Levator Auris Longus Muscle Research Articles

Modulation of ACh release by presynaptic muscarinic autoreceptors in the neuromuscular junction of the newborn and adult rat.

We studied the presynaptic muscarinic autoreceptor subtypes controlling ACh release and their relationship with voltage-dependent calcium channels in the neuromuscular synapses of the Levator auris longus muscle from adult (30-40 days) and newborn (3-6 and 15 days postnatal) rats. Using intracellular recording, we studied how several muscarinic antagonists affected the evoked endplate potentials. In some experiments we previously incubated the muscle with calcium channel blockers (nitrendipine, omega-conotoxin-GVIA and omega-Agatoxin-IVA) before determining the muscarinic response. In the adult, the M1 receptor-selective antagonist pirenzepine (10 micro m) reduced evoked neurotransmission ( approximately 47%). The M2 receptor-selective antagonist methoctramine (1 micro m) increased the evoked release ( approximately 67%). Both M1- and M2-mediated mechanisms depend on calcium influx via P/Q-type synaptic channels. We found nothing to indicate the presence of M3 (4-DAMP-sensitive) or M4 (tropicamide-sensitive) receptors in the muscles of adult or newborn rats. In the 3-6-day newborn rats, pirenzepine reduced the evoked release ( approximately 30%) by a mechanism independent of L-, N- and P/Q-type calcium channels, and the M2 antagonist methoctramine (1 micro m) unexpectedly decreased the evoked release ( approximately 40%). This methoctramine effect was a P/Q-type calcium-channel-dependent mechanism. However, upon maturation in the first two postnatal weeks, the M2 pathway shifted to perform the calcium-dependent release-inhibitory activity found in the adult. We show that the way in which M1 and M2 muscarinic receptors modulate neurotransmission can differ between the developing and adult rat neuromuscular synapse.

Pre- and postsynaptic maturation of the neuromuscular junction during neonatal synapse elimination depends on protein kinase C.

The distribution of acetylcholine receptors (AChRs) within and around the neuromuscular junction changes dramatically during the first postnatal weeks, a period during which polyneuronal innervation is eliminated. We reported previously that protein kinase C (PKC) activation accelerates postnatal synapse loss. Because of the close relationship between axonal retraction and AChR cluster dispersal, we hypothesize that PKC can modulate morphological maturation changes of the AChR clusters in the postsynaptic membrane during neonatal axonal reduction. We applied substances affecting PKC activity to the neonatal rat levator auris longus muscle in vivo. Muscles were then stained immunohistochemically to detect both AChRs and axons. We found that, during the first postnatal days of normal development, substantial axonal loss preceded the formation of areas in synaptic sites that were free of AChRs, implying that axonal loss could occur independently of changes in AChR cluster organization. Nevertheless, there was a close relationship between axonal loss and AChR organization; PKC modulates both, although differently. Block of PKC activity with calphostin C prevented both AChR loss and axonal loss between postnatal days 4 and 6. PKC may act primarily to influence AChR clusters and not axons, insofar as phorbol ester activation of PKC accelerated changes in receptor aggregates but produced relatively little axon loss.

Decreased calcium influx into the neonatal rat motor nerve terminals can recruit additional neuromuscular junctions during the synapse elimination period

Individual skeletal muscle fibers in newborn vertebrates are innervated at a single endplate by several motor axons. During the first postnatal weeks, the polyneuronal innervation decreases in an activity-dependent process of synaptic elimination by axonal competition. Because synaptic activity depends strongly on the influx of calcium from the external media via presynaptic voltage-dependent calcium channels, we investigate the relationship between calcium channels, synaptic activity and developmental axonal elimination. We studied how several calcium channel blockers affect (after 1 h of incubation) the total number of functional axons per muscle fiber (poly-innervation index) of the Levator auris longus muscle of 6-day-old rats. We determined the poly-innervation index by gradually raising the stimulus amplitude and recorded the recruitment of one or more axons that produced a stepwise increment of the endplate potential. The L-type channel blocker nitrendipine (1 μM) increased the mean poly-innervation index (35.79%±3.91; P<0.05). This effect was not washed out with normal Ringer, although the poly-innervation index returned to the control value when high-calcium Ringer (5 mM) was used. The P-type channel blocker ω-agatoxin-IVA (100 nM) also increased the number of recruitable endplate potentials (27.49%±1.78; P<0.05), whereas N-type channel blocker ω-conotoxin-GVIA (1 μM) was ineffective ( P>0.05). However, neither nitrendipine nor ω-agatoxin-IVA modified the poly-innervation index on high-calcium Ringer ( P>0.05 in both cases). A more intense inhibition of calcium influx (by the sequential use of two calcium channel blockers) did not recruit any additional silent synapses. Moderately increasing the magnesium ions (by 500 μM) in the physiological solution produces a synaptic recruitment (36.78%±2.1; P<0.05) similar to that with L- and P-type calcium channel blockers incubation. This magnesium effect was not washed with normal Ringer but a Ringer that is high in calcium can reverse it. The recruited endings were identified by selective activity-dependent loading with styryl dyes. Rhodaminated α-bungarotoxin-labeled acetylcholine receptors were present in the postsynaptic counterpart. Based on these findings we suggest that, before their complete retraction, functionally silent nerve terminals can be manifested or recovered if calcium influx is reduced by a calcium channel blocker or if external magnesium is increased. The normal activation of this calcium-dependent silencing mechanism during development may be related to the final loss of the supernumerary axons.

