Embryonic Spinal Motoneurons Research Articles

Transplantation of embryonic spinal motor neurons into peripheral nerves enables functional reconstruction of a denervated diaphragm

Respiratory muscle paralysis due to trauma or neurodegenerative diseases can have devastating consequences. Only a few studies have investigated the reconstruction of motor function in denervated diaphragms caused by such conditions. Here, we studied the efficacy of transplanting E14 embryonic spinal motor neurons (SMNs) into peripheral nerve grafts for functionally reconstructing a denervated diaphragm in a rat model. The diaphragms of 8-week-old male Fischer 344 rats were first denervated by transecting the phrenic nerves. Subsequently, peripheral nerve grafts taken from the lower limb were used for neurotization of the denervated diaphragms. One week later, fetal E14 SMNs were transplanted into the peripheral nerve grafts. After 3 months, we observed functional contraction of the diaphragm following neuromuscular electrical stimulation (NMES) of the peripheral nerve graft. Additionally, we confirmed that SMN transplantation into the peripheral nerve graft had an inhibitory effect on diaphragm muscle atrophy. The SMNs transplanted into the peripheral nerve grafts formed a structure similar to the spinal cord, and the neuromuscular junction of the denervated diaphragm was reinnervated. These findings suggest the establishment of an ectopic motor neuron pool in the peripheral nerve graft. Free peripheral intra-nerve SMN transplantation in combination with NMES, which can be applied for diaphragmatic pacing, offers novel insights into the development of neuroregenerative therapies for treating life-threatening and intractable respiratory muscle paralysis caused by severe nerve damage and degenerative diseases.

Excitatory action of low frequency depolarizing GABA/glycine synaptic inputs is prevalent in prenatal spinal SOD1G93A motoneurons.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onsetneurodegenerative disease characterized by progressive motor neuron degeneration and muscle paralysis. Recent evidence suggests the dysfunction of inhibitory signalling in ALS motor neurons. We have shown that embryonic day (E)17.5spinal motoneurons (MNs) of the SOD1G93A mouse model of ALS exhibit an altered chloride homeostasis. At this prenatal stage, inhibition of spinal motoneurons (MNs) is mediated by depolarizing GABAergic/glycinergic postsynaptic potentials (dGPSPs). Here, using an ex vivo preparation and patch clamp recording from MNs with a chloride equilibrium set below spike threshold, we report that low input resistance (Rin ) E17.5 MNs from the SOD1G93A ALS mouse model do not correctly integrate dGPSPs evoked by electrical stimulations of GABA/glycine inputs at different frequencies. Indeed, firing activity of most wild-type (WT) MNs with low Rin was inhibited by incoming dGPSPs, whereas low Rin SOD1G93A MNs were excited or exhibited a dual response (excited by low frequency dGPSPs and inhibited by high frequency dGPSPs). Simulation highlighted the importance of the GABA/glycine input density and showed that pure excitation could be obtained in SOD-like MNs by moving GABA/glycine input away from the cell body to dendrites. This was in agreement with confocal imaging showing a lack of peri-somatic inhibitory terminals in SOD1G93A MNs compared to WT littermates. Putative fast ALS-vulnerable MNs with low Rin are therefore lacking functional inhibition at the near-term prenatal stage. KEY POINTS: We analysed the integration of GABAergic/glycinergic synaptic events by embryonic spinal motoneurons (MNs) in a mouse model of the amyotrophic lateral sclerosis (ALS) neurodegenerative disease. We found that GABAergic/glycinergic synaptic events do not properly inhibit ALS MNs with low input resistance, most probably corresponding to future vulnerable MNs. We used a neuron model to highlight the importance of the GABA/glycine terminal location and density in the integration of the GABAergic/glycinergic synaptic events. Confocal imaging showed a lack of GABA/glycine terminals on the cell body of ALS MNs. The present study suggests that putative ALS vulnerable MNs with low Rin lack functional inhibition at the near-term stage.

