Activity-dependent plastic changes in the motor nerve terminals of the adult rat

Summary— Small and short-lasting physiologic variations in the locomotor activity of normal adult rats can induce remodelling in the motor nerve endings of the fast extensor digitorum longus muscle. The specificity and relative importance of the different plastic adaptations occurring in the presynaptic axonal tree have been studied, in silver impregnated nerve endings, by using an automatic image analysis treatment of the nerve terminals' geometric properties and a discriminant analysis of the morphometric parameters. Changes observed, like selective length variations in certain terminal segments and positional rearrangements, agree with a mechanism of neural connectivity regulation in the adult that arises as a consequence of normal neuromuscular activity.     

Morphological differences between excitatory and inhibitory nerve terminals in cockroach coxal muscles

Two morphologically distinct types of neuromuscular junction on the coxal leg muscles of the cockroach, Periplaneta americana, which have been physiologically described as innervated by fast, slow and inhibitory nerve fibers, have been found. In one type of neuromuscular junction the axon terminal contains many round clear synaptic vesicles and contacts several sarcoplasmic extensions from the muscle fiber. The muscle processes adhere to the axon terminal for a short distance (short contact or SC type). The axon terminal of the other type of neuromuscular junction directly contacts the muscle fiber and no extensions of the muscle fiber are formed. The contact region is comparatively long (long contact or LC type). The nerve terminal contains many polymorphic synaptic vesicles. From a correlation of the present morphological findings and the previous physiological results, it may be suggested that the SC type of nerve terminal represents both fast and slow nerve terminals and the inhibitory terminal is of the LC type.     

Peculiarities of synaptic vesicle recycling in frog and mouse motor nerve terminals

Using electrophysiology and fluorescence microscopy with dye FM 1-43, a comparative study of peculiarities of neurotransmitter secretion, synaptic vesicle exo-endocytosis and recycling has been carried out in nerve terminals (NT) of the skin-sternal muscle of the frog Rana ridibunda and of the white mouse diaphragm muscle during a long-term high-frequency stimulation (20 imp/s). The obtained data have allowed identifying three synaptic vesicle pools and two recycling ways in the motor NT. In the frog NT, the long-term high-frequency stimulation induced consecutive expenditure of the pool ready to release, the mobilizational, and reserve vesicle pools. The exocytosis rate exceeded markedly the endocytosis rate; the slow synaptic vesicle recycling with replenishment of the reserve pool was predominant. In the mouse NT, only the vesicles of the ready to release and the mobilizational pools, which are replenished predominantly by fast recycling, were exocytosed. The exo- and endocytosis occurred practically in parallel, while vesicles of the reserve pool did not participate in the neurotransmitter secretion. It is suggested that evolution of the motor NT from the poikilothermal to homoiothermal animals went by the way of a decrease of the vesicle pool size, the more economic expenditure and the more effective reuse of synaptic vesicles owing to the high rates of endocytosis and recycling. These peculiarities can provide in NT of homoiothermal animals a long maintenance of neurotransmitter secretion at the steady and sufficiently high level to preserve reliability of synaptic transmission in the process of the high-frequency activity.     

Fine structure,origin, and distribution density of the autonomic nerve endings in the tarsal muscle in the eyelid of the mouse

Summary The fine structure, origin, and distribution density of the autonomic nerve endings in the tarsal muscle of the mouse were studied by histochemistry and electron microscopy. With histochemical methods, the fine nerve plexus in the normal muscle shows both catecholamine-positive varicose fibers and acetylcholinesterase-active varicose fibers. The former are distributed more densely than the latter. After superior cervical ganglionectomy, the catecholamine-positive fibers disappear, while after pterygopalatine ganglionectomy, the acetylcholinesterase-active fibers vanish. In electron micrographs, the varicosities appear as expansions containing many synaptic vesicles. The axonal expansions partly lack a Schwann sheath and directly face the pinocytotic vesicle-rich zones of the smooth muscle cells. A relatively wide space, 0.1 to 1.0 m in width, lies between nerve expansion and muscle cell. The expansions can be classified into two types: Type I having small granular synaptic vesicles, and Type II having agranular vesicles instead of small granular synaptic vesicles. Type I undergoes degeneration after superior cervical ganglionectomy, while Type II degenerates after pterygopalatine ganglionectomy. This indicates that Type I corresponds to the synaptic ending of the adrenergic fiber originating from the superior cervical ganglion, and Type II to the synaptic ending of the cholinergic nerve fiber derived from the pterygopalatine ganglion. Type I is more frequent (88/10⁴ m² area of muscle) than Type II (17/10⁴ m²).     

