Regulation of putative muscle-derived neurotrophic factors by muscle activity and innervation: in vivo and in vitro studies

The normal embryonic development of spinal cord motoneurons (MNs) involves the proliferation of precursor cells followed by the degeneration of approximately 50% of postmitotic MNs during the period when nerve-muscle connections are being established. The death of MNs in vivo can be ameliorated by activity blockade and by treatment with muscle extracts. Muscle activity and innervation have been suggested to regulate the availability of putative muscle-derived neurotrophic agent(s), and MNs are thought to compete for limited amounts of these trophic agents during normal development. Thus, activity and innervation are thought to regulate MN survival by modulating trophic factor availability. We have tested this notion by examining MN survival in vivo and ChAT development in spinal cord neurons in vitro following treatments with partially purified muscle extracts from normally active, paralyzed (genetically or pharmacologically), aneural, denervated, slow tonic, and fast-twitch muscles from embryonic and postnatal animals. Extracts from active and chronically inactive embryonic avian and mouse muscles were found to be equally effective in promoting the in vivo survival of MNs in the chick embryo. Similarly, extracts from fast-twitch and slow tonic postnatal avian muscles did not differ in their ability to promote both MN survival in vivo and ChAT activity in vitro. Although aneural and control embryonic muscle extract had similar effects on ChAT development in vitro, aneural muscle extract contained somewhat less MN survival-promoting activity when tested in vivo. By contrast, denervated postnatal muscle extract was more effective in promoting both MN survival in vivo and ChAT activity in vitro than age-matched control muscle extract.(ABSTRACT TRUNCATED AT 250 WORDS)     

Overexpression of glial cell line-derived neurotrophic factor in the CNS rescues motoneurons from programmed cell death and promotes their long-term survival following axotomy

To study the role of one of the most potent motoneuron (MN) survival factors, glial cell line-derived neurotrophic factor (GDNF) derived from the CNS, we generated transgenic animals overexpressing GDNF under the control of an astrocyte-specific GFAP promoter. In situ hybridization revealed that GDNF was expressed at high levels in astrocytes throughout the brain and spinal cord. We analyzed the effects of CNS-derived GDNF on MN survival during the period of programmed cell death (PCD) and after nerve axotomy. In GFAP-GDNF mice at E15, E18, and P1, the survival of brachial MNs was increased on average by 30%, lumbar MNs by 20%, and thoracic MNs at P1 by 33%. GDNF also prevented MN PCD in several cranial motor nuclei. We demonstrated for the first time that the number of MNs in the mouse abducens nucleus was also increased by 40%, thus extending known MN populations that are responsive to GDNF. Next, we tested if GDNF could support complete and relatively long-term survival of MNs following neonatal facial nerve axotomy. We found that virtually all MNs (91%) in GFAP-GDNF mice survived for up to 18 weeks post-axotomy. This is the longest GDNF-mediated survival of neonatal MNs reported following axotomy. Most of surviving MNs were not atrophic, and MN-specific ChAT and neurofilament immunoreactivity (IR) were preserved. Furthermore, GDNF attenuated axotomy-induced astroglial activation. These data demonstrate that overexpression of GDNF in the CNS has very profound effects on MN survival both during the PCD period and after neuronal injury. GFAP-GDNF mice will be valuable to study the effects of CNS-derived GDNF in mouse models of MN degenerative diseases and axonal regeneration in vivo.     

Reduction of neuromuscular activity is required for the rescue of motoneurons from naturally occurring cell death by nicotinic-blocking agents.

