Interactions between spinal cord stimulation and activity blockade in the regulation of synaptogenesis and motoneuron survival in the chick embryo

The present study investigated the effects of spinal cord stimulation, neuromuscular blockade, or a combination of the two on neuromuscular development both during and after the period of naturally occurring motoneuron death in the chick embryo. Electrical stimulation of the spinal cord was without effect on motoneuron survival, synaptogenesis, or muscle properties. By contrast, activity blockade rescued motoneurons from cell death and altered synaptogenesis. A combination of spinal cord stimulation and activity blockade resulted in a marked increase in motoneuron death, and also altered synaptogenesis similar to that seen with activity blockade alone. Perturbation of normal nerve–muscle interactions by activity blockade may increase the vulnerability of developing motoneurons to excessive excitatory afferent input (spinal cord stimulation) resulting in excitotoxic-induced cell death. © 1993 John Wiley & Sons, Inc.     

Cell death of motoneurons in the chick embryo spinal cord. XI. Acetylcholine receptors and synaptogenesis in skeletal muscle following the reduction of motoneuron death by neuromuscular blockade

Treatment of chick embryos with neuromuscular blocking agents such as curare during periods of naturally occurring motoneuron death results in a striking reduction of this normal cell loss. Inactivity-induced changes in motoneuron survival were found to be associated with increased levels of AChRs and AChR-clusters in skeletal muscle and with increased focal sites of AChE that are innervated ('synaptic sites'). Treatment of embryos with curare after the normal cell death period (E12-E15) resulted in no change in motoneuron survival. Although AChR-clusters and focal sites of AChE were increased in these embryos on E16, many of these sites were uninnervated. Treatment of embryos with nicotine or decamethonium (E6-E10) also reduced neuromuscular activity but did not alter motoneuron survival nor did such treatment alter AChRs. The different effects of curare vs nicotine and decamethoniam on motoneuron survival and AChRs may be related to the fact that the former is a competitive blocker whereas the latter two drugs are depolarizing blockers. Finally, treatment of embryos (E6-9) with doses of curare (1 mg daily) that allow for the almost complete recovery of neuromuscular activity a few days following treatment (by E16) resulted in the gradual loss of the excess motoneurons that were present on E10, and by E16 the number of remaining AChR clusters and focal sites of AChE were also decreased to levels comparable to control values. Inactivity-induced changes in AChRs or AChR-clusters may be an important factor in the reduced motoneuron death that accompanies neuromuscular blockade during critical stages of development. These receptor changes very likely reflect increased synaptogenesis in the muscles of paralyzed embryos which in turn may act to reduce motoneuron death by providing increased access to muscle-derived neurotrophic molecules.     

Chronic application of curare does not increase the level of motoneuron survival-promoting activity in limb muscle extracts during the naturally occurring motoneuron cell death period

Naturally occurring motoneuron cell death during development is a well-described phenomenon and the existence of a survival factor provided by target muscles has been postulated. Blockade of activity by chronic application of a neuromuscular junction blocker rescues almost all motoneurons from cell death. The present study was conducted in order to examine the possibility that the motoneuron survival-promoting activity in muscles increases following activity blockade. Cell culture was used to assess the degree of motoneuron survival-promoting activity present in muscle extracts. Embryonic chick motoneurons were labeled by injecting the water-insoluble fluorescent dye, DiI (Molecular Probes, Inc.) into the spinal nerves both before and during the cell death period. The labeled cells extending long neurites were counted after 2 days of culture as viable motoneurons in low-density heterogeneous cell cultures. The culture medium, Ham F12/DMEM (1:1 mixture) supplemented with 10% horse serum, 5% chick serum, and 5% fetal calf serum, was employed as a basic culture medium for assessing motoneuron survival factor, since it supported the survival of a significantly higher number of motoneurons derived from embryos before cell death than those during the cell death period, thus representing the motoneuron's requirement for survival factor in vivo. The number of surviving motoneurons clearly increased in proportion to the amount of muscle extract added to the culture medium. In comparison with control chick embryos, the dose-response relation between the number of surviving motoneurons and the amount of muscle extract added did not change when embryos were used after chronic application of curare. These results therefore indicate that survival factor derived from target muscle is crucial to the in vitro motoneurons during the cell death period, but do not support the idea that inactive muscle contains a higher amount of the survival factor.     

