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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.

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.

Several studies have reported the neuroprotective effects of lithium (Li) suggesting its potential in the treatment of neurological disorders, among of them amyotrophic lateral sclerosis (ALS). Although the cause of motoneuron (MN) death in ALS remains unknown, there is evidence that glutamate-mediated excitotoxicity plays an important role. In the present study we used an organotypic culture system of chick embryo spinal cord to explore the presumptive neuroprotective effects of Li against kainate-induced excitotoxic MN death. We found that chronic treatment with Li prevented excitotoxic MN loss in a dose dependent manner and that this effect was mediated by the inhibition of glycogen synthase kinase-3beta (GSK-3beta) signaling pathway. This neuroprotective effect of Li was potentiated by a combined treatment with riluzole. Nevertheless, MNs rescued by Li displayed structural changes including accumulation of neurofilaments, disruption of the rough endoplasmic reticulum and free ribosome loss, and accumulation of large dense core vesicles and autophagic vacuoles. Accompanying these changes there was an increase in immunostaining for (a) phosphorylated neurofilaments, (b) calcitonin gene-related peptide (CGRP) and (c) the autophagic marker LC3. Chronic Li treatment also resulted in a reduction in the excitotoxin-induced rise in intracellular Ca(2+) in MNs. In contrast to the neuroprotection against excitotoxicity, Li was not able to prevent normal programmed (apoptotic) MN death in the chick embryo when chronically administered in ovo. In conclusion, these results show that although Li is able to prevent excitotoxic MN death by targeting GSK-3beta, this neuroprotective effect is associated with conspicuous cytopathological changes.

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.

Motoneuron (MN) cell death is the histopathologic hallmark of spinal muscular atrophy (SMA), although MN loss seems to be a late event. Conversely, disruption of afferent synapses on MNs has been shown to occur early in SMA. Using a mouse model of severe SMA (SMNΔ7), we examined the mechanisms involved in impairment of central synapses. We found that MNs underwent progressive degeneration in the course of SMA, with MN loss still occurring at late stages. Loss of afferent inputs to SMA MNs was detected at embryonic stages, long before MN death. Reactive microgliosis and astrogliosis were present in the spinal cord of diseased animals after the onset of MN loss. Ultrastructural observations indicate that dendrites and microglia phagocytose adjacent degenerating presynaptic terminals. Neuronal nitric oxide synthase was upregulated in SMNΔ7 MNs, and there was an increase in phosphorylated myosin light chain expression in synaptic afferents on MNs; these observations implicate nitric oxide in MN deafferentation and suggest that the RhoA/ROCK pathway is activated. Together, our observations suggest that the earliest change occurring in SMNΔ7 mice is the loss of excitatory glutamatergic synaptic inputs to MNs; reduced excitability may enhance their vulnerability to degeneration and death.

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease affecting upper and lower motoneurons (MNs). Although the motor phenotype is a hallmark for ALS, there is increasing evidence that systems other than the efferent MN system can be involved. Mutations of superoxide dismutase 1 (SOD1) gene cause a proportion of familial forms of this disease. Misfolding and aggregation of mutant SOD1 exert neurotoxicity in a noncell autonomous manner, as evidenced in studies using transgenic mouse models. Here, we used the SOD1(G93A) mouse model for ALS to detect, by means of conformational-specific anti-SOD1 antibodies, whether misfolded SOD1-mediated neurotoxicity extended to neuronal types other than MNs. We report that large dorsal root ganglion (DRG) proprioceptive neurons accumulate misfolded SOD1 and suffer a degenerative process involving the inflammatory recruitment of macrophagic cells. Degenerating sensory axons were also detected in association with activated microglial cells in the spinal cord dorsal horn of diseased animals. As large proprioceptive DRG neurons project monosynaptically to ventral horn MNs, we hypothesise that a prion-like mechanism may be responsible for the transsynaptic propagation of SOD1 misfolding from ventral horn MNs to DRG sensory neurons. The authors would like to thank Montse Ortega for her technical assistance and Claudia Cervero, Alexandra Eritja and Ariadna Salvador for their help with some of the experiments in this study. This work was supported by grants from the Ministerio de Ciencia y Tecnologia and Ministerio de Economia y Competitividad and cofinanced by FEDER (SAF2011-22908; SAF2012-31831).

