Electrophysiological Properties of Motor Neurons in a Mouse Model of Severe Spinal Muscular Atrophy: In Vitro versus In Vivo Development

We examined the electrophysiological activity of motor neurons from the mouse model of severe spinal muscular atrophy (SMA) using two different methods: whole cell patch clamp of neurons cultured from day 13 embryos; and multi-electrode recording of ventral horns in spinal cord slices from pups on post-natal days 5 and 6. We used the MED64 multi-electrode array to record electrophysiological activity from motor neurons in slices from the lumbar spinal cord of SMA pups and their unaffected littermates. Recording simultaneously from up to 32 sites across the ventral horn, we observed a significant decrease in the number of active neurons in 5–6 day-old SMA pups compared to littermates. Ventral horn activity in control pups is significantly activated by serotonin and depressed by GABA, while these agents had much less effect on SMA slices. In contrast to the large differences observed in spinal cord, neurons cultured from SMA embryos for up to 21 days showed no significant differences in electrophysiological activity compared to littermates. No differences were observed in membrane potential, frequency of spiking and synaptic activity in cells from SMA embryos compared to controls. In addition, we observed no difference in cell survival between cells from SMA embryos and their unaffected littermates. Our results represent the first report on the electrophysiology of SMN-deficient motor neurons, and suggest that motor neuron development in vitro follows a different path than in vivo development, a path in which loss of SMN expression has little effect on motor neuron function and survival.     

Astrocytic production of nerve growth factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis

Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75(NTR)) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75(NTR) was inhibited with blocking antibodies. In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75(NTR +/+) mouse embryos but had no effect in p75(NTR -/-) knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75(NTR) blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.     

Sonic hedgehog signaling is required during the appearance of spinal cord oligodendrocyte precursors

Spinal cord oligodendrocyte precursors arise in the ventral ventricular zone as a result of local signals. Ectopic oligodendrocyte precursors can be induced by sonic hedgehog (Shh) in explants of chick dorsal spinal cord over an extended developmental period. The role of Shh during normal oligodendrocyte development is, however, unclear. Here we demonstrate that Shh is localized to the ventral spinal cord immediately prior to, and during the appearance of oligodendrocyte precursors. Continued expression of Shh is required for the appearance of spinal cord oligodendrocyte precursors as neutralization of Shh signaling both in vivo and in vitro during a defined developmental period blocked their emergence. The inhibition of oligodendrocyte precursor emergence in the absence of Shh signaling was not the result of inhibiting precursor cell proliferation, and the neutralization of Shh signaling after the emergence of oligodendrocyte precursors had no effect on the appearance of additional cells or their subsequent differentiation. Similar concentrations of Shh induce motor neurons and oligodendrocytes in dorsal spinal cord explants. However, in explants from early embryos the motor neuron lineage is preferentially expanded while in explants from older embryos the oligodendrocyte lineage is preferentially expanded.     

Mesodermal and neuronal retinoids regulate the induction and maintenance of limb innervating spinal motor neurons

During embryonic development, the generation, diversification and maintenance of spinal motor neurons depend upon extrinsic signals that are tightly regulated. Retinoic acid (RA) is necessary for specifying the fates of forelimb-innervating motor neurons of the Lateral Motor Column (LMC), and the specification of LMC neurons into medial and lateral subtypes. Previous studies implicate motor neurons as the relevant source of RA for specifying lateral LMC fates at forelimb levels. However, at the time of LMC diversification, a significant amount of retinoids in the spinal cord originates from the adjacent paraxial mesoderm. Here we employ mouse genetics to show that RA derived from the paraxial mesoderm is required for lateral LMC induction at forelimb and hindlimb levels, demonstrating that mesodermally synthesized RA functions as a second source of signals to specify lateral LMC identity. Furthermore, reduced RA levels in postmitotic motor neurons result in a decrease of medial and lateral LMC neurons, and abnormal axonal projections in the limb; invoking additional roles for neuronally synthesized RA in motor neuron maintenance and survival. These findings suggest that during embryogenesis, mesodermal and neuronal retinoids act coordinately to establish and maintain appropriate cohorts of spinal motor neurons that innervate target muscles in the limb.     

