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Peripheral nerve section with subsequent disconnection of motor neuron (MN) cell bodies from their skeletal muscle targets leads to a rapid reactive response involving the recruitment and activation of microglia. In addition, the loss of afferent synapses on MNs occurs in concomitance with microglial reaction by a process described as synaptic stripping. However, the way in which postaxotomy‐activated microglia adjacent to MNs are involved in synaptic removal is less defined. Here, we used confocal and electron microscopy to examine interactions between recruited microglial cells and presynaptic terminals in axotomized MNs between 1 and 15 days after sciatic nerve transection in mice. We did not observe any bulk engulfment of synaptic boutons by microglia. Instead, microglial cells internalized small membranous‐vesicular fragments which originated from the acute disruption of synaptic terminals involving the activation of the necroptotic pathway. The presence of abundant extracellular vesicles in the perineuronal space after axotomy, together with the increased expression of phospho‐mixed lineage kinase domain‐like protein and, later, of extracellular vesicle markers, such as CD9, CD63, and flotillin, indicate that the vesicles mainly originated in synapses and were transferred to microglia. The upregulation of Rab7 and Rab10 in microglia interacting with injured MNs, indicated the activation of endocytosis. As activated microglia and synaptic boutons displayed positive C1q immunoreactivity, a complement‐mediated opsonization may also contribute to microglial‐mediated synaptic disruption. In addition to the relevance of our data in the context of neuroinflammation and MN disease, they should also be taken into account for understanding functional recovery after peripheral nerve injury., Main Points Early after axotomy, microglia recruited near injured motor neurons, emit processes that tend to contact their afferent synaptic terminals.Extracellular vesicles resulting from necroptotic synaptic disruption are removed by microglia.

Background The cellular mechanisms underlying the age‐associated loss of muscle mass and function (sarcopenia) are poorly understood, hampering the development of effective treatment strategies. Here, we performed a detailed characterization of age‐related pathophysiological changes in the mouse neuromuscular system. Methods Young, adult, middle‐aged, and old (1, 4, 14, and 24-30 months old, respectively) C57BL/6J mice were used. Motor behavioural and electrophysiological tests and histological and immunocytochemical procedures were carried out to simultaneously analyse structural, molecular, and functional age‐related changes in distinct cellular components of the neuromuscular system. Results Ageing was not accompanied by a significant loss of spinal motoneurons (MNs), although a proportion (~15%) of them in old mice exhibited an abnormally dark appearance. Dark MNs were also observed in adult (~9%) and young (~4%) animals, suggesting that during ageing, some MNs undergo early deleterious changes, which may not lead to MN death. Old MNs were depleted of cholinergic and glutamatergic inputs (~40% and ~45%, respectively, P < 0.01), suggestive of age‐associated alterations in MN excitability. Prominent microgliosis and astrogliosis [~93% (P < 0.001) and ~100% (P < 0.0001) increase vs. adults, respectively] were found in old spinal cords, with increased density of pro‐inflammatory M1 microglia and A1 astroglia (25‐fold and 4‐fold increase, respectively, P < 0.0001). Ageing resulted in significant reductions in the nerve conduction velocity and the compound muscle action potential amplitude (~30%, P < 0.05, vs. adults) in old distal plantar muscles. Compared with adult muscles, old muscles exhibited significantly higher numbers of both denervated and polyinnervated neuromuscular junctions, changes in fibre type composition, higher proportion of fibres showing central nuclei and lipofuscin aggregates, depletion of satellite cells, and augmented expression of different molecules related to development, plasticity, and maintenance of neuromuscular junctions, including calcitonin gene‐related peptide, growth associated protein 43, agrin, fibroblast growth factor binding protein 1, and transforming growth factor‐β1. Overall, these alterations occurred at varying degrees in all the muscles analysed, with no correlation between the age‐related changes observed and myofiber type composition or muscle topography. Conclusions Our data provide a global view of age‐associated neuromuscular changes in a mouse model of ageing and help to advance understanding of contributing pathways leading to development of sarcopenia. This work was supported by Abbott and a grant from the Ministerio de Ciencia, Innovación y Universidades cofinancedby Fondo Europeo de Desarrollo Regional (RTI2018-099278-B-I00 to J.C. and J.E.)