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by defective levels of the survival motor neuron (SMN) protein. SMA causes spinal motoneuron (MN) loss, and progressive muscle weakness and paralysis. Currently, there is no effective therapy to cure this disease. Although different strategies focused on increasing the expression of functional SMN protein have been assayed, numerous SMN-independent therapeutic approaches have been demonstrated to have potential effectiveness in improving the SMA phenotype in mouse models and clinical trials. Recent works have shown that compounds which inhibit GSK-3β activity are effective in promoting MN survival and ameliorating lifespan in models of MN diseases including SMA. Taking into account the reported neuroprotective actions of lithium (Li) through the inhibition of GSK-3β in different studies, we tested here its potential efficiency as a therapeutic agent in a mouse model of severe SMA (SMNΔ7 mice). We show that the chronic treatment with Li initiated before the appearance of disease symptoms, although inhibited GSK-3β, did not improve the median survival, motor behavior, and spinal MN loss linked to SMA. Li administration did not either ameliorate the microglial and astroglial reaction in the spinal cord or the depletion of glutamatergic synapses on MNs observed in SMNΔ7 animals. Moreover, Li treatment did not mitigate muscle atrophy or calcitonin gene-related peptide (CGRP) downregulation in the neuromuscular junctions linked to the disease. However, a significant reduction in apoptotic cell death found in the skeletal muscle of SMA mice was observed after Li treatment.

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.

A detailed pathologic analysis was performed on Smn(-/-);SMN2 mice as a mouse model for human type I spinal muscular atrophy (SMA). We provide new data concerning changes in the spinal cord, neuromuscular junctions and muscle cells, and in the organs of the immune system. The expression of 10 synaptic proteins was analyzed in 3-dimensionally reconstructed neuromuscular junctions by confocal microscopy. In addition to defects in postsynaptic occupancy, there was a marked reduction in calcitonin gene-related peptide and Rab3A in the presynaptic motor terminals of some, but not all, of the skeletal muscles analyzed. Defects in the organization of presynaptic nerve terminals were also detected by electron microscopy. Moreover, degenerative changes in muscle cells, defective postnatal muscle growth, and prominent muscle satellite cell apoptosis were also observed. All of these changes occurred in the absence of massive loss of spinal cord motoneurons. On the other hand, astroglia, but not microglia, increased in the ventral horn of newborn SMA mice. In skeletal muscles, the density of interstitial macrophages was significantly reduced, and monocyte chemotactic protein-1 was downregulated. These findings raise questions regarding the primary contribution of a muscle cell defect to the SMA phenotype.

The role of microglia during normal development of the nervous system is still not well understood. In the present study, a chick embryo model was used to examine the development of microglia in the spinal cord and characterize their changes in response to naturally occurring and pathological death of motoneurons (MNs). The microglial response to MN axotomy and the effects of microglial activation on MN survival were also studied. We found that: 1) macrophages/microglial cells were present in the spinal cord at early developmental stages (E3) and that they were recruited after normal and induced MN apoptosis; 2) although many microglial cells were seen phagocytosing apoptotic bodies, a proportion of dying cells were devoid of engulfing microglia; 3) axotomy of mature MNs was accompanied by microglial activation in the absence of MN death; 4) excitotoxic (necrotic) MN death provoked a rapid and massive microglial recruitment with phagocytic activity; 5) lipopolysaccharide-induced microglial activation in vivo resulted in the death of immature, but not mature, microglia; and 6) overactivation of microglia modulated the survival of mature MNs, either by killing them or by enhancing their vulnerability to die in response to a mild injury. Taken together, these observations indicate that normal microglia do not play an active role in triggering apoptosis of developing MNs. Rather, they act as phagocytes for the removal of dying cells during the process of programmed cell death.

We previously showed that, in contrast to the acute administration of NMDA, chronic treatment of chick embryos from embryonic day (E) 5 to E9 with this excitotoxin rescues motoneurons (MNs) from programmed cell death. Following this protocol, MNs are also protected against later acute excitotoxic cell death. Previously, we found that MNs treated from E5 to E9 develop long-lasting changes involving vesicular trafficking and other organelle pathology similar to the abnormalities observed in certain chronic neurological diseases including amyotrophic lateral sclerosis (ALS). Here we extend these previous results by showing that protein aggregation within the endoplasmic reticulum (ER) takes place selectively in MNs as an early event of chronic excitotoxicity. Although protein aggregates do not induce appreciable MN death, they foreshadow the activation of a conspicuous autophagic response leading to long-lasting degenerative changes that causes dysfunction but not immediate cell death. Chronic early treatment with NMDA results in a transient (between E6 and E10) lack of vulnerability to undergo cell death induced by different types of stimuli. It is suggested that blockade of protein translation in stressed ER may inhibit apoptosis in NMDA-treated MNs. However, in embryos older than E12, degenerating MNs are sensitized to die after limb ablation (axotomy) and accumulate hyperphosphorylated neurofilaments. Moreover, chronic NMDA treatment does not induce the upregulation of molecular chaperones in spinal cord. These results represent a new model of glutamate receptor-mediated neurotoxicity that selectively occurs in spinal cord MNs and also demonstrate an experimental system that may be valuable for understanding the mechanisms involved in chronic MN degeneration and in certain cytological hallmarks of ALS-diseased MNs such as inclusion bodies.

