Effect of castration on the morphology of the motor end-plates of the rat levator ani muscle.

The levator ani (L. A.) muscle, part of the genital apparatus of rodents, atrophies after castration. Changes in end-plate structure in the L. A. muscle of castrated male rats were examined with correlated light and electron microscopic methods. Four months after castration acetylcholinesterase staining reveals, in some muscle fibres, the presence of subneural gutters composed of a succession of cuplets whereas the subneural gutters are continuous and ramified in control muscles. Six months after castration most of the end-plates are further modified. Their terminal arborization, as revealed by silver nitrate staining, is more tortuous and irregular than in controls. At the ultrastructural level, reduced sole-plate and superimposed axonal endings are seen in some end-plates three months after castration. Our findings demonstrate that the changes (reduction of muscular activity and atrophy of muscle) are accompanied by adaptations of the neuromuscular junctions. As receptors for testosterone are known to be present in these motoneurons and muscle fibres, the observed morphological changes might be under the control of testosterone acting on both muscle and motoneurons.

Synaptophysin (p38) immunolabelling at the mouse neuromuscular junction.

The synaptophysin (p38), a transmembrane glycoprotein of synaptic vesicles, has been used as a marker in order to study the membrane events that take place during transmitter release at the mouse neuromuscular junction (NMJ). p38 has been labelled by immunofluorescence using a monoclonal anti-p38 antibody and fluorescein-conjugated IgG on dissociated muscle fibres (biceps brachialis m.). Its localization has been compared to that of the acetylcholine (ACh) receptors labelled with rhodaminated alpha-bungarotoxin. A weak labelling was obtained in nerve-muscle preparations at rest only when the muscle fibres were permeabilized with Triton X-100. By contrast, an intense immunofluorescence of the NMJ was observed after an exhaustive ACh release induced by Cd2+ in Ca(2+)-free medium, which leads to a synaptic vesicle depletion and an increase in the membranous structures in nerve terminals. Treatment with Cd2+ in Ca(2+)-free solution leads to both synaptic vesicle depletion and p38 immunolabelling, which is in favour of synaptic vesicle fusion and incorporation into the axolemma.

Effects of carbonyl cyanide m-chlorophenylhydrazone on quantal transmitter release and ultrastructure of frog motor nerve terminals.

The quantal acetylcholine release and the ultrastructural effects of the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone have been examined at frog neuromuscular junctions. Carbonyl cyanide m-chlorophenylhydrazone (2 microM) caused a temperature-dependent block of evoked quantal transmitter release accompanied by an increase in the rate of spontaneous quantal release. The carbonyl cyanide m-chlorophenylhydrazone-induced increase in miniature endplate potential frequency was neither antagonized nor prevented by tetrodotoxin. It also occurred in a Ca2+-free medium and after replacement of Ca2+ by Sr2+, indicating that it does not depend upon a Na+ or Ca2+ influx from the external medium but may act by releasing Ca2+ from intraterminal stores. Spontaneous quantal transmitter release was exhausted irreversibly within 4 h of carbonyl cyanide m-chlorophenylhydrazone (2 microM) action, during which time an average of 4.7 x 10(5) acetylcholine quanta were released per junction. The morphologic analysis revealed a significant temperature and time-dependent reduction in the number of synaptic vesicles with swelling and dispersion of mitochondria within the motor nerve terminals. Changes in synaptic vesicle number appear to be directly related to the intensity of transmitter release. The good correlation observed between the number of quanta secreted and the number of vesicles lost by nerve terminals in the absence of vesicle recycling provides an estimate of the initial store of transmitter quanta.

Persistence of functional neuromuscular junctions formed in a denervated skeletal muscle of the adult rat by axons that have regrown from the injured spinal cord through a peripheral nerve autograft.

In previous 'short-term' (2 to 7 months) experiments, we had demonstrated, in the adult rat, that motoneurons of the injured cervical spinal cord could extend lengthy axons into an autologous peripheral nerve segment which was connected to a nearby denervated skeletal muscle. In addition, we had shown that new functional motor endplates were formed by these axons both at the original sites of innervation and at ectopic locations of the denervated muscle. This substitution motor system, although quite functional, was anatomically very different from the original model of innervation in the intact animal, relating to its motoneuronal pool, the course of its motor axons and the sites of terminal innervation. The present 'long-term' (11 to 21 months) experiments demonstrate the anatomical and functional permanency of the new motor circuitry, despite a lack of strict specificity in the new neuromuscular connections. However, some minor modifications or adjustments were observed with time: (i) the maintenance of functional ectopic endplates could be consistently demonstrated, while functional reinnervated endplates at the initial sites of innervation were rare or even lacking; (ii) there was a definitive withdrawal of all non target-specific regenerated axons from the vicinity of the muscle. It is now necessary to address the question to what extent this substitution motor system is actually controlled by central and/or peripheral inputs.