Calcium channels coupled to neurotransmitter release at dually innervated neuromuscular junctions in the newborn rat

We studied the effect of several calcium channel blockers (ω-Conotoxin-GVIA, 1 and 3 μM; ω-Agatoxin-IVA, 100 nM; Nitrendipine, 1 and 10 μM) on evoked transmitter release at singly and dually innervated endplates of the levator auris longus muscle from three- to six-day-old rats. In dually innervated fibers, a second endplate potential may appear after the first one when we increase the stimulation intensity. The lowest and highest endplate potential amplitudes are designated “small endplate potential” and “large endplate potential”, respectively. The percentage of doubly innervated junctions remains almost constant throughout the age range examined. Nevertheless, the percentage of junctions innervated by three or more terminal axons drops, whereas the singly innervated junctions increase. Therefore, between postnatal days 3 and 6, roughly half the neuromuscular junctions may experience the final process of axonal elimination. The synaptic efficacy of the large endplate potential in dual junctions, measured as the mean amplitude of the synaptic potential and mean quantal content, was the same as in the junctions that had become recently mono-innervated in the same postnatal period. In singly innervated fibers, the endplate potential size was strongly reduced by both the P/Q-type voltage-dependent calcium channel blocker ω-Agatoxin-IVA (79.17±4.02%; P<0.05) and the N-type voltage-dependent calcium channel blocker ω-Conotoxin-GVIA (56.31±7.80%; P<0.05), whereas endplate potential amplitude was not significantly changed by the L-type voltage-dependent calcium channel blocker Nitrendipine. In dually innervated fibers, the P/Q-type voltage-dependent calcium channel blocker ω-Agatoxin-IVA and L-type voltage-dependent calcium channel blocker Nitrendipine increased the size of the small endplate potential (161.29±47.87% and 109.32±11.03%, respectively; P<0.05 in both cases) and reduced the large endplate potential (74.42±15.32% and 70.91±10.04%, respectively; P<0.05 in both cases). The N-type voltage-dependent calcium channel blocker ω-Conotoxin-GVIA significantly increased the small endplate potential in the first few minutes after toxin application (at 10 min: 90.23±17.38%; P<0.05). This increase was not maintained, while the large endplate potential was strongly inhibited (69.25±7.5%; P<0.05). In conclusion, in the dually innervated endplates of the newborn rat, presynaptic calcium channel types can have different roles in transmitter release from each of the two inputs, which suggests that nerve terminal voltage-dependent calcium channels are involved in neonatal synaptic maturation.

Pertussis toxin-sensitive G-protein and protein kinase C activity are involved in normal synapse elimination in the neonatal rat muscle.

Individual skeletal muscle fibers in most new-born rodents are innervated at a single endplate by several motor axons. During the first postnatal weeks, the polyneuronal innervation decreases in a process of synaptic elimination. Previous studies showed that the naturally occurring serine-protease thrombin mediates the activity-dependent synapse reduction at the neuromuscular junction (NMJ) in vitro and that thrombin-receptor activation may modulate nerve terminal consolidation through a protein kinase mechanism. To test whether these mechanisms may be operating in vivo, we applied external thrombin and its inhibitor hirudin, and several substances affecting the G protein-protein kinase C system (GP-PKC) directly over the external surface of the neonatal rat Levator auris longus muscle. Muscles were processed for immunocytochemistry to simultaneously detect acetylcholine receptors (AChRs) and axons for counting the percentage of polyinnervated NMJ. We found that exogenous thrombin accelerated synapse loss and hirudin blocked axonal removal. Phorbol-12-myristate-13-acetate, a potent PKC activator, had a similar effect as thrombin, whereas the PKC inhibitors, calphostin C and staurosporine, prevented axonal removal. Pertussis toxin, an effective blocker of GP function, blocked synapse elimination. These findings suggest that the normal synapse elimination in the neonatal rat muscle may be modulated, at least in part, by the pertussis-sensitive G-protein and PKC activity and that thrombin could play a role in the postnatal synaptic maturation in vivo.

