Embryonic Spinal Motoneurons Research Articles

Whole-cell patch-clamp recordings from identified spinal neurons in the zebrafish embryo.

We describe a preparation for obtaining patch-clamp recordings from identified embryonic spinal cord interneurons, motoneurons and sensory neurons in an in vivo zebrafish preparation. This preparation is used to study the spatial and temporal patterns of spontaneous and touch-evoked electrical activity during the initial development of circuitry in the spinal cord. The combination of these physiological techniques with the powerful genetic and molecular tools available in the zebrafish has the potential to increase our understanding of the complex interactions between genes and electrical activity during the development of the vertebrate nervous system.

Rabies virus is not cytolytic for rat spinal motoneurons in vitro.

Cultures of purified rat embryonic spinal cord motoneurons were used to investigate the capacity of the neurons to survive rabies virus infection in vitro. In crude primary spinal cord cultures, neurons did not survive more than 2 days after rabies virus infection with the fixed strain Challenge Virus Standard. In contrast, virus-infected purified motoneurons resisted cytolysis for at least 7 days, as also did infected motoneurons treated with conditioned medium sampled from rabies virus-infected crude spinal cord cultures. This survival rate was also observed when motoneurons were grown in the presence of astrocytes or fibroblasts and it was not dependent on the presence of growth factors in the culture medium. Moreover, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling experiments showed that only 30% of infected motoneurons were apoptotic after 7 days of infection. In vivo, despite the massive infection of the spinal cord in infected rat neonates, the moderate number of apoptotic cells in the ventral horn suggests that only a few motoneurons were affected by this mechanism of cell death. Morphometric analyses showed that motoneurons' axon elongated at a comparable rate in virus-infected and noninfected cultures, a sign of high metabolic activity maintained in rabies virus-infected motoneurons. In contrast, hippocampus neurons were susceptible to rabies virus infection, because 70% of infected neurons were destroyed within 3 days, a large proportion of them being apoptotic. These experiments suggest that spinal cord motoneurons consist in a neuronal population that survive rabies virus infection because the viral induction of apoptosis is delayed in these neurons. They suggest also that paralyses frequently observed in rabid animals could be the consequence of dysfunctions of the locomotor network or of the spinal cord motoneurons themselves, whose parameters could be studied in vitro.

Acetylcholine secretion enhanced by glutamate in rat embryonic spinal motoneurons: respective involvement of NMDA and AMPA receptors.

The spontaneous acetylcholine secretion and endogenous acetylcholine content were measured by means of chemiluminescent assay from isolated embryonic rat spinal motoneurons. The sensitivity of the detection allows to study the kinetics of the acetylcholine secretion with short time intervals. Following the demonstration of the presence of acetylcholine and glutamate in embryonic motoneurons, the aim of this work was to study the characteristics of acetylcholine secretion and the effect of glutamate in its modulation. The involvement of NMDA and AMPA glutamatergic receptors was mainly studied. Our data show that spontaneously acetylcholine secretion, is not calcium-dependent and is significantly enhanced by glutamate (1 mM). Pharmacological approaches show that glutamate effect on acetylcholine secretion is decreased in presence of APV (50 microM and 100 microM), or in presence of GYKI 53655 (10 microM), demonstrating that both NMDA and AMPA receptors are present at the membrane of embryonic spinal motoneurons and involved in the modulation of acetylcholine secretion. Presence of glutamate in the embryonic motoneuron and secretion may represent a mechanism of control of extracellular acetylcholine concentration, which was shown to control neuritic growth at early embryonic stage.