Integrity of developing spinal motor columns is regulated by neural crest derivatives at motor exit points

Spinal motor neurons must extend their axons into the periphery through motor exit points (MEPs), but their cell bodies remain within spinal motor columns. It is not known how this partitioning is established in development. We show here that motor neuron somata are confined to the CNS by interactions with a neural crest subpopulation, boundary cap (BC) cells that prefigure the sites of spinal MEPs. Elimination of BC cells by surgical or targeted genetic ablation does not perturb motor axon outgrowth but results in motor neuron somata migrating out of the spinal cord by translocating along their axons. Heterologous neural crest grafts in crest-ablated embryos stop motor neuron emigration. Thus, before the formation of a mature transitional zone at the MEP, BC cells maintain a cell-tight boundary that allows motor axons to cross but blocks neuron migration.     

The neuroanatomy of an amphibian embryo spinal cord

Horseradish peroxidase has been used to stain spinal cord neurons in late embryos of the clawed toad (Xenopus laevis). It has shown clearly the soma, dendrites and axonal projections of spinal sensory, motor and interneurons. On the basis of light microscopy we describe nine differentiated spinal cord neuron classes. These include the Rohon-Beard cells and extramedullary cells which are both primary sensory neurons, one class of motoneurons that innervate the segmental myotomes, two classes of interneurons with decussating axons, three classes of interneurons with ipsilateral axons and a previously undescribed class of ciliated ependymal cells with axons projecting ipsilaterally to the brain. We believe that all differentiated neuron classes are described and that this anatomical account is the most complete for any vertebrate spinal cord.     

BDNF heightens the sensitivity of motor neurons to excitotoxic insults through activation of TrkB

The survival promoting and neuroprotective actions of brain-derived neurotrophic factor (BDNF) are well known but under certain circumstances this growth factor can also exacerbate excitotoxic insults to neurons. Prior exploration of the receptor through which BDNF exerts this action on motor neurons deflects attention away from p75. Here we investigated the possibility that BDNF acts through the receptor tyrosine kinase, TrkB, to confer on motor neurons sensitivity to excitotoxic challenge. We blocked BDNF activation of TrkB using a dominant negative TrkB mutant or a TrkB function blocking antibody, and found that this protected motor neurons against excitotoxic insult in cultures of mixed spinal cord neurons. Addition of a function blocking antibody to BDNF to mixed spinal cord neuron cultures is also neuroprotective indicating that endogenously produced BDNF participates in vulnerability to excitotoxicity. We next examined the intracellular signaling cascades that are engaged upon TrkB activation. Previously we found that inhibition of the phosphatidylinositide-3'-kinase (PI3'K) pathway blocks BDNF-induced excitotoxic sensitivity. Here we show that expression of a constitutively active catalytic subunit of PI3'K, p110, confers excitotoxic sensitivity (ES) upon motor neurons not incubated with BDNF. Parallel studies with purified motor neurons confirm that these events are likely to be occuring specifically within motor neurons. The abrogation of BDNF's capacity to accentuate excitotoxic insults may make it a more attractive neuroprotective agent.     

GDP-bound Gαi2 regulates spinal motor neuron differentiation through interaction with GDE2

Gαi proteins play major roles in the developing and mature nervous system, ranging from the control of cellular proliferation to modulating synaptic plasticity. Although best known for transducing signals from activated seven transmembrane G-protein coupled receptors (GPCRs) when bound to GTP, key cellular functions for Gαi-GDP are beginning to emerge. Here, we show that Gαi2 is expressed in motor neuron progenitors that are differentiating to form postmitotic motor neurons in the developing spinal cord. Ablation of Gαi2 causes deficits in motor neuron generation but no changes in motor neuron progenitor patterning or specification, consistent with a function for Gαi2 in regulating motor neuron differentiation. We show that Gαi2 function is mediated in part by its interaction with GDE2, a known regulator of motor neuron differentiation, and that disruption of the GDE2/Gαi2 complex in vivo causes motor neuron deficits analogous to Gαi2 ablation. Gαi2 preferentially associates with GDE2 when bound to GDP, invoking GPCR-independent functions for Gαi2 in the control of spinal motor neuron differentiation.     

