month) old female C57Bl/6J mice. There was no significant difference in the size or number of motoneuron cell bodies at the lumbar level (L1-L5) of the spinal cord at 3 and 29 months. However, in geriatric mice, there was a striking increase (by ～2.5 fold) in the percentage of fully denervated neuromuscular junctions (NMJs) and associated deterioration of Schwann cells in fast extensor digitorum longus (EDL), but not in slow soleus muscles. There were also distinct changes in myofibre composition of lower limb muscles (tibialis anterior (TA) and soleus) with a shift at 29 months to a faster phenotype in fast TA muscle and to a slower phenotype in slow soleus muscle. Overall, we demonstrate complex changes at the NMJ and muscle levels in geriatric mice that occur despite the maintenance of motoneuron cell bodies in the spinal cord. The challenge is to identify which components of the neuromuscular system are primarily responsible for the marked changes within the NMJ and muscle, in order to selectively target future interventions to reduce sarcopenia.     

Modulation of vertebrate and invertebrate neuromuscular junctions

Advances in our understanding of how the neuromuscular junction is modulated include an expanded appreciation of the many different types of modulatory influences, from soluble factors to second-messenger systems, to specific proteins in nerve and muscle. Recent studies indicate that modulation of neuromuscular function is effected on both the presynaptic and postsynaptic sides of the neuromuscular junction.