Relevance of motoneuron specification and programmed cell death in embryos to therapy of ALS

The molecular cues that generate spinal motoneurons in early embryonic development are well defined. Motoneurons are generated in excess and consequently undergo a natural period of programmed cell death. Although it is not known exactly how motoneurons compete for survival in embryonic development, it is hypothesized that they rely on the ability to access limited amounts of trophic factors from peripheral tissues, a process that is tightly regulated by skeletal muscle activity. Attempts to elucidate the molecular mechanisms that underlie motoneuron generation and programmed cell death in embryos have led to various effective strategies for treating injury and disease in animal models. Such studies provide great hope for the amelioration of human amyotrophic lateral sclerosis (ALS), a devastating progressive motoneuron degenerative disease. Here we review the clinical relevance of studying motoneuron specification and death during embryonic development.     

The role of interactions in determining cell fate of two identified motoneurons in the embryonic zebrafish.

The role of cellular interactions in determining the fates of two identified motoneurons in the embryonic zebrafish was investigated by transplanting individual motoneurons from labeled donor embryos to unlabeled hosts. The results suggest that although these cells normally adopt different fates, they form an equivalence group in which one fate is primary and the other is secondary. Both cells are able to adopt the primary fate. A cell that has adopted the secondary fate can be induced to switch to the primary fate by ablating the cell that has adopted the primary fate, even many hours after axogenesis. Although interactions between the two cells appear to regulate which cell adopts the secondary fate, these interactions seem to be independent of neuromuscular activity.     

Elucidating the molecular mechanisms that underlie the target control of motoneuron death

Approximately half of the motoneurons generated during normal embryonic development undergo programmed cell death. Most of this death occurs during the time when synaptic connections are being formed between motoneurons and their target, skeletal muscle. Subsequent muscle activity stemming from this connection helps determine the final number of surviving motoneurons. These observations have given rise to the idea that motoneuron survival is dependent upon access to muscle derived trophic factors, presumably through intact neuromuscular synapses. However, it is not yet understood how the muscle regulates the supply of such trophic factors, or if there are additional mechanisms operating to control the fate of the innervating motoneuron. Recent observations have highlighted target independent mechanisms that also operate to support the survival of motoneurons, such as early trophic-independent periods of motoneuron death, trophic factors derived from Schwann cells and selection of motoneurons during pathfinding. Here we review recent investigations into motoneuron cell death when the molecular signalling between motoneurons and muscle has been genetically disrupted. From these studies, we suggest that in addition to trophic factors from muscle and/or Schwann cells, specific adhesive interactions between motoneurons and muscle are needed to regulate motoneuron survival. Such interactions, along with intact synaptic basal lamina, may help to regulate the supply and presentation of trophic factors to motoneurons.     

Regulation of motoneuron death in the spinal nucleus of the bulbocavernosus.

A sexual dimorphism in the number of motoneurons in the spinal nucleus of the bulbocavernosus (SNB) of rats is engendered by a sex difference in ontogenetic cell death. Testicular secretions, specifically androgenic steroids, reduce SNB motoneuron death in males. The fate of the target muscles generally mirrors that of the motoneurons, and androgens appear to exert their effects upon the target muscles, sparing the motoneurons as a secondary consequence. Treatment with ciliary neurotrophic factor can also spare SNB motoneurons in newborn females, raising the possibility that this factor normally mediates androgen's effect upon motoneuron survival. The ontogeny of calcitonin gene-related peptide immunoreactivity is delayed in SNB cells compared with other motoneurons and is further delayed in the SNB cells of females. In both sexes, calcitonin gene-related peptide is detected after the period of SNB motoneuron death is complete. A sex difference in motoneuron number is also seen in the human homologue of the SNB and, because ontogenetic death of motoneurons in humans overlaps the period of androgen secretion, may arise in a manner similar to that in the rat SNB.     

Brachially innervated ectopic hindlimbs in the chick embryo. I. Limb motility and motor system anatomy during the development of embryonic behavior