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. J. Neurosci. Res. 59:692–697, 2000 © 2000 Wiley-Liss, Inc.

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.

Topological differences along mammalian motor nerve terminals for spontaneous and alpha-bungarotoxin-induced sprouting.

Spontaneous sproutings can be observed in end plates from normal adult vertebrate muscles and motor end plates develop increased growth signs and sprouts when target muscle cells become less active or paralysed. Nevertheless, very little is known about where in the motor nerve terminal arborization spontaneous and experimentally induced sprouts originate, their similarities and differences and also about their final maturation or elimination. In this study we investigate the topological properties of both spontaneous and alpha-bungarotoxin-induced sprouts (during different periods of intoxication and after recovery) along the motor nerve terminal branches of the Levator auris longus muscle of Swiss mice (between 48-169 day old). Muscles were processed for immunocytochemistry to simultaneously detect postsynaptic AChRs and axons. This procedure permits us to make an accurate identification of the fine sprouts and a morphometric study of the presynaptic branching pattern profile in control muscles, during the toxin action and after recovery from paralysis. The results show that in normal muscles, the initial and trunk segments (those between branch points) of the terminal arborization sprouted proportionally more branches when taking their relative lengths into account than the distal free-end segments. In contrast, every micrometer of alpha-bungarotoxin-treated muscles throughout the full terminal arborization have the same probability of generating a sprout. Moreover, the toxin-induced sprouts can consolidate as new branches once recovered from the paralysis without changing the total length of the nerve terminal arborization.

Multiple types of calcium channels mediate transmitter release during functional recovery of botulinum toxin type A-poisoned mouse motor nerve terminals

The involvement of different types of voltage-dependent calcium channels in nerve-evoked release of neurotransmitter was studied during recovery from neuromuscular paralysis produced by botulinum toxin type A intoxication. For this purpose, a single subcutaneous injection of botulinum toxin (1 IU; dl 50) on to the surface of the mouse levator auris longus muscle was performed. The muscles were removed at several time-points after injection (i.e. at one, two, three, four, five, six and 12 weeks). Using electrophysiological techniques, we studied the effect of different types of calcium channel blockers (nitrendipine, ω-conotoxin-GVIA and ω-agatoxin-IVA) on the quantal content of synaptic transmission elicited by nerve stimulation. Morphological analysis using the conventional silver impregnation technique was also made. During the first four weeks after intoxication, sprouts were found at 80% of motor nerve terminals, while at 12 weeks their number was decreased and the nerve terminals were enlarged. The L-type channel blocker nitrendipine (1 μM) inhibited neurotransmitter release by 80% and 30% at two and five weeks, respectively, while no effects were found at later times. The N-type channel blocker ω-conotoxin-GVIA (1 μM) inhibited neurotransmitter release by 50–70% in muscles studied at two to six weeks, respectively, and had no effect 12 weeks after intoxication. The P-type channel blocker ω-agatoxin-IVA (100 nM) strongly reduced nerve-evoked transmitter release (>90%) at all the time-points studied. Identified motor nerve terminals were also sensitive to both nitrendipine and ω-conotoxin-GVIA. This study shows that multiple voltage-dependent calcium channels were coupled to release during the period of sprouting and consolidation, suggesting that they may be involved in the nerve ending functional recovery process.

Association of adhesive macromolecules with terminal sprouts at the neuromuscular junction after botulinum treatment

Small quantities of botulinum toxin (BTX) are useful in the treatment of certain movement disorders, such as laryngeal spasmodic dysphonia, blepharospasm, and cervical dystonia. However, the corrective paralytic effects of BTX are only temporary, in part because of the formation of remodeled neuromuscular junctions. Here, we questioned whether various factors within and near the neuromuscular junction could contribute to the remodeling seen after BTX treatment. BTX was injected subcutaneously in the region of the levator auris longus muscle. At 1-week intervals, levator auris longus muscles were removed and examined histochemically. As previously described, BTX treatment results in a progressive elongation of end plates. The neural cell adhesion molecule was not associated with the elongated end plates but was associated with the BTX-induced nerve sprouts after long intervals (3 to 4 weeks). Similarly, after BTX, laminin-1 (composed of α1, β1, and γ1 chains) reactivity was associated with the nerve sprouts, but not with the end plates. Laminin β2 reactivity at the end plate dispersed somewhat within 1 week but remained diffusely associated with the elongating end plates for up to 5 weeks. Together these results suggest that neural cell adhesion molecule and laminins may participate in the sprouting observed after BTX treatment and that alterations in laminin β2 expression may participate in initial loss of contacts. (Otolaryngol Head Neck Surg 1999;120:255-61.)