Spinal motoneurons (MNs) in the chick embryo undergo programmed cell death coincident with the establishment of nerve-muscle connections and the onset of synaptic transmission at the neuromuscular junction. Chronic treatment of embryos during this period with nicotinic acetylcholine receptor (nAChR)-blocking agents [e.g., curare or alpha-bungarotoxin (alpha-BTX)] prevents the death of MNs. Although this rescue effect has been attributed previously to a peripheral site of action of the nAChR-blocking agents at the neuromuscular junction (NMJ), because nAChRs are expressed in both muscle and spinal cord, it has been suggested that the rescue effect may, in fact, be mediated by a direct central action of nAChR antagonists. By using a variety of different nAChR-blocking agents that target specific muscle or neuronal nAChR subunits, we find that only those agents that act on muscle-type receptors block neuromuscular activity and rescue MNs. However, paralytic, muscular dysgenic mutant chick embryos also exhibit significant increases in MN survival that can be further enhanced by treatment with curare or alpha-BTX, suggesting that muscle paralysis may not be the sole factor involved in MN survival. Taken together, the data presented here support the argument that, in vivo, nAChR antagonists promote the survival of spinal MNs primarily by acting peripherally at the NMJ to inhibit synaptic transmission and reduce or block muscle activity. Although a central action of these agents involving direct perturbations of MN activity may also play a contributory role, further studies are needed to determine more precisely the relative roles of central versus peripheral sites of action in MN rescue.     

Involvement of the alpha 7 subunit of the nicotinic receptor in morphogenic and trophic effects of acetylcholine on embryonic rat spinal motoneurons in culture

The morphogenic and trophic effects of acetylcholine (ACh) on embryonic cultured rat spinal cord motoneurons (MNs) through nicotinic alpha7 autoreceptors were assessed. Alpha7 Subunits of the nicotinic cholinergic receptor were detected in cultures of purified rat spinal embryonic MNs sampled at E15, by both immunocytochemistry and alpha-bungarotoxin binding. According to these two methods, alpha7 subunits are located mainly at somatic and axonal membrane. Functional involvement of the alpha7 subunit in survival and development of morphological properties of growing cultured MNs was tested using an antisense strategy. The antisense oligonucleotide significantly decreases the expression of the alpha7 protein compared with control and mismatch oligonucleotide-treated cultures. This decrease in the expression of the alpha7 protein leads to a significant increase in the number of axonal branches and in the length of the axon. The antisense treatment also induces, as early as the first day in culture, a decrease of MN survival, leading to total cell death at day 5. TUNEL staining revealed that the MNs are dying through apoptotic processes. Thus, our study shows that ACh is a morphogenic and trophic factor. These effects are directly linked to the membrane expression level of alpha7 protein. Indeed, the lower the alpha7 expression, the lower the inhibition of axonal growth (i.e., axonal elongation) and the lower the MN survival.     

Excitotoxic motoneuron degeneration induced by glutamate receptor agonists and mitochondrial toxins in organotypic cultures of chick embryo spinal cord

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. J. Comp. Neurol. 516:277–290, 2009. © 2009 Wiley‐Liss, Inc.     

Development of microglia in the chick embryo spinal cord: Implications in the regulation of motoneuronal survival and death

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. © 2009 Wiley‐Liss, Inc.     

Growth factors in combination, but not individually, rescue rd mouse photoreceptors in organ culture

The rd mouse retina is an animal model for human retinal dystrophy in which the rod photoreceptors undergo apoptosis during the first 4 weeks in vivo or in organ culture. We have examined the effect of different families of trophic factors on the survival of rd mouse photoreceptors in organ culture. Retinas were harvested from rd mice at postnatal day 2 and grown in organ culture for 27 days in vitro (DIV) in DMEM with 10% fetal calf serum. Ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF2), glial cell line-derived neurotrophic factor (GDNF), neurturin, and persephon were added individually or in combination to the medium at a dose of 50 ng/ml or less. CNTF + BDNF in combination resulted in photoreceptor survival comparable to wild-type retinas after 27 DIV. CNTF + FGF2 or CNTF + GDNF produced a partial prevention of photoreceptor death. Photoreceptor degeneration was not blocked by any of the trophic factors added individually. A significant increase in photoreceptor survival was seen with forskolin added to CNTF, but not to BDNF, FGF2, or GDNF. These results demonstrate that trophic factors promote photoreceptor survival through a synergistic interaction. Increased understanding of receptor interactions and signaling pathways may lead to a potential therapeutic role for combinatorial trophic factors in treatment of photoreceptor dystrophies.     

