Abnormal reinnervation of skeletal muscle in a tenascin-C-deficient mouse.

The possible involvement of tenascin-C in the reinnervation of a skeletal muscle was investigated in the tenascin-C-deficient mouse (T-/-) produced by Saga et al. (1992; Genes Dev 6:1821-1831). The pattern of reinnervation, observed after denervation of the triangularis sterni muscle, differs in T-/- and wild-type muscles in several traits. Axonal growth and stability of terminal arbors are impaired in the T-/- muscle: Some axons in mutant muscles grow beyond their original targets and reinnervate other synaptic sites, which may become dually innervated. In contrast to wild type, polyinnervation increases with time after denervation in T-/- muscles and is still present 7 months after nerve crush. The expression of a tenascin-C mRNA product disappears between 1 and 2 months after nerve crush. Of interest is that this transcriptional regulation in T-/- muscles occurs when major alterations in the morphology of regenerating endings become obvious. These observations strongly implicate tenascin-C in the formation, maturation, and stabilization of the neuromuscular junction.

Fiber types in the mouse levator auris longus muscle: a convenient preparation to study muscle and nerve plasticity.

The histochemical composition of the levator auris longus (LAL) muscle has been investigated in adult NMRi mice. Histochemical reaction for myofibrillar adenosine triphosphatase (ATPase) after preincubation in alkaline and acidic media, nicotine amideadenine-dinucleotide dehidrogenase (NADH-dehydrogenase), and alpha-glycerophosphate dehydrogenase were performed on cryosections of LAL muscle. Expression of myosin heavy chain (MyHC) isoforms was detected with the immunoperoxidase method applying monoclonal antibodies against MyHC isoforms -1, -2a, -2x/d, and -2b, as well as by sodium dodecylsulfate (SDS) glycerol gel electrophoresis. The muscle was proven to be a pure fast-twitch muscle. The most numerous fibers in LAL muscles contained MyHC-2b and some MyHC-2a. Histochemically, pure IIA fibers with oxidative metabolism and pure IIB fibers with glycolytic metabolism were detected. In contrast to the majority of mature control muscles, numerous hybrid fibers coexpressing MyHC-2x/d with MyHC-2a or MyHC-2b were present. Both hybrids were oxidative-glycolytic; additionally, some hybrids containing MyHC-2a were oxidative. In one out of six muscles, traces of MyHC-1 were detected both with immunoperoxidase staining and with SDS glycerol gel electrophoresis. Rare fibers that exceptionally expressed small amounts of MyHC-1 always coexpressed MyHC-2a, which is an additional proof that pure type I fibers do not exist in LAL. Due to these histochemical characteristics and to its previously described morphological features, the use of the LAL muscle as a model for various studies, particularly muscle and nerve interactions, is emphasized.

Enhanced neurotransmitter release is associated with reduction of neuronal branching in a Drosophila mutant overexpressing frequenin.

Frequenin is a Drosophila Ca2+ binding protein whose overexpression causes a chronic facilitation of transmitter release at the larval neuromuscular junction and multiple firing of action potentials. These functional abnormalities are similar to those found in other hyperexcitable mutants (Shaker, ether-a-gogo, Hyperkinetic) which, in turn, exhibit increased branching at the motor nerve endings. We report here that mutants which overexpress frequenin have motor nerve terminals with reduced number and length of branches as well as number of synaptic boutons. Similar defects are observed in transgenic flies which have additional copies of the frequenin gene indicating that the phenotype can be adscribed to the overexpression of the protein. The ultrastructure of boutons, however, appears indistinguishable from wild type. In addition, we show here that frequenin overexpression leads also to a down regulation of Shaker proteins expression. The contrast between the observations in frequenin and the other hyperexcitable mutants indicates that nerve terminal morphology and enhanced transmitter release do not have a direct causal relationship.

Incorporation of synaptotagmin II to the axolemma of botulinum type-A poisoned mouse motor endings during enhanced quantal acetylcholine release.