Androgens rescue avian embryonic lumbar spinal motoneurons from injury-induced but not naturally occurring cell death

The regulation of survival of spinal motoneurons (MNs) has been shown to depend during development and after injury on a variety of neurotrophic molecules produced by skeletal muscle target tissue. Increasing evidence also suggests that other sources of trophic support prevent MNs from undergoing naturally occurring or injury-induced death. We have examined the role of endogenous and exogenous androgens on the survival of developing avian lumbar spinal MNs during their period of programmed cell death (PCD) between embryonic day (E)6 and E11 or after axotomy on E12. We found that although treatment with testosterone, dihydrotestosterone (DHT), or the androgen receptor antagonist flutamide (FL) failed to affect the number of these MNs during PCD, administration of DHT from E12 to E15 following axotomy on E12 significantly attenuated injury-induced MN death. This effect was inhibited by cotreatment with FL, whereas treatment with FL alone did not affect MN survival. Finally, we examined the spinal cord at various times during development and following axotomy on E12 for the expression of androgen receptor using the polyclonal PG-21 antibody. Our results suggest that exogenously applied androgens are capable of rescuing MNs from injury-induced cell death and that they act directly on these cells via an androgen receptor-mediated mechanism. By contrast, endogenous androgens do not appear to be involved in the regulation of normal PCD of developing avian MNs.

The Spatial–Temporal Gradient of Naturally Occurring Motoneuron Death Reflects the Time of Prior Exit from the Cell Cycle and Position within the Lateral Motor Column

Embryonic lumbar spinal motoneurons (MNs) are characterized by a period of programmed cell death (PCD) that spans several days and occurs in a rostrocaudal gradient. The generation of these MNs also takes place in a temporal–spatial gradient, such that MNs within rostral lumbar segments exit the cell cycle earlier and MNs within progressively caudal regions are born later. In vitro studies have shown that the latest born spinal MNs, presumably through the possession of endogenous “survival properties,” are also the last to acquire their trophic dependence. If the birth date and therefore spinal cord location of lumbar spinal MNs influence the spatial–temporal pattern of PCD, then earlier born MNs should die sooner and be located more rostrally than those generated later. Alternatively, if the time at which MNs die during development is unrelated to their prior exit from the cell cycle, those born at various phases should die throughout the period of PCD. We report here that lumbar MNs generated during the earliest part (embryonic day 2–3) of the proliferative period in the developing chick spinal cord tend to die during the earliest stages of the PCD period and that MNs born in successive 12-h intervals die at correspondingly later periods during PCD. Furthermore, the spatial progression of PCD of these subpopulations of MNs occurs in a rostrocaudal gradient. Finally, while MNs do appear to die in a mediolateral gradient during the period of MN PCD, this pattern is only partly accounted for by MNs born in consecutive intervals. These data support the notion that the timing and rostrocaudal location of MNs undergoing PCD reflect their time of exit from the cell cycle.

Androgens rescue avian embryonic lumbar spinal motoneurons from injury-induced but not naturally occurring cell death.

The regulation of survival of spinal motoneurons (MNs) has been shown to depend during development and after injury on a variety of neurotrophic molecules produced by skeletal muscle target tissue. Increasing evidence also suggests that other sources of trophic support prevent MNs from undergoing naturally occurring or injury-induced death. We have examined the role of endogenous and exogenous androgens on the survival of developing avian lumbar spinal MNs during their period of programmed cell death (PCD) between embryonic day (E)6 and E11 or after axotomy on E12. We found that although treatment with testosterone, dihydrotestosterone (DHT), or the androgen receptor antagonist flutamide (FL) failed to affect the number of these MNs during PCD, administration of DHT from E12 to E15 following axotomy on E12 significantly attenuated injury-induced MN death. This effect was inhibited by cotreatment with FL, whereas treatment with FL alone did not affect MN survival. Finally, we examined the spinal cord at various times during development and following axotomy on E12 for the expression of androgen receptor using the polyclonal PG-21 antibody. Our results suggest that exogenously applied androgens are capable of rescuing MNs from injury-induced cell death and that they act directly on these cells via an androgen receptor-mediated mechanism. By contrast, endogenous androgens do not appear to be involved in the regulation of normal PCD of developing avian MNs.

Anomalous L-type calcium channels of rat spinal motoneurons.