Cell death of motoneurons in the chick embryo spinal cord. IX. The loss of motoneurons following removal of afferent inputs

The primary aim of this study was to clarify the role of supraspinal, propriospinal, and primary sensory afferents in motoneuron (MN) development in the lateral motor column (LMC) of the lumbar spinal cord of the chick embryo. For this purpose three types of operations were carried out on embryonic day (E) 2. (1) The spinal cord was transected at the cervical (C-gap) or at the thoracic (T-gap) level so as to eliminate supraspinal and/or propriospinal inputs to the lumbar cord. (2) The entire lumbar neural crest was removed (NCR) in order to eliminate primary sensory inputs arising from the dorsal root ganglia (DRG). (3) A combined operation of T-gap and lumbar NCR was performed. The numbers of MNs in the LMC of the lumbar spinal cord were counted in embryos sacrificed between E10 and E18. The number of MNs on E10, when naturally occurring neuron death is almost complete, was not changed following either operation 1 or 2 described above. However, by E16, when naturally occurring neuron death is over, these same deafferented groups had 20 to 25% fewer MNs than did controls. Thus, the removal of either descending or sensory (DRG) afferents results in a significant increased loss of MNs that appears to take place only during the final stages of natural neuronal death or later. By contrast, the removal of both sources of input (T-gap + NCR) results in an additional 37% loss of MNs by E10 compared to controls. Thus, in this group deafferentation significantly increases cell loss during the major period of naturally occurring MN death (E5 to E10). No further loss of MNs occurs in this group after E10. Chronic treatment of deafferented embryos with curare from E6 to E9 or from E10 to E14 prevented the naturally occurring MN loss during these stages but was without effect on the increased cell loss induced by deafferentation. These results imply that the cellular mechanisms involved in target- versus afferent-regulated cell death are different. Collectively, these results indicate that the regulation of MN numbers is more complicated than previously thought. Both targets and afferents appear to be involved in controlling the survival of this population of neurons during the period of naturally occurring MN death.     

Response of motoneurons to neonatal sciatic nerve axotomy in Bax-knockout mice

Neonatal motoneurons (MNs) die rapidly after axotomy, a response that is mediated by the pro-apoptotic gene Bax and is followed by a mitochondria-mediated apoptotic cascade. Although motoneurons in neonatal Bax-deficient mice fail to degenerate following axotomy, it has not been previously examined whether the rescued MNs can regenerate following injury. We report here that although spinal MNs in Bax-knockout (Bax-KO) mice survive indefinitely, they undergo severe atrophy by 14 days after axotomy. By 1 month following axotomy, MN regeneration was observed and cellular atrophy was partially reversed. Interestingly, we observed that all MNs, including those previously rescued from normal developmental cell death in the embryo by Bax deletion, exhibit a regenerative response to peripheral nerve injury. The regenerative response may be mediated by specific trophic factors because the expression of glial cell line-derived neurotrophic factor (GDNF) was greatly increased in the proximal stump of injured nerves and application of a GDNF-blocking antibody greatly reduced regeneration/regrowth of rescued MNs in Bax-KO mice. These results indicate that MNs rescued from developmental or injury-induced cell death by Bax deletion have the potential to regenerate or regrow in response to nerve-derived signals following neonatal axotomy.     

Muscle-derived but not centrally derived transgene GDNF is neuroprotective in G93A-SOD1 mouse model of ALS

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for motoneurons (MNs), and is considered a potential agent for the treatment of amyotrophic lateral sclerosis (ALS) and other MN diseases. The effectiveness of GDNF may depend significantly upon its route of delivery to MNs. In this study we tested the neuroprotective effects of target-derived and centrally derived GDNF in the G93A-SOD1 mouse model of ALS using a transgenic approach. We found that overexpression of GDNF in the skeletal muscle (Myo-GDNF mice) significantly delayed the onset of disease and increased the life span of G93A-SOD1 mice by 17 days. The duration of disease also increased by 8.5 days, indicating that GDNF slowed down the progression of disease. Locomotor performance in Myo-GDNF/G93A-SOD1 mice was also significantly improved. The behavioral improvement correlated well with anatomical and histological data. We demonstrated that muscle-derived GDNF resulted in increased survival of spinal MNs, and twice as many MNs survived in end-stage double transgenic mice compared to end-stage G93A-SOD1 mice. Muscle-derived GDNF also had profound effects on muscle innervation and axonal degeneration. Significantly higher numbers of completely or partially innervated NMJs and large caliber myelinated axons were found in double transgenic mice. In contrast, we demonstrated that overexpression of GDNF in astrocytes in the CNS (GFAP-GDNF mice) failed to demonstrate any neuroprotective effects in G93A-SOD1 mice both on behavioral and histological levels. These data indicate that retrograde transport and signaling of GDNF is more physiological and effective for ALS treatment than anterogradely transported GDNF.     

