Ethanol influences on the chick embryo spinal cord motor system. II. Effects of neuromuscular blockade and period of exposure

The study described below was performed as a continuation of a previous study in which we found reduced motoneuron number in lumbar spinal cord of the chick embryo following chronic ethanol administration from embryonic day 4 (E4) to E11. We sought to determine whether this reduction was due to primary ethanol toxicity or to enhancement of naturally occurring cell death (NOCD) and to determine whether administration of ethanol at a later period of development could also reduce motoneuron number. Earlier studies have shown that curare suspends NOCD in the chick embryo. By administering both ethanol and curare to these embryos from E4 to E11 and examining the lumbar spinal cord on E12, we determined that ethanol was directly toxic to motoneurons and reduced motoneuron number in the absence of NOCD. By administering ethanol from E10 to E15 and examining the lumbar spinal cord on E16, we determined that ethanol can reduce motoneuron number without altering spinal cord length during more than one stage of chick embryo development, and that ethanol toxicity is not dependent on NOCD. In addition, we demonstrated that ethanol does not affect the neurotrophic content of chick muscle when it is administered from E10 to E15. © 1997 John Wiley & Sons, Inc. J Neurobiol 32 : 684–694, 1997     

Short- and long-term effects of ciliary neurotrophic factor on androgen-sensitive motoneurons in the lumbar spinal cord

Motoneuron death in the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN) of the lumbar spinal cord is androgen regulated. As a result, many more SNB and DLN motoneurons die in perinatal female rats than in males, whereas treatment of newborn females with androgen results in a permanent sparing of the motoneurons and their target muscles. We previously observed that a neurotrophic molecule, ciliary neurotrophic factor (CNTF), also arrests the death of SNB motoneurons and their target musculature, at least in the short term. The present study compares the short- and long-term consequences of perinatal CNTF treatment on motoneuron number in the SNB, the DLN, and the retrodorsolateral nucleus (RDLN), a motor pool in the lower lumbar cord that does not exhibit hormone-regulated cell death. Female pups were treated with CNTF or vehicle alone from embryonic day 22 through postnatal day 6 (P6). Motoneuron number in each nucleus was then determined immediately after treatment on P7, or 10 weeks later (P77). CNTF treatment significantly elevated motoneuron number in the SNB and DLN on P7; the volume of SNB target muscles on P7 was also greater in the CNTF-treated group. These effects were transient, however, as motoneuron number and ratings of muscle size were not different in CNTF- and vehicle-treated females on P77. Perinatal CNTF treatment did not alter cell number in the RDLN at either age. The finding that effects of CNTF on SNB and DLN motoneuron number are short lived contrasts with the permanent effects of early androgen treatment, and has implications for molecular models of the actions of androgen and neurotrophic factors on the developing spinal cord. © 1996 John Wiley & Sons, Inc.     

Developmental motoneuron cell death and neurotrophic factors

During the development of higher vertebrates, motoneurons are generated in excess. In the lumbar spinal cord of the developing rat, about 6000 motoneurons are present at embryonic day 14. These neurons grow out axons which make contact with their target tissue, the skeletal muscle, and about 50% of the motoneurons are lost during a critical period from embryonic day 14 until postnatal day 3. This process, which is called physiological motoneuron cell death, has been the focus of research aiming to identify neurotrophic factors which regulate motoneuron survival during this developmental period. Motoneuron cell death can also be observed in vitro when the motoneurons are isolated from the embryonic avian or rodent spinal cord. These isolated motoneurons and other types of primary neurons have been a useful tool for studying basic mechanisms underlying neuronal degeneration during development and under pathophysiological conditions in neurodegenerative disorders. Accumulating evidence from such studies suggests that some specific requirements of motoneurons for survival and proper function may change during development. The focus of this review is a synopsis of recent data on such specific mechanisms.     

