The development of functional neuromuscular junctionsin vitro: An ultrastructural and physiological study

Neuromuscular junctions were formedin vitro between rat spinal cord explants and myotubes. At various intervals after the spinal cord explants were added to the myotube culture (7 hr to 15 days of coculture), the presence of functional neuromuscular junctions was determined by recording miniature endplate potentials (mepps) from the myotubes contacted by a few neurites. Electron microscopical studies were conducted on identified myotubes in which mepps were recorded. Mepps were already found as early as 7 hr after coculture. The fine structure of these newly formed neuromuscular junctions was simple. No synaptic specializations were observed except the presence of a small number of synaptic vesicles in the nerve. The neuromuscular junctions differentiated during the coculture period. Synaptic vesicles formed a cluster at the prejunctional membrane with a localized density in the middle. Basal lamina started to form in 4-day-old cocultures and became continuous in cocultures of 10 days or longer. Clear postjunctional foldings were observed in 15-day-old cocultures. Higher mepp frequencies were correlated with more advanced ultrastructure.     

Synaptic junctions in the sinus gland of the freshwater prawn Palaemon paucidens

Summary Two types of neurosecretory fibers, designated as Type 5 and Type 6 axons, in the sinus gland of the freshwater prawn, Palaemon, establish contact with other neurosecretory axons by means of synaptic junctions. This finding strongly supports the view that release of some neurohormones from the eyestalk may be regulated by neurosecretory neurons through synaptic transmission.The author wishes to express his sincere appreciation to Prof. T. Aoto for his invaluable advice during the course of this study, to Mr. S. Kubota for collecting the material, and to Mr. Y. Takakuwa for his excellent assistance in electron microscopy     

α-Tocopherol Modifies Lead Induced Functional Changes at Murine Neuromuscular Junction

Lead impacts neuromuscular junction and might induce skeletal muscle weakness. Antioxidants may prevent toxic actions of lead on muscle. In this study, resting membrane potentials, endplate potentials, miniature endplate potentials (MEPPs) and isometric twitch tensions were recorded to investigate effects of α-tocopherol (Vitamin E) on lead induced changes at murine dorsiflexor muscle. Moreover, levels of endplate nicotinic receptors were measured by receptor autoradiography. Forty rats were divided into four groups (lead alone, α-tocopherol, lead plus α-tocopherol and saline). Lead (1?mg/kg, i.p.), was administered daily for 2 weeks and α-tocopherol (100?mg/kg, i.p.) was given daily for 3 weeks. Lead treatment significantly reduced twitch tension (from 4.4±0.4 to 2.2±0.3?g) and delayed half time of decay. MEPP frequencies and quantal content were also significantly reduced after lead treatment. Pretreatment with α-tocopherol reversed twitch tension reduction (4.1±0.3?g) and modified lead induced delay in half time of decay. Similarly, α-tocopherol modified the negative actions of lead exposure on MEPP frequencies and quantal content. Receptor autoradiographic studies revealed significant increase of nicotinic receptor levels at the endplate region of flexor muscle in lead treated mice. However, animals treated with lead plus α-tocopherol showed significantly decreased levels of nicotinic receptors. α-Tocopherol appears to protect against lead induced neuromuscular dysfunction. These effects of α-tocopherol are possibly mediated via a free radical mechanism or modification of calcium homeostasis.     

In vitro formation of neuromuscular junctions between adult Rana muscle fibres and embryonic Xenopus neurons

Adult muscle fibres of the frog Rana temporaria were cultured with neurons from embryos of the frog Xenopus laevis. Electron microscopical and electro-physiological examination of the cultures showed that hetero-specific (Xenopus-Rana) neuromuscular junctions were formed in vitro. Nerve processes, without any Schwann cell covering, made contacts anywhere along a muscle fibre, and the junctions resembled those seen during early regeneration of neuromuscular synapses in situ. Functional contacts, as inferred by the presence of spontaneous miniature endplate potentials, or currents, were more common if the muscle fibres were denervated prior to culturing with neurons. Miniature endplate currents (m.e.p.cs) had a skewed amplitude distribution, with many small events lost in the recording noise, and their mean amplitude was much smaller than that of m.e.p.cs in the original lumbricalis muscle. The time constant of decay of m.e.p.cs in the hetero-specific junctions formed in vitro was several times longer than the decay of m.e.p.cs in the original muscle. Analysis of membrane current noise elicited by ionophoretically applied acetylcholine (ACh) suggests that the slower decay of m.e.p.cs in the junctions formed in vitro is due to a prolonged lifetime of the channels opened by ACh and to repetitive activation of ACh-receptors, which becomes possible because of a comparative lack of cholinesterase in the junctions.     

