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.

Electrical activity of mouse motor endings during muscle reinnervation.

An in vitro study of electrical activity of regenerating motor endings was performed 11-15 days after the crushing of one motor nerve supplying the triangularis sterni muscle in the adult mouse. For this purpose, presynaptic membrane currents elicited by electrical stimulation of the regenerating nerve were recorded by external electrodes. Ionic channel distribution along the length of the endings was deduced from wave form configuration in normal perfusing fluid together with changes produced by application of specific channel blocking agents. The sharp negative deflection which was shown to correspond to inward Na+ current by its sensitivity to tetrodotoxin application could be recorded along most of the length of the endings indicating a widespread distribution of Na channels. Frequent absence of the late wave form component which signals K+ current was taken to indicate an even K+ current density in the few last nodes, the heminode and the distal part of the endings. Therefore, it appears that regenerating motor endings are characterized by an overlap of Na and K conductances all along their length. In the course of regeneration, the heminode loses the sensitivity to K channel blocking agents and the remainder of the terminal becomes insensitive to tetrodotoxin, the former change occurring usually earlier than the latter.

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.

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.

Advantages of the triangularis sterni muscle of the mouse for investigations of synaptic phenomena.

The triangularis sterni muscle (TS) of the mouse is a thin trapezoidal sheet of fibres in which individual neuromuscular junctions are easily observed with Nomarski optics. Thus, microelectrodes are readily positioned to accurately record various synaptic phenomena. For example, miniature end-plate currents were easily recorded with a focally positioned extracellular electrode and the end-plate sensitivity to acetylcholine averaged 2062 mV/nC. In addition, the intercostal nerves segmentally innervate the TS. Electrophysiologic and histologic analysis showed that each nerve innervates a sharply defined territory of the muscle surface. These preliminary observations suggest that the TS may be ideal for studies of synaptic function and the processes underlying synapse stabilization in the mammal.

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+.

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.

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.

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.

Inability of regenerating mouse motor axons to innervate a denervated target.

We used the triangularis sterni muscle of the adult mouse to study the influence of the connective sheaths that surround a nerve on the regeneration of its crushed axons. Our data indicate that regenerating axons: invade all the branches of a denervated pathway, independent of the presence of endplates; but are unable to escape from the perineurium even when vacant endplate sites are present in the immediate vicinity. Although regenerating axons do not share the first feature with intact sprouting axons, the second feature is common to both.

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.

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.

The dorsal column system: I. Existence of long ascending postsynaptic fibres in the cat's fasciculus gracilis.

Microelectrode recordings were made from dorsal column fibres at the 12th thoracic segment in cats. Two kinds of activity were elicited by electric shocks applied on the sciatic nerve in the popliteal fossa: primary afferent activity and trans-synaptically evoked activity. The dorsal column postsynaptic (DCPS) fibres represented 9.3% of the total population of fibres studied in the fasciculus gracilis and 14.5% of those with receptors in the skin. They were found to lie between the primary fibres of cutaneous and those of deep origin. The fastest fibres of the alpha range contributed to their activation and it is likely that C fibres contributed as well. 87% of the DCPS fibres studied at Th 12 were antidromically activated from the first cervical segment, and their conduction velocities measured between cervical and thoracic levels ranged from 16 to 71 m/sec.

The dorsal column system: II. Functional properties and bulbar relay of the postsynaptic fibres of the cat's fasciculus gracilis.

Microelectrode recordings were made from dorsal column postsynaptic (DCPS) fibres, in the fasciculus gracilis of the cat, at thoracic level Th 12, and from single cells in the nucleus gracilis. The sensitivity of the fibres (Th 12) and cells (bulbar level) to both gentle and noxious stimuli was studied and a classification of the units was made on the basis of their responses to these natural stimuli. The DCPS fibres have been classified into three groups: i) 16.3% of them were activated only by light mechanical stimuli, ii) a few responded to nothing but noxious mechanical stimuli, iii) 77% were characterized by their sensitivity to both gentle and noxious stimuli and constituted the polymodal group. The study of the nucleus gracilis cells revealed that 31.2% of the total number of cells investigated in the nucleus were also characterized by a high degree of modality convergence (polymodal cells). Besides their convergence, the polymodal DCPS fibres and the polymodal cells of the nucleus gracilis were functionally in contrast to the properties of the specific fibres and cells of the dorsal column system in other respects: i) they responded with a slowly adapting response to constant mechanical stimulation of the skin, ii) they were sensitive to noxious mechanical and thermal stimuli. It is argued that the polymodal cells could be the bulbar relay of the impulses conveyed through the DCPS fibres of the fasciculus gracilis. The possible role of the dorsal column system in nociception is discussed in the light of the results.