Changes in large dense-core vesicles during maturation of the rat neuromuscular junction.

The proportion of large dense-core vesicles (LDCVs) in motor nerve terminals of the rat biceps brachialis muscle was evaluated from embryonic day 20 to 4 weeks postpartum as well as in the adult. A progressive decrease was observed up to 3 weeks postpartum when maturation of the endplates is achieved. Differences compared with the adult were no longer significant at 4 weeks postpartum. Three types of LDVCs, classified according to their size and the core density, were detected. Their relative proportion did not vary significantly during the period of life examined. The high proportion of LDCVs during early development and their persistence at a low level in the adult suggest that they might play a role in the maturation and maintenance of the endplates.

Ultrastructure of botulinum type-A poisoned frog motor nerve terminals after enhanced quantal transmitter release caused by carbonyl cyanide m-chlorophenylhydrazone.

The effects of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on spontaneous quantal transmitter release and nerve terminal ultrastructure were studied on isolated cutaneous pectoris nerve-muscle preparations from frogs that were completely paralysed by a single sublethal dose of Clostridium botulinum type A toxin (BoTx). CCCP enhanced miniature endplate potential frequency at poisoned junctions and caused a reduction in the density of clear synaptic vesicles and of large dense core vesicles in motor nerve terminals. However, the intensity of these effects was much less important than that previously reported at unpoisoned junctions. The moderate depletion of synaptic vesicles can be related to the low levels of transmitter release detected with CCCP at BoTx-poisoned terminals.

Sprouting of frog motor nerve terminals after long-term paralysis by botulinum type A toxin.

A single sublethal injection of botulinum type A toxin (BoTx-A) to winter frogs induced a general and complete paralysis of skeletal muscles, which lasted several months. Quantitative analysis of 483 end-plates from 8 BoTx-A poisoned muscles and 495 endplates from 8 control muscles revealed a higher and significant incidence of terminal and ultraterminal sprouts in poisoned junctions when taking into account the normal remodelling of motor innervation. We conclude that prolonged neuromuscular blockade by BoTx-A results in the extension of the nerve terminal arborization.

Formation of functional endplates by spinal axons regenerating through a peripheral nerve graft. A study in the adult rat.

Peripheral nerve (PN) autografts were used in the adult rat to join the midcervical spinal cord to a nearby denervated skeletal muscle. Retrograde tracing, morphological and electrophysiological studies indicated the following: 1) a great number of neurons, located bilaterally, between C3 and C7 in most laminae of the grey matter, extended axons into the PN grafts, 2) a lesser number of neurons regenerated up to the reconnected muscle, but most of them were typical motoneurons, 3) neuromuscular junctions were formed in ectopic locations, around the tip of the grafted nerve, and at the sites of original endplates, 4) these junctions were functional and formed by axons that had regenerated into the PN bridges, as muscle contraction was obtained by electrical stimulation of the grafted nerves, 5) they were proved to be cholinergic since endplate potentials, evoked by stimulating the PN graft, were suppressed by curare. These results strongly suggest that spinal neurons, and especially motoneurons, are involved in the formation, through PN bridges, of new functional cholinergic connections with denervated skeletal muscles.

Selective motor hyperreinnervation using motor rootlet transfer: an experimental study in rat brachial plexus.

Misdirection of sensory fibers into motor pathways is, in part, responsible for the poor results obtained after peripheral nerve repair. After avulsion of the C-5 root in rats, the authors connected a C-4 ventral rootlet to the musculocutaneous nerve by means of a sural nerve graft. In this way, they were able to increase the number of regenerating motor fibers and avoid growth of sensory fibers into the nerve grafts. Functional recovery was evaluated electrophysiologically and histologically. The origin of the axons that reinnervated the nerve graft was analyzed by means of morphological studies including retrograde labeling procedures. Motor neurons survived and regenerated after the rootlet transfer and there was no functional impairment. Many neurons were retrograde labeled in the ventral horn and widespread biceps muscle reinnervation was demonstrated with recovery of nearly normal electrophysiological properties. Motor hyperreinnervation of the musculocutaneous nerve was observed. This high degree of reinnervation in a long (40-mm) graft was attributed to the good chance that a muscle fiber can be reinnervated by a motor fiber when the number of regenerating motor neurons is increased and when competitive sensory fibers are excluded from reinnervation.

Increase in polyneuronal innervation in frog muscle after muscle injury.