A method for testing an extended poisson hypothesis of spontaneous quantal transmitter release at neuromuscular junctions

A statistical method for testing the Poisson hypothesis of spontaneous quantal transmitter release at neuromuscular junctions has been proposed. The notion of the Poisson hypothesis is extended so as to allow for nonstationarity in the data, since nonstationarity is commonly seen in the occurrence of spontaneous miniature potentials. Special emphasis has been put on the nonstationary analysis of the quantal release. A time scaling technique has been introduced and is discussed for the analysis. Artificially generated data, which simulate three types of nonstationary spontaneous quantal release, i.e., Poisson, non-Poisson-clustered, and non-Poisson-ordered types, were analyzed to demonstrate the effectiveness of the method. Some sets of miniature endplate potentials, intracellularly recorded at frog sartorius neuromuscular junctions in low Ca++ and high Mg++ solutions showing apparent nonstationarities, were analyzed as illustrative examples. The proposed method will extend the range of applicable data for the statistical analysis of spontaneous quantal transmitter release.     

Endocochlear potential generation is associated with intercellular communication in the stria vascularis: Structural analysis in the viable dominant spotting mouse mutant

Summary Deafness in the viable dominant spotting mouse mutant is due to a primary defect of the stria vascularis which results in absence of the positive endocochlear potential in scala media. Endocochlear potentials were measured and the structure of stria vascularis of mutants with potentials close to zero was compared with that in normal littermate controls by use of morphometric methods. The stria vascularis was significantly thinner in mutants. Marginal cells were not significantly differnet from controls in terms of volume density or intramembrane particle density but the network density of tight junctions was significantly reduced in the mutants. A virtual absence of gap junctions between basal cells and marginal or intermediate cells was observed, but intramembrane particle density and junctional complexes between adjacent basal cells were not different from controls. The volume density of basal cells was significantly greater in mutants. Intermediate cells accounted for a significantly smaller volume density of the stria vascularis in mutants and had a lower density of intramembrane particles than controls. Melanocytes were not identified in the stria vascularis of mutants. These results suggest that communication between marginal, intermediate and basal cells might be important to the normal function of the stria vascularis.     

Unresponsive, a behavioral mutant in Xenopus laevis: electrophysiological studies of the neuromuscular system

Normal Xenopus laevis embryos begin movements at 1 day after fertilization. Embryos homozygous for the unresponsive mutation fail to move until 4 days after fertilization (just prior to feeding), after which they recover slowly. Electrophysiological studies were undertaken to determine the focus of this mutation. Formamide treatment of normal embryos was used to produce a phenocopy of the unresponsive condition, permitting direct comparisons between mutant and normal embryos. Intracellular recordings from muscle cells were obtained in formamide-treated and untreated preparations with both normal and unresponsive animals. Local electrical stimulation evoked either isolated endplate potentials and action potentials or after-discharges of these events in all preparations. A decrease in illumination also caused a burst of endplate potentials and action potentials. Therefore, the electrophysiology of the neuromuscular junction in unresponsive appears qualitatively normal; the effect of the mutation on the motor system is probably distal to the neuromuscular junction, either at or subsequent to excitation-contraction coupling.     

Intercellular junctions during development and in tissue cultures ofDrosophila melanogaster: An electron-microscopic study

Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis.The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos.Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development.All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae.Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.     

Discrepancies between spontaneous and evoked synaptic potentials at normal, regenerating and botulinum toxin poisoned mammalian neuromuscular junctions

