Large dense-core vesicles at the frog neuromuscular junction: characteristics and exocytosis.

The population of large dense-core vesicles (LDCVs) in motor nerve terminals of the frog cutaneous pectoris muscle was analysed after various experimental protocols leading to large acetylcholine release. Three types of LDCVs classified according to their size and the core density were detected. Vesicles, 100-150 nm in diameter, with a large and very dense core (type 1) or with an irregular and diffuse dense core (type 2) were present in similar proportions (45 and 50% respectively) in controls.

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.

[Post-traumatic reconnection of the cervical spinal cord with skeletal striated muscles. Study in adult rats and marmosets].

In an attempt at repairing the injured spinal cord of adult mammals (rat, dog and marmoset) and its damaged muscular connections, we are currently using: 1) peripheral nerve autografts (PNG), containing Schwann cells, to trigger and direct axonal regrowth from host and/or transplanted motoneurons towards denervated muscular targets; 2) foetal spinal cord transplants to replace lost neurons. In adult rats and marmosets, a PNG bridge was used to joint the injured cervical spinal cord to a denervated skeletal muscle (longissimus atlantis [rat] or biceps brachii [rat and marmoset]). The spinal lesion was obtained by the implantation procedure of the PNG.

Ultrastructural distribution of synaptophysin and synaptic vesicle recycling at the frog neuromuscular junction.

Synaptic vesicle recycling after intense acetylcholine (ACh) release was studied at the frog neuromuscular junction (NMJ) using the synaptic vesicle transmembrane protein synaptophysin as immunocytochemical marker of the synaptic vesicle membrane during the process of exo-endocytosis. ACh release in cutaneous pectoris nerve-muscle preparations was stimulated by three different means: K+, Cd2+ in Ca(2+)-free medium, and electrical stimulation in the presence of 4-aminopyridine (4-AP). Cd2+ stimulation produced synaptic vesicle depletion and nerve terminal swelling.

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 rat brachial plexus and its terminal branches: an experimental model for the study of peripheral nerve regeneration.

Despite the introduction of microsurgical techniques into clinical practice, the results of surgical procedures involving the brachial plexus and peripheral nerves are still far from spectacular. We therefore studied the rat brachial plexus and its terminal branches in 203 rats. Detailed anatomic and morphologic analyses of the biceps brachii and musculocutaneous nerve, finger flexors, flexor carpi radialis, and the median nerve were performed.

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.

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.

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.

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.).

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.

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.

Nerve sprouting induced by a piece of peripheral nerve placed over a normally innervated frog muscle.

1. The effects of a segment of peripheral nerve on the innervation of a skeletal muscle were investigated in the cutaneous pectoris muscle of the frog Rana esculenta. An explant of the sciatic nerve was placed in an aneural region of the muscle at a distance of 1-3 mm from the zone of neuromuscular junctions.

Presynaptic actions of botulinal neurotoxins at vertebrate neuromuscular junctions.

1. In the present paper we review some presynaptic aspects of the mode of action of botulinal toxins (BoTxs) at vertebrate neuromuscular junctions with emphasis on studies carried out in our laboratories using electrophysiological and morphological techniques. 2.

Terminal nerve sprouting at the frog neuromuscular junction induced by prolonged tetrodotoxin blockade of nerve conduction.

The effects of a prolonged blockade of nerve conduction by tetrodotoxin on frog motor innervation were studied in the cutaneous pectoris muscle of Rana esculenta. Prolonged nerve blockade (up to 22 days) was obtained by repeated subperineural injections of tetrodotoxin. Changes in morphological parameters of neuromuscular junctions were investigated in muscles after staining with a combined cholinesterase-silver method.

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.

Effects of cadmium on quantal transmitter release and ultrastructure of frog motor nerve endings.

Exposure of frog cutaneous pectoris nerve-muscle preparations to cadmium (0.1-1 mM) results in an increase in miniature end-plate potential (m.e.

Portable stimulator for direct electrical stimulation of denervated muscles in laboratory animals.

A portable lightweight stimulator for small animals is described. It delivers pulse trains of high intensity and is convenient for denervated muscle studies. It does not cause discomfort and does not restrict activity.

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.

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.

[Functional reinnervation of a skeletal muscle by means of a peripheral nerve autograft to the spinal cord by dorsal approach. Study in the adult rat].

Peripheral nerve grafts were used in adult rats to join the spinal cord to a denervated skeletal muscle. From 2 to 5 months later, results of electrophysiological, histological and retrograde horseradish peroxidase labelling studies showed that spinal neurons, and especially motoneurons, were probably involved in the formation of new functional cholinergic neuromuscular junctions through these bridges.