Shock waves as treatment of mouse myofascial trigger points.

INTRODUCTION: An abnormal increase in spontaneous neurotransmission can induce subsynaptic knots in the myocyte called myofascial trigger points. The treatment of choice is to destroy these trigger points by inserting needles. However, 10% of the population has a phobia of needles, blood, or injuries. Therefore, the objective of this study is to verify the usefulness of shock waves in the treatment of myofascial trigger points. METHODS: Two groups of mice have been developed for this: healthy muscles treated with shock waves; trigger points affected muscles artificially generated with neostigmine and subsequently treated with shock waves. Muscles were stained with methylene blue, PAS-Alcian Blue, and labeling the axons with fluorescein and the acetylcholine receptors with rhodamine. Using intracellular recording the frequency of miniature endplate potentials (mEPPs) was recorded and endplate noise was recorded with electromyography. RESULTS: No healthy muscles treated with shock waves showed injury. Twitch knots in mice previously treated with neostigmine disappeared after shock wave treatment. Several motor axonal branches were retracted. On the other hand, shock wave treatment reduces the frequency of mEPPs and the number of areas with endplate noise. DISCUSSION: Shock waves seem to be a suitable treatment for myofascial trigger points. In the present study, with a single session of shock waves, very relevant results have been obtained, both functional (normalization of spontaneous neurotransmission) and morphological (disappearance of myofascial trigger points). Patients with a phobia of needles, blood, or injuries who cannot benefit from dry needling may turn to noninvasive radial shock wave treatment.

Epigenetic Changes Governing Scn5a Expression in Denervated Skeletal Muscle.

The SCN5A gene encodes the α-subunit of the voltage-gated cardiac sodium channel (NaV1.5), a key player in cardiac action potential depolarization. Genetic variants in protein-coding regions of the human SCN5A have been largely associated with inherited cardiac arrhythmias. Increasing evidence also suggests that aberrant expression of the SCN5A gene could increase susceptibility to arrhythmogenic diseases, but the mechanisms governing SCN5A expression are not yet well understood. To gain insights into the molecular basis of SCN5A gene regulation, we used rat gastrocnemius muscle four days following denervation, a process well known to stimulate Scn5a expression. Our results show that denervation of rat skeletal muscle induces the expression of the adult cardiac Scn5a isoform. RNA-seq experiments reveal that denervation leads to significant changes in the transcriptome, with Scn5a amongst the fifty top upregulated genes. Consistent with this increase in expression, ChIP-qPCR assays show enrichment of H3K27ac and H3K4me3 and binding of the transcription factor Gata4 near the Scn5a promoter region. Also, Gata4 mRNA levels are significantly induced upon denervation. Genome-wide analysis of H3K27ac by ChIP-seq suggest that a super enhancer recently described to regulate Scn5a in cardiac tissue is activated in response to denervation. Altogether, our experiments reveal that similar mechanisms regulate the expression of Scn5a in denervated muscle and cardiac tissue, suggesting a conserved pathway for SCN5A expression among striated muscles.

Presynaptic membrane receptors in acetylcholine release modulation in the neuromuscular synapse.

Over the past few years, we have studied, in the mammalian neuromuscular junction (NMJ), the local involvement in transmitter release of the presynaptic muscarinic ACh autoreceptors (mAChRs), purinergic adenosine autoreceptors (P1Rs), and trophic factor receptors (TFRs; for neurotrophins and trophic cytokines) during development and in the adult. At any given moment, the way in which a synapse works is largely the logical outcome of the confluence of these (and other) metabotropic signalling pathways on intracellular kinases, which phosphorylate protein targets and materialize adaptive changes. We propose an integrated interpretation of the complementary function of these receptors in the adult NMJ. The activity of a given receptor group can modulate a given combination of spontaneous, evoked, and activity-dependent release characteristics. For instance, P1Rs can conserve resources by limiting spontaneous quantal leak of ACh (an A1 R action) and protect synapse function, because stimulation with adenosine reduces the magnitude of depression during repetitive activity. The overall outcome of the mAChRs seems to contribute to upkeep of spontaneous quantal output of ACh, save synapse function by decreasing the extent of evoked release (mainly an M2 action), and reduce depression. We have also identified several links among P1Rs, mAChRs, and TFRs. We found a close dependence between mAChR and some TFRs and observed that the muscarinic group has to operate correctly if the tropomyosin-related kinase B receptor (trkB) is also to operate correctly, and vice versa. Likewise, the functional integrity of mAChRs depends on P1Rs operating normally.