Vascular endothelial growth factor protects spinal cord motoneurons against glutamate-induced excitotoxicity via phosphatidylinositol 3-kinase

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motoneurons. Recently, vascular endothelial growth factor (VEGF) has been identified as a neurotrophic factor and has been implicated in the mechanisms of pathogenesis of ALS and other neurological diseases. The potential neuroprotective effects of VEGF in a rat spinal cord organotypic culture were studied in a model of chronic glutamate excitotoxicity in which glutamate transporters are inhibited by threohydroxyaspartate (THA). Particularly, we focused on the effects of VEGF in the survival and vulnerability to excitotoxicity of spinal cord motoneurons. VEGF receptor-2 was present on spinal cord neurons, including motoneurons. Chronic (3 weeks) treatment with THA induced a significant loss of motoneurons that was inhibited by co-exposure to VEGF (50 ng/mL). VEGF activated the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) signal transduction pathway in the spinal cord cultures, and the effect on motoneuron survival was fully reversed by the specific PI3-K inhibitor, LY294002. VEGF also prevented the down-regulation of Bcl-2 and survivin, two proteins implicated in anti-apoptotic and/or anti-excitotoxic effects, after THA exposure. Together, these findings indicate that VEGF has neuroprotective effects in rat spinal cord against chronic glutamate excitotoxicity by activating the PI3-K/Akt signal transduction pathway and also reinforce the hypothesis of the potential therapeutic effects of VEGF in the prevention of motoneuron degeneration in human ALS.     

The development of motoneurons in the embryonic spinal cord of the mouse mutant, muscular dysgenesis (mdg/mdg): survival, morphology, and biochemical differentiation

Motoneuron development was studied in the spinal cord of the mouse mutant, muscular dysgenesis, between embryonic days (E) 13 and 18. Dysgenic embryos are characterized by the absence of neuromuscular activity (motility) and exhibit a number of other striking changes in neuromuscular development. Many of these changes have also been observed in chick embryos chronically treated with neuromuscular blocking agents that suppress motility. Motoneuron survival, as well as several other aspects of neuronal development, was examined in the thoracic and lumbar spinal cords of mutant and control embryos. There was a significant decrease in motoneuron numbers in control embryos indicating the presence of naturally occurring cell death in the mouse spinal cord. At all ages examined, the dysgenic embryos had significantly more healthy and significantly fewer degenerating motoneurons than controls. There were no differences in the number of dorsal root ganglion neurons or in any of the other morphometric parameters examined between mutant and control embryos. Creatine kinase activity, a marker for myofiber maturation, was significantly reduced in the limb musculature of mutant embryos. Choline acetyltransferase activity was significantly increased in the spinal cord of mutant embryos. No significant differences were observed in spinal cord levels of acetylcholinesterase activity between control and mutant embryos. The absence of muscle contractions in the dysgenic mouse is associated with a number of changes in neuromuscular development, including a substantial reduction of naturally occurring motoneuron death.     

Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in vitro.

We present evidence that the cholinergic differentiation factor (CDF), originally purified from cardiac and skeletal muscle cell-conditioned medium and found to be identical to leukemia inhibitory factor (LIF), promotes survival of embryonic day 14 rat motoneurons in vitro. These neurons were retrogradely labeled with the fluorescent tracer Dil and enriched on a density gradient or purified to homogeneity by fluorescence-activated cell sorting. Subnanomolar concentrations of CDF/LIF supported the survival of 85% of the motoneurons that would have died between days 1 and 4 of culture. The enhanced survival was accompanied by a 4-fold increase in choline acetyltransferase (ChAT) activity per culture. CDF/LIF also increased ChAT activity in dorsal spinal cord cultures, but had no detectable effect on ChAT levels in septal or striatal neuronal cultures. For comparison, other neurotrophic molecules were tested on motoneuron cultures. Ciliary neurotrophic factor had effects on motoneuron survival similar to those of CDF/LIF, whereas basic fibroblast growth factor was somewhat less effective. Nerve growth factor had no effect on the survival of rat motoneurons.     