CBP and p300 coactivators contribute to the maintenance of Isl1 expression by the Onecut transcription factors in embryonic spinal motor neurons

Onecut transcription factors are required to maintain Islet1 (Isl1) expression in developing spinal motor neurons (MNs), and this process is critical for proper MN differentiation. However, the mechanisms whereby OC stimulate Isl1 expression remain unknown. CREB-binding protein (CBP) and p300 paralogs are transcriptional coactivators that interact with OC proteins in hepatic cells. In the embryonic spinal cord, CBP and p300 play key roles in neurogenesis and MN differentiation. Here, using chromatin immunoprecipitation and in ovo electroporation in chicken spinal cord, we provide evidence that CBP and p300 contribute to the regulation of Isl1 expression by the OC factors in embryonic spinal MNs. CBP and p300 are detected on the CREST2 enhancer of Isl1 where OC factors are also bound. Inhibition of CBP and p300 activity inhibits activation of the CREST2 enhancer and prevents the stimulation of Isl1 expression by the OC factors. These observations suggest that CBP and p300 coactivators cooperate with OC factors to maintain Isl1 expression in postmitotic MNs.

Single-copy expression of an amyotrophic lateral sclerosis-linked TDP-43 mutation (M337V) in BAC transgenic mice leads to altered stress granule dynamics and progressive motor dysfunction

Mutations in the gene encoding the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS), clinically and pathologically indistinguishable from the majority of ‘sporadic’ cases of ALS, establishing altered TDP-43 function and distribution as a primary mechanism of neurodegeneration. Transgenic mouse models in which TDP-43 is overexpressed only partially recapitulate the key cellular pathology of human ALS, but may also lead to non-specific toxicity. To avoid the potentially confounding effects of overexpression, and to maintain regulated spatio-temporal and cell-specific expression, we generated mice in which an 80 kb genomic fragment containing the intact human TDP-43 locus (either TDP-43WT or TDP-43M337V) and its regulatory regions was integrated into the Rosa26 (Gt(ROSA26)Sor) locus in a single copy. At 3 months of age, TDP-43M337V mice are phenotypically normal but by around 6 months develop progressive motor function deficits associated with loss of neuromuscular junction integrity, leading to a reduced lifespan. RNA sequencing shows that widespread mis-splicing is absent prior to the development of a motor phenotype, though differential expression analysis reveals a distinct transcriptional profile in pre-symptomatic TDP-43M337V spinal cords. Despite the presence of clear motor abnormalities, there was no evidence of TDP-43 cytoplasmic aggregation in vivo at any timepoint. In primary embryonic spinal motor neurons and in embryonic stem cell (ESC)-derived motor neurons, mutant TDP-43 undergoes cytoplasmic mislocalisation, and is associated with altered stress granule assembly and dynamics. Overall, this mouse model provides evidence that ALS may arise through acquired TDP-43 toxicity associated with defective stress granule function. The normal phenotype until 6 months of age can facilitate the study of early pathways underlying ALS.

Smn-Deficiency Increases the Intrinsic Excitability of Motoneurons.

During development, motoneurons experience significant changes in their size and in the number and strength of connections that they receive, which requires adaptive changes in their passive and active electrical properties. Even after reaching maturity, motoneurons continue to adjust their intrinsic excitability and synaptic activity for proper functioning of the sensorimotor circuit in accordance with physiological demands. Likewise, if some elements of the circuit become dysfunctional, the system tries to compensate for the alterations to maintain appropriate function. In Spinal Muscular Atrophy (SMA), a severe motor disease, spinal motoneurons receive less excitation from glutamatergic sensory fibers and interneurons and are electrically hyperexcitable. Currently, the origin and relationship among these alterations are not completely established. In this study, we investigated whether Survival of Motor Neuron (SMN), the ubiquitous protein defective in SMA, regulates the excitability of motoneurons before and after the establishment of the synaptic contacts. To this end, we performed patch-clamp recordings in embryonic spinal motoneurons forming complex synaptic networks in primary cultures, and in differentiated NSC-34 motoneuron-like cells in the absence of synaptic contacts. Our results show that in both conditions, Smn-deficient cells displayed lower action potential threshold, greater action potential amplitudes, and larger density of voltage-dependent sodium currents than cells with normal Smn-levels. These results indicate that Smn participates in the regulation of the cell-autonomous excitability of motoneurons at an early stage of development. This finding may contribute to a better understanding of motoneuron excitability in SMA during the development of the disease.

Elevated intracellular Na+ concentrations in developing spinal neurons.

