[A reminder of the structure and function of the skeletal neuromuscular junction]. / Rappels structuraux et fonctionnels de la jonction neuromusculaire squelettique.

The skeletal neuromuscular junction has been considered as a model of chemical synapses due to its relatively simple organization. It is made up of three cellular partners including the motoneuron nerve terminals, the peri-synaptic Schwann cells and a specialized region of skeletal muscle fibers. It has been extensively studied revealing its ultrastructural complexity involving many molecular actors. The neuromuscular junction is a highly specialized structure, optimized for the rapid transmission of information from the presynaptic nerve terminal to the post-synaptic muscle fiber. This rapid transmission requires a very close apposition of plasmic membranes of pre- and post-synaptic partners, and a strict structural and molecular arrangement on both sides of the narrow synaptic cleft separating nerve terminal and muscle membranes. In this short review, we summarize the knowledge regarding pre- and post-synaptic ultrastructural specializations and give an overview of some functional aspects of neuromuscular transmission, including the quantal acetylcholine release process, which will help to better understand the pharmacological actions of botulinum toxins in esthetic and corrective dermatology.

Behavioral depression in the swim test causes a biphasic, long-lasting change in accumbens acetylcholine release, with partial compensation by acetylcholinesterase and muscarinic-1 receptors.

The nucleus accumbens may play a role in acquisition and expression of behavioral depression as measured using the inescapable swim test. Previous work shows that a local injection of a cholinergic muscarinic-1 receptor agonist increases immobility and a specific muscarinic-1 antagonist acts as an antidepressant-like drug by increasing swimming escape efforts. The present study used microdialysis to monitor extracellular acetylcholine levels in the accumbens, fluorescent labeled toxins to monitor changes in acetylcholinesterase and muscarinic-1 receptors, and semiquantitative-polymerase chain reaction to detect changes in gene expression for the muscarinic-1 receptor. Microdialysis showed that acetylcholine levels did not change while an animal was swimming; however, a significant transient decrease occurred when the rat was returned to the dialysis cage, followed by a long-lasting increase that reached a maximum three hours after the test. Acetylcholine levels stayed high even 24 h after the initial test as evidenced by a significant elevation in basal level prior to the second swim. This increase in neurotransmitter may have been partially compensated by a significant increase in the degradative enzyme, acetylcholinesterase, and by a decrease in muscarinic-1 receptors and their gene expression. These results further demonstrate the importance of accumbens cholinergic function in the appearance of a depression-like state.

Drug induced weight gain, an impediment to successful pharmacotherapy: focus on antipsychotics.

The antipsychotic drugs (APDs) are fundamental tools in current psychiatric practice. A new generation of agents, the atypical APDs, represents an important progress in the treatment of psychotic disorders. Unfortunately, some of them induce excessive body weight gain (BWG), obesity, hyperglycemia and dyslipidemia in the following order: clozapine approximately equal to olanzapine > quetiapine > risperidone > ziprasidone = aripiprazole. Appetite stimulation is probably the main mechanism of BWG and this is strongly correlated with the APD affinity for H1 (histaminergic) and alpha1 (adrenergic) receptors. A composed ratio of the APD affinity for diverse neurotransmitters involved in food intake (FI) regulation correlates with BWG as well. Endocrine/metabolic mechanisms, such as the activation of the hypothalamus-pituitary-adrenal axis, changes in insulin sensitivity (by conventional and atypical agents), hyperprolactinemia and gonadal dysfunction (by conventional APDs and risperidone) may also be involved. Importantly, patients with schizophrenia may have a genetically-based predisposition to appetite dysregulation, insulin resistance and endocrine imbalance involving gonadal steroids. Excessive BWG must be prevented or attenuated by proper drug selection, combining or switching agents, nutritional assistance and physical exercise. Amantadine. metformin and reboxetine proved to significantly lessen APD-induced BWG. Notwithstanding this, novel strategies are necessary to treat this side effect in a clinical population particularly prone to poor compliance and under a high risk of negative drug interaction.

Nocardia otitidiscaviarum (GAM-5) induces parkinsonian-like alterations in mouse.