CDP-choline protects motor neurons against apoptotic changes in a model of chronic glutamate excitotoxicity in vitro

Cytidine-5-diphosphocholine (CDP-choline, citicoline) is an endogenous nucleoside involved in generation of phospholipids, membrane formation and its repair. It demonstrates beneficial effects in certain central nervous system injury models, including cerebral ischaemia, neurodegenerative disorders and spinal cord injury. Defective neuronal and/or glial glutamate transport is claimed to contribute to progressive loss of motor neurons (MNs) in amyotrophic lateral sclerosis (ALS). Our previous ultrastructural studies, performed on an organotypic tissue culture model of chronic glutamate excitotoxicity, documented a subset of various modes of MN death including necrotic, apoptotic and autophagocytic cell injury. The aim of this ultrastructural study was to determine the potential neuroprotective effect of CDP-choline on neuronal changes in a glutamate excitotoxic ALS model in vitro. Organotypic cultures of the rat lumbar spinal cord subjected to 100 microM DL-threo-beta-hydroxyaspartate (THA) were pretreated with 100 microM of CDP-choline. The exposure of spinal cord cultures to CDP-choline and THA distinctly reduced the development of typical apoptotic changes, whereas both necrotic and autophagocytic THA-induced MN injury occurred. These results indicate that CDP-choline treatment might exert a neuroprotective effect against neuronal apoptotic changes in a model of chronic excitotoxicity in vitro.     

Motor neurone metabolism.

The cell and molecular mechanisms which determine the motor neurone (MN) phenotype are unclear. Tissue culture models offer a unique system for the study of a wide variety of MN features. For instance, since the neurone-astrocyte metabolic interactions play a critical role in the selective MN loss observed in amyotrophic lateral sclerosis (ALS), the glutamatergic MN toxicity could be reanalyzed in vitro, after a careful evaluation of the role of astrocytes. Ca(2+) appears to be important in inducing MN loss from in vitro studies. It was shown primarily in culture that apoptotic or necrotic death of neurones after injury depends upon the cell energetic status. Also, SOD-1 mutations were successfully expressed in cultured MNs, providing a critical assay to sequence the molecular processes responsible for MN degeneration due to an identified genetic defect. Purified human developing MNs and astrocytes were recently obtained from the spinal cord anterior horn. The effects of molecules affecting MN survival, neurite extension, and metabolism can easily be tested in long-term cultures. Interactions at the single cell level can be studied today using a series of RNA amplification techniques. Understanding the properties of human MNs in vitro may represent a critical tool in defining regional metabolic changes that could constitute the first pathogenic event of cell degeneration in ALS.     

Protein retention in the endoplasmic reticulum, blockade of programmed cell death and autophagy selectively occur in spinal cord motoneurons after glutamate receptor-mediated injury

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.     

TGFbeta trophic factors differentially modulate motor axon outgrowth and protection from excitotoxicity

Transforming growth factor (TGF) beta-like trophic factors have been shown to be protective in acute neuronal injury paradigms. In the current study, we analyzed and compared members of this growing family, including glial cell line-derived neurotrophic factor (GDNF), neurturin, nodal, persephin, and TGFbeta1, for protection against chronic glutamate toxicity. In parallel, we developed a organotypic spinal cord culture system to study the ability of these factors to promote motor axon outgrowth across white matter. Using these systems, we were able to differentiate the neuroprotective effect of the TGFbeta-like factors from their motor axon outgrowth-promoting activity. GDNF, neurturin, persephin, nodal, and TGFbeta1 all protected against excitotoxic motor neuron degeneration. Low amounts of GDNF (1 ng/ml) and high concentrations of neurturin induced vigorous motor axon outgrowth. In contrast, nodal, persephin, and TGFbeta1 did not induce motor axon outgrowth. Both GDNF and neurturin bind to Ret receptor complexes and were capable of activating the MAP kinase pathway. A specific inhibitor of MAP kinase kinase, PD98059, inhibited the motor axon outgrowth-promoting activity of the GDNF but not the neuroprotective activity. Similarly, the specific PI3K inhibitors, LY294002 and wortmannin, were able to inhibit the promotion of motor axon outgrowth by GDNF, but did not affect neuroprotective activity. Our results suggest that the neurite outgrowth-promoting effect of GDNF is mediated through the PI3K and MAP kinase pathways. The neuroprotective effect of GDNF appears to be through a separate pathway.     