Single channel patch-clamp recordings show that embryonic rat spinal motoneurons express anomalous L-type calcium channels, which reopen upon repolarization to resting potentials, displaying both short and long reopenings. The probability of reopening increases with increasing voltage of the preceding depolarization without any apparent correlation with inactivation during the depolarization. The probability of long with respect to short reopenings increases with increasing length of the depolarization, with little change in the total number of reopenings and in their delay. With less negative repolarization voltages, the delay increases, while the mean duration of both short and long reopenings decreases, remaining longer than that of the openings during the preceding depolarization. Open times decrease with increasing voltage in the range -60 to +40 mV. Closed times tend to increase at V > 20 mV. The open probability is low at all voltages and has an anomalous bell-shaped voltage dependence. We provide evidence that short and long reopenings of anomalous L-type channels correspond to two gating modes, whose relative probability depends on voltage. Positive voltages favor both the transition from a short-opening to a long-opening mode and the occupancy of a closed state outside the activation pathway within each mode from which the channel reopens upon repolarization. The voltage dependence of the probability of reopenings reflects the voltage dependence of the occupancy of these closed states, while the relative probability of long with respect to short reopenings reflects the voltage dependence of the equilibrium between modes. The anomalous gating persists after patch excision, and therefore our data rule out voltage-dependent block by diffusible ions as the basis for the anomalous gating and imply that a diffusible cytosolic factor is not necessary for voltage-dependent potentiation of anomalous L-type channels.

Cyclic AMP Elevation Is Sufficient to Promote the Survival of Spinal Motor NeuronsIn Vitro

The short-term survival of highly purified embryonic spinal motor neurons (SMNs) in culture can be promoted by many peptide trophic factors, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF), glial-derived neurotrophic factor (GDNF), and hepatocyte growth factor (HGF). We have asked whether these peptides are sufficient to promote the long-term survival of purified E15 SMNs. Contrary to previous reports, we find that when SMNs are cultured in serum-free medium containing a single peptide trophic factor only approximately one-third of the cells survive for 3 d in culture. When multiple factors are combined, additive effects on survival are observed transiently, but by 7 d of culture the majority of SMNs has died. Surprisingly, when cAMP levels are elevated, the majority of SMNs extend processes and survive for 1 week in culture in the absence of peptide trophic factors, even in low-density cultures. A combination of five peptide trophic factors, together with cAMP elevation, promotes the long-term survival of most of the SMNs in serum-free culture for 3 weeks. These findings provide useful culture conditions for studying the properties of SMNs and have implications for the treatment of motor neuron diseases.

Thrombin Perturbs Neurite Outgrowth and Induces Apoptotic Cell Death in Enriched Chick Spinal Motoneuron Cultures through Caspase Activation

Increasing evidence indicates several roles for thrombin-like serine proteases and their cognate inhibitors (serpins) in normal development and/or pathology of the nervous system. In addition to its prominent role in thrombosis and/or hemostasis, thrombin inhibits neurite outgrowth in neuroblastoma and primary neuronal cells in vitro, prevents stellation of glial cells, and induces cell death in glial and neuronal cell cultures. Thrombin is known to act via a cell surface protease-activated receptor (PAR-1), and recent evidence suggests that rodent neurons express PAR-1. Previously, we have shown that the thrombin inhibitor, protease nexin-1, significantly prevents neuronal cell death both in vitro and in vivo. Here we have examined the effects of human alpha-thrombin and the presence and/or activation of PAR-1 on the survival and differentiation of highly enriched cultures of embryonic chick spinal motoneurons. We show that thrombin significantly decreased the mean neurite length, prevented neurite branching, and induced motoneuron death by an apoptosis-like mechanism in a dose-dependent manner. These effects were prevented by cotreatment with hirudin, a specific thrombin inhibitor. Treatment of the cultures with a synthetic thrombin receptor-activating peptide (SFLLRNP) mimicked the deleterious effects of thrombin on motoneurons. Furthermore, cotreatment of the cultures with inhibitors of caspase activities completely prevented the death of motoneurons induced by either thrombin or SFLLRNP. These findings indicate that (1) embryonic avian spinal motoneurons express functional PAR-1 and (2) activation of this receptor induces neuronal cell degeneration and death via stimulation of caspases. Together with previous reports, our results suggest that thrombin, its receptor(s), and endogenous thrombin inhibitors may be important regulators of neuronal cell fate during development, after injury, and in pathology of the nervous system.