Over 25years ago it was first reported that intracellular chloride levels (Cl-in ) were higher in developing neurons than in maturity. This finding has had significant implications for understanding the excitability of developing networks and recognizing the underlying causes of hyperexcitability associated with disease and neural injury. While there is some evidence that intracellular sodium levels (Na+in ) change during the development of non-neural cells, it has largely been assumed that Na+in is the same in developing and mature neurons. Here, using the sodium indicator SBFI, we test this idea and find that Na+in is significantly higher in embryonic spinal motoneurons and interneurons than in maturity. We find that Na+in reaches ~60mM in mid-embryonic development and is then reduced to ~30mM in late embryonic development. By retrogradely labeling motoneurons with SBFI we can reliably follow Na+in levels invitro for hours. Bursts of spiking activity, and blocking voltage-gated sodium channels did not influence observed motoneuron sodium levels. On the other hand, Na+in was reduced by blocking the Na+ -K+ -2Cl- cotransporter NKCC1, and was highly sensitive to changes in external Na+ and a blocker of the Na+ /K+ ATPase. Our findings suggest that the Na+ gradient is weaker in embryonic neuronal development and strengthens in maturity in a manner similar to that of Cl- .

VEGF modulates synaptic activity in the developing spinal cord.

Although it has been documented that the nervous and the vascular systems share numerous analogies and are closely intermingled during development and pathological processes, interactions between the two systems are still poorly described. In this study, we investigated whether vascular endothelial growth factor (VEGF), which is a key regulator of vascular development, also modulates neuronal developmental processes. We report that VEGF enhances the gamma-aminobutyric acid (GABA)/glycinergic but not glutamatergic synaptic activity in embryonic spinal motoneurons (MNs), without affecting MNs excitability. In response to VEGF, the frequency of these synaptic events but not their amplitude was increased. Blocking endogenous VEGF led to an opposite effect by decreasing frequency of synaptic events. We found that this effect occurred specifically at early developmental stages (E13.5 and E15.5) and vanished at the prenatal stage E17.5. Furthermore, VEGF was able to increase vesicular inhibitory amino acid transporter density at the MN membrane. Inhibition of single VEGF receptors did not modify electrophysiological parameters indicating receptor combinations or an alternative pathway. Altogether, our findings identify VEGF as a modulator of the neuronal activity during synapse formation and highlight a new ontogenic role for this angiogenic factor in the nervous system.

Activity blockade and GABAA receptor blockade produce synaptic scaling through chloride accumulation in embryonic spinal motoneurons and interneurons.

Synaptic scaling represents a process whereby the distribution of a cell's synaptic strengths are altered by a multiplicative scaling factor. Scaling is thought to be a compensatory response that homeostatically controls spiking activity levels in the cell or network. Previously, we observed GABAergic synaptic scaling in embryonic spinal motoneurons following in vivo blockade of either spiking activity or GABAA receptors (GABAARs). We had determined that activity blockade triggered upward GABAergic scaling through chloride accumulation, thus increasing the driving force for these currents. To determine whether chloride accumulation also underlies GABAergic scaling following GABAAR blockade we have developed a new technique. We expressed a genetically encoded chloride-indicator, Clomeleon, in the embryonic chick spinal cord, which provides a non-invasive fast measure of intracellular chloride. Using this technique we now show that chloride accumulation underlies GABAergic scaling following blockade of either spiking activity or the GABAAR. The finding that GABAAR blockade and activity blockade trigger scaling via a common mechanism supports our hypothesis that activity blockade reduces GABAAR activation, which triggers synaptic scaling. In addition, Clomeleon imaging demonstrated the time course and widespread nature of GABAergic scaling through chloride accumulation, as it was also observed in spinal interneurons. This suggests that homeostatic scaling via chloride accumulation is a common feature in many neuronal classes within the embryonic spinal cord and opens the possibility that this process may occur throughout the nervous system at early stages of development.

Establishment of in vitro co-culture system for embryonic rat spinal motoneurons and C2C12 myotubes

Establishment of in vitro co-culture system for embryonic rat spinal motoneurons and C2C12 myotubes

Stages of neuronal morphological development in vitro—An automated assay

Following plating in vitro, neurons pass through a series of morphological stages as they adhere and mature. These morphological stage transitions can be monitored as a function of time to evaluate the relative health and development of neuronal cultures under different conditions. While morphological development is usually quite obvious to the experienced eye, it can often be difficult to quantify in a meaningful way. Morphology quantification typically relies on manual image measurement and can therefore be tedious, time consuming and prone to human error. Here we report the successful development of an automated process using the commercially available image analysis program MetaMorph® to analyze the morphology and quantify the growth of embryonic spinal motor neurons in vitro. Our process relied on the Neurite Outgrowth and Cell Scoring modules included in MetaMorph® and on analyzing the exported data with an algorithm written in MATLAB®. We first adopted a series of stages of motor neuron development in vitro. Neurons were classified into these stages directly from the available output of MetaMorph® using the algorithm written in MATLAB®. We validated the results of the automated analysis against a manual analysis of the same images and found no statistically significant difference between the two methods. When properly configured, automated image analysis with MetaMorph® is a rapid and reliable alternative to manual measurement and has the potential to accelerate the research process.