The proportion of polyneuronal innervation was evaluated electrophysiologically in curare-blocked frog cutaneous pectoris muscles after local injury to the muscle fibres on one side. Focal polyneuronal innervation was revealed by recording end-plate potentials evoked by a gradual increase in the stimulus intensity applied to the motor nerve. An increase in the proportion of focally polyneuronally innervated muscle fibres appeared in the injured muscle 3-5 days after injury. The difference between the values obtained 3-5 days and 7-9 days (31 and 38%, respectively) and the control value (18%) was highly significant. A similar increase in the proportion of pluri-innervated muscle fibres was observed in the contralateral muscle, but after a longer period. The different components of complex end-plate potentials (e.p.p.s) usually had similar latencies and rise times in control and experimental muscles. This may indicate that the axons had similar conduction velocities and that synapses were located close to each other. A repeated muscle fibre section 24 h after the initial injury resulted in an enhanced polyneuronal innervation (52%) 7-9 days after the first section. The experiments were repeated on partially blocked muscles in order to detect small e.p.p.s with an amplitude similar to that of spontaneous miniature end-plate potentials (m.e.p.p.s). The proportion of polyneuronally innervated fibres estimated by this technique in control muscles approximated 40%. Polyneuronal innervation was also found to be significantly increased in cut muscles 7-9 days after muscle injury and a week later in contralateral muscles. Combined silver and cholinesterase staining was used to detect morphologically polyneuronal innervation. The comparison of morphological and electrophysiological data indicated that the increase in polyneuronal innervation after muscle injury is likely due to nerve sprouting and formation of new synapses. The results suggest that the signal for nerve sprouting originates from the damaged muscle cell and that it is transferred transneuronally to the contralateral side.

Morphological evidence for tracer uptake at the active zones of stimulated frog neuromuscular junction.

Lanthanum or ferritin added to the fixative were found in small and non-coated vesicles located at active zones in nerve-muscle preparations stimulated by potassium during cold aldehyde fixation. The presence of labeled vesicles at active zones supports the hypothesis that a double process of exo-endocytosis might occur under moderate stimulation conditions.

Action of vinblastine on the spontaneous release of acetylcholine at the frog neuromuscular junction.

1. Vinblastine induces reversible changes of the spontaneous release of acetylcholine (ACh) at the frog neuromuscular junction as characterized by the appearance of giant potentials. These large potentials occur soon after soaking the muscle in vinblastine and are not consequent to a large increase in the frequency of spontaneous release. Their number, relative to the total number of spontaneous potentials, increases with the duration of soaking. 2. Large potentials appear even in the presence of tetrodotoxin or in low Ca2+-high Mg2+ Ringer solution. 3. Following vinblastine treatment,the amplitude histogram of spontaneous potentials recorded from a number of fibres display an evident periodicity with peaks occurring regularly at simple multiples of the model amplitude of the unitary potentials. It is suggested that giant potentials are produced by the release of preformed pluriquantal packets of ACh. 4. Comparison of the amplitude distribution of spontaneous potentials and end-plates potentials show that only an insignificant number of large quanta are released by nerve stimuli. The absolute frequency of giant potentials does not markedly change when spontaneous discharge is accelerated by hypertonic solution. 5. The mechanism by which vinblastine induces the appearance of giant potentials is discussed.

Electrophysiological evaluation of the effects of naftidrofuryl on skeletal muscle reinnervation in the rat.

The effects of naftidrofuryl on the reinnervation of the rat gastrocnemius muscle after its denervation by localized freezing of the sciatic nerve were tested with electrophysiological techniques. Daily intraperitoneal injections of 30 mg/kg of naftidrofuryl do not increase the rate of axonal regeneration since early signs of reinnervation appeared as in controls around the 10th day after surgery. However, axonal sprouting is markedly increased since the percentage of muscle fibers with polyneuronal innervations was almost twice as high as in controls at the 15 and 21 day postoperative stages. The promoting effects of naftidrofuryl on polyneuronal innervation which gives rise to a redundant innervation during the first period of reinnervation constitutes an improvement of motor function which might be efficient for treatment of nerve injury and neuropathies.

Electrophysiological and morphological study of polyneuronal innervation in the cutaneous pectoris muscle of adult frog (Rana esculenta).