The functional status of brachially innervated hindlimbs, produced by transplanting hindlimb buds of chick embryos in place of forelimb buds, was quantified by analyzing the number and temporal distribution of spontaneous limb movements. Brachially innervated hindlimbs exhibited normal motility until E10 but thereafter became significantly less active than normal limbs and the limb movements were more randomly distributed. Contrary to the findings with axolotls and frogs, functional interaction between brachial motoneurons and hindlimb muscles cannot be sustained in the chick embryo. Dysfunction is first detectable at E10 and progresses to near total immobility by E20 and is associated with joint ankylosis and muscular atrophy. Although brachially innervated hindlimbs were virtually immobile by the time of hatching (E21), they produced strong movements in response to electrical stimulation of their spinal nerves, suggesting a central rather than peripheral defect in the motor system. The extent of motoneuron death in the brachial spinal cord was not significantly altered by the substitution of the forelimb bud with the hindlimb bud, but the timing of motoneuron loss was appropriate for the lumbar rather than brachial spinal cord, indicating that the rate of motoneuron death was dictated by the limb. Measurements of nuclear area indicated that motoneuron size was normal during the motoneuron death period (E6-E10) but the nuclei of motoneurons innervating grafted hindlimbs subsequently became significantly larger than those of normal brachial motoneurons. Although the muscle mass of the grafted hindlimb at E18 was significantly less than that of the normal hindlimb (and similar to that of a normal forelimb), electronmicroscopic examination of the grafted hindlimbs and brachial spinal cords of E20 embryos revealed normal myofiber and neuromuscular junction ultrastructure and a small increase in the number of axosomatic synapses on cross-sections of motoneurons innervating grafted hindlimbs compared to motoneurons innervating normal forelimbs. The anatomical data indicate that, rather than being associated with degenerative changes, the motor system of the brachial hindlimb of late-stage embryos is intact, but inactive. © 1993 John Wiley & Sons, Inc.     

Development and survival of thoracic motoneurons and hindlimb musculature following transplantation of the thoracic neural tube to the lumbar region in the chick embryo: functional aspects

Following heterotopic transplantation of the thoracic neural tube to the lumbar region on embryonic day (E) 2, the transplanted cord differentiates normally and establishes neuroanatomical connections with the host central nervous system and hindlimb muscles. Beginning on about E12, however, the neuromuscular system begins to undergo regressive changes resulting in motoneuron degeneration and muscle atrophy (O'Brien and Oppenheim, 1990). In the present paper, we have examined the development of neuromuscular function in thoracic transplant embryos from E6 to the time of hatching on E20-21. The onset of hindlimb movements and reflexes occurred at the same time (E6-E8) in both control and thoracic transplant embryos. Further, both the nature (pattern) and frequency of these movements appeared normal in the thoracic transplants up to E10-E12, after which there was a gradual and marked reduction in the frequency, and an alteration in the pattern, of both spontaneous and reflex-evoked hindlimb movements. After E16 normal movements were virtually absent in many of the thoracic transplant cases. By contrast, movements of the head, trunk and wings were normal in these embryos throughout the observation period. Hindlimbs innervated partly by the thoracic transplant and partly by remaining host lumbar cord did not exhibit the regressive changes in function after E10 that occurred in hindlimbs innervated exclusively by the thoracic transplant. EMG recordings from specific hindlimb muscles innervated solely by thoracic motoneurons demonstrated that the activation pattern of both flexors and extensors was similar to the repetitive pattern observed in normal thoracically innervated intercostal muscles (i.e., extensor-like). Muscles did not show distinguishable EMG burst patterns with inhibitory periods as do control lumbar innervated muscles. We conclude that the development of the pattern generating circuitry in the transplanted thoracic cord was similar to normal thoracic cord and thus appeared to be uninfluenced by having contacted the foreign hindlimb muscle targets early in development. Activity blockade with curare from E6 to E14 suppressed the loss of motoneurons that occurs in the thoracic transplant after E10. Thus, the abnormal thoracic-like activation pattern of thoracically innervated hindlimbs may be a critical signal in the initiation of the neuromuscular regression that occurs after E10 in these preparations. Finally, although the innervation and formation of neuromuscular endplates in thoracic transplants appeared normal up to E12, by E14 both the intramuscular nerves and the endplates exhibited signs of degeneration and regression. Thoracic motoneurons are initially able to innervate and functionally activate hindlimb muscles in a manner similar to that of thoracically innervated intercostal muscles.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Developmental expression of voltage-dependent calcium currents in identified mouse motoneurons.

Previous work has shown that during chick embryonic development, large changes occur in the density of specific, motoneuronal calcium currents just prior to the period of naturally occurring motoneuron cell death. Here we report on calcium currents in mouse motoneurons isolated from embryos at the time of peak cell death and also during a subsequent developmental stage when supernumerary synapses are being eliminated. In mouse motoneurons, the density of high-voltage-activated calcium current increases significantly after the phase of cell death, during the period of synapse elimination.     

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.     