Parkinson's disease, a major neurodegenerative disorder in humans whose etiology is unknown, may be associated with some environmental factors. Nocardia otitidiscaviarum (GAM-5) isolated from a patient with an actinomycetoma produced signs similar to Parkinson's disease following iv injection into NMRI mice. NMRI mice were infected intravenously with a non-lethal dose of 5 x 10(6) colony forming units of N. otitidiscaviarum (GAM-5). Fourteen days after bacterial infection, most of the 60 mice injected exhibited parkinsonian features characterized by vertical head tremor, akinesia/bradykinesia, flexed posture and postural instability. There was a peak of nocardial growth in the brain during the first 24 h followed by a decrease, so that by 14 days nocardiae could no longer be cultured. At 24 h after infection, Gram staining showed nocardiae in neurons in the substantia nigra and occasionally in the brain parenchyma in the frontal and parietal cortex. At 21 days post-infection, tyrosine hydroxylase immunolabeling showed a 58% reduction of tyrosine hydroxylase in the substantia nigra, and a 35% reduction of tyrosine hydroxylase in the ventral tegmental region. Dopamine levels were reduced from 110 +/- 32.5 to 58 +/- 16.5 ng/mg protein (47.2% reduction) in brain from infected mice exhibiting impaired movements, whereas serotonin levels were unchanged (191 +/- 44 protein in control and 175 +/- 39 ng/mg protein in injected mice). At later times, intraneuronal inclusion bodies were observed in the substantia nigra. Our observations emphasize the need for further studies of the potential association between Parkinson's disease or parkinsonism-like disease and exposure to various nocardial species.

Nocardia otitidiscaviarum (GAM-5) induces parkinsonian-like alterations in mouse

Parkinson's disease, a major neurodegenerative disorder in humans whose etiology is unknown, may be associated with some environmental factors. Nocardia otitidiscaviarum (GAM-5) isolated from a patient with an actinomycetoma produced signs similar to Parkinson's disease following iv injection into NMRI mice. NMRI mice were infected intravenously with a non-lethal dose of 5 x 10(6) colony forming units of N. otitidiscaviarum (GAM-5). Fourteen days after bacterial infection, most of the 60 mice injected exhibited parkinsonian features characterized by vertical head tremor, akinesia/bradykinesia, flexed posture and postural instability. There was a peak of nocardial growth in the brain during the first 24 h followed by a decrease, so that by 14 days nocardiae could no longer be cultured. At 24 h after infection, Gram staining showed nocardiae in neurons in the substantia nigra and occasionally in the brain parenchyma in the frontal and parietal cortex. At 21 days post-infection, tyrosine hydroxylase immunolabeling showed a 58 percent reduction of tyrosine hydroxylase in the substantia nigra, and a 35 percent reduction of tyrosine hydroxylase in the ventral tegmental region. Dopamine levels were reduced from 110 ± 32.5 to 58 ± 16.5 ng/mg protein (47.2 percent reduction) in brain from infected mice exhibiting impaired movements, whereas serotonin levels were unchanged (191 ± 44 protein in control and 175 ± 39 ng/mg protein in injected mice). At later times, intraneuronal inclusion bodies were observed in the substantia nigra. Our observations emphasize the need for further studies of the potential association between Parkinson's disease or parkinsonism-like disease and exposure to various nocardial species.

Selective amino acids changes in the medial and lateral preoptic area in the formalin test in rats.

A combination of microdialysis in freely moving rats and capillary zone electrophoresis coupled to laser induced fluorescence detection was used to measure extracellular concentrations of amino acid neurotransmitters in different hypothalamic areas during noxious stimulation. Arginine, glutamate and aspartate were monitored every 30 s before and after a s.c. injection of formalin (5%, 50 microl) or saline (0.9%) in the right hind paw. In the medial and lateral preoptic area, calcium and nerve impulse dependent increases of arginine, glutamate and aspartate were observed during the first 2 min after formalin injection. However, amino acid changes were not detected in the lateral hypothalamus or in the ventromedial nucleus when compared with pre-injection levels or with the levels from animals injected with saline in the hind paw. Flinching behavior was also scored during the first 10 min following the formalin or saline injection. Flinching frequency was maximum at minute 2 after formalin injection, whereas saline injection did not elicited any flinching behavior. These results show that nociceptive stimulation induces rapid and differential amino acids changes in discrete areas of the hypothalamus that can be associated with pain-related behavior.

Resistance to Fas-mediated apoptosis of human T-cell lines expressing human T-lymphotropic virus type-2 (HTLV-2) Tax protein.

The susceptibility to Fas-mediated apoptosis was evaluated in seven T-cell lines (two infected with HTLV-2, one with HTLV-1, and four HTLV-free) as well as in Jurkat cells transfected with a Tax-2 expressing vector. Fas-mediated apoptosis was significantly reduced in the HTLV-1- and HTLV-2-infected lines in comparison with the HTLV-free lines regardless of the surface expression of Fas antigen (which was no different in the infected and uninfected cells). Fas-mediated apoptosis was also significantly inhibited in Jurkat cells transfected with the Tax-2 expressing vector without any modification in Fas expression. There was significantly more antiapoptotic Bcl-x(L) mRNA and protein in the transfected than in the untransfected Jurkat T cells. In conclusion, our results suggest that HTLV-2 is capable of inhibiting Fas-mediated apoptosis by means of a mechanism involving the tax-2 gene and probably the expression of bcl-x(L) messenger and protein.