Amplitudes and times to peak of spontaneous miniature endplate potentials (m.e.p.ps) and evoked quantal endplate potentials (e.p.ps) were compared at normal, regenerating and botulinum toxin poisoned neuromuscular junctions of the extensor digitorum longus muscle of the rat. At normal junctions the mean time to peak of m.e.p.ps was longer and more variable than that of similar-sized e.p.ps. At endplates where nerve regeneration was induced by mechanical crushing of the motor nerve the frequency of m.e.p.ps was reduced and their amplitude distribution was broader than normal. The distribution of times to peak of m.e.p.ps was considerably broader than that of quantal e.p.ps recorded at the same endplates. At neuromuscular junctions poisoned with botulinum toxin type A, spontaneous and evoked transmitter release were greatly reduced. The amplitude distribution of m.e.p.ps was wider than that of e.p.ps and the time to peak of e.p.ps was about twice as fast as and less variable than that of m.e.p.ps. To explain the observed differences in time to peak among m.e.p.ps and between m.e.p.ps and quantal e.p.ps we suggest that some m.e.p.ps, but not e.p.ps, originate from transmitter quanta released from sites at a greater distance from postsynaptic receptors or that the release or diffusion process for acetylcholine is more prolonged when producing some of the m.e.p.ps. Such mechanisms produce at normal junctions a small population of m.e.p.ps with prolonged times to peak, at regenerating junctions a greater proportion of such m.e.p.ps and in botulinum toxin poisoning a majority.     

Immuno-localization of ferritin polypeptides in oocytes and somatic tissue of the freshwater snails Lymnaea stagnalis L. and Planorbarius corneus L.

Summary Antibodies were raised in rabbits against the 19000 Mr and 24000 Mr polypeptides of snail ferritin from Lymnaea stagnalis L. Anti-24000 Mr polypeptide antibodies were purified by an affinity-purification step and were made monospecific for their antigen by preabsorption with the 19000 Mr antigen. These purified antibodies were then used for in situ detection of their respective antigens by the indirect immunofluorescence method. The 19000 Mr polypeptide was found widely distributed in tissues of both pulmonate snails investigated (Lymnaea stagnalis L. and Planorbarius corneus L.) with the most intense antigen-directed fluorescence in certain connective tissue cells, secretory cells of the midgut gland and Sertoli cells and epithelia of the gonadal acini. In contrast, the 24000 Mr polypeptide could be detected only in yolk platelets of vitellogenic oocytes. The results indicate that yolk and somatic cell ferritins differ in immunoreactivity and structure and, accordingly may differ in function.This investigation was supported by the Deutsche Forschungsgemeinschaft. I greatly appreciate the advice given to me by Drs. U. Mays and V. Riedel, Münster.     

Induction of action potentials in fish red muscle by denervation

The effects of denervation on the electrical membrane properties of fish red muscle were investigated. Forty to fifty hours after denervation, miniature endplate potentials disappeared abruptly and field stimulation of the nerve within the muscle failed to evoke endplate potentials, indicating that transmission failure occurred at this time. The membrane resistance of the red muscle fibre increased after denervation. Normally innervated fish red muscles do not generate action potentials in response to either nerve or direct muscle stimulation. However, approximately 3 weeks after nerve sectioning, action potentials could be induced in the muscles. The action potential was sodium-dependent, and was sensitive to tetrodotoxin. Actinomycin D injected in the early phase after operation suppressed the induction of the action potential. These results indicate that RNA synthesis is preliminary to the induction of the action potential mechanism, and that this mechanism is under neural control.     

The fine structure of the kidney of the hagfish (Myxine glutinosa L.)

Summary The fine structure of the kidney (glomerulus and archinephric duct) of the hagfish, Myxine glutinosa (L.) was studied in thin sections and by freeze-fracture technique.The glomerular filtration barrier is similar to that of mammalian kidneys. However, endothelial fenestrations are relatively scanty and the basement membranes of endothelial cells and podocytes always appear separated by a layer of collagen fibrils and microfibrils often surrounding numerous and extended mesangial cells. Between podocytes and their processes maculae occludentes and peculiar junctions of another type occur.The zonulae occludentes between epithelial cells of the archinephric duct are composed of five or more strands, occasionally of only one or two.Supported in part by Deutsche Forschungsgemeinschaft (SFB 146, STO B4) and NIH (ISOI-RR 05764).We are grateful to Dipl.-Ing. G. Wermbter for her helpful criticism and to Mr. H. Heidreich for his excellent technical assistance.     