Adenosine A2B and A3 receptor location at the mouse neuromuscular junction.

To date, four subtypes of adenosine receptors have been cloned (A(1)R, A(2A)R, A(2B)R, and A(3)R). In a previous study we used confocal immunocytochemistry to identify A(1)R and A(2A)R receptors at mouse neuromuscular junctions (NMJs). The data shows that these receptors are localized differently in the three cells (muscle, nerve and glia) that configure the NMJs. A(1)R localizes in the terminal teloglial Schwann cell and nerve terminal, whereas A(2A)R localizes in the postsynaptic muscle and in the axon and nerve terminal. Here, we use Western blotting to investigate the presence of A(2B)R and A(3)R receptors in striated muscle and immunohistochemistry to localize them in the three cells of the adult neuromuscular synapse. The data show that A(2B)R and A(3)R receptors are present in the nerve terminal and muscle cells at the NMJs. Neither A(2B)R nor A(3)R receptors are localized in the Schwann cells. Thus, the four subtypes of adenosine receptors are present in the motor endings. The presence of these receptors in the neuromuscular synapse allows the receptors to be involved in the modulation of transmitter release.

Protein kinase C isoforms at the neuromuscular junction: localization and specific roles in neurotransmission and development.

The protein kinase C family (PKC) regulates a variety of neural functions including neurotransmitter release. The selective activation of a wide range of PKC isoforms in different cells and domains is likely to contribute to the functional diversity of PKC phosphorylating activity. In this review, we describe the isoform localization, phosphorylation function, regulation and signalling of the PKC family at the neuromuscular junction. Data show the involvement of the PKC family in several important functions at the neuromuscular junction and in particular in the maturation of the synapse and the modulation of neurotransmission in the adult.

Adenosine A1 and A2A receptor-mediated modulation of acetylcholine release in the mice neuromuscular junction.

Immunocytochemistry shows that purinergic receptors (P1Rs) type A1 and A2A (A1 R and A2 A R, respectively) are present in the nerve endings at the P6 and P30 Levator auris longus (LAL) mouse neuromuscular junctions (NMJs). As described elsewhere, 25 µm adenosine reduces (50%) acetylcholine release in high Mg(2+) or d-tubocurarine paralysed muscle. We hypothesize that in more preserved neurotransmission machinery conditions (blocking the voltage-dependent sodium channel of the muscle cells with µ-conotoxin GIIIB) the physiological role of the P1Rs in the NMJ must be better observed. We found that the presence of a non-selective P1R agonist (adenosine) or antagonist (8-SPT) or selective modulators of A1 R or A2 A R subtypes (CCPA and DPCPX, or CGS-21680 and SCH-58261, respectively) does not result in any changes in the evoked release. However, P1Rs seem to be involved in spontaneous release (miniature endplate potentials MEPPs) because MEPP frequency is increased by non-selective block but decreased by non-selective stimulation, with A1 Rs playing the main role. We assayed the role of P1Rs in presynaptic short-term plasticity during imposed synaptic activity (40 Hz for 2 min of supramaximal stimuli). Depression is reduced by micromolar adenosine but increased by blocking P1Rs with 8-SPT. Synaptic depression is not affected by the presence of selective A1 R and A2 A R modulators, which suggests that both receptors need to collaborate. Thus, A1 R and A2 A R might have no real effect on neuromuscular transmission in resting conditions. However, these receptors can conserve resources by limiting spontaneous quantal leak of acetylcholine and may protect synaptic function by reducing the magnitude of depression during repetitive activity.

Stress increases the spontaneous release of ACh and may be involved in the generation and maintenance of myofascial trigger points in mouse.

An increase in spontaneous neurotransmission may be related to myofascial pain. Sympathetic neurons innervate most of the neuromuscular junction sand are involved in the modulation of synaptic transmission. Therefore, a direct action of stress on acetylcholine release is expected. For this reason, this study aims to evaluate the relationship between stress and spontaneous neurotransmission. Five acute stressors (immobilization, forced swimming, food and water deprivation, social isolation and ultrasound) were tested in 6 weeks adult Swiss male mice. Subsequently, these types of stress were combined to generate a model of chronic stress. The study of ACh release was evaluated before and after the application of stress by intracellular recording of spontaneous neurotransmission (mEPPs). In each one of the stressors, an increase in the frequency of mEPPs was obtained immediately after treatment, which remained elevated for 5 days and thereafter returned to control values after a week. With chronic stress, a much higher increase in the frequency of mEPPs was obtained and it was maintained for 15 days. In summary, stress, both in its acute and chronic forms, increased spontaneous neurotransmission significantly. There is a possibility that chronic stress is related with the genesis or maintenance of myofascial pain.