Molecular epidemiology of TT virus in Italy and phylogenesis of viral isolates from subjects at different risk for parenteral exposure.

The prevalence and genotype distribution of human TT virus (TTV) in Italy were analysed in 593 subjects at different risk of parenteral infection who included blood donors, patients with chronic type C hepatitis (HCV), thalassemic patients, patients on haemodialysis, human immunodeficiency virus type 1 (HIV-1)-negative intravenous drug users (IVDUs), and HIV-1-infected subjects (IVDUs, heterosexual contacts and homosexual males). Plasma TTV-DNA was detected using nested PCR with primers deduced from the N22 region of the open reading frame 1 (ORF-1) and from the untranslated region (UTR) of the viral genome. Phylogenetic analysis of the sequences obtained from ORF-1 was also undertaken. A high prevalence of plasma TTV-DNA was observed using the UTR primers, with rates varying from 83-100% in the study groups. Using the N22 primers, HIV-1 positive IVDUs and homosexual males, haemodialysed patients and thalassemic patients had a significantly higher TTV prevalence (range: 23.0-86.1%) than blood donors, who displayed a high frequency of positivity (10.6%). Sequence analysis of 127 N22-positive isolates revealed that 42.5% were of type 1, 53.5% of type 2, 2.4% of type 3, and that two isolates (1.6%) were closely related to genotypes 1-2 but distinct from the other major genotypes. TTV-2 was significantly more prevalent in patients at high risk for parenteral infection and in HIV-1 positive homosexuals. In sequential samples from 15 TTV-infected subjects, N22 sequences were detectable persistently in 12 (80.0%) and UTR sequences persisted in all 15 patients over a mean period of 29.6 months. This data indicates that TTV is widespread in Italy in parenterally exposed subjects, and that the infection frequently persists.

Aspectos patológicos del músculo esquelético debido a la infección experimental por trypanosoma cruzi / Pathological aspects of skeletal muscle in experimental trypanosoma cruzi infection

En este estudio se muestran los signos clínicos desarrollados por ratones durante la fase aguda de la infección producida por tres cepas de trypanosoma cruzi. Los ratones infectados con la cepa Dm74 sufrieron alteración de la mobilidad de los miembros posteriores y murieron durante la fase aguda de la infección. El análisis histológico del músculo esquelético mostró infiltrado inflamatorio de leucocitos mononucleares y polimorfonucleares, fibroblastos, eritrocitos libres y depósitos de IgG en el espacio intersticial del Gastrocnemius (G). La desintegración de las microfibrillas y cambios en la microvasculatura, fibras nerviosas y en la unión neuromuscular del G fueron también observados. Estos resultados indican que la infección aguda producida por T. cruzi causa daño progresivo en la fibra muscular esquelética y alteración de la actividad motora

Effects of feeding on extracellular levels of glutamate in the medial and lateral portion of the globus pallidus of freely moving rats.

The globus pallidus (GP) is considered to be part of the basal ganglia and previous research has determined that it might be involved in feeding behavior. For example, it has been shown that the GP becomes active during feeding and that disinhibition of this nucleus, by locally injecting a GABA antagonist, is sufficient to induce feeding in a satiated rat. However, few studies have measured extracellular levels of glutamate during free feeding in the GP of rats. For this reason brain microdialysis coupled to capillary electrophoresis with laser-induced fluorescence was used to determine FTC-glutamate levels, either in the medial or lateral portion of the GP, of freely moving rats. Retrograde labeling of the neurons projecting to the two areas was also examined in an attempt to gain some insight on the identity of the neurons that released glutamate in the GP during feeding. Extracellular levels of glutamate-FTC differentially increased in both portions of the GP during a 2-min interval of free feeding. Retrograde labeling also showed that both areas received projections from different brain areas suggesting that each of the GP portions could be involved in separate aspects of the feeding behavior.

Naltrexone does not prevent the weight gain and hyperphagia induced by the antipsychotic drug sulpiride in rats.