Pattern of arborization of the motor nerve terminals in the fast and slow mammalian muscles.

A silver impregnation method and a morphometric approach were used to define differences existing in the motor nerve terminal branching pattern between a fast-twitch muscle (extensor digitorum longus) and a slow-twitch one (soleus) of the normal adult rat. Because no single measure can describe precisely all geometrical properties (ie both topology and metrics) of the nerve terminals, we evaluated morphologic parameters defining length and angular characteristics in the different terminal segments classified according to their centrifugal order. The main results indicate that the distal free-end segments in the extensor digitorum longus muscle are shorter and less divergent than in the soleus nerve terminals. The endings in the two muscles have different fractal dimensions. Findings are discussed in the context of the hypothetical mechanisms governing motor nerve terminal size and complexity.     

Cathepsin B-dependent motor neuron death after nerve injury in the adult mouse

There are significant differences in the rate of neuronal death after peripheral nerve injury between species. The rate of neuronal death of motor neurons after nerve injury in the adult rats is very low, whereas that in adult mice is relatively high. However, the understanding of the mechanism underlying axotomy-induced motor neuron death in adult mice is limited. Cathepsin B (CB), a typical cysteine lysosomal protease, has been implicated in three major morphologically distinct pathways of cell death; apoptosis, necrosis and autophagic cell death. The possible involvement of CB in the neuronal death of hypogrossal nucleus (HGN) neurons after nerve injury in adult mice was thus examined. Quantitative analyses showed the mean survival ratio of HGN neurons in CB-deficient (CB−/−) adult mice after nerve injury was significantly greater than that in the wild-type mice. At the same time, proliferation of microglia in the injured side of the HGN of CB−/− adult mice was markedly reduced compared with that in the wild-type mice. On the injured side of the HGN in the wild-type adult mice, both pro- and mature forms of CB markedly increased in accordance with the increase in the membrane-bound form of LC3 (LC3-II), a marker protein of autophagy. Furthermore, the increase in CB preceded an increase in the expression of Noxa, a major executor for axotomy-induced motor neuron death in the adult mouse. Conversely, expression of neither Noxa or LC3-II was observed in the HGN of adult CB−/− mice after nerve injury. These observations strongly suggest that CB plays a critical role in axotomy-induced mortor neuron death in adult mice.     

Rapid, stimulation-induced reduction of C12-resorufin in motor nerve terminals: linkage to mitochondrial metabolism

The Alamar blue (resazurin) assay of cell viability monitors the irreversible reduction of non-fluorescent resazurin to fluorescent resorufin. This study focused on the reversible reduction of C₁₂-resorufin to non-fluorescent C₁₂-dihydroresorufin in motor nerve terminals innervating lizard intercostal muscles. Resting C₁₂-resorufin fluorescence decreased when the activity of the mitochondrial electron transport chain (ETC) was accelerated with carbonyl cyanide m -chloro phenyl hydrazone, and increased when ETC activity was inhibited with cyanide. Trains of action potentials (50 Hz for 20–50 s), which reversibly decreased NADH fluorescence and partially depolarized the mitochondrial membrane potential, produced a reversible decrease in C₁₂-resorufin fluorescence which had a similar time course. The stimulation-induced decrease in C₁₂-resorufin fluorescence was blocked by inhibitors of ETC complexes I, III, and IV and by carbonyl cyanide m -chloro phenyl hydrazone, but not by inhibiting mitochondrial ATP synthesis with oligomycin. Mitochondrial depolarization and the decreases in C₁₂-resorufin and NADH fluorescence depended on Ca²⁺ influx into the terminal, but not on vesicular transmitter release. These results suggest that the reversible reduction of C₁₂-resorufin in stimulated motor nerve terminals is linked, directly or indirectly, to the reversible oxidation of NADH and to Ca²⁺ influx into mitochondria, and provides an assay for rapid changes in motor terminal metabolism.     