Can Myofascial Trigger Points Involve Nociplastic Pain? A Scoping Review on Animal Models.

Nociplastic pain is a non-specific, regional pain lasting more than three months, characterised by the onset of hypersensitivity, despite no clear evidence of tissue damage. It is a relatively new classified type of pain. As a result, there has not yet been much work describing its precise modelling. The mechanism of its formation needs to be clearly explained. Authors point out that the occurrence of myofascial trigger points (MTrPs) can lead to this type of pain as one possibility. This paper summarises the available literature on modelling nociplastic pain and MTrPs. It complies with studies describing animal model creation and presents the results of performed experiments. The literature search was conducted in December 2022 and included the following databases: PubMed, Scopus, and Web of Science. In this scoping review, six studies were included. Two described the creation of animal models of nociplastic pain, one adapted old models to nociplastic pain, and three described the modelling of MTrPs. This is the first paper pointing in the possible direction of detecting and studying the correlation between MTrPs and nociplastic pain in animal models. However, there is currently insufficient evidence to describe MTrPs as nociplastic, as few studies with animal models exist.

Muscarinic Receptors in Developmental Axonal Competition at the Neuromuscular Junction.

In recent years, we have studied by immunohistochemistry, intracellular recording, and western blotting the role of the muscarinic acetylcholine receptors (mAChRs; M1, M2, and M4 subtypes) in the mammalian neuromuscular junction (NMJ) during development and in the adult. Here, we evaluate our published data to emphasize the mAChRs' relevance in developmental synaptic elimination and their crosstalk with other metabotropic receptors, downstream kinases, and voltage-gated calcium channels (VGCCs). The presence of mAChRs in the presynaptic membrane of motor nerve terminals allows an autocrine mechanism in which the secreted acetylcholine influences the cell itself in feedback. mAChR subtypes are coupled to different downstream pathways, so their feedback can move in a broad range between positive and negative. Moreover, mAChRs allow direct activity-dependent interaction through ACh release between the multiple competing axons during development. Additional regulation from pre- and postsynaptic sites (including neurotrophic retrograde control), the agonistic and antagonistic contributions of adenosine receptors (AR; A1 and A2A), and the tropomyosin-related kinase B receptor (TrkB) cooperate with mAChRs in the axonal competitive interactions which lead to supernumerary synapse elimination that achieves the optimized monoinnervation of musculoskeletal cells. The metabotropic receptor-driven balance between downstream PKA and PKC activities, coupled to developmentally regulated VGCC, explains much of how nerve terminals with different activities finally progress to their withdrawal or strengthening.

Exogenous ciliary neurotrophic factor (CNTF) reduces synaptic depression during repetitive stimulation.

It has been shown that ciliary neurotrophic factor (CNTF) has trophic and maintenance effects on several types of peripheral and central neurons, glia, and cells outside the nervous system. Both CNTF and its receptor, CNTF-Rα, are expressed in the muscle. We use confocal immunocytochemistry to show that the trophic cytokine and its receptor are present in the pre- and post-synaptic sites of the neuromuscular junctions (NMJs). Applied CNTF (7.5-200 ng/ml, 60 min-3 h) does not acutely affect spontaneous potentials (size or frequency) or quantal content of the evoked acetylcholine release from post-natal (in weak or strong axonal inputs on dually innervated end plates or in the most mature singly innervated synapses at P6) or adult (P30) NMJ of Levator auris longus muscle of the mice. However, CNTF reduces roughly 50% the depression produced by repetitive stimulation (40 Hz, 2 min) on the adult NMJs. Our findings indicate that, unlike neurotrophins, exogenous CNTF does not acutely modulate transmitter release locally at the mammalian neuromuscular synapse but can protect mature end plates from activity-induced synaptic depression.

Dry Needling Produces Mild Injuries Irrespective to Muscle Stiffness and Tension in Ex Vivo Mice Muscles.