Simulation system of spinal cord motor nuclei and associated nerves and muscles,in a Web-based architecture

A Web-based simulation system of the spinal cord circuitry responsible for muscle control is described. The simulator employs two-compartment motoneuron models for S, FR and FF types, with synaptic inputs acting through conductance variations. Four motoneuron pools with their associated interneurons are represented in the simulator, with the possibility of inclusion of more than 2,000 neurons and 2,000,000 synapses. Each motoneuron action potential is followed, after a conduction delay, by a motor unit potential and a motor unit twitch. The sums of all motor unit potentials and twitches result in the electromyogram (EMG), and the muscle force, respectively. Inputs to the motoneuron pool come from populations of interneurons (Ia reciprocal inhibitory interneurons, Ib interneurons, and Renshaw cells) and from stochastic point processes associated with descending tracts. To simulate human electrophysiological experiments, the simulator incorporates external nerve stimulation with orthodromic and antidromic propagation. This provides the mechanisms for reflex generation and activation of spinal neuronal circuits that modulate the activity of another motoneuron pool (e.g., by reciprocal inhibition). The generation of the H-reflex by the Ia-motoneuron pool system and its modulation by spinal cord interneurons is included in the simulation system. Studies with the simulator may include the statistics of individual motoneuron or interneuron spike trains or the collective effect of a motor nucleus on the dynamics of muscle force control. Properties associated with motor-unit recruitment, motor-unit synchronization, recurrent inhibition and reciprocal inhibition may be investigated.     

Mimicking the neurotrophic factor profile of embryonic spinal cord controls the differentiation potential of spinal progenitors into neuronal cells

Recent studies have indicated that the choice of lineage of neural progenitor cells is determined, at least in part, by environmental factors, such as neurotrophic factors. Despite extensive studies using exogenous neurotrophic factors, the effect of endogenous neurotrophic factors on the differentiation of progenitor cells remains obscure. Here we show that embryonic spinal cord derived-progenitor cells express both ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) mRNA before differentiation. BDNF gene expression significantly decreases with their differentiation into the specific lineage, whereas CNTF gene expression significantly increases. The temporal pattern of neurotrophic factor gene expression in progenitor cells is similar to that of the spinal cord during postnatal development. Approximately 50% of spinal progenitor cells differentiated into astrocytes. To determine the effect of endogenous CNTF on their differentiation, we neutralized endogenous CNTF by administration of its polyclonal antibody. Neutralization of endogenous CNTF inhibited the differentiation of progenitor cells into astrocytes, but did not affect the numbers of neurons or oligodendrocytes. Furthermore, to mimic the profile of neurotrophic factors in the spinal cord during embryonic development, we applied BDNF or neurotrophin (NT)-3 exogenously in combination with the anti-CNTF antibody. The exogenous application of BDNF or NT-3 promoted the differentiation of these cells into neurons or oligodendrocytes, respectively. These findings suggest that endogenous CNTF and exogenous BDNF and NT-3 play roles in the differentiation of embryonic spinal cord derived progenitor cells into astrocytes, neurons and oligodendrocytes, respectively.     

Growth factor dependent cholinergic function and survival in primary mouse spinal cord cultures

In primary embryonic spinal cord cultures, synaptic transmission can be conveniently studied by monitoring radiolabeled neurotransmitter release or by recording of electrophysiological responses. However, while the mature spinal cord contains an appreciable number of cholinergic motoneurons, cultures of embryonic spinal cord have a paucity of these neurons and release little or no acetylcholine upon stimulation. To determine whether the proportion of cholinergic neurons in primary mouse spinal cord cultures can be augmented, the effects of several classes of growth factors were examined on depolarization- and Ca(2+)-evoked release of choline/acetylcholine (Ch/ACh). In the absence of growth factors, little or no evoked release of radiolabeled Ch/ACh could be demonstrated. Media supplemented with brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF) were examined for their ability to preserve the population of neurons in culture. CNTF was found to increase the number of surviving neurons and to enhance the release of radiolabeled Ch/ACh; the other factors were without effect. The action of CNTF was transient, and the neuronal population decreased to levels observed in cultures lacking growth factor after 20 days in vitro. The correlation between enhanced neuron survival and increased Ch/ACh release suggests that CNTF protected cholinergic neurons, albeit transiently, from cell death.     