Muscle-Specific Cell Ablation Conditional upon Cre-Mediated DNA Recombination in Transgenic Mice Leads to Massive Spinal and Cranial Motoneuron Loss

We describe here a binary transgenic system based on Cre-mediated DNA recombination for genetic cell ablation in mice that enabled us to obtain skeletal muscle-deficient embryos by mating two phenotypically normal transgenic lines. In those embryos, skeletal muscles are eliminated as a consequence of the expression of the gene encoding the diphtheria toxin A fragment. Cell ablation occurs gradually beginning approximately on embryonic day (E) 12.5, and by E18.5 almost all skeletal muscle is absent. Analysis of the consequences of muscle cell ablation revealed that almost all spinal motoneurons are lost by E18.5, providing strong evidence that survival of spinal motoneurons during embryogenesis is dependent on signals from their target tissue, skeletal muscle, and that trophic signals produced by nonmuscle sources are sufficient to support survival of no more than 10% of embryonic spinal motoneurons in the absence of muscle-derived signals. There was also substantial loss of cranial (hypoglossal and facial) motoneurons in the muscle-deficient embryos, thus indicating that cranial motoneuron survival is also dependent on trophic signals produced by their target tissue. Although spinal motoneurons are a major target of spinal interneurons, the loss of motoneurons did not affect interneuron survival. Muscle-deficient embryos had a cleft palate and abnormalities of the lower jaw, raising the possibility that they might serve as a mouse model for the human disorder, Robin sequence. The data reported here demonstrate the utility of a binary transgenic system for obtaining mouse embryos in which a specific cell population has been ablated, so that its role in embryonic development can be studied.

Expression of MHC Class I and β2-Microglobulin in Rat Spinal Motoneurons: Regulatory Influences by IFN-Gamma and Axotomy

The low expression of MHC antigens is believed to be one factor of importance contributing to the immune-privileged status of CNS neurons. We here describe that motoneurons, in contrast to other nerve cells in the lumbar spinal cord of the adult rat, express both MHC class I and β2-microglobulin mRNA. The motoneurons also displayin situhybridization signal for IFN-gamma receptor mRNA. After a peripheral axotomy, the motoneurons show a clear upregulation of β2-microglobulin mRNA. IFN-gamma treatment of cultured rat embryonic spinal motoneurons causes a similar upregulation of especially β2-microglobulin. Based on these facts, we propose that spinal motoneurons can be influenced by IFN-gamma and recognized by cytotoxic CD8+ T-cells. These findings could be of relevance in the search for pathogenetic mechanisms in motoneuron-specific diseases, such as ALS.

Embryonic rat motoneurons express a functional P-type voltage-dependent calcium channel

Only L- and N-type high voltage-activated calcium currents (HVA ICa) have been demonstrated in identified embryonic spinal motoneurons. However, pharmacological experiments suggest that other HVA ICa, including P-type, govern neurotransmitter release at the adult neuromuscular junction. We sought to analyse if embryonic motoneurons express these other ICa, using the whole-cell voltage-clamp method on motoneurons purified by a new metrizamide-panning technique from E15 rat embryos. In addition to L-type dihydropyridine-sensitive and N-type ω-GVIA-sensitive currents, motoneurons express two other HVA ICa. One has properties related to the P-type channel currents described in Purkinje cells: it is inhibited by the peptide ω-agatoxin-IVA with a maximal effect at 100–200 nM. The inhibited current has a characteristic sustained component during depolarizing test pulses. Furthermore, 50–100 nM concentration of ω-agatoxin-IVA reduce the increase in cytoplasmic calcium concentration observed after depolarization. The other HVA ICa is resistant to saturating concentrations of verapamil, ω-conotoxin GVIA and ω-agatoxin-IVA which block L, N and P-type HVAICa, respectively. These results suggest that it is now possible to dissect, using a simple method of purification, the properties of the ICa in embryonic mammalian motoneurons and to provide pharmacological evidence for multiple calcium channels which may be involved in regulation of their activity during development.