Numerous studies have suggested that the myofascial trigger points are responsible for most of the myofascial pain syndrome, so it seems reasonable that its destruction is a good therapeutic solution. The effectiveness of dry needling (DN) has been confirmed in muscles with myofascial trigger points, hypertonicity, and spasticity. The objective of this study is to analyze the need of repetitive punctures on muscles in different situations. The levator auris longus (LAL) muscle and gastrocnemius muscle from adult male Swiss mice were dissected and maintained alive, while being submerged in an oxygenated Ringer's solution. DN was evaluated under four animal models, mimicking the human condition: normal healthy muscles, muscle fibers with contraction knots, muscles submerged in a depolarizing Ringer solution (KCl-CaCl2), and muscles submerged in Ringer solution with formalin. Thereafter, samples were evaluated with optical microscopy (LAL) and scanning electron microscopy (gastrocnemius). Healthy muscles allowed the penetration of needles between fibers with minimal injuries. In muscles with contraction knots, the needle separated many muscle fibers, and several others were injured, while blood vessels and intramuscular nerves were mostly not injured. Muscles submerged in a depolarizing solution inducing sustained contraction showed more injured muscular fibers and several muscle fibers separated by the needle. Finally, the muscles submerged in Ringer solution with formalin showed a few number of injured muscular fibers and abundant muscle fibers separated by the needle. Scanning electron microscopy images confirm the optical analyses. In summary, dry needling is a technique that causes mild injury irrespective of the muscle tone.

The interaction between tropomyosin-related kinase B receptors and presynaptic muscarinic receptors modulates transmitter release in adult rodent motor nerve terminals.

The neurotrophin brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and the receptors tropomyosin-related kinase B (trkB) and p75(NTR) are present in the nerve terminals on the neuromuscular junctions (NMJs) of the levator auris longus muscle of the adult mouse. Exogenously added BDNF or NT-4 increased evoked ACh release after 3 h. This presynaptic effect (the size of the spontaneous potentials is not affected) is specific because it is not produced by neurotrophin-3 (NT-3) and is prevented by preincubation with trkB-IgG chimera or by pharmacological block of trkB [K-252a (C27H21N3O5)] or p75(NTR) [Pep5 (C86H111N25O19S2] signaling. The effect of BDNF depends on the M1 and M2 muscarinic acetylcholine autoreceptors (mAChRs) because it is prevented by atropine, pirenzepine and methoctramine. We found that K-252a incubation reduces ACh release (~50%) in a short time (1 h), but the p75(NTR) signaling inhibitor Pep5 does not have this effect. The specificity of the K-252a blocking effect on trkB was confirmed with the anti-trkB antibody 47/trkB, which reduces evoked ACh release, like K-252a, whereas the nonpermeant tyrosine kinase blocker K-252b does not. Neither does incubation with the fusion protein trkB-IgG (to chelate endogenous BDNF/NT-4), anti-BDNF or anti-NT-4 change ACh release. Thus, the trkB receptor normally seems to be coupled to ACh release when there is no short-term local effect of neurotrophins at the NMJ. The normal function of the mAChR mechanism is a permissive prerequisite for the trkB pathway to couple to ACh release. Reciprocally, the normal function of trkB modulates M1- and M2-subtype muscarinic pathways.

Blocking p75 (NTR) receptors alters polyinnervationz of neuromuscular synapses during development.

High-resolution immunohistochemistry shows that the receptor protein p75(NTR) is present in the nerve terminal, muscle cell, and glial Schwann cell at the neuromuscular junction (NMJ) of postnatal rats (P4-P6) during the synapse elimination period. Blocking the receptor with the antibody anti-p75-192-IgG (1-5 µg/ml, 1 hr) results in reduced endplate potentials (EPPs) in mono- and polyinnervated synapses ex vivo, but the mean number of functional inputs per NMJ does not change for as long as 3 hr. Incubation with exogenous brain-derived neurotrophic factor (BDNF) for 1 hr (50 nM) resulted in a significant increase in the size of the EPPs in all nerve terminals, and preincubation with anti-p75-192-IgG prevented this potentiation. Long exposure (24 hr) in vivo of the NMJs to the antibody anti-p75-192-IgG (1-2 µg/ml) results in a delay of postnatal synapse elimination and even some regrowth of previously withdrawn axons, but also in some acceleration of the morphologic maturation of the postsynaptic nicotinic acetylcholine receptor (nAChR) clusters. The results indicate that p75(NTR) is involved in both ACh release and axonal retraction during postnatal axonal competition and synapse elimination.