S100 is present in developing chicken neurons and schwann cell 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 S100β 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. © 1992 John Wiley & Sons, Inc.     

Genetic disruption of the growth hormone receptor does not influence motoneuron survival in the developing mouse

In the rodent central nervous system (CNS) during the five days prior to birth, both growth hormone (GH) and its receptor (GHR) undergo transient increases in expression to levels considerably higher than those found postnatally. This increase in expression coincides with the period of neuronal programmed cell death (PCD) in the developing CNS. To evaluate the involvement of growth hormone in the process of PCD, we have quantified the number of motoneurons in the spinal cord and brain stem of wild type and littermate GHR-deficient mice at the beginning and end of the neuronal PCD period. We found no change in motoneuron survival in either the brachial or lumbar lateral motor columns of the spinal cord or in the trochlear, trigeminal, facial or hypoglossal nuclei in the brain stem. We also found no significant differences in spinal cord volume, muscle fiber diameter, or body weight of GHR-deficient fetal mice when compared to their littermate controls. Therefore, despite considerable in vitro evidence for GH action on neurons and glia, genetic disruption of GHR signalling has no effect on prenatal motoneuron number in the mouse, under normal physiological conditions. This may be a result of compensation by the signalling of other neurotrophic cytokines.     

Rescue of motoneurons from cell death by a purified skeletal muscle polypeptide: effects of the ChAT development factor, CDF

Rat skeletal muscle contains a 22 kd polypeptide that increases the level of choline acetyltransferase (ChAT) activity in cultures of embryonic rat spinal cord neurons and has been purified to homogeneity. The application of this factor, ChAT development factor or CDF, to developing chick embryos during the period of naturally occurring motoneuron cell death significantly increased the survival of motoneurons but did not affect the survival of dorsal root ganglion neurons or sympathetic preganglionic neurons (column of Terni). These results provide the first demonstration that an isolated, skeletal muscle-derived molecule can selectively enhance the survival of motoneurons in vivo and suggest that CDF may function in vivo to regulate the survival and development of motoneurons.     

Brain-derived proteins that rescue spinal motoneurons from cell death in the chick embryo: Comparisons with target-derived and recombinant factors

Spinal motoneurons that normally die during early development can be rescued by a variety of purified growth or neurotrophic factors and target tissue extracts. There is also indirect evidence that brain or supraspinal afferent input may influence lumbar motoneuron survival during development and that this effect may be mediated by central nervous system–derived trophic agents. This report examines the biological and biochemical properties of motoneuron survival activity obtained from extracts of the embryonic chick brain. Treatment with an ammonium sulfate (25% to 75%) fraction of embryonic day 16 (E16) or E10 brain extracts rescued many spinal motoneurons that otherwise die during the normal period of cell death in vivo (E6 to E10). The same fractions also enhanced lumbar motoneuron survival following deafferentation. There were both similarities and differences between the active fractions derived from brain extracts (BEX) when compared with extracts derived from target muscles (MEX) or with purified neurotrophic factors. Survival activity from E10 BEX was as effective in promoting motoneuron survival as E10 MEX and more effective than astrocyte-conditioned media. Unlike MEX, the active fractions from BEX also rescued placode-derived nodose ganglion cells. In addition, unlike nerve growth factor and brain-derived neurotrophic factor, active BEX fractions did not rescue neural crest-derived dorsal root ganglion cells or sympathetic ganglion neurons. Interestingly, among many cranial motor and other brainstem nuclei examined, only the survival of motoneurons from the abducens nucleus was enhanced by BEX. Active proteins obtained from BEX were further separated by gel filtration chromatography and by preparative isolelectric focusing techniques. Activity was recovered in a basic (pI8) and an acidic (pI5) small molecular weight protein fraction (20 kD or less). The specific activity of the basic fraction was increased ×66 when compared with the specific activity of crude BEX, and the basic fraction had a slightly higher specific activity than the acidic fraction. The biological and biochemical properties of these fractions are discussed in the context of known neurotrophic factors and their effects on normal and lesion-induced motoneuron death during development. © 1995 John Wiley & Sons, Inc.     