Expression of Sorting Nexin 18 (SNX18) Is Dynamically Regulated in Developing Spinal Motor Neurons

The sorting nexin (SNX) family proteins, which contain a Phox homology (PX) domain, play crucial roles in regulating the intracellular membrane trafficking of the endocytic pathway. The proper coordination of this pathway is important for axonal elongation; however, little is known about the expression and intracellular dynamics of the SNX members during the formation of the nervous system. Here the authors found that SNX18, which belongs to the Src-homology-3-PX-Bin/Amphiphysin/Rvs domain-containing SNX subfamily, was specifically expressed in differentiating motor neurons in the chick and mouse embryonic spinal cord. The expression of SNX18 in embryonic spinal motor neurons was transient and was downregulated as the neurons matured. The authors further demonstrated that the localization of EGFP-SNX18 in growth cones was dynamically regulated and accumulated especially at areas in contact with permissive substrates. These findings collectively suggest that SNX18 may play an active role in axonal elongation.

Müllerian inhibiting substance is anterogradely transported and does not attenuate avulsion-induced death of hypoglossal motor neurons

Müllerian Inhibiting Substance (MIS, Anti-Müllerian hormone) is a gonadal hormone that contributes to the subtle sex-biases in the nervous system. Mature neurons of both sexes also produce MIS, suggesting that MIS may be a paracrine regulator of adult neural networks. We report here that murine hypoglossal motor neurons produce MIS and its receptors, MISRII and bone morphogenetic protein receptor 1A (BMPR1A, ALK3), but differentially transport them, with only MIS being detectable in axons. The production of MIS and its receptors were rapidly down regulated after axonal damage, which is a characteristic of genes involved in mature neuronal function. MIS is a survival factor for embryonic spinal motor neurons, but the rate of cell loss after hypoglossal nerve avulsion was normal in Mis −/− mice and was not attenuated by intraventricular administration of MIS. These observations suggest that MIS may be involved in anterograde rather than autocrine or retrograde regulation of neurons.

Integration of grafted embryonic spinal cord motoneurons into the circuitry of the injured lumbar cord

Event Abstract Back to Event Integration of grafted embryonic spinal cord motoneurons into the circuitry of the injured lumbar cord Antal Nógrádi1*, Gábor Márton1 and Urszula Slawinska2 1 University of Szeged, Department of Ophthalmology, Faculty of Medicine, Hungary 2 Nencki Institute of Experimental Biology, Polish Academy of Sciences, Department of Neurophysiology, Poland Previous studies have shown that embryonic motoneurons transplanted into the host spinal cord survive, extend their axons into a peripheral nerve conduit and reinnervate the denervated muscles. In this study we investigated whether grafted motoneurons that reinnervated hindlimb muscles via a reimplanted ventral root were able to establish appropriate connections with the neurons of the host spinal cord and thus to integrate into the circuitry of the host cord. The experiments were performed on adult Sprague-Dawley rats in which the L4 ventral root was avulsed from the spinal cord and gently reinserted dorsolaterally into the host cord. In addition to this procedure embryonic spinal cord tissue derived from GFP transgenic rats was grafted into the L4 segment close to the place of the reimplanted ventral root. In control animals only the L4 ventral root was avulsed and reimplanted. After 5-6 months survival the L4 spinal nerve was labelled with retrograde tracers, the position of retrogradely labelled cells was mapped, their origin identified and connections traced. Retrogradely labelled neurons of graft origin were typically located at the host-graft interface and many of them received numerous 5-HT innervation. Serotonergic inervation may have arised both from host and grafted neurons as several (4-5) 5-HT immunoreactive neurons were found in the L4 spinal segment, located both within the graft and in the host cord. Abundant reciprocal connections were found between host and grafted neurons after microiontophoretic injections of Phaseolus vulgaris applied into the graft or into the host tissue. Our results suggest that the grafted motoneurons, which were at a very early stage of development when transplanted into the adult spinal cord, were able to integrate into the host's circuitry and receive an innervation pattern similar to that of host motoneurons. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Disorders of the nervous system Citation: Nógrádi A, Márton G and Slawinska U (2010). Integration of grafted embryonic spinal cord motoneurons into the circuitry of the injured lumbar cord. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00052 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 20 Apr 2010; Published Online: 20 Apr 2010. * Correspondence: Antal Nógrádi, University of Szeged, Department of Ophthalmology, Faculty of Medicine, Szeged, Hungary, nogradi.antal@med.u-szeged.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Antal Nógrádi Gábor Márton Urszula Slawinska Google Antal Nógrádi Gábor Márton Urszula Slawinska Google Scholar Antal Nógrádi Gábor Márton Urszula Slawinska PubMed Antal Nógrádi Gábor Márton Urszula Slawinska Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