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 α-latrotoxin or La 3+ 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 Ca 2+-independent exocytosis.

Axonal and nerve terminal sprouting induced in vivo by injection of botulinum type C 1toxin in the mouse levator auris longus muscle

Axonal and nerve terminal sprouting induced in vivo by injection of botulinum type C 1toxin in the mouse levator auris longus muscle

Upregulation of calcitonin gene-related peptide at mouse motor nerve terminals poisoned with botulinum type-A toxin.

Calcitonin gene-related peptide (CGRP)-like immunoreactivity of motor nerve terminals was investigated at different times after local in vivo injection of botulinum type-A toxin (BoNT/A) close to the mouse levator auris longus muscle. CGRP expression in most of control nerve terminals was undetectable, but markedly increased during muscle paralysis and synaptic remodelling and, declined once functional recovery occurred.

Uptake of immunoglobulin G from amyotrophic lateral sclerosis patients by motor nerve terminals in mice

Clinical and experimental evidence support an autoimmune etiopathogenesis for amyotrophic lateral sclerosis (ALS). We have shown that local application of ALS-IgG onto nerve terminals induces dysfunction in transmission at the neuromuscular junction. It has been established that IgG and other circulating serum proteins can be taken up by motor nerve terminals, being immunolocalized in the soma where they accumulate following retrograde axonal transport. In the present study, we investigated the presence of human ALS and control IgG in the soma of mouse motoneurons. IgG was applied onto motor nerve terminals of mice by subcutaneous injections on the left levator auris longus muscle which is innervated by a branch of the facial nerve. After several injections, sections of the brainstem containing the facial nuclei were immunoprocessed to detect human IgG. For all IgG tested, motoneuron labeling was significantly more intense in the facial nucleus ipsilateral to the site of injection. In ALS-IgG-treated animals, ipsilateral labeling was significantly stronger than that found on the ipsilateral side of control IgG-treated animals. Our results are compatible with the concept that motoneurons preferentially take up, transport and/or accumulate ALS-IgG. Uptake of pathogenic antibodies by motoneuron terminals may play a role in the pathogenesis of motoneuron disease.

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.

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.

Sprouting of mammalian motor nerve terminals induced by in vivo injection of botulinum type-D toxin and the functional recovery of paralysed neuromuscular junctions

Paralysis of the mouse levator auris longus muscle by in vivo injection of Clostridium botulinum type-D neurotoxin (BoNT/D) triggered a marked outgrowth of the motor nerve from the original terminal arborization. The increase in total nerve terminal length was due to both increase in the number of terminal branches and in average branch length. Asynchronous quantal transmitter release in response to nerve impulses was a prominent feature in paralysed junctions that started 24 h after poisoning and lasted for about 15 days. The functional recovery of poisoned junctions occurred 25–30 days after poisoning and was characterized by the synchronous quantal transmitter release upon nerve stimulation that triggered synaptically evoked action potentials and muscle fibre contraction.

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. Ca 2+ influx and Ca 2+-dependent K + currents in the sprout membrane were found to be similar to those described in unpoisoned endings. The presence of normal Ca 2+ 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.

A study of synchronization of quantal transmitter release from mammalian motor endings by the use of botulinal toxins type A and D.

1. The effects of botulinum toxin (BoTx) types A and D on spontaneous and evoked phasic transmitter release were studied in the isolated extensor digitorum longus muscle of the rat or the levator auris longus muscle of mice. 2. The toxins were injected subcutaneously into the hindleg of adult rats or the dorsal aspect of the neck of mice. At various times after the injection the muscles were removed from the anaesthetized animal and neuromuscular transmission examined in vitro by conventional intracellular techniques. 3. Both toxins reduced spontaneous transmitter release recorded as the frequency of miniature end-plate potentials but BoTx type D was less effective in that respect than the type A toxin. 4. With both toxins the block of evoked phasic transmitter release, recorded as end-plate potentials, was almost complete. As previously reviewed by Simpson (1986) the block produced by BoTx type A was partially reversed by procedures which elevate the intraterminal level of calcium ions. However, in BoTx type D-paralysed muscles such procedures failed to restore phasic transmitter release but caused a period of high-frequency asynchronous transmitter release following each nerve impulse. 5. To investigate if the lack of synchronization of evoked transmitter release observed in BoTx type D-paralysed muscles was due to alterations in presynaptic currents we examined, by perineural recordings, the Na+, fast K+, slow K+, K+-Ca2+-dependent and the Ca2+ currents in BoTx type D-paralysed muscles. These presynaptic currents were not altered as compared to unpoisoned controls. 6. We suggest that there exists a presynaptic process, which in addition to Ca2+ influx participates in transmitter synchronization and which is a main target for BoTx type D action.

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.