C-type synaptic boutons (C-boutons) provide cholinergic afferent input to spinal cord motor neurons (MNs), which display an endoplasmic reticulum (ER)–related subsurface cistern (SSC) adjacent to their postsynaptic membrane. A constellation of postsynaptic proteins is clustered at C-boutons, including M2 muscarinic receptors, potassium channels, and s-1 receptors. In addition, we previously found that neuregulin (NRG)1 is associated with C-boutons at postsynaptic SSCs, whereas its ErbB receptors are located in the presynaptic compartment. Cbouton–mediated regulation of MN excitability has been implicated in MN disease, but NRG1-mediated functions and the impact of various pathologic conditions on C-bouton integrity have not been studied in detail. Here, we investigated changes inC-boutons after electrical stimulation,pharmacological treatment, and peripheral nerve axotomy. SSC-linked NRG1 clusters were severely disrupted in acutely stressedMNs and after tunicamycin-induced ER stress. In axotomized MNs, C-bouton loss occurred in concomitance with microglial recruitment and was prevented by the ER stress inhibitor salubrinal.Activatedmicroglia displayed apositive chemotaxis to C-boutons.Analysis of transgenicmice overexpressing NRG1 type I and type III isoforms in MNs indicated that NRG1 type III acts as an organizer of SSC-like structures, whereas NRG1 type I promotes synaptogenesis of presynaptic cholinergic terminals.Moreover,MN-derived NRG1 signals may regulate the activity of perineuronal microglial cells. Together, these data provide new insights into the molecular and cellular pathology of C-boutons in MN injury and suggest that distinct NRG1 isoform–mediated signaling functions regulate the complex matching between pre- and postsynaptic C-bouton elements. The authors thank Klaus A. Nave (Max-Planck-Institute of Experimental Medicine, Göttingen, Germany) for advice and for supplying neuregulin-1–mutant mice; Jesús María López (Universidad Complutense de Madrid, Madrid, Spain), Ester Desfilis, and José Antonio Moreno for providing spinal cord samples from nonrodent animals; Anaïs Panosa and Xavier Calomarde (all from Universitat de Lleida−Institut de Recerca Biomèdica de Lleida) for technical support with confocal and electron microscopy; and the Serveis Científico-Tècnics Anima Facility of the University of Lleida for mouse care and housing. This work was supported by grants to J.E.E. and J.C. from the Spanish Ministerio de Economía y Competitividad cofinanced by the Fondo Europeo de Desarrollo Regional (FEDER; SAF2015-70801-R). S.S. holds a grant from Spanish Ministerio de Educación, Cultura, y Deporte (FPU). M.H.S. holds a Heisenberg Fellowship from the Deutsche Forschungsgemeinschaft (DFG) and acknowledges funding by a DFG research grant (SCHW741/4-1). The authors declare no conflicts of interest.

C boutons are large, cholinergic, synaptic terminals that arise from local interneurons and specifically contact spinal α-motoneurons (MNs). C boutons characteristically display a postsynaptic specialization consisting of an endoplasmic reticulum-related subsurface cistern (SSC) of unknown function. In the present work, by using confocal microscopy and ultrastructural immunolabeling, we demonstrate that neuregulin-1 (NRG1) accumulates in the SSC of mouse spinal MNs. We also show that the NRG1 receptors erbB2 and erbB4 are presynaptically localized within C boutons, suggesting that NRG1-based retrograde signaling may occur in this type of synapse. In most of the cranial nuclei, MNs display the same pattern of NRG1 distribution as that observed in spinal cord MNs. Conversely, MNs in oculomotor nuclei, which are spared in amyotrophic lateral sclerosis (ALS), lack both C boutons and SSC-associated NRG1. NRG1 in spinal MNs is developmentally regulated and depends on the maintenance of nerve-muscle interactions, as we show after nerve transection experiments. Changes in NRG1 in C boutons were also investigated in mouse models of MN diseases: i.e., spinal muscular atrophy (SMNΔ7) and ALS (SOD1(G93A)). In both models, a transient increase in NRG1 in C boutons occurs during disease progression. These data increase our understanding of the role of C boutons in MN physiology and pathology.