GABA A transmission is a critical step in the process of triggering homeostatic increases in quantal amplitude

When activity levels are altered over days, a network of cells is capable of recognizing this perturbation and triggering several distinct compensatory changes that should help to recover and maintain the original activity levels homeostatically. One feature commonly observed after activity blockade has been a compensatory increase in excitatory quantal amplitude. The sensing machinery that detects altered activity levels is a central focus of the field currently, but thus far it has been elusive. The vast majority of studies that reduce network activity also reduce neurotransmission. We address the possibility that reduced neurotransmission can trigger increases in quantal amplitude. In this work, we blocked glutamatergic or GABA(A) transmission in ovo for 2 days while maintaining relatively normal network activity. We found that reducing GABA(A) transmission triggered compensatory increases in both GABA and AMPA quantal amplitude in embryonic spinal motoneurons. Glutamatergic blockade had no effect on quantal amplitude. Therefore, GABA binding to the GABA(A) receptor appears to be a critical step in the sensing machinery for homeostatic synaptic plasticity. The findings suggest that homeostatic increases in quantal amplitude may normally be triggered by reduced levels of activity, which are sensed in the developing spinal cord by GABA, via the GABA(A) receptor. Therefore, GABA appears to be serving as a proxy for activity levels.

Boundary cap cells constrain spinal motor neuron somal migration at motor exit points by a semaphorin-plexin mechanism

BackgroundIn developing neurons, somal migration and initiation of axon outgrowth often occur simultaneously and are regulated in part by similar classes of molecules. When neurons reach their final destinations, however, somal translocation and axon extension are uncoupled. Insights into the mechanisms underlying this process of disengagement came from our study of the behaviour of embryonic spinal motor neurons following ablation of boundary cap cells. These are neural crest derivatives that transiently reside at motor exit points, central nervous system (CNS):peripheral nervous system (PNS) interfaces where motor axons leave the CNS. In the absence of boundary cap cells, motor neuron cell bodies migrate along their axons into the periphery, suggesting that repellent signals from boundary cap cells regulate the selective gating of somal migration and axon outgrowth at the motor exit point. Here we used RNA interference in the chick embryo together with analysis of null mutant mice to identify possible boundary cap cell ligands, their receptors on motor neurons and cytoplasmic signalling molecules that control this process.ResultsWe demonstrate that targeted knock down in motor neurons of Neuropilin-2 (Npn-2), a high affinity receptor for class 3 semaphorins, causes their somata to migrate to ectopic positions in ventral nerve roots. This finding was corroborated in Npn-2 null mice, in which we identified motor neuron cell bodies in ectopic positions in the PNS. Our RNA interference studies further revealed a role for Plexin-A2, but not Plexin-A1 or Plexin-A4. We show that chick and mouse boundary cap cells express Sema3B and 3G, secreted semaphorins, and Sema6A, a transmembrane semaphorin. However, no increased numbers of ectopic motor neurons are found in Sema3B null mouse embryos. In contrast, Sema6A null mice display an ectopic motor neuron phenotype. Finally, knockdown of MICAL3, a downstream semaphorin/Plexin-A signalling molecule, in chick motor neurons led to their ectopic positioning in the PNS.ConclusionWe conclude that semaphorin-mediated repellent interactions between boundary cap cells and immature spinal motor neurons regulates somal positioning by countering the drag exerted on motor neuron cell bodies by their axons as they emerge from the CNS at motor exit points. Our data support a model in which BC cell semaphorins signal through Npn-2 and/or Plexin-A2 receptors on motor neurons via a cytoplasmic effector, MICAL3, to trigger cytoskeletal reorganisation. This leads to the disengagement of somal migration from axon extension and the confinement of motor neuron cell bodies to the spinal cord.