Regulation of motoneuron death in the spinal nucleus of the bulbocavernsus

A sexual dimorphism in the number of motoneurons in the spinal nucleus of the bulbocavernosus (SNB) of rats is engendered by a sex difference in ontogenetic cell death. Testicular secretions, specifically androgenic steroids, reduce SNB motoneuron death in males. The fate of the target muscles generally mirrors that of the motoneurons, and androgens appear to exert their effects upon the target muscles, sparing the motoneurons as a secondary consequence. Treatment with ciliary neurotrophic factor can also spare SNB motoneurons in newborn females, raising the possibility that this factor normally mediates androgen's effect upon motoneuron survival. The ontogeny of calcitonin gene-related peptide immunoreactivity is delayed in SNB cells compared with other motoneurons and is further delayed in the SNB cells of females. In both sexes, calcitonin gene-related peptide is detected after the period of SNB motoneuron death is complete. A sex difference in motoneuron number is also seen in the human homologue of the SNB and, because ontogenetic death of motoneurons in humans overlaps the period of androgen secretion, may arise in a manner similar to that in the rat SNB. © 1992 John Wiley & Sons, Inc.     

Mechanisms of insulin-like growth factor regulation of programmed cell death of developing avian motoneurons

During development of the avian neuromuscular system, lumbar spinal motoneurons (MNs) innervate their muscle targets in the hindlimb coincident with the onset and progression of MN programmed cell death (PCD). Paralysis (activity blockade) of embryos during this period rescues large numbers of MNs from PCD. Because activity blockade also results in enhanced axonal branching and increased numbers of neuromuscular synapses, it has been postulated that following activity blockade, increased numbers of MNs can gain access to muscle-derived trophic agents that prevent PCD. An assumption of the access hypothesis of MN PCD is the presence of an activity-dependent, muscle-derived sprouting or branching agent. Several previous studies of sprouting in the rodent neuromuscular system indicate that insulin-like growth factors (IGFs) are candidates for such a sprouting factor. Accordingly, in the present study we have begun to test whether the IGFs may play a similar role in the developing avian neuromuscular system. Evidence in support of this idea includes the following: (a) IGFs promote MN survival in vivo but not in vitro; (b) neutralizing antibodies against IGFs reduce MN survival in vivo; (c) both in vitro and in vivo, IGFs increase neurite growth, branching, and synapse formation; (d) activity blockade increases the expression of IGF-1 and IGF-2 mRNA in skeletal muscles in vivo; (e) in vivo treatment of paralyzed embryos with IGF binding proteins (IGF-BPs) that interfere with the actions of endogenous IGFs reduce MN survival, axon branching, and synapse formation; (f) treatment of control embryos in vivo with IGF-BPs also reduces synapse formation; and (g) treatment with IGF-1 prior to the major period of cell death (i.e., on embryonic day 6) increases subsequent synapse formation and MN survival and potentiates the survival-promoting actions of brain-derived neurotrophic factor (BDNF) and glial cell-line-derived neurotrophic factor (GDNF) administered during the subsequent 4- to 5-day period of PCD. Collectively, these data provide new evidence consistent with the role of the IGFs as activity-dependent, muscle-derived agents that play a role in regulating MN survival in the avian embryo. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 379–394, 1998     

Hyaluronidase activity and hyaluronate content of the developing chick embryo heart.

Hyaluronidase activity and hyaluronate content were measured in the developing chick heart from embryonic day 3 through posthatching stages. High levels of both enzyme and substrate were found during the earliest stages examined. Hyaluronidase activity gradually declined to 63% of the initial (day 3) level by embryonic day 16. Enzyme activity decreased more sharply during the next 4 days to 30% of the initial level and remained constant through 2 weeks after hatching. Low levels of enzyme activity (about 10% initial levels) were still detectable in 10-week-old chicken hearts. The heart hyaluronidase is an endoglycosidase with an estimated molecular weight of 62,000, which degrades hyaluronate and, to a lesser extent, chondroitin sulfate at an acid pH optimum. Hyaluronate constituted approximately 50% of the total glycosaminoglycan content at embryonic day 5. Between embryonic days 5 and 12, the concentration of hyaluronate decreased to 25–30% of the initial level and remained constant thereafter. The level of other glycosaminoglycans decreased more gradually than hyaluronate and did not reach a constant level until hatching. This pattern of hyaluronidase activity and hyaluronate concentration presumably reflects the extensive tissue remodeling which transforms the developing heart from a thin-walled tube containing extensive regions of extracellular matrix to a compact, thick-walled myocardium having a limited extracellular compartment.     