Glutamate receptor-mediated excitotoxicity and mitochondrial dysfunction appear to play an important role in motoneuron (MN) degeneration in amyotrophic lateral sclerosis (ALS). In the present study we used an organotypic slice culture of chick embryo spinal cord to explore the responsiveness of mature MNs to different excitotoxic stimuli and mitrochondrial inhibition. We found that, in this system, MNs are highly vulnerable to excitotoxins such as glutamate, N-methyl-D-aspartate (NMDA), and kainate (KA), and that the neuroprotective drug riluzole rescues MNs from KA-mediated excitotoxic death. MNs are also sensitive to chronic mitochondrial inhibition induced by malonate and 3-nitropropionic acid (3-NP) in a dose-dependent manner. MN degeneration induced by treatment with mitochondrial toxins displays structural changes similar to those seen following excitotoxicity and can be prevented by applying either the antiexcitotoxic drug 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX) or riluzole. Excitotoxicity results in an increased frequency of normal spontaneous Ca2+ oscillations in MNs, which is followed by a sustained deregulation of intracellular Ca2+. Tolerance to excitotoxic MN death resulting from chronic exposure to excitotoxins correlates with a reduced excitotoxin-induced increase in intracellular Ca2+ and increased thapsigargin-sensitive Ca2+ stores.

In an attempt to determine whether the rescue of developing motoneurons (MNS) from programmed cell death (PCD) in the chick embryo following reductions in neuromuscular function involves muscle or neuronal nicotinic acetylcholine receptors (nAChRs), we have employed a novel cone snail toxin αA-OIVA that acts selectively to antagonize the embryonic/fetal form of muscle nAChRs. The results demonstrate that αA-OIVA is nearly as effective as curare or α-bungarotoxin (α-BTX) in reducing neuromuscular function and is equally effective in increasing MN survival and intramuscular axon branching. Together with previous reports, we also provide evidence consistent with a transition between the embryonic/fetal form to the adult form of muscle nAChRs in chicken that involves the loss of the gamma subunit in the adult receptor. We conclude that selective inhibition of the embryonic/fetal form of the chicken muscle nAChR is sufficient to rescue MNs from PCD without any involvement of neuronal nAChRs. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008

In the chick embryo, in ovo application of NMDA from embryonic day (E) 5 to E9 results in selective damage to spinal cord motoneurons (MNs) that undergo a long-lasting degenerative process without immediate cell death. This contrasts with a single application of NMDA on E8, or later, which induces massive necrosis of the whole spinal cord. Chronic MN degeneration after NMDA implies transient incompetence to develop programmed cell death, altered protein processing within secretory pathways, and late activation of autophagy. Chronic NMDA treatment also results in an enlargement of thapsigargin-sensitive Ca(2+) stores. In particular MN pools, such as sartorius-innervating MNs, the neuropeptide CGRP is accumulated in somas, peripheral axons and neuromuscular junctions after chronic NMDA treatment, but not in embryos paralyzed by chronic administration of curare. Intramuscular axonal branching is also altered severely after NMDA: it usually increases, but in some cases a marked reduction can also be observed. Moreover, innervated muscle postsynaptic sites increase by NMDA, but to a lesser extent than by curare. Because some of these results show interesting homologies with MN pathology in human sporadic ALS, the model presented here provides a valuable tool for advancing in the understanding of some cellular and molecular processes particularly involved in this disease.

We have developed an organotypic culture technique that uses slices of chick embryo spinal cord, in which trophic requirements for long-term survival of mature motoneurons (MNs) were studied. Slices were obtained from E16 chick embryos and maintained for up to 28 days in vitro (DIV) in a basal medium. Under these conditions, most MNs died. To promote MN survival, 14 different trophic factors were assayed. Among these 14, glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor were the most effective. GDNF was able to promote MN survival for at least 28 DIV. K(+) depolarization or caspase inhibition prevented MN death but also induced degenerative-like changes in rescued MNs. Agents that elevate cAMP levels promoted the survival of a proportion of MNs for at least 7 DIV. Examination of dying MNs revealed that, in addition to cells exhibiting a caspase-3-dependent apoptotic pattern, some MNs died by a caspase-3-independent mechanism and displayed autophagic vacuoles, an extremely convoluted nucleus, and a close association with microglia. This organotypic spinal cord slice culture may provide a convenient model for testing conditions that promote survival of mature-like MNs that are affected in late-onset MN disease such as amyotrophic lateral sclerosis.