Few pharmacological tools are currently available to counteract the excessive body weight gain often observed during prolonged administration of antipsychotic drugs. Most antipsychotic drugs block dopamine receptors, and both the brain dopaminergic and opioid systems appear to be involved in initiation and maintenance of feeding behavior, respectively. We evaluated whether the opioid antagonist naltrexone (NAL, 0.5-16 mg/kg/ip for 21 days) (a) affects body weight and food intake in gonadally-intact and drug-free female rats, (b) prevents obesity, hyperphagia, hyperprolactinemia and vaginal cycle disruption induced by long-term administration of the antipsychotic drug sulpiride (SUL, 20 mg/kg/ip for 21 days), or (c) reverses the acute hyperphagia induced by SUL (15 microg bilaterally), when directly applied in the perifornical lateral hypothalamus (PFLH). In drug-free rats, only NAL doses above 4 mg/kg, significantly decreased weight gain and food intake. Even though NAL (1 and 8 mg/kg) significantly attenuated SUL-induced hyperphagia and hyperprolactinemia, it did not reverse at any dose the weight gain and permanent diestrous induced by SUL. In addition, local NAL did not prevent the hyperphagia and polidypsia observed after acute intrahypothalamic SUL. Unexpectedly, the cumulative and 24 h food intake in SUL-treated rats was significantly increased by NAL. Collectively, these results do not support a role for endogenous opiates in the neural and endocrine mechanisms involved in weight gain during prolonged antipsychotic drug administration in rats.

Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+.

Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct.

Recycling and refilling of transmitter quanta at the frog neuromuscular junction.

1. Fluorescent dyes have been used at the frog neuromuscular junction to label synaptic vesicular membrane. Retrieved membrane is reformed into vesicles, which are released along with pre-existing vesicles. Consequently, if vesicular refilling with acetylcholine (ACh) is depressed by inhibitors, two sizes of quanta should be released: normal and smaller. As recycling continues the fraction of smaller size quanta should increase exponentially. 2. We enhanced the rate of quantal release by elevating the K+ concentration. The principal inhibitors were (-)-vesamicol (VES), hemicholinium-3 (HC3), and NH4+. Quantal size measurements were fitted to one and to two cumulative lognormal probability distribution functions. When two fitted better, the statistical significance assessment took into account the three additional parameters used in calculating the fit. 3. After recycling in the presence of inhibitor, many sets were fitted better by two lognormal functions. As recycling continued, the fraction of the miniature endplate potential voltage-time integrals ( MEPPs) in the larger sub-population decreased exponentially. 4. The size of the releasable pool was estimated by counting the quanta released by carbonyl cyanide m-chlorophenylhydrazone (CCCP). This was compared to pool sizes calculated from the inhibitor experiments. The two estimates of pool size were indistinguishable, with mean values ranging from about 170,000 to 270,000. 5. With all of the treatments tested, the means of the sizes in the smaller sub-population of MEPPs were about 1/3 those of the larger sub-populations. 6. Recycling synaptic vesicles appear to be incorporated into the releasable pool from which they have roughly the same probability of release as the pre-existing vesicles.

Ultrastructural investigation of human sperm using atomic force microscopy.

Ultrastructural investigation of human sperm in its natural environment (without fixation, dehydration, embedding, sectioning, etc.) was carried out by using atomic force microscope (AFM) in its tapping mode. This technique permits the examination of fine structural details of undamaged sperm and its topography with precision. Moreover, it allows 3D reconstruction of images and enhances the contrast to resolve details such as mitochondria that surround the axoneme at the sperm middle piece. An organized structure has been found in the flageller axoneme region. Ultrastructure also reveals folding and details of the depression of the membrane that cannot be examined with conventional techniques.

Human T-lymphotropic virus type 2 (HTLV-2) provirus in circulating cells of the monocyte/macrophage lineage in patients dually infected with human immunodeficiency virus type 1 and HTLV-2 and having predominantly sensory polyneuropathy.

We investigated the presence of human T-lymphotropic virus type 2 (HTLV-2) DNA in the peripheral blood mononuclear cell subsets obtained from 18 patients coinfected with human immunodeficiency virus type 1 and HTLV-2, 6 of whom also had predominantly sensory polyneuropathy (PSP). HTLV-2 DNA and RNA were found in CD8- and CD19-positive cells, and, for patients with PSP, in CD14-positive cells as well. Furthermore, the patients with PSP had higher proviral loads than those without PSP.

Sodium-dependent increase in quantal secretion induced by brevetoxin-3 in Ca2+-free medium is associated with depletion of synaptic vesicles and swelling of motor nerve terminals in situ.