Blockade of neuromuscular activity in the chick embryo during the period of programmed cell death of motoneurons results in a complete rescue of these cells. Understanding the cellular mechanisms that mediate this counterintuitive effect is of considerable interest with respect to the regulation of motoneuron survival during development as well as for understanding why motoneurons die pathologically. Although considerable evidence supports the role of a peripheral site of action at the neuromuscular junction in mediating the rescue of motoneurons following activity blockade, some evidence also supports a role for central nervous system (CNS) neurons. For example, the rescue of motoneurons by curare has been reported to be blocked by the GABA(A) agonist muscimol via its actions on CNS neurons. We have carried out a series of studies to further investigate this interesting observation. Surprisingly, we find that: (1) muscimol blocks activity and rescues MNs in a dose-dependent manner, similar to curare; (2) muscimol's effects on MN survival appear to be mediated by its action on intramuscular nerve branching, similar to curare; and (3) although muscimol acts centrally, the effects of muscimol on MN survival and axon branching are mediated peripherally at the neuromuscular junction, similar to curare. Because muscimol reduces MN depolarization these data also suggest that the depolarization of MNs by afferents is not required for promoting MN survival. Taken together, these data provide further evidence in support of a peripheral site of action of activity blockade in rescuing motoneurons from developmental cell death.

Unilateral limb-bud removal (LBR) before the outgrowth of sensory or motor neurons to the leg of chick embryos was used to examine the role of limb (target)-derived signals in the development and survival of lumbar motoneurons and sensory neurons in the dorsal root ganglia (DRG). After LBR, motor and sensory neurons underwent normal initial histological differentiation, and cell growth in both populations was unaffected. Before their death, target-deprived motoneurons also expressed a cell-specific marker, the homeodomain protein islet-1. Proliferation of sensory and motor precursor cells was also unaffected by LBR, and the migration of neural crest cells to the DRG and of motoneurons into the ventral horn occurred normally. During the normal period of programmed cell death (PCD), increased numbers of both sensory and motor neurons degenerated after LBR. However, whereas motoneuron loss increased by 40–50% (90% total), only ∼25% more sensory neurons degenerated after LBR. A significant number of the surviving sensory neurons projected to aberrant targets in the tail after LBR, and many of these were lost after ablation of both the limb and tail. Treatment with neurotrophic factors (or muscle extract) rescued sensory and motor neurons from cell death after LBR without affecting precursor proliferation of either population. Activity blockade with curare failed to rescue motoneurons after LBR, and combined treatment with curare plus muscle extract was no more effective than muscle extract alone. Treatment with the antioxidantN-acetylcysteine rescued motoneurons from normal cell death but not after LBR. Two specific inhibitors of the interleukin β1 converting enzyme (ICE) family of cysteine proteases also failed to prevent motoneuron death after LBR. Taken together these data provide definitive evidence that the loss of spinal neurons after LBR cannot be attributed to altered proliferation, migration, or differentiation. Rather, in the absence of limb-derived trophic signals, the affected neurons fail to survive and undergo PCD. Although normal cell death and cell death after target deprivation share many features in common, the intracellular pathways of cell death in the two may be distinct.

In the present work, we show that chick embryo Schwann cells die by apoptosis both during normal development and after axonal degeneration induced by neurotoxin treatment. Schwann cell apoptosis during development takes place during a period roughly coincidental with normally occurring motoneuron death. Administration of NMDA to chick embryos on embryonic day 7 induces extensive excitotoxic motoneuronal damage in the spinal cord without any apparent effects on neurons in the dorsal root ganglia (DRG). The death of Schwann cells inventralnerve roots after NMDA treatment causes degenerative changes that display ultrastructural features of apoptosis and exhibitin situdetectable DNA fragmentation. By contrast, NMDA treatment does not increase the death of Schwann cells indorsalnerve roots.In situdetection of DNA fragmentation in combination with the avian Schwann cell marker 1E8 antibody demonstrates that dying cells in ventral nerve roots are in the Schwann cell lineage. Administration of cycloheximide does not prevent the toxic effects of NMDA on motoneurons, but dramatically reduces the number of pyknotic Schwann cells and DNA fragmentation profiles in the ventral nerve roots.In ovoadministration of various tissue extracts (muscle, brain, and spinal cord) from the chick embryo or of the motoneuron conditioned medium fails to prevent Schwann cell apoptosis in NMDA-treated embryos. Intramuscular administration of the snake toxin β-bungarotoxin produces a massive death of both lateral motor column motoneurons and DRG neurons, resulting in a substantial increase in the number of pyknotic Schwann cells in both ventral and dorsal nerve roots. It is concluded that during development, axonal-derived trophic signals are involved in the regulation of Schwann cell survival in peripheral nerves.