The involvement of terminal sprouts in neurotransmitter release by in vivo botulinum type-A toxin poisoned motor endings was investigated 15 to 40 days after a single injection of the toxin onto the levator auris longus muscle of the mouse. Enhanced quantal acetylcholine release was induced by alpha-latrotoxin or La3+ in conditions that prevent endocytosis, and an antibody directed against the lumenal domain of synaptotagmin II (Syt II) was used in the presence or absence of Triton X-100. We showed that, under resting conditions, the intravesicular domain of Syt II requires Triton X-100 to be labelled, whereas it becomes exposed to the outside of the axolemma of both the original terminal arborization and the newly formed sprouts during enhanced exocytosis. These data were taken to indicate that, when sprouting is prominent, the whole modified terminal arborization, including the original branches and the sprouts, possesses the machinery for Ca2+-independent exocytosis.

The peripheral nerve and the neuromuscular junction are affected in the tenascin-C-deficient mouse.

A thorough examination of the structure and plasticity of the neuromuscular system was performed in tenascin-C mutant mice deficient in tenascin-C. The study of the peripheral nerve revealed a number of abnormal features. In the motor nerve, numerous unmyelinated and myelinated fibers with degraded myelin were present. Schwann cell processes often enclosed degenerative terminals. Transgene (beta-galactosidase) expression analyzed at the ultrastructural level was found to be unequally distributed in the mutant's neuromuscular tissues. At the NMJ, preterminal disorganization was prevalent. Some axon terminals exhibited abnormal overgrowth. A surprising lack of beta-galactosidase expression at some cellular sites known to possess tenascin-C in wild type mice correlated best with marked changes in the cytoarchitecture of the peripheral nerve and NMJ. In some other -but not all- cellular sites which normally express the molecule, immunofluorescence analysis suggested the presence of significant but low levels of tenascin-C-like immunoreactivity together with beta-galactosidase expression. Messenger RNA detection by RT-PCR confirmed the presence of low amounts of tenascin-C mRNA in skeletal muscle suggesting that the mice deficient in tenascin-C are not complete knock-outs of this gene, but low-expression mutants. Following in vivo injections of botulinum type-A toxin, we observed a greatly reduced sprouting response of the motor nerves in tenascin-C mutant mice. We also observed that N-CAM and beta-catenin were overexpressed in the mutant. Our results suggest that tenascin-C is involved both in stabilization and in plasticity of the NMJ.

Nitric oxide and peroxynitrite affect differently acetylcholine release, choline acetyltransferase activity, synthesis, and compartmentation of newly formed acetylcholine in Torpedo marmorata synaptosomes.

Recent reports proposed that nitric oxide was a modulator of cholinergic transmission. Here, we examined the role of NO on cholinergic metabolism in a model of the peripheral cholinergic nervous synapse: synaptosomes from Torpedo electric organ. The presence of NO synthase was immunodetected in the cell bodies, in the nerve ending area of nerve-electroplate tissue and in the electroplates. Exogenous source of NO was provided from SIN1, a donor of NO and O2-., and an end-derivative peroxynitrite (ONOO-). SIN1 increased calcium-dependent acetylcholine (ACh) release induced by KCl depolarization or a calcium ionophore A23187. The formation of ONOO- was continuously followed by a new chemiluminescent assay. The addition of superoxide dismutase, that decreases the formation of ONOO-, did not impair the stimulation of ACh release, suggesting that NO itself was the main stimulating agent. When the endogenous source of NO was blocked by proadifen, an inhibitor of cytochrome P450 activity of NO synthase, both KCl- and A23187-induced ACh release were abolished; nevertheless, the inhibitor Ng-monomethyl-L-arginine did not modify ACh release when applied in a short time duration of action. Both NO synthase inhibitors reduced the synthesis of ACh from the radioactive precursor acetate and its incorporation into synaptic vesicles as did ONOO- chemically synthesized or formed from SIN1. In addition, choline acetyltransferase activity was strongly inhibited by ONOO- and SIN1 but not by the NO donors SNAP and SNP or, by NO synthase inhibitors. Altogether these results indicate that NO and ONOO modulate presynaptic cholinergic metabolism in the micromolar range, NO (up to 100 microM) being a stimulating agent of ACh release and ONOO- being an inhibitor of ACh synthesis and choline acetyltransferase activity.

Nerve terminal sprouting in botulinum type-A treated mouse levator auris longus muscle.