Brevetoxin-3 at nanomolar concentrations markedly enhanced spontaneous quantal transmitter release from neuromuscular junctions equilibrated in a Ca2+-free EGTA medium. After about 3 h, the sustained increase in miniature endplate potential frequency led to an exhaustion of transmitter release. This increase still occurred after loading the nerve terminals with the Ca2+ chelator bis-(aminophenoxy)ethanetetra-acetate or after pretreatment with various pharmacological agents known to prevent Ca2+ release from intracellular pools, but was completely prevented by the Na+ channel blocker tetrodotoxin. Brevetoxin-3 also increased miniature endplate potential frequency from junctions treated with botulinum type-A toxin, but to a smaller extent than at normal junctions. At normal junctions, brevetoxin-3 exposure for 2 h increased the three-dimensional projected area of living motor nerve terminals in situ by about 74% while at botulinum type-A poisoned junctions a similar toxin exposure caused only a 29% increase. Tetrodotoxin prevented such effects, indicating that they are related to both Na+ entry into the terminals and increased quantal transmitter release. Ultrastructural examination of nerve terminals from junctions exposed for 3 h to brevetoxin-3 revealed profound depletions of clear and large dense core synaptic vesicles and an increase in coated vesicles and axolemma infoldings. These results indicate that brevetoxin-3 impairs the recycling of clear synaptic vesicles and are consistent with our immunofluorescent observations showing that synaptophysin epitopes can be revealed without nerve terminal permeabilization. In contrast, no such changes were detected in nerve terminals poisoned with botulinum type-A toxin which, after 3 h exposure to brevetoxin-3, retained their synaptic vesicles and had a normal appearance. We conclude that tetrodotoxin-sensitive Na+ entry into motor nerve terminals induced by brevetoxin-3 triggers external Ca2+-independent asynchronous quantal transmitter release, blocks synaptic vesicle recycling and induces swelling of the terminals. We suggest that an excess of cytoplasmic Na+ per se can activate the asynchronous neurotransmitter release process.

Selective depletion of clear synaptic vesicles and enhanced quantal transmitter release at frog motor nerve endings produced by trachynilysin, a protein toxin isolated from stonefish (Synanceia trachynis) venom.

Our previous observation that low concentrations of stonefish (Synanceia trachynis) venom elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals prompted our present study to purify the quantal transmitter-releasing toxin present in the venom and to characterize the toxin's ability to alter the ultrastructure and immunoreactivity of frog motor nerve terminals. Fractionation of S. trachynis venom by sequential anion exchange fast protein-liquid chromatography (FPLC) and size-exclusion FPLC yielded a highly purified preparation of a membrane-perturbing (haemolytic) protein toxin, named trachynilysin. Trachynilysin (2-20 micrograms/ml) significantly increased spontaneous quantal acetylcholine release from motor endings, as detected by recording miniature endplate potentials from isolated frog cutaneous pectoris neuromuscular preparations. Ultrastructural analysis of nerve terminals in which quantal acetylcholine release was stimulated to exhaustion by 3 h exposure to trachynilysin revealed swelling of nerve terminals and marked depletion of small clear synaptic vesicles. However, trachynilysin did not induce a parallel depletion of large dense-core vesicles. Large dense core vesicles contained calcitonin gene-related peptide (CGRP), as revealed by colloidal gold immunostaining, and trachynilysin-treated nerve endings exhibited CGRP-like immunofluorescence similar to that of untreated terminals. Our results indicate that the ability of stonefish venom to elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals is a function of trachynilysin, which selectively stimulates the release of small clear synaptic vesicles and impairs the recycling of small clear synaptic vesicles but does not affect the release of large dense-core vesicles. Trachynilysin may be a valuable tool for use in other secretory terminals to discriminate between neurotransmitter and neuropeptide release.

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. Electrical stimulation in the presence of 4-AP produced a reduction in the number of synaptic vesicles, deep axolemmal infoldings, coated pits, and coated vesicles. K+ stimulation did not produce any observable ultrastructural changes. Synaptophysin was labeled using silver-intensified immunogold in dissociated muscle fibers. Unstimulated and K(+)-stimulated preparations showed synaptophysin immunolabeling associated only with synaptic vesicles. In contrast, in Cd(2+)-stimulated preparations, synaptophysin appeared along the axolemma, mainly at the active zones, and after electrical stimulation it appeared in both axolemmal infoldings and the remaining synaptic vesicles. The results show that when synaptic vesicle recycling is inhibited by Cd2+ in Ca(2+)-free medium, or when 4-AP is present during electrical stimulation, synaptic vesicle fusion is accompanied by translocation and incorporation of synaptic vesicle membrane proteins into the axolemma. However, during the latter condition, synaptic vesicles are recycled through coated vesicles arising from the axolemmal infoldings. Conversely, during physiological-like stimulation of ACh release by K+ the synaptic vesicles are rapidly recycled at the active zones, by a double and rapid process of exo-endocytosis, without collapse into the axolemma.

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.