We have examined c-Jun protein expression by immunocytochemistry in normal and pathologically induced cell death by focusing primarily on the developing neuromuscular system of the chick embryo. Several commercially available antibodies against c-Jun were used in combination with the TUNEL technique or propidium iodide staining for detection of cells undergoing programmed cell death (PCD). Among these, a rabbit polyclonal antibody raised against the amino acids 91-105 mapping to the amino terminal domain of mouse c-Jun p39 (c-Jun/sc45) transiently immunostained the cytoplasm of dying spinal cord motoneurons at a time coincident with naturally occurring motoneuron death. Late apoptotic bodies were devoid of c-Jun/sc45 immunoreactivity. A monoclonal antibody directed against a region corresponding to the amino acids 26-175 of c-Jun p39 (c-Jun/mAB) did not specifically immunostain dying neurons, but, rather, showed nuclear immunolabeling in almost all healthy motoneurons. Experimentally induced programmed death of motoneurons by means of early limb bud ablation, axotomy, or in ovo injection of the neurotoxin β-bungarotoxin increased the number of dying cells showing positive c-Jun/sc45 immunoreactivity. Immunoelectron microscopy with c-Jun/sc45 antibody showed that the signal was present in the cytoplasm without a specific association with organelles, and was also present in large lysosome-like dense bodies inside neuritic profiles. Similar findings were obtained in different types of cells undergoing normal or experimentally induced PCD. These include dorsal root ganglion neurons, Schwann cells, muscle cells, neural tube and neural crest cells during the earliest stages of spinal cord development, and interdigital mesenchymal cells of hindlimbs. In all these cases, cells showed morphological and histochemical characteristics of apoptotic-like PCD. By contrast, motoneurons undergoing necrotic cell death induced by the excitotoxin N-methyl-D-aspartate did not show detectable c-Jun/sc45 immunoreactivity, although they displayed an increase in nuclear c-Jun/mAB immunostaining. In Western blot analysis of spinal cord extracts, c-Jun/sc45 antibody weakly detected a 39-kD band, corresponding to c-Jun, and more strongly detected two additional bands of 66 and 45 kD which followed developmental changes coincident with naturally occurring or experimentally stimulated apoptotic motoneuron death. By contrast, c-Jun/mAB only recognized a single p39 band as expected for c-Jun, and did not display changes associated with neuronal apoptosis. From these data, we conclude that the c-Jun/sc45 antibody recognizes apoptosis-related proteins associated with the early stages of morphological PCD in a variety of neuronal and nonneuronal cells, and that c-Jun/sc45 is a reliable marker for a variety of developing cells undergoing programmed cell death. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 171–190, 1999

The aim of this study was to examine whether changes in rat motoneuronal calcitonin gene-related peptide (CGRP) can be correlated with axonal growth and plasticity of neuromuscular synapses. Nerve terminal outgrowth was induced by local paralysis with botulinum toxin. Normal adult soleus and tibialis anterior did not show detectable CGRP content at the motor endplates. Following botulinum toxin injection there was a progressive, transient and bimodal increase in CGRP in both motoneuron cell bodies which innervated poisoned muscles and their motor endplates. CGRP content was moderately increased 1 day after paralysis and, after an initial decline, reached a peak 20 days after injection. This was followed by a gradual decrease and a return to normal levels at the 200th day. CGRP changes in intoxicated endplates were less evident in the tibialis anterior than in the soleus muscle. The CGRP content in motoneurons was positively correlated with the degree of intramuscular nerve sprouting found by silver staining. In situ hybridization revealed an increase in CGRP mRNA in spinal cord motoneurons 20 days after toxin administration. We conclude that motoneurons regulate their CGRP in situations in which peripheral synapse remodelling and plasticity occur.