The incidence of polyneuronal innervation in the cutaneous pectoris muscle of the adult frog, Rana esculenta, was determined quantitatively using electrophysiological and morphological techniques. The mean percentages of multiple innervated endplates obtained with both techniques from a series of 19 muscles examined at all times of the year were in very good correlation: 30.7% (196/639 endplates) by electrophysiology and 30.5% (478/1569 endplates) by morphology. In nine muscles examined during the period from December to March the mean percentages, 36.8% (110/299) by electrophysiology and 38.6% (281/727) by morphology, were significantly higher than those obtained for 10 muscles investigated during the period from May to November, 25.3% (86/340) and 23.4% (197/842) with both techniques respectively. The higher incidence of collateral sprouted branches detected at polyinnervated endplate sites in muscles of winter frogs might be related to these seasonal variations. Most of the 1688 fibres from 26 muscles examined throughout the year exhibited one centrally located endplate. However, around 11% of them were found to be innervated at two separate endplate sites. Muscle fibres exhibiting this type of innervation were invariably the largest fibres in each muscle tested, having an apparent diameter greater than 48 micron. The distance between the endplates of these fibres represented between 10 and 30% of their total length. No significant seasonal variations were observed in the incidence of these dually innervated fibres. In conclusion, both electrophysiological and morphological results show that the normal incidence of polyneuronal innervation in the cutaneous pectoris muscle of adult Rana esculenta is affected by seasonal related factors which influence the nodal sprouting activity. Moreover, they show that a dual pattern of innervation is a common feature in large fibres of this muscle.

Cd(2+)-and K(+)-evoked ACh release induce different synaptophysin and synaptobrevin immunolabelling at the frog neuromuscular junction.

Synaptophysin and synaptobrevin, two integral proteins of synaptic vesicles, have been used as immunocytochemical markers of the synaptic vesicle membrane during Cd(2+)- or K(+)-induced ACH release at the frog neuromuscular junction. ACh release was stimulated in cutaneous pectoris nerve-muscle preparations by: (1) 1 mM Cd2+ in Ca(2+)-free medium for a period of 3 h, (2) 25 or 40 mM K+ in normal Ringer's solution. Synaptophysin and synaptobrevin were immunolabelled in single fibres teased from fixed muscles using rabbit antisera raised against synaptophysin and synaptobrevin revealed with fluorescein-conjugated IgG. The postsynaptic ACh receptors were simultaneously labelled with rhodaminated alpha-bungarotoxin. Unstimulated and K(+)-stimulated preparations showed synaptophysin and synaptobrevin immunolabelling only after membrane permeabilization with 0.1% Triton X-100. In preparations stimulated with Cd2+ in Ca(2+)-free medium, the immunofluorescence was also observed in non Triton X-100 treated muscle fibres. Confocal laser scanning microscopy analysis revealed that in unstimulated and K(+)-stimulated preparations, synaptophysin and synaptobrevin immunofluorescence appears as bands regularly spaced along the permeabilized nerve terminals and that their distribution corresponds to clusters of synaptic vesicles. After Cd2+ stimulation in Ca(2+)-free medium, labelling for both proteins is irregularly distributed, being more intense at the lateral margins of swollen nerve terminals, suggesting a translocation of synaptic vesicle proteins to the axolemma. At the electron microscopic level, Cd2+ stimulation in Ca(2+)-free medium produces nerve terminal swelling and synaptic vesicle depletion. The results show that when ACh release is stimulated under an impairment of synaptic vesicle recycling, which leads to synaptic vesicle depletion, synaptophysin and synaptobrevin translocation occurs. These findings are in favour of a permanent incorporation of synaptic vesicle membrane into the axolemma. In contrast, after K+ stimulation, the immunofluorescence and the normal synaptic vesicle population observed, suggest that a double process of synaptic vesicle exo-endocytosis rapidly occurs, without incorporation of synaptic vesicle components into the axolemma.

Membrane events related to transmitter release in mouse motor nerve terminals captured by ultrarapid cryofixation.

The sequence of structural changes occurring in the presynaptic membrane during transmitter release was studied at the mouse neuromuscular junction using the combined quick-freezing and cryosubstitution techniques. The mouse levator auris longus (LAL) muscle was stimulated by two means: either, chemically, by soaking 5 min before freezing in a physiological solution containing 25 mM potassium chloride or, electrically, by applying, 10 ms before freezing, a single supramaximal stimulus to the nerve-muscle preparation treated with 50 microM 3,4-diaminopyridine (3,4-DAP) and 100 microM (+)tubocurarine. In both cases, the preparations were maintained at approximately 5 degrees C, 5 min prior to freezing, in order to prolong nerve membrane changes. In most experiments, tannic acid (0.1%) was added to the substitution medium for better preservation of membranes. The different steps of warming in the substitution medium were strictly controlled from -90 degrees C to 4 degrees C. When fixed under chemical stimulation, the presynaptic membrane appeared very sinuous and synaptic vesicles were seen apposed to specialized sites facing subjunctional folds. When submitted to a single electrical stimulus, after treatment with 3,4-diaminopyridine, features of synaptic vesicle fusion were observed at these specialized sites which appear similar by their morphology, their macromolecular organization (already described) and their functional changes to active zones of the frog neuromuscular junction. Other images suggested that with 3,4-diaminopyridine which causes a pronounced and long-lasting release of transmitter, some vesicles collapse after exocytosis instead of being locally reformed by endocytosis.