Cell death of motoneurons in the chick embryo spinal cord. X. Synapse formation on motoneurons following the reduction of cell death by neuromuscular blockade

Chronic treatment of chick embryos with neuromuscular blocking agents, such as curare, rescues motoneurons from naturally occurring cell death. In the present study, embryos treated with curare from E6 to E9 had 35% more motoneurons than controls on E10 and 42% more than controls on E16. Previous studies have shown that several aspects of motoneuron differentiation occur normally in curare-treated embryos. We report here that dendrite growth and arborization is also unaltered on E10 and E16 following curare treatment. A quantitative analysis of afferent synapses on motoneurons shows that the packing density of both axosomatic and axodendritic synapses is also normal on E10 in curare-treated embryos, despite the greater number of motoneurons present. This indicates that the interneurons that provide presynaptic input to motoneurons are able to compensate for the increased number of synaptic sites made available by curare treatment. However, by E16 the packing density of synapses is reduced by about half. Because motoneurons and their dendrites continue to grow between E10 and E16, the further increase in synaptic sites made available in curare-treated embryos apparently exceeds the compensatory capacity of presynaptic interneurons on E16. One can conclude from these results that the increased survival of motoneurons in curare-treated embryos is not owing to an increase in afferent synapses. Motoneurons in these embryos continue to survive in the face of either no change (E10) or a reduction (E16) in the number of axodendritic and axosomatic synapses. Therefore, increased motoneuron survival in this situation is very likely regulated primarily by motoneuron-target interactions.     

Antagonistic roles for Ultrabithorax and Antennapedia in regulating segment-specific apoptosis of differentiated motoneurons in the Drosophila embryonic central nervous system

The generation of morphological diversity among segmental units of the nervous system is crucial for correct matching of neurons with their targets and for formation of functional neuromuscular networks. However, the mechanisms leading to segment diversity remain largely unknown. We report here that the Hox genes Ultrabithorax (Ubx) and Antennapedia (Antp) regulate segment-specific survival of differentiated motoneurons in the ventral nerve cord of Drosophila embryos. We show that Ubx is required to activate segment-specific apoptosis in these cells, and that their survival depends on Antp. Expression of the Ubx protein is strongly upregulated in the motoneurons shortly before they undergo apoptosis, and our results indicate that this late upregulation is required to activate reaper-dependent cell death. We further demonstrate that Ubx executes this role by counteracting the function of Antp in promoting cell survival. Thus, two Hox genes contribute to segment patterning and diversity in the embryonic CNS by carrying out opposing roles in the survival of specific differentiated motoneurons.     

The relationship of intramuscular nerve branching and synaptogenesis to motoneuron survival.

The target has been considered for some time to play a major role in allowing neurons to survive the period of naturally occurring cell death. For the motoneurons that innervate the chick limb, evidence is presented that suggests access to target-derived trophic factor via intramuscular nerve branches and synapses may be important in regulating neuronal survival. Alterations in branching and synapse formation produced by activity blockade as well as by alteration of adhesion molecule function are shown to result in changes in motoneuron survival consistent with the proposed hypothesis. The relevance of these observations to the numerical-matching hypothesis of vertebrate neuronal cell death is also considered.     

Naturally occurring motoneuron cell death in rat upper respiratory tract motor nuclei: A histological,fast dil and immunocytochemical study in the nucleus ambiguus

The mammalian upper respiratory tract (URT) serves as the common modality for aspects of respiration, deglutition, and vocalization. Although these actions are dependent on coordinated and specific neuromuscular control, little is known about the development of URT control centers. As such, this study investigated the occurrence of naturally occurring motoneuron cell death (MCD) in the nucleus ambiguus (NA) of a developmental series of rats. Standard histological techniques were used to count motoneurons in the ventrolateral brainstem where the mature NA is found. In addition, the neural tracer, fast Dil, was used to determine whether motoneurons were still migrating into the region of the NA during the period that cell counts were first taken. Furthermore, to elucidate the potential effect of inadvertently counting large interneurons on the assessment of motoneuron numbers, an antibody to γ-aminobutyric acid (GABA) was used. The results of this study have, for the first time, demonstrated that MCD occurs in a URT-related motor nucleus. Approximately a 50% cell death was observed during the prenatal development of NA, with no further loss seen postnatally. The fast DiI studies showed that by embryonic day 17, NA was fully formed, suggesting that motoneuron migration from the basal plate was complete. In addition, use of the GABA antibody showed a lack of inhibitory interneurons within the NA. The finding of MCD in the NA helps define a critical period in the formation of URT neuromuscular control. As the course of MCD is modifiable by epigenetic signals, insult to the organism during this prenatal period may compromise future URT control. © 1995 John Wiley & Sons, Inc.