Silent synapses in neuromuscular junction development.

In the last few years, evidence has been found to suggest that some synaptic contacts become silent but can be functionally recruited before they completely retract during postnatal synapse elimination in muscle. The physiological mechanism of developmental synapse elimination may be better understood by studying this synapse recruitment. This Mini-Review collects previously published data and new results to propose a molecular mechanism for axonal disconnection. The mechanism is based on protein kinase C (PKC)-dependent inhibition of acetylcholine (ACh) release. PKC activity may be stimulated by a methoctramine-sensitive M2-type muscarinic receptor and by calcium inflow though P/Q- and L-type voltage-dependent calcium channels. In addition, tropomyosin-related tyrosine kinase B (trkB) receptor-mediated brain-derived neurotrophic factor (BDNF) activity may oppose the PKC-mediated ACh release depression. Thus, a balance between trkB and muscarinic pathways may contribute to the final functional suppression of some neuromuscular synapses during development.

Safety analysis of percutaneous needle electrolysis: a study of needle composition, morphology, and electrical resistance.

BACKGROUND: Percutaneous needle electrolysis (PNE) consists of a galvanic current combined with the insertion of a solid needle into the tissues of the musculoskeletal system. The application of a galvanic current through a needle can alter the morphology and composition during treatment application. This procedure may also provoke a localized temperature increase. AIM: The aim was to evaluate the safety of the PNE procedure by analyzing possible alterations of the needles employed. METHODS: Physio Invasiva® and AguPunt EPI® brand needles, commonly used for the application of this technique, were analyzed in response to three different treatment protocols. Temperature changes were evaluated with the needles immersed in a test tube containing Ringer's solution, and electrical resistance was evaluated with a multimeter. The morphology of the needles, pre- and post-treatment, was examined with a scanning electron microscope (FEI Quanta 600), and the composition of the needles was evaluated using RX diffusion with Oxford Instruments software. RESULTS: Ringer's solution contained in the test tubes examined did not present temperature changes. No changes were observed in the needles under investigation with respect to electrical resistance, morphology, or composition with a protocol applying 3-mA intensity for 3 s and three applications. However, important morphological alterations were observed that affected needle composition after 50 applications (at 3 mA for 3 s). CONCLUSION: PNE, applied according to conventional protocols, appeared to be safe and athermal, and did not provoke a loss of metal particles or modify the morphology of the needles used when studied in vitro.

Localization of brain-derived neurotrophic factor, neurotrophin-4, tropomyosin-related kinase b receptor, and p75 NTR receptor by high-resolution immunohistochemistry on the adult mouse neuromuscular junction.

Neurotrophins and their receptors, the trk receptor tyrosine kinases (trks) and p75(NTR), are differentially expressed among the cell types that make up synapses. It is important to determine the precise location of these molecules involved in neurotransmission. Here we use immunostaining and Western blotting to study the localization and expression of neurotrophin brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) and the receptors tropomyosin-related kinase b (trkB) and p75(NTR) at the adult neuromuscular junction. Our confocal immunofluorescence results on the whole mounts of the mouse Levator auris longus muscle and on semithin cross-sections showed that BDNF, NT-4, trkB, and p75(NTR) were localized on the three cells in the neuromuscular synapse (motor axons, post-synaptic muscle and Schwann cells).

Effects of a Fat-Rich Diet on the Spontaneous Release of Acetylcholine in the Neuromuscular Junction of Mice.

Western societies are facing a clear increase in the rate of obesity and overweight which are responsible for musculoskeletal pain. Some of the substances described in the environment of myofascial trigger points (MTrPs) are the same as those found in the skeletal muscle of obese people, such as cytokines. Furthermore, elevated neuromuscular neurotransmission has been associated with MTrPs. The main objective of this study is to assess whether obesity or overweight may be a facilitator of myofascial pain. The experiments were performed on male Swiss mice. One experimental group was given a typical "cafeteria" diet and another group a commercial high-fat diet for six weeks. Intramuscular adipocytes were assessed with Sudan III. The functional study was performed with electromyographic recording to determine the plaque noise and intracellular recording of miniature endplate potentials (MEPPs). The intake of a cafeteria diet showed the presence of more adipocytes in muscle tissue, but not with the fat-supplemented diet. Both experimental groups showed an increase in the plaque noise and an increase in the frequency of MEPPs that lasted several weeks after interrupting diets. In summary, the supply of a hypercaloric diet for six weeks in mice increases spontaneous neurotransmission, thus facilitating the development of MTrPs.