Cloning, expression during development, and evidence for release of a trophic factor for ciliary ganglion neurons.

Ciliary ganglion (CG) neurons undergo a period of cell death during development that may be regulated by the limited availability of trophic factor produced by their target tissues. We have previously reported the purification of a ciliary neurotrophic factor from adult chick sciatic nerve that we called growth promoting activity (GPA). Here we demonstrate that GPA can be purified and cloned from embryonic day 15 (E15) chick eyes, which contain all the target tissues of the CG. Our studies show the following: GPA mRNA is induced in embryonic chick eyes during the period of CG neuron cell death; GPA mRNA is expressed specifically in the layer of the eye that contains the targets of the CG and in primary cultures of smooth muscle cells isolated from the choroid layer of the eye; and biologically active GPA is released from cells transfected with a GPA cDNA.     

Necdin protects embryonic motoneurons from programmed cell death

NECDIN belongs to the type II Melanoma Associated Antigen Gene Expression gene family and is located in the Prader-Willi Syndrome (PWS) critical region. Necdin-deficient mice develop symptoms of PWS, including a sensory and motor deficit. However, the mechanisms underlying the motor deficit remain elusive. Here, we show that the genetic ablation of Necdin, whose expression is restricted to post-mitotic neurons in the spinal cord during development, leads to a loss of 31% of specified motoneurons. The increased neuronal loss occurs during the period of naturally-occurring cell death and is not confined to specific pools of motoneurons. To better understand the role of Necdin during the period of programmed cell death of motoneurons we used embryonic spinal cord explants and primary motoneuron cultures from Necdin-deficient mice. Interestingly, while Necdin-deficient motoneurons present the same survival response to neurotrophic factors, we demonstrate that deletion of Necdin leads to an increased susceptibility of motoneurons to neurotrophic factor deprivation. We show that by neutralizing TNFα this increased susceptibility of Necdin-deficient motoneurons to trophic factor deprivation can be reduced to the normal level. We propose that Necdin is implicated through the TNF-receptor 1 pathway in the developmental death of motoneurons.     

Motoneuron projection patterns in chick embryonic limbs with a double complement of dorsal thigh musculature

During the normal development of the chick, lateral motoneurons within the lumbosacral motor column of the spinal cord consistently project to muscles of dorsal origin within the limb while medial motoneurons project to muscles of ventral origin. To determine if specific cues arising from each type of target are the dominant guidance cues used by lateral and medial motoneurons to create this pattern, I examined motoneuron projections in embryonic chick limbs with a double complement of dorsal thigh musculature and no ventral musculature. Results indicate that cues associated with muscles of a specific developmental origin do not invariably dominate. Before and after the major period of motoneuron death, all muscles in dorsal limb regions (host) were innervated by lateral or dorsal pool neurons. Most ventrally positioned (donor) muscles were innervated by medial or ventral pool neurons. Only the donor iliofibularis, a muscle located very near to its original source of innervation, received projections from some lateral neurons. Within the limb proper, medial or ventral pool neurons projected to donor muscles in a patterned manner suggesting that they were following nonspecific regional cues and perhaps also responding to the availability of uninnervated target tissue. I conclude that axon sorting into distinct lateral and medial classes is independent of limb target complement and that subsequent pathway choice is a separate event governed by both specific target cues and other guidance mechanisms.     