The marked outgrowth of the motor nerve terminal arborization triggered by an in vivo local injection of Clostridium botulinum type-A toxin in the mouse levator auris longus muscle was studied with morphological and immunochemical approaches. The increase in total nerve terminal length depended on the time elapsed after toxin administration and was due to both increased number of terminal branches and branch length as revealed by a quantitative morphological analysis of whole mounts using the combined cholinesterase-silver stain. Nerve terminal sprouts increased in number, length and complexity even after the functional recovery of neuromuscular transmission had occurred as revealed by electrophysiological examination. Although we cannot exclude that transmitter release sites from the original nerve terminal arborization may still be functional after botulinum type-A toxin (BoTx-A) treatment, it is likely that newly formed functional release sites on the sprouts play a major role in the functional recovery of neuromuscular transmission. The presence of an immunoreactivity to synaptophysin and synaptotagmin-II, integral proteins of synaptic vesicles, gives support to our previous findings suggesting that nerve terminal sprouts have the molecular machinery for acetylcholine release.

Synaptotagmin II immunoreactivity in normal and botulinum type-A treated mouse motor nerve terminals.

The distribution of synaptotagmin II, a synaptic vesicle protein, was examined by immunohistochemistry at normal mouse motor nerve terminals and after botulinum type-A treatment. An immunoreactivity to synaptotagmin II was detected in both control and botulinum type-A treated motor nerve terminals including newly formed sprouts. These data, together with other reports showing the absence of synaptotagmin I at the neuromuscular junction, suggest that synaptotagmin II is the isoform involved in transmitter release at motor nerve terminals.

Mouse motor nerve terminal immunoreactivity to synaptotagmin II during sustained quantal transmitter release.

An antibody directed against the lumenal NH2-terminus of synaptotagmin II was used to examine the distribution of this vesicular protein either after spontaneous acetylcholine release or after sustained release induced by La3+ or alpha-latrotoxin, in conditions that prevent endocytosis. The detection of the epitope was examined in the presence or absence of Triton X-100. We show that, in resting conditions of transmitter release, permeabilization of nerve terminal membranes is required for obvious detection of synaptotagmin Ii immunoreactivity whereas during sustained rates of quantal release, permeabilization is not necessary. These data indicate that, in the latter conditions, synaptotagmin II is incorporated into the terminal axolemma and its intravesicular domain exposed at the extracellular nerve terminal surface.

Plasticity of motor nerve terminals in Drosophila T (X,Y)V7 mutant: effect of deregulation of the novel calcium-binding protein frequenin.

The Drosophila T(X,Y)V7 mutant is characterized by abnormally large motor responses that build up upon repetitive stimulation. Genetically it is characterized by a chromosomal breakpoint located at the proximal end of the Shaker gene complex. This mutation affects a gene which encodes a novel calcium-binding protein: the frequenin. Since neuronal activity is known to affect neurite elongation we looked for the geometry of motor terminal arborization in this mutant. Our results show a significant reduction in number and length of motor terminal branches in mutants as compared to wild type. This observation is opposite to the effect of other hyperexcitable mutations such as Shaker or ether-a-gogo or Hyperkinetic. Thus the V7 phenotype cannot be interpreted as a result of changes in motoneuron firing pattern. According to results obtained on transformed larvae in which frequenin cDNA expression was under the control of a heat shock promoter, it appears that the morphological phenotype of V7 may be due to specific effects of deregulation of this calcium-binding protein.

Changes of quantal transmitter release caused by gadolinium ions at the frog neuromuscular junction.