Intense ultraterminal sprouting from motor nerves and ultrastructural aspects of the neuromuscular junction and non-junctional sarcolemma of the soleus (slow-twitch) muscle in motor endplate disease in the mouse

Motor end-plate disease (med) in the mouse is an hereditary defect of the neuromuscular system, with partial functional denervation and muscle inactivity in late stages of the disease. Motor end-plate disease is characterized by an intense ultraterminal sprouting of the motor nerves from swollen nerve terminal branches in the soleus muscle. At the ultrastructural level, the neuromuscular junctions extend to very wide territories, often outside the original motor end-plate, in regions where the nerve sprouts are in simple apposition to the muscle fiber, with no secondary synaptic folds. The nerve terminals are rich in neurofilaments and poor in synaptic vesicles.Freeze fracture analysis of the pre-synaptic and post-synaptic membrane specializations fails to reveal any important structural alteration which could suggest a defect in acetylcholine release or in muscle membrane excitability. However, the non-junctional sarcolemmal specializations (the so-called ‘square arrays’) arc found with a frequency slightly higher than in normal muscle.The nerve abnormalities at the neuromuscular junction may be either a consequence of muscle inactivity or the morphological expression of some primary nerve abnormality. Further studies of the soleus muscle at early stages of the disease may provide evidence in favor of either possibility.     

Shared and unique roles of CAP23 and GAP43 in actin regulation, neurite outgrowth, and anatomical plasticity

CAP23 is a major cortical cytoskeleton-associated and calmodulin binding protein that is widely and abundantly expressed during development, maintained in selected brain structures in the adult, and reinduced during nerve regeneration. Overexpression of CAP23 in adult neurons of transgenic mice promotes nerve sprouting, but the role of this protein in process outgrowth was not clear. Here, we show that CAP23 is functionally related to GAP43, and plays a critical role to regulate nerve sprouting and the actin cytoskeleton. Knockout mice lacking CAP23 exhibited a pronounced and complex phenotype, including a defect to produce stimulus-induced nerve sprouting at the adult neuromuscular junction. This sprouting deficit was rescued by transgenic overexpression of either CAP23 or GAP43 in adult motoneurons. Knockin mice expressing GAP43 instead of CAP23 were essentially normal, indicating that, although these proteins do not share homologous sequences, GAP43 can functionally substitute for CAP23 in vivo. Cultured sensory neurons lacking CAP23 exhibited striking alterations in neurite outgrowth that were phenocopied by low doses of cytochalasin D. A detailed analysis of such cultures revealed common and unique functions of CAP23 and GAP43 on the actin cytoskeleton and neurite outgrowth. The results provide compelling experimental evidence for the notion that CAP23 and GAP43 are functionally related intrinsic determinants of anatomical plasticity, and suggest that these proteins function by locally promoting subplasmalemmal actin cytoskeleton accumulation.     

Developmental change in the calcium sensor for synaptic vesicle endocytosis in central nerve terminals

Synaptic vesicle endocytosis is stimulated by calcium influx in mature central nerve terminals via activation of the calcium-dependent protein phosphatase, calcineurin. However, in different neuronal preparations calcineurin activity is either inhibitory, stimulatory or irrelevant to the process. We addressed this inconsistency by investigating the requirement for calcineurin activity in synaptic vesicle endocytosis during development, using vesicle recycling assays in isolated nerve terminals. We show that endocytosis occurs independently of calcineurin activity in immature nerve terminals, and that a calcineurin requirement develops 2-4 weeks after birth. Calcineurin-independent endocytosis is not due to the absence of calcineurin activity, since calcineurin is present in immature nerve terminals and its substrate, dynamin I, is dephosphorylated on depolarization. Calcineurin-independent endocytosis is calcium-dependent, since substitution of the divalent cation, barium, inhibits the process. Finally, we demonstrated that in primary neuronal cultures derived from neonatal rats, endocytosis that was initially calcineurin-independent developed a calcineurin requirement on maturation in culture. Our data account for the apparent inconsistencies regarding the role of calcineurin in synaptic vesicle endocytosis, and we propose that an unidentified calcium sensor exists to couple calcium influx to endocytosis in immature nerve terminals.     

Transients in acetylcholine receptor site density and degradation during reinnervation of mouse sternomastoid muscle

The degradation rates of acetylcholine receptors (AchRs) were evaluated at the neuromuscular junction during and just after reinnervation of denervated muscles. When mouse sternomastoid muscles are denervated by multiple nerve crush, reinnervation begins 2-4 days later and is complete by day 7-9 after the last crush. In fully innervated muscles, the AChR degradation rate is stable and slow (t1/2 approximately 10 days), whereas after denervation the newly inserted receptors degrade rapidly (t1/2 approximately 1.2 days). The composite profile of degradation, which a mixture of the stable and the rapid receptors would give, is not observed during reinnervation. Instead, the receptors inserted between 2.5 and 7.5 days after the last crush all have an intermediate degradation rate of t1/2 approximately 3.7 days with standard error +/- 0.3 days. The total receptor site density at the endplate was evaluated during denervation and during reinnervation. As predicted theoretically, the site density increased substantially, but temporarily, after denervation. An analogous deleterious substantial decrease in density would be expected during reinnervation, without the intermediate receptor. This decrease is not observed, however, because of a large insertion rate at intermediate times (3000 +/- 700 receptor complexes per micro m2 per day). The endplate density of receptors thus remains relatively constant.     