Biological studies of a putative avian muscle-derived neurotrophic factor that prevents naturally occurring motoneuron death in vivo

A series of in vivo studies have been carried out using the chick embryo to address several critical questions concerning the biological, and to a lesser extent, the biochemical characteristics of a putative avian muscle-derived trophic agent that promotes motoneuron survival in vivo. A partially purified fraction of muscle extract was shown to be heat and trypsin sensitive and rescued motoneurons from naturally occurring cell death in a dose-dependent fashion. Muscle extract had no effect on mitotic activity in the spinal cord and did not alter cell number when administered either before or after the normal cell death period. The survival promoting activity in the muscle extract appears to be developmentally regulated. Treatment with muscle extract during the cell death period did not permanently rescue motoneurons. The motoneuron survival-promoting activity found in skeletal muscle was not present in extracts from a variety of other tissues, including liver, kidney, lung, heart, and smooth muscle. Survival activity was also found in extracts from fetal mouse, rat, and human skeletal muscle. Conditioned medium derived from avian myotube cultures also prevented motoneuron death when administered in vivo to chick embryos. Treatment of embryos in ovo with muscle extract had no effect on several properties of developing muscles. With the exception of cranial motoneurons, treatment with muscle extract did not promote the survival of several other populations of neurons in the central and peripheral nervous system that also exhibit naturally occurring cell death. Initial biochemical characterization suggests that the activity in skeletal muscle is an acidic protein between 10 and 30 kD. Examination of a number of previously characterized growth and trophic agents in our in vivo assay have identified several molecules that promote motoneuron survival to one degree or another. These include S100β, brain-derived neurotrophic factor (BDNF), neurotrophin 4/5 (NT-4/5), ciliary neurotrophic factor (CNTF), transforming growth factor β (TGFβ), platelet-derived growth factor-AB (PDGF-AB), leukemia inhibitory factor (CDF/LIF), and insulin-like growth factors I and II (IGF). By contrast, the following agents were ineffective: nerve growth factor (NGF), neurotrophin-3 (NT3), epidermal growth factor (EGF), acidic and basic fibroblast growth factors (aFGF, bFGF), and the heparin-binding growth-associated molecule (HB-GAM). Of those agents that were effective, CDF/LIF, IGF-1 and -2, BDNF, and TGF are reported to be expressed in developing or adult muscle. Studies are underway to determine whether the survival activity found in avian muscle extract can be accounted for by one or more of these growth factors. Of all the tissue extracts and purified proteins tested here, only the neurotrophins—NGF, NT-3, and BDNF (but not NT-4/5)—rescured sensory neurons from naturally occurring cell death. © 1993 John Wiley & Sons, Inc.     

A morphometric analysis of the spinal motor pool in relation to its target muscle during growth in the trout, Salmo gairdneri Richardson

The relation between number and size of spinal motoneurons and the dimension of myotomal muscle has been investigated in trout at different stages of embryonic, larval and postlarval development (body length 1–15 cm). Three spinal segments have been analysed (cervical, trunk and caudal) and the following parameters were determined by means of a Micromeasurements Image Analyzer: (a) mean cross-sectional myotomal area; (b) mean soma size of principal (or dorsomedian, DM) and secondary (or ventrolateral, VL) motoneurons; (c) DM and VL motoneuron density per segment. Myotomal muscle and motor pool growth was evaluated by percent increments of a, b and c parameters at each stage. Their relationships were denned by equations of computed regression lines.
The analysis provided evidence that: (1) a continuous exponential growth of mean myotomal area takes place in the three segments, with the same trend and with the lowest values in the caudal segment; (2) DM and VL motoneuron size and density per segment also increase during development, with the least value in the caudal segment, VL parameters being of lesser value than DM; (3) motoneuron pool and its target myotomal muscle parameters bear a linear relationship as defined by equations of computer regression lines; (4) motoneuron number percent increment at early eleutherembryonic stage precedes myotomal area increment which takes place during late eleutherembryonic stage.
It is apparent that spinal motor pool and target myotomal muscle grow at the same rate in the trout during the considered stages. The discussion links this fact with the hypothesis of a neuronal influence on muscle fibre type differentiation.