Dual innervation of end-plate sites and its consequences for neuromuscular transmission in muscles of adult Xenopus laevis.

1. Electrophysiological study of dually innervated end-plate sites was carried out in Xenopus laevis pectoral muscle fibres. End-plate potentials (e.p.p.s) and miniature end-plate potentials (m.e.p.p.s) have been recorded in Mg-blocked preparations. 2. The mean quantal content (m) of each e.p.p. at dually innervated end-plates was significantly smaller than the corresponding values obtained at singly innervated ones. At a given doubly innervated end-plate site the values of m tended to be inversely related, so that the compound value of m (obtained by adding them) was in the same range as that found in singly innervated junctions. These findings were taken to suggest the existence of an upper limit in the average amount of transmitter released at a synaptic site. 3. It was found that neither intermittent presynaptic conduction block, nor particular muscle fibre properties could account for the low values of m in dual end plates. The small size of the nerve terminals appears to be the most likely explanation. 4. The sensitivity to acetylcholine and muscle fibre electrical properties were investigated; no differences were found between fibres with sub- or suprathreshold e.p.p.s. 5. The nature of the factors responsible for this presumed small size of the nerve endings (competition between nerve endings for a limited synaptic space on the muscle membrane or reciprocal interaction between closely located terminals) as well as the possible origins of polyinnervation are discussed.

Ionic channel distribution in regenerating mouse motor endings.

One of the nerves supplying the Triangularis sterni muscle of the adult mouse was crushed and focal extracellular recordings were made from the regenerating motor nerve terminals. Stimulation of the nerve elicited 4 types of signals depending upon the position of the electrode along the newly formed terminal branches. Specific ionic channel blockers were applied in the bath or iontophoretically to identify the nature of the different signal components. Sodium and potassium channels appeared to be located along most of the length of the newly formed terminals. This overlapping distribution differs considerably from their segregated localization previously described for mature endings. During maturation of regenerating endings, potassium channels disappear from the heminodal area and the density of sodium channels is greatly reduced along the remainder of the branches. Presynaptic evidence for polyneuronal reinnervation of single endplates is presented.

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.

Reflex modulation of motoneurone activity in the cheliped of the crayfish Astacus leptodactylus.

1. The reflex activity elicited by movement of the mero-carpopodite (M-C) joint in the cheliped of the crayfish Astacus leptodactylus is investigated and the role of the different proprioceptors (chordotonal and myochordotonal organs) separately studied. 2. The reflex discharge involves mainly the tonic motoneurones of the extensor (E), the flexor (F) and the accessory flexor (AF) muscles. 3. M-C joint posture is also regulated by the cuticular stress detector (CSD2) afferents: they increase mainly the F discharge and secondarily the AF command. 4. The activity of the motor axons supplying the muscles of the meropodite can be also influenced by a variety of natural stimuli applied to other appendages. The effect usually produced is a general flexion reaction which is characterized by a reciprocity between E and F involving both central and peripheral mechanisms. 5. The AF muscle is innervated by two antagonistic motoneurones, an excitatory neurone functionally linked in its discharge with one of the four excitors supplying F and an inhibitory motoneurone, common with E. The resulting competitive effect between these two neurones has been recorded intracellularly in AF muscle fibres. 6. The role of the myochordotonal organ (MCO) in the crayfish is discussed. In particular the modulation of the AF command in relation to the discharges of the motor nerves to the main muscle E and F is studied.