Increasing the Frequency of Spontaneous Rhythmic Activity Disrupts Pool-Specific Axon Fasciculation and Pathfinding of Embryonic Spinal Motoneurons

Rhythmic spontaneous bursting activity, which occurs in many developing neural circuits, has been considered to be important for the refinement of neural projections but not for early pathfinding decisions. However, the precise frequency of bursting activity differentially affects the two major pathfinding decisions made by chick lumbosacral motoneurons. Moderate slowing of burst frequency was shown previously to cause motoneurons to make dorsoventral (D-V) pathfinding errors and to alter the expression of molecules involved in that decision. Moderate speeding up of activity is shown here not to affect these molecules or D-V pathfinding but to strongly perturb the anteroposterior (A-P) pathfinding process by which motoneurons fasciculate into pool-specific fascicles at the limb base and then selectively grow to muscle targets. Resumption of normal frequency allowed axons to correct the A-P pathfinding errors by altering their trajectories distally, indicating the dynamic nature of this process and its continued sensitivity to patterned activity.

Release of glutamate by the embryonic spinal motoneurons of rat positively regulated by acetylcholine through the nicotinic and muscarinic receptors

It has been shown that mature neurons in adult vertebrates can co-express glutamate and acetylcholine. Furthermore, interactions at the synaptic level have been demonstrated. In a previous study we found that also motoneurons at early embryonic stages, thus well prior to synapse formation, release acetylcholine, and that glutamate increases this release. We now report the existence of a glutamate release from embryonic motoneurons and the increase of glutamate release by acetylcholine. This effect is mediated by nicotinic and muscarinic cholinergic receptors present on embryonic motoneurons. Using conditions of partial or total depletion of calcium, we show that the glutamate release has two components: one is calcium-dependent and the other calcium-independent. Furthermore, we show that extracellular glutamate can be taken up by motoneurons, probably via the neuronal glutamate transporter EAAC1, which we find to be expressed at this stage. Monitoring of the glutamate release kinetics showed that extracellular glutamate concentration reached a steady-state level, strongly suggesting the establishment of equilibrium between glutamate release and uptake. Altogether, these results support the idea that glutamate can act as a neurotransmitter in embryonic motoneurons. We hypothesise that, glutamate acts as a regulator of motoneuron maturation and spinal cord development.

The LRR and Ig domain-containing membrane protein SST273 is expressed on motoneurons

We performed signal sequence trap cDNA cloning using a cDNA library of enriched chicken embryonic spinal motoneurons and identified a novel transmembrane molecule, SST273 ( Signal Sequence Trap clone 273). SST273 has five leucine-rich repeats (LRRs) and one immunoglobulin (Ig) domain in its extracellular domain. The human (KIAA1465) and mouse (BC059068) homologues of SST273 were already cloned, but had not yet been analyzed. The amino acid homologies between chick SST273 and human or mouse homologue are 52.1 and 51.5%, respectively. SST273 mRNA and its protein product were uniquely expressed in the embryonic spinal and cranial motoneurons at early developmental stages.

Foxo3a induces motoneuron death through the Fas pathway in cooperation with JNK

BackgroundProgrammed cell death of motoneurons in the developing spinal cord is thought to be regulated through the availability of target-derived neurotrophic factors. When deprived of trophic support, embryonic spinal motoneurons in vitro over-express FasL, a ligand activating a Fas-mediated death pathway. How trophic factors regulate the expression of FasL is presently unclear, but two regulators of FasL, FOXO3a (FKHRL1) and JNK have been described to play a role in other cell types. Thus, their potential function in motoneurons was investigated in this study.ResultsWe show here that as a result of removal of neurotrophic factors and the consequent reduction in signalling through the PI3K/Akt pathway, Foxo3a translocates from the cytoplasm to the nucleus where it triggers cell death. Death is reduced in Fas and FasL mutant motoneurons and in the presence of JNK inhibitors indicating that a significant part of it requires activation of the Fas/FasL pathway through JNK.ConclusionsTherefore, in motoneurons as in other cell types, FOXO transcriptional regulators provide an important link between other signalling pathways and the cell death machinery.

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.