We have studied in living chick embryos the effects of an extracellular calcium load on the induction of apoptosis in spinal cord motoneurons. The action of a calcium ionophore, A23187, that does not raise extracellular calcium was also evaluated in order to explore the role of endogenous calcium in determining developmentally-regulated cell death of motoneurons. The application of a single dose of 50 microliters of 1.8 M CaCl2 onto the chorioallantoic membrane of E7 chick embryos produce a transient elevation of intraembryonic calcium concentration that was followed by a transitory rise in the number of apoptotic cells in the lateral motor column. Administration of 250 microM of the ionophore A23187 (100 microliters), also results in an increase in apoptosis of motoneurons in the lateral motor column on E6 and E7 but this effect is progressively lost following treatment at more advanced stages of development. Neither of these effects can be explained by unspecific calcium cytotoxicity since they can be inhibited by prior administration of the protein synthesis inhibitor cycloheximide or the neuromuscular blocking agent (+)-tubocurarine. After calcium loading, degenerating cells display similar ultrastructural characteristics as during physiologically occurring motoneuron death and exhibit histochemically detectable DNA fragmentation. Chronic administration of CaCl2 or A23187 does not reduce the total number of surviving motoneurons at the end of the normal period of naturally occurring motoneuron death (E10). It is suggested that calcium loading stimulates and accelerates the physiological degeneration of a restricted subpopulation of motoneurons which will undergo the process of natural cell death.

Acute administration of a single dose of NMDA on embryonic day (E) 7 or later induces a marked excitotoxic injury in the chick spinal cord, including massive necrotic motoneuron (MN) death. When the same treatment was performed before E7, little, if any, excitotoxic response was observed. Chronic treatment with NMDA starting on E5 prevents the excitotoxic response produced by a later “acute” administration of NMDA. Additionally, chronic NMDA treatment also prevents the later excitotoxic injury induced by non-NMDA glutamate receptor agonists, such as kainate or AMPA. Chronic NMDA treatment also reduces normal MN death when treatment is maintained during the period of naturally occurring programmed cell death (PCD) of MNs and rescues MNs from PCD induced by early peripheral target deprivation. The trophic action of chronic NMDA treatment appears to involve a downregulation of glutamate receptors as shown by both a reduction in the obligatory NR1 subunit protein of the NMDA receptor and a decrease in the kainate-induced Co2+uptake in MNs. Both tolerance to excitotoxicity and trophic effects of chronic NMDA treatment are prevented by the NMDA receptor antagonist MK-801. Additionally, administration of MK-801 alone results in an increase in MN PCD. These data indicate for the first time that early activation of NMDA receptors in developing avian MNsin vivohas a trophic, survival-promoting effect, inhibiting PCD by a target-independent mechanism that involves NMDA receptor downregulation.

The electric activity of lower motor neurons (MNs) appears to play a role in determining cell-vulnerability in MN diseases. MN excitability is modulated by cholinergic inputs through C-type synaptic boutons, which display an endoplasmic reticulum-related subsurface cistern (SSC) adjacent to the postsynaptic membrane. Besides cholinergic molecules, a constellation of proteins involved in different signal-transduction pathways are clustered at C-type synaptic sites (M2 muscarinic receptors, Kv2.1 potassium channels, Ca2+ activated K+ [SK] channels, and sigma-1 receptors [S1R]), but their collective functional significance so far remains unknown. We have previously suggested that neuregulin-1 (NRG1)/ErbBs-based retrograde signalling occurs at this synapse. To better understand signalling through C-boutons, we performed an analysis of the distribution of C-bouton-associated signalling proteins. We show that within SSC, S1R, Kv2.1 and NRG1 are clustered in highly specific, non-overlapping, microdomains, whereas ErbB2 and ErbB4 are present in the adjacent presynaptic compartment. This organization may define highly ordered and spatially restricted sites for different signal-transduction pathways. SSC associated proteins are disrupted in axotomised MNs together with the activation of microglia, which display a positive chemotactism to C-bouton sites. This indicates that C-bouton associated molecules are also involved in neuroinflammatory signalling in diseased MNs, emerging as new potential therapeutic targets. We would like to thank Dr. Ronald W. Oppenheim for his critical reading of the manuscript and for helpful comments and suggestions. We would also like to thank Marta Hereu for her technical assistance, Lidia Delgado, Gema Marta Martínez and M. Yolanda Muela, from the Unitat de Criomicroscòpia Electrònica (Centres Científics i Tecnològics de la Universitat de Barcelona), for their technical support with ultrastructural immunolabelling, Daniel Cabezas for his help in some experiments, and the SCT animal facility of the Universitat de Lleida for mouse care and housing. This work was supported by grants from the Ministerio de Economía y Competitividad cofinanced by FEDER (SAF2015–70801-R to J.E.E. and J.C.), and from Jack Van den Hoek a la investigació de l’ELA - Fundació Miquel Valls (to J.E.E.).