Involution of the caudal musculature during metamorphosis in the ascidian,Botryllus schlosseri

Summary The caudal musculature of the free-swimming tadpole of the ascidian, B. schlosseri consists of cylindrical mononucleated cells connected in longitudinal rows flanking the axial notochord. During resorption of the larval tail, which is apparently induced by the contraction of the epidermis, muscle cells are dissociated and pushed into the body cavity where most of them are rapidly engulfed by phagocytes. In the initial stages of tail withdrawal muscle cells display surface alterations due to the disruption of intercellular junctions and disarrangement of myofibrils. Extensive degenerative changes, with shrinkage of mitochondria and disintegration of the contractile material are subsequently observed. Lysosomes and autophagic vacuoles are rarely seen and appear to play a secondary role in the degradation of the muscle cells, which occurs predominantly within the phagocytes. Myofilaments and myofibrils have never been observed within autophagic vacuoles. Clumps of muscle fragments and degenerated phagocytes undergo eventual dissolution in the blood lacunae, concomitantly with the differentiation of the young oozooid.This investigation was supported in part by a grant from the Muscular Dystrophy Associations of America and by CNR contract No. 7100396/04115542 from the Istituto di Biologia del Mare, Venice. We gratefully acknowledge the skillful assistance of Mr. G. Gallian, Mr. M. Fabbri and Mr. G. Tognon. We also thank the staff of the Stazione Idrobiologica at Chioggia for collecting the colonies.     

Intercellular junctions in the hypodermis,salivary gland and gené's organ of the cattle tick,Boophilus microplus

Summary The intercellular junctions that occur in the hypodermis, Gené's organ, and the salivary glands of the tick, B. microplus, are described. The epithelial cells of the hypodermis are connected by spot desmosomes and septate junctions and the secretory cells of Gené's organ by septate and gap junctions. The cap cells in the alveoli of the salivary gland connect to adjacent cells by gap junctions, hemidesmosomes and septate junctions into which microtubules are inserted.The authors would like to thank Mr. R. Lamb for preparing the plates. M.W.J. Megaw was supported by an S.R.C. Studentship     

T-DNA is organized predominantly in inverted repeat structures in plants transformed with Agrobacterium tumefaciens C58 derivatives

Summary The detailed structural organization of DNA sequences transferred to the plant genome via Agrobacterium tumefaciens has been determined in 11 transgenic tomato plants that carry the transferred DNA (T-DNA) at a single genetic locus. The majority (seven) of these plants were found to carry multiple copies of T-DNA arranged in inverted repeat structures. Such a high frequency of inverted repeats among transgenotes has not been previously reported and appears to be characteristic of transformation events caused by C58/pGV3850 strains of Agrobacterium. The inverted repeats were found to be centered on either the left or the right T-DNA boundary and both types were observed at similar frequency. In several plants both types of inverted repeat were found to coexist in the same linear array of elements. Direct repeats were observed in two plants, each time at the end of an array of inverted repeat elements, and at a lower frequency than inverted repeats. The junctions between T-DNA elements and plant DNA sequences and the junctions between adjacent T-DNA elements were mapped in the same 11 plants, allowing the determination of the distribution of junction points at each end for both types of junction. Based on a total of 17 distinct junctions at the right end of T-DNA and 19 at the left end, the distribution of junction points was found to be much more homogeneous at the right end than at the left end. Left end junctions were found to be distributed over a 3 kb region of T-DNA with two thirds of the junctions within 217 bp of the left repeat. Two thirds of the right end junctions were found to lie within 11 bp of the right repeat with the rest more than 39 bp from the right repeat. T-DNA::plant DNA junctions and T-DNA::T-DNA inverted repeat junctions showed similar distributions of junction points at both right and left ends. The possibilities that T-DNA inverted repeats are unstable in plants and refractory to cloning in wild type Escherichia coli is discussed. Two distinct types of mechanisms for inverted repeat formation are contrasted, replication and ligation mechanisms.     

Habitual exercise enhances neuromuscular transmission efficacy of rat soleus muscle in situ.

Rat motor nerve terminals and the endplates they interact with exhibit changes to varying patterns of use, as when exposed to increased activation in the form of endurance exercise training. The extent to which these changes affect neuromuscular transmission efficacy is uncertain. In this study, the effects of habitual exercise on the electrophysiological properties of neuromuscular transmission in rat soleus muscle were investigated using a novel in situ approach. Consistent with previous reports, miniature endplate potential frequency was enhanced by habitual exercise. Other passive properties, such as resting membrane potential, miniature endplate potential amplitude, and "giant" miniature endplate potential characteristics were unaltered by the training program. Full-size endplate potentials were obtained by blocking soleus muscle action potentials with mu-conotoxin GIIIb. Quantal content values were 91.5 and 119.9 for control and active groups, respectively (P < 0.01). We also measured the rate and extent of endplate potential amplitude rundown during 3-s trains of continuous stimulation at 25, 50, and 75 Hz; at 50 and 75 Hz, we found both the rate and extent of rundown to be significantly attenuated (10--20%) in a specific population of cells from active rats (P < 0.05). The results establish the degree of activity-dependent plasticity as it pertains to neuromuscular transmission in a mammalian slow-twitch muscle.     