Excitotoxic motoneuron disease in chick embryo evolves with autophagic neurodegeneration and deregulation of neuromuscular innervation

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.     

Lineage, arrangement, and death of clonally related motoneurons in chick spinal cord

We have used recombinant retroviruses as lineage markers to study the genealogy of motoneurons (MNs) in the chick spinal cord. We infected individual progenitors by injecting virions into the neural tube at stages 11-18, a few cell divisions before MNs are born. The descendants of infected cells were subsequently detected with a histochemical stain for beta-galactosidase (lacZ), the product of the retrovirally introduced gene. Clonally related, lacZ-positive cells formed clusters that were usually radial or planar in shape. The cells that comprised these clones were classified by morphology, size, and location. About 15% of the clones in the spinal cord contained MNs, and these were studied further. Multicellular clones that contained only MNs were infrequent. Instead, close relatives of MNs included a variety of other neurons, as well as glia and ependymal cells. Most non-MNs in these clones were found in the ventral and intermediate parts of the spinal cord. Neurons included interneurons and autonomic preganglionic neurons in the column of Terni. Labeled glia were found in both the gray and white matter and included astrocytes and cells tentatively identified as oligodendrocytes. Thus, even shortly before MNs are born, their progenitors are multipotential. Clonally related MNs were not restricted to a single motor pool. Some clones contained MNs in both the medial and lateral parts of the lateral motor column, which are known to innervate distinct groups of limb muscles. Furthermore, some clones contained MNs in the medial motor column (which innervate axial muscles) as well as in the lateral motor column. In contrast, the dispersal of clonally related MNs (and other neurons) was restricted in the rostrocaudal axis; most clones were less than one-quarter segment in length. Thus, MNs derived from a single progenitor are more likely to share rostrocaudal position than synaptic targets. To investigate the fate of clonally related MNs, we counted the number of MNs per clone at times before, during, and after the major period of MN death. The number of MNs per clone declined in precise parallel with the total number of MNs during this period, suggesting that neurons are eliminated without regard to their clone of origin. This result implies that the decision to live or die occurs on a cell-by-cell rather than a clone-by-clone basis.     

Osteopontin is an alpha motor neuron marker in the mouse spinal cord

Motor neurons (MNs) are designated as alpha/gamma and fast/slow based on their target sites and the types of muscle fibers innervated; however, few molecular markers that distinguish between these subtypes are available. Here we report that osteopontin (OPN) is a selective marker of alpha MNs in the mouse spinal cord. OPN was detected in approximately 70% of postnatal choline acetyltransferase (ChAT)-positive MNs with relatively large somas, but not in those with smaller somas. OPN+/ChAT+ MNs were also positive for NeuN, an alpha MN marker, but were negative for Err3, a gamma MN marker. The size distribution of OPN+/ChAT+ cells was nearly identical to that of NeuN+/ChAT+ alpha MNs. Group Ia proprioceptive terminals immunoreactive for vesicular glutamate transporter-1 were selectively detected on the OPN+/ChAT+ cells. OPN staining was also detected at motor axon terminals at neuromuscular junctions, where the OPN+ terminals were positive or negative for SV2A, a marker distinguishing fast/slow motor endplates. Finally, retrograde labeling following intramuscular injection of fast blue indicated that OPN is expressed in both fast and slow MNs. Collectively, our findings show that OPN is an alpha MN marker present in both the soma and the endplates of alpha MNs in the postnatal mouse spinal cord.