1. The actions of the trivalent cation, gadolinium (Gd3+), were studied on frog isolated neuromuscular preparations by conventional electrophysiological techniques. 2. Gd3+ (450 microM) applied to normal or formamide-treated cutaneous pectoris nerve-muscle preparations induced, after a short delay, a complete block of neuromuscular transmission. The reversibility of the effect was dependent on the time of exposure. 3. Gd3+ (5-450 microM) had no consistent effect on the resting membrane potential of the muscle fibres. 4. Gd3+ (5-40 microM) applied to preparations equilibrated in solutions containing high Mg2+ and low Ca2+ reduced the mean quantal content of endplate potentials (e.p.ps) in a dose-dependent manner. Under those conditions, 3,4-diaminopyridine (10 microM) consistently reversed the depression of evoked quantal release. 5. The calcium current entering motor nerve terminals, revealed after blocking presynaptic potassium currents with tetraethylammonium (10 mM) in the presence of elevated extracellular Ca2+ (8 mM), was markedly reduced by Gd3+ (0.2-0.5 mM). 6. Gd3+ (40-200 microM) increased the frequency of spontaneous miniature endplate potentials (m.e.p.ps) in junctions bathed either in normal Ringer solution or in a nominally Ca(2+)-free medium supplemented with 0.7 microM tetrodotoxin. This effect may be due to Gd3+ entry into the nerve endings since it is not reversed upon removal of extracellular Gd3+ with chelators (1 mM EGTA or EDTA). Gd3+ also enhanced the frequency of me.p.ps appearing after each nerve stimulus in junctions bathed in a medium containing high Mg2+ and low Ca2+. 7. Gd3+, in concentrations higher than 100 microM, decreased reversibly the amplitude of m.e.p.ps suggesting a postsynaptic action. 8. It is concluded that the block of nerve-impulse evoked quantal release caused by Gd3 + is related to its ability to block the calcium current entering the nerve endings, supporting the view that Gd3 + blocks N-type Ca2+ channels; while the enhancement of spontaneous quantal release is probably the result of Gd3 + entry into motor nerve endings. Besides its dual prejunctional effects on quantal release it is suggested that Gd3 + exerts a postsynaptic action on the endplate acetylcholine receptor-channel complex.

Nerve terminal excitability and neuromuscular transmission in T(X;Y)V7 and Shaker mutants of Drosophila melanogaster.

We investigated the neuromuscular transmission in relation with genetic neuronal excitability changes in mutants T(X;Y)V7 and ShK,S133 of Drosophila. These mutations affect two different genes belonging to the Shaker gene complex which encode different yet functionally related proteins. Experiments were performed on neuromuscular junctions from Drosophila larvae by recording pre- and postsynaptic membrane currents using external electrodes. It was found that the neuromuscular electrophysiological phenotype of T(X;Y)V7 is caused by presynaptic membrane hyperexcitability probably in relation with a Ca2(+)-dependent down regulation of voltage dependent K channels. By contrast, the ShKS133 phenotype can be explained solely by action potential widening due to the absence of type-A K channels.

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. Spontaneous quantal transmitter release recorded as miniature end-plate potentials is drastically affected by BoTxs. The low probability of release at poisoned terminals can be enhanced by carbonyl cyanide m-chlorophenylhydrazone (CCCP), Cd2+ and La3+. However, CCCP and La3+ which drastically deplete clear synaptic vesicles from unpoisoned terminals failed to markedly affect the density of synaptic vesicles at poisoned terminals. It is concluded that poisoned terminals have a reduced sensitivity to the release-promoting action of Ca2+, Cd2+ and La3+. 3. When comparing the effect of the various BoTxs on nerve-impulse evoked transmitter release it appears that increasing phasic Ca2+ entry into the terminals enhances evoked synchronized quantal release only from terminals poisoned with serotypes A and E. In contrast, enhanced Ca2+ entry into terminals poisoned with serotypes B, D and F induced a period of high frequency asynchronous release suggesting that these BoTxs may affect a presynaptic step beyond the influx of Ca2+, that may be involved in the synchronization of transmitter quanta. These data suggest that the actions of BoTxs involve several steps of the acetylcholine release process. 4. The analysis of presynaptic currents which depend on both Ca2+ entry and intraterminal background Ca2+ levels strongly suggests that neither Ca2+ entry nor intraterminal Ca2+ levels are altered by BoTxs. Furthermore, poisoned terminals are no more efficient than unpoisoned ones in dealing with Ca2+ overloads. 5. Finally, the morphological examination of junctions paralysed by BoTx-A indicates that the toxin triggers a particularly important overgrowth of the nerve terminals and suggests that the in vivo functional recovery may occur from an extension of the original nerve terminal arborization and the concomitant remodelling of postsynaptic structures.

Terminal sprouting in mouse neuromuscular junctions poisoned with botulinum type A toxin: morphological and electrophysiological features.