Ultrastructural features of the synaptic complex of the vestibular nuclei of Lampetra planeri (Bloch)

Summary The vestibular fibres of ammocoetes of Lampetra planeri form a peculiar type of synaptic contact with the large nerve cells of the vestibular nuclei. In fact, a large expansion of the fibre is enveloped by the nerve cell cytoplasm, which makes a sort of spoon or actual channel by sealing its own plasma membrane with adhesion plaques around the fibre.In the whole area of juxtapposition between the fibre and the cell membranes, three different types of specialized contacts occur: desmosomoid junctions, which are the most numerous; zones having the characteristics of chemically active synapses on account of the presence of vesicles clustered on the presynaptic side; gap-junctions, where electrical transmission might occur. A puzzling feature is the presence of an enormous number of presynaptic vesicles located in the central part of the axoplasm of the fibre expansion.Supported in part by a grant to the Centre of Neuroembryology from the National Research Council.We wish to thank Prof. Aldo Rossi for supplying the perfusion technique and Mr. Dino Scorsini for his skilful technical assistance.     

Local development of action potentials in slow muscle fibres after complete or partial denervation.

Pyriformis muscles of Rana temporaria were completely or partially denervated by cutting the sciatic nerve or some of the small nerve branches entering the muscle. One stimulating and one to three recording microelectrodes were inserted along the fibres in order to compare the electrical activity at these points. In an early period following denervation action potentials of variable size and shape could be observed; these action potentials were often composed of two, sometimes of three or four, components. The size of individual components depended on the position of the recording microelectrode. Individual components could occasionally be triggered separately by adjusting the strength of the stimulating current pulse; propagation of these "all or none" responses was absent. In other fibres one component of the action potential could trigger another one several millimetres apart, thus indicating propagation. Conduction velocities were approximately 0.4 m/s. In partially denervated slow fibres, endplate potentials were confined to one lateral segment of the fibres, while the action potential occupied the denervated part of the membrane. The amplitudes of endplate and action potentials varied inversely with distance. Rough estimates of the length constant of the slow fibre membrane were calculated from the spatial decay of action potentials, endplate potentials and hyperpolarizing electrotonic potentials; mean values obtained were 2.5, 4.8 and 7.7 mm respectively. The results suggest that following denervation Na channels are built into discrete areas of the slow fibre membrane and that this process depends on the amount of denervation in individual fibres.     

Effects of α-Bungarotoxin and Reversible Cholinergic Ligands on Normal and Denervated Mammalian Skeletal Muscle

α-Bungarotoxin (BuTX; 5 μg/ml) completely blocked the endplate potential and extrajunctional acetylcholine (ACh) sensitivity of surface fibers in normal and chronically denervated mammalian muscles, respectively, in about 35 min. A 0.72 ± 0.033 mV amplitude endplate potential returned in normal muscle fibers after 6.5 hr. of washout of α-BuTX, and an ACh sensitivity of 41.02 ± 3.95 mV/nC was recorded in denervated muscle after 6.5 hr of wash (control being 1215 ± 197 mV/nC). A two-step reaction of BuTX with binding sites which may allosterically interact is postulated.

Several pharmacologic differences were noted between the ACh receptors at the normal endplate and those appearing extrajunctionally following denervation. In normal innervated muscles exposed to BuTX in the presence of 20 μM carbamylcholine or decamethonium, washout of both drugs restored twitch to control levels within 2 hr. Endplate potentials large enough to initiate action potentials were also recorded in most surface fibers. In contrast, these agents, in much higher concentrations (50 μM), were almost ineffective in preventing BuTX blockade of ACh sensitivity in denervated muscle. Hexamethonium (10 and 50 mM) depressed neuromuscular transmission and blocked the action of BuTX in normal muscle in a dose-dependent fashion. On the extrajunctional receptors, hexamethonium (50 mM) was ineffective in protecting against BuTX. We may conclude that at the normal endplate region there are two distinct populations of ACh receptors, both of which react with cholinergic ligands and BuTX, but that a small population (representing ± 1% of the total) reacts with BuTX reversibly. Our findings further suggest a clear distinction between ACh receptors located at the normal endplate region and those of the extrajunctional region of the chronically denervated mammalian muscle.