A postmitotic role for Isl-class LIM homeodomain proteins in the assignment of visceral spinal motor neuron identity

LIM homeobox genes have a prominent role in the regulation of neuronal subtype identity and distinguish motor neuron subclasses in the embryonic spinal cord. We have investigated the role of Isl-class LIM homeodomain proteins in motor neuron diversification using mouse genetic methods. All spinal motor neuron subtypes initially express both Isl1 and Isl2, but Isl2 is rapidly downregulated by visceral motor neurons. Mouse embryos lacking Isl2 function exhibit defects in the migration and axonal projections of thoracic level motor neurons that appear to reflect a cell-autonomous switch from visceral to somatic motor neuron character. Additional genetic mutations that reduce or eliminate both Isl1 and Isl2 activity result in more pronounced defects in visceral motor neuron generation and erode somatic motor neuron character. Thus, an early phase of high Isl expression and activity in newly generated motor neurons permits the diversification of visceral and somatic motor neuron subtypes in the developing spinal cord.     

S100 is present in developing chicken neurons and Schwann cells and promotes motor neuron survival in vivo.

We used polyclonal antisera recognizing S100, a small acidic protein highly enriched in nervous tissue, to stain sections of embryonic chicken lumbosacral spinal cord and hindlimb. S100 immunoreactivity was detected in developing sensory neurons of the dorsal root ganglia (DRG) and motor neurons of the ventral spinal cord as early as embryonic day (E) 5, and staining persisted through hatching. In contrast, expression of S100 first became apparent in Schwann cells at E13, just before myelination, and was not detected in developing skin or muscle. Since S100 beta was present in motor and sensory neurons and is known to promote neuronal survival and neurite extension in vitro (Winningham-Major, Staecker, Barger, Coats, and Van Eldik, 1989), we tested the ability of S100 to promote neuron survival in an in ovo survival assay. Addition of S100 to chick embryos in ovo during the period of naturally occurring motor neuron cell death resulted in a significant increase in motor neuron survival, but had no effect on the in vivo survival of sensory neurons in the DRG. The findings that S100 is present in spinal motor neurons and that the addition of S100 enhances the survival of these cells in vivo are consistent with the possibility that S100 may act as a naturally occurring neuron survival factor during development.     

Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis.

The mechanisms for motor neuron degeneration and regeneration in adult spinal cord following axotomy and target deprivation are not fully understood. We used a unilateral sciatic nerve avulsion model in adult rats to test the hypothesis that retrograde degeneration of motor neurons resembles apoptosis. By 21 days postlesion, the number of large motor neurons in lumbar spinal cord was reduced by approximately 30%. The death of motor neurons was confirmed using the terminal transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling method for detecting fragmentation of nuclear DNA. Motor neuron degeneration was characterized by aberrant accumulation of perikaryal phosphorylated neurofilaments. Structurally, motor neuron death was apoptosis. Apoptotic motor neurons undergo chromatolysis followed by progressive cytoplasmic and nuclear condensation with chromatin compaction into uniformly large round clumps. Prior to apoptosis, functionally active mitochondria accumulate within chromatolytic motor neurons, as determined by cytochrome c oxidase activity. These dying motor neurons sustain oxidative damage to proteins and nucleic acids within the first 7 days after injury during the progression of apoptosis, as identified by immunodetection of nitrotyrosine and hydroxyl-modified deoxyguanosine and guanosine. We conclude that the retrograde death of motor neurons in the adult spinal cord after sciatic nerve avulsion is apoptosis. Accumulation of active mitochondria within the perikaryon and oxidative damage to nucleic acids and proteins may contribute to the mechanisms for apoptosis of motor neurons in the adult spinal cord.