Functional properties of terminal sprouts elicited by an in vivo injection of Clostridium botulinum type A toxin were studied in endplates of the Levator auris longus muscle of the mouse poisoned from a few days to 28 days beforehand. For this purpose, morphological observations of the extent of terminal sprouts and localization of acetylcholine receptors was performed in whole mount preparations. Sprouts appeared as thin unmyelinated filaments that run usually parallel to the longitudinal axis of the muscle fibres; labelling acetylcholine receptors revealed their line-shaped accumulation co-localized with the sprouts. In addition, presynaptic membrane currents elicited by nerve stimulation were recorded by external electrodes applied under visual control onto the membrane of pre-existing motor endings and newly formed sprouts. These recordings showed the presence of widespread triphasic waveforms which indicated active impulse propagation of the action potential over most of the length of the poisoned endings. Ca2+ influx and Ca2(+)-dependent K+ currents in the sprout membrane were found to be similar to those described in unpoisoned endings. The presence of normal Ca2+ influx, upon active depolarization, in the terminal sprout membranes together with the localization of acetylcholine receptors in front of these membranes, indicates that the terminal sprouts may play a role in the recovery of neuromuscular transmission after Clostridium botulinum poisoning.

Membrane currents in lizard motor nerve terminals and nodes of Ranvier.

Presynaptic membrane currents were recorded by external electrodes and nodal membrane currents were obtained by the voltage clamp technique in motor nerve endings and nodes of Ranvier of the lizard Anolis carolinensis. Although of compact shape, lizard motor endings display relatively long terminal branches; they exhibit, in agreement with previous findings in mouse and frog motor terminals, Na, Ca and K conductances, the latter consisting of a voltage- and a Ca-dependent type. Lizard nodes of Ranvier, like those of the frog, but unlike those of the mouse, exhibit a K conductance. These observations provide an explanation for the differences and similarities in presynaptic wave form configuration between the lizard and the other two species.

The interference of truncated with normal potassium channel subunits leads to abnormal behaviour in transgenic Drosophila melanogaster.

The Shaker locus of Drosophila melanogaster encodes a family of A-type potassium channel subunits. Shaker mutants behave as antimorphs in gene dosage tests. This behaviour is due to the production of truncated A-channel subunits. We propose that they interfere with the function of their normal counterpart by forming multimeric A-channel structures. This hypothesis was tested by constructing transgenic flies carrying a heat-inducible gene encoding a truncated A-type potassium channel subunit together with a normal wild type doses of A-type potassium channel subunits. The altered subunit leads at larval, pupal or adult stages to the transformation of wild type into Shaker flies. The transformed flies exhibited a heat-inducible abnormal leg shaking behaviour and a heat-inducible facilitated neurotransmitter release at larval neuromuscular junctions. By the overexpression of an aberrant A-channel subunit the normal behaviour of transgenic D. melanogaster can be altered in a predictable way.

Is the internal calcium regulation altered in type A botulinum toxin-poisoned motor endings?

The hypothesis according to which botulinum A toxin blocks acetylcholine release from motor endings by stimulating intracellular Ca2+ disposal systems was tested by recording presynaptic membrane currents from poisoned muscles. Calcium and calcium-activated potassium currents displayed amplitudes, time courses and stimulation frequency-dependent inactivation similar to those observed in unpoisoned preparations. This indicates that poisoned endings are no more efficient than normal ones in dealing with Ca2+ overloads.

The levator auris longus muscle of the mouse: a convenient preparation for studies of short- and long-term presynaptic effects of drugs or toxins.

We describe here the levator auris longus muscle of the mouse as a convenient neuromuscular preparation for the in vitro study of presynaptic effects of drugs and toxins applied in vivo in young or adult mice. The good visibility of its motor axons and terminals using Nomarski optics allows accurate electrophysiological studies of presynaptic signals. In addition, the levator auris longus muscle is sufficiently thin to be stained as a whole mount preparation. Preliminary results indicate that some correlation can be established between changes in time course of the presynaptic signal and the morphology of motor endings after poisoning the levator auris longus muscle with botulinum type A toxin.

In botulinum type A-poisoned frog motor endings ouabain induces phasic transmitter release through Na+-Ca2+ exchange.

Ouabain (100 microM) applied for 60 min to botulinum A (BoTx) poisoned motor junctions increases, in a time-dependent manner, the mean number of acetylcholine quanta released by nerve stimulation and enhances the delayed transmitter release. The drug does not affect spontaneous quantal release. The observed effects on evoked transmitter release cannot be explained by changes in the configuration of presynaptic currents recorded from motor terminals. They suggest that in BoTx-poisoned motor endings the level of intraterminal Ca2+, lower than that required for the activation of quantal transmitter release, can be effectively increased through the reversed operation of an Na+-Ca2+ exchange system that normally uses the Na+ gradient to extrude Ca2+.