Experimental myofascial trigger point creation in rodents.

Myofascial pain syndrome is one of the most common forms of muscle pain. In this syndrome, pain is originated by the so-called trigger points, which consists of a set of palpable contraction knots in the muscle. It has been proposed that a high, spontaneous neurotransmission may be involved in the generation of these contraction knots. To confirm this hypothesis, we exposed mouse muscles to an anticholinesterasic agent to increase the neurotransmision in the synaptic cleft in two different conditions, in vivo and ex vivo experiments. Using intracellular recordings, a sharp increase in the spontaneous neurotransmission in the levator auris longus muscle and a lower increase in the diaphragm muscle could be seen. Likewise, electromyography recordings reveal an elevated endplate noise in gastrocnemius muscle of treated animals. These changes are associated with structural changes such as abundant neuromuscular contracted zones observed by rhodaminated α-bungarotoxin and the presence of abundant glycosaminoglycans around the contraction knots, as shown by Alcian PAS staining. In a second set of experiments, we aimed at demonstrating that the increases in the neurotransmission reproduced most of the clinical signs associated to a trigger point. We exposed rats to the anticholinesterase agent neostigmine, and 30 min afterward we observed the presence of palpable taut bands, the echocardiographic presence of contraction knots, and local twitch responses upon needle stimulation. In summary, we demonstrated that increased neurotransmission induced trigger points in both rats and mice, as evidenced by glycosaminoglycans around the contraction zones as a novel hallmark of this pathology. NEW & NOTEWORTHY In rodents, when neostigmine was injected subcutaneously, the neuromuscular neurotransmission increased, and several changes can be observed: an elevated endplate noise compared with normal endplate noise, as evidenced by electromyographyc recording; many muscular fibers with contraction knots (narrower sarcomeres and locally thickened muscle fiber) surrounded by infiltration of connective tissue like glycosaminoglycans molecules; and palpable taut bands and local twitch responses upon needle stimulation. Several of these signs are also observed in humans with muscle pain.

Short-term effects of beta-amyloid25-35 peptide aggregates on transmitter release in neuromuscular synapses.

The beta-amyloid (AB) peptide25-35 contains the functional domain of the AB precursor protein that is both required for neurotrophic effects in normal neural tissues and is involved in the neurotoxic effects in Alzheimer disease. We demonstrated the presence of the amyloid precursor protein/AB peptide in intramuscular axons, presynaptic motor nerve terminals, terminal and myelinating Schwann cells, and the postsynaptic and subsarcolemmal region in the Levator auris longus muscle of adult rats by immunocytochemistry. Using intracellular recording, we investigated possible short-term functional effects of the AB fragment (0.1-10 micromol/L) on acetylcholine release in adult and newborn motor end plates. We found no change in evoked, spontaneous transmitter release or resting membrane potential of the muscle cells. A previous block of the presynaptic muscarinic receptor subtypes and a previous block or stimulation of protein kinase C revealed no masked effect of the peptide on the regulation of transmitter release. The aggregated form of AB peptide25-35, however, interfered acutely with acetylcholine release (quantal content reduction) when synaptic activity was maintained by electric stimulation. The possible relevance of this inhibition of neurotransmission by AB peptide25-35 to the pathogenesis of Alzheimer remains to be determined.

Anti-GM2 gangliosides IgM paraprotein induces neuromuscular block without neuromuscular damage.

We analyzed the effect on the mouse neuromuscular synapses of a human monoclonal IgM, which binds specifically to gangliosides with the common epitope [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-]. We focused on the role of the complement. Evoked neurotransmission was partially blocked by IgM both acutely (1 h) and chronically (10 days). Transmission electron microscopy shows important nerve terminal growth and retraction remodelling though axonal injury can be ruled out. Synapses did not show mouse C5b-9 immunofluorescence and were only immunolabelled when human complement was added. Therefore, the IgM-induced synaptic changes occur without complement-mediated membrane attack.