Structure of cells and nerve endings in abdominal vagal paraganglia of the rat

Summary The abdominal vagal paraganglia of the rat consist of small groups of cells, interspersed by blood vessels and nerve bundles and lying close to, or within, the vagus nerve or its branches. Each cell group consists of 2–10 Type I cells incompletely invested by 1–3 satellite cells. Type I cells are characterised by the presence of numerous dense-cored vesicles in their cytoplasm and may exhibit synaptic-like contact with each other.Small efferent nerve endings make synaptic contacts with Type I cells. Larger cup-shaped afferent nerve endings also make synaptic contacts of two kinds with Type I cells. Nerve-nerve synapses are often seen within or close to paraganglia.Attention is drawn to the close similarity of fine structure of abdominal vagal paraganglia, carotid body and small intensely fluorescent cells of the superior cervical ganglion in rats. Possible functional implications of this morphological similarity are discussed.     

Computer reconstruction of the spread of excitation in nerve terminals with inhomogeneous channel distribution

A direct numerical integration method, as modified by Du Fort and Frankel (1953), has been used to solve the partial differential equation system which describes the spread of action potential in a mammalian nerve terminal. Branching of the terminal as well as inhomogeneous distributions of Na⁺ and K⁺ voltage-dependent channels (Brigant and Mallart 1982) have been incorporated in the model.Using the channel densities and the kinetic parameters measured in the node of Ranvier, the depolarization in the terminal branches has an amplitude of only 60% of the action potential in the node. Furthermore, the time courses of the calculated membrane currents differ considerably from the ones measured by Brigant and Mallart (1982) and by Konishi and Sears (1984).Increasing the Na⁺ and K⁺ channel densities may considerably increase the terminal depolarization and also reproduce qualitatively the current waveforms observed experimentally. The model can also reproduce some of the effects of pharmacological channel blocks.The simulation allows a new interpretation of the different components of membrane current along the terminal.     

Exercise-dependent regulation of glial cell line-derived neurotrophic factor (GDNF) expression in skeletal muscle and its importance for the neuromuscular system

The focus of this review is to highlight the importance of glial cell line-derived neurotrophic factor (GDNF) for the motor nervous system. GDNF is the most potent survival factor for motor neurons, where it enhances maintenance and survival of both developing and mature motor neurons in vivo and in vitro. GDNF aids in neuromuscular junction formation, maintenance, and plasticity, where skeletal muscle-derived GDNF may be responsible for this phenomenon. Increased levels of physical activity can increase GDNF protein levels in skeletal muscle, where alterations in acetylcholine and acetylcholine receptor activation may be involved in regulation of these changes observed. With inactivity and disuse, GDNF expression shows different patterns of regulation in the central and peripheral nervous systems. Due to its potent effects for motor neurons, GDNF is being extensively studied in neuromuscular diseases.     

Ultrastructure of motor nerve terminals on different types of muscle fibers in the atlantic hagfish (Myxine glutinosa,L.)

Summary White and intermediate parietal muscle fibers of Myxine are innervated focally at one end. Most synaptic vesicles are empty. These terminals also contain 1–2% large 800–1.100 Å dense-core vesicles. Red fibers of parietal and craniovelar muscle are innervated in a distributed fashion, and the presynaptic profiles contain a higher number of large dense-core vesicles (averaging 9% and 15%, respectively; up to 37%). For all terminals the synaptic gap is 450–600 Å wide, and postsynaptic folds are absent.Empty synaptic vesicles exist as round or elongated profiles. The proportion of elongated profiles increases by formation from round ones when increasing the molarity of the buffer in the aldehyde fixative. Furthermore, the proportion of elongated vesicle profiles in terminals on Myxine white fibers at different buffer molarities, is identical with that in mammalian motor terminals at similar molarities. On this basis the significance and mode of formation of elongated vesicle profiles is discussed. The conclusion is made that the susceptibility of flattening depends on the osmotic pressure of the vesicle contents once the aldehyde has influenced the vesicle membrane.The different vesicle populations in terminals on different types of muscle fibers are significant. Terminals on red fibers probably contain serotonin (5-HT) either as sole transmitter or in addition to acetylcholine.The author is indebted to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supply of hagfishes, and to Mrs. Jorunn Line Vaaland for expert technical assistance.