Viral Transduction of trkA into Cultured Nodose and Spinal Motor Neurons Conveys NGF Responsiveness

The trkA gene encodes the tyrosine kinase receptor that transduces the NGF signal. We have used a defective herpes simplex virus-1 (HSV-1) vector containing a full-length rat trkA sequence (pHSVtrkA) to express NGF receptors in primary cultures of neonatal nodose neurons and embryonic spinal motor neurons which are normally unresponsive to NGF. Transduction of rat neonatal nodose ganglion neurons and spinal motor neurons with pHSVtrkA resulted in expression of functional NGF receptors. Stimulation of these receptors by NGF resulted in the survival and the morphologic and biochemical differentiation of these neurons. These observations indicate that nodose and spinal motor neurons can express the downstream signal transduction apparatus necessary to mediate the survival and differentiating effects mediated by ligand binding to trkA and that transduction with pHSVtrkA can effectively convert NGF nonresponsive neurons into responsive ones.

Reinnervation of Adult Rat Gastrocnemius Muscle by Embryonic Motoneurons Transplanted into the Axotomized Tibial Nerve

In some cases of spinal cord injury and in certain motoneuron diseases, such as amyotrophic lateral sclerosis and spinal muscular atrophies, lower motoneurons are destroyed and muscle function cannot be restored except by reinnervation from alternate motoneuron sources. We have tested the feasibility of employing local transplantation of embryonic motoneurons to restore innervation to denervated somatic muscle as a first step in salvaging muscle function and enabling use of functional electric stimulation. Dissociated ventral spinal cord cells from Embryonic Days 14 and 15 rats were transplanted into the distal stump of axotomized tibial nerves of adult rats. Animals were killed 3-18 weeks after transplantation. After 3 weeks large multipolar cells, resembling alpha motoneurons, were observed within the transplant site surrounded by myelinated and unmyelinated axons and dendrites. Axons emanating from these transplanted motoneurons were identified within the nerve stump and within the previously denervated gastrocnemius muscle, forming neuromuscular junctions. Transplanted motoneurons survived up to 18 weeks and were labeled after intramuscular injection of fast blue. This study demonstrates that embryonic spinal motoneurons, transplanted into the distal adult peripheral nerve stump, are able to survive and reinnervate the denervated target muscle. We are now exploring the possibility of using this experimental approach to retard the atrophy of denervated skeletal muscle, thus providing a muscle capable of useful response to functional electrical stimulation.

Neurotrophins promote motor neuron survival and are present in embryonic limb bud.

Embryonic spinal motor neurons are thought to depend for survival on unidentified factors secreted both by their peripheral targets and by cells within the central nervous system. The neurotrophins are a family of polypeptides required for survival of discrete central and peripheral neuronal populations in vivo and in vitro. In spite of their ability to reduce motor neuron death in vivo, the known neurotrophins have been thought to be without direct effect on motor neurons. Here we show that picomolar concentrations of three of them, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-5, can prevent the death of cultured embryonic rat spinal motor neurons. Furthermore, messenger RNA coding for neurotrophins is present at appropriate stages in spinal cord and limb bud, and mRNA for their receptors is found in motor neurons. These neurotrophins may therefore be physiological motor neuron growth factors.

Cell surface distribution of endogenous and effects of exogenous gangliosides on neuronal survival, cell shape and growth In vitro

In vitro immunostaining of neurons from spinal cord or brain of embryonic chicken by means of monoclonal anti-ganglioside antibodies (Q211, D21b) revealed a fluorescence-labeling of c-polysialogangliosides and GD1b evenly distributed over the entire neuronal surface including filopodia at the growth cones. On electronmicroscopical level the gold-stained ganglioside-antigens were found more or less densely packed in small adjacent areas suggesting a concentration in local “domains”. Survival in serum-free or serum-containing medium of embryonic spinal cord motoneurons, which normally died if not cultivated in muscle conditioned medium or in contact to myotubes, was remarkably improved in the presence of a ganglioside mixture (10 μM) from bovine brain. If embryonic neurons from optic lobes were cultivated at low Ca 2+-concentration (<20 μM) they developed flat, broad cell bodies with many filopodia and only a few flat-shaped short processes. A very weak cytoskeleton-staining by means of rhodamine-linked phalloidine indicated that polymerization of actin was impaired in these neurons. At the same low Ca 2+-concentration of <20 μM but in the presence of ganglioside GM1 (up to 100 μM) most of the neurons developed a “normal” cell shape with rounded perikarya and thin neurites with “normal” shaped growth cones. In this case rhodamine-linked phalloidine revealed a much more intense staining mainly concentrated within the growing tips. The morphology and growth of the ganglioside-treated neurons resembled that of neurons cultivated at a higher Ca 2+-concentration of at least 600 μM.

Distinct neurotrophic factors from skeletal muscle and the central nervous system interact synergistically to support the survival of cultured embryonic spinal motor neurons

Motor neurons isolated from 6-day-old embryonic chick spinal cords require muscle extract for survival in culture; however, it was found, that some motor neurons, identified by retrograde labeling with rhodamine, will survive in mixed spinal cell cultures in the absence of the extract. The motor neuron survival-promoting activity produced by spinal cells is soluble and differs from the factor present in muscle extract, the two activities acting in a synergistic manner: the spinal cell activity potentiated that of muscle to decrease its ED 50 by an order of magnitude, the motor neuronal survival (30%) seen in the presence of both factors being more than the sum of their individual activities. This synergism was shown to be restricted to the action of the spinal cell factor on motor neurons, no effect of the factor being noted with sympathetic neurons. As a series of defined growth and survival factors present in the central nervous system (nerve growth factor, brain-derived neurotrophic factor, acidic and basic fibroblast growth factors) had no effect on motor neuron survival, we conclude that the molecule responsible for the motor neuron survival-promoting activity of the spinal cells is a previously undefined factor.

Muscle-derived factors that support survival and promote fiber outgrowth from embryonic chick spinal motor neurons in culture

The purpose of the experiments reported is to provide an unambiguous demonstration that embryonic skeletal muscle contains factors that act directly on embryonic spinal motor neurons both to support their survival and to stimulate the outgrowth of neurites. Cells of lumbar and brachial ventral spinal cords from 6-day-old chick embryos were separated by centrifugation in a two-step metrizamide gradient, and a motor neuron enriched fraction was obtained. Motor neurons were identified by retrogradely labeling with rhodamine isothiocyanate, and were enriched fourfold in the motor neuron fraction relative to unfractionated cells. In culture, the isolated motor neurons died within 3–4 days unless they were supplemented with embryonic chick skeletal muscle extract. Two functionally distinct entities separable by ammonium sulfate precipitation were responsible for the effects of muscle extracts on motor neurons. The 0–25% ammonium sulfate precipitate contained molecules that alone had no effect on neuronal survival but when bound to polyornithine-coated culture substrata, stimulated neurite outgrowth and potentiated the survival activity present in muscle. Most of this activity was due to a laminin-like molecule being immunoprecipitated with antisera against laminin, and immunoblotting demonstrated the presence of both the A and B chains of laminin. A long-term survival activity resided in the 25–70% ammonium sulfate fraction, and its apparent total and specific activities were strongly dependent on the culture substrate. In contrast to the motor neurons, the cells from the other metrizamide fraction (including neuronal cells) could be kept in culture for a prolonged time without addition of exogenous factor(s).