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L T Landmesser

Publications and source records attributed to L T Landmesser.

15 recordsLinked to original sources
PubMed1991

Relationship of primary and secondary myogenesis to fiber type development in embryonic chick muscle.

The formation of fast and slow myotubes was investigated in embryonic chick muscle during primary and secondary myogenesis by immunocytochemistry for myosin heavy chain and Ca2(+)-ATPase. When antibodies to fast or slow isoforms of these two molecules were used to visualize myotubes in the posterior iliotibialis and iliofibularis muscles, one of the isoforms was observed in all primary and secondary myotubes until very late in development. In the case of myosin, the fast antibody stained virtually all myotubes until after stage 40, when fast myosin expression was lost in the slow myotubes of the iliofibularis. In the case of Ca2(+)-ATPase, the slow antibody also stained all myotubes until after stage 40, when staining was lost in secondary myotubes and in the fast primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis. In contrast, the antibodies against slow muscle myosin heavy chain and fast muscle Ca2(+)-ATPase stained mutually exclusive populations of myotubes at all developmental stages investigated. During primary myogenesis, fast Ca2(+)-ATPase staining was restricted to the primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis, whereas slow myosin heavy chain staining was confined to all of the primary myotubes of the slow region of the iliofibularis. During secondary myogenesis, the fast Ca2(+)-ATPase antibody stained nearly all secondary myotubes, while primaries in the slow region of the iliofibularis remained negative. Thus, in the slow region of the iliofibularis muscle, these two antibodies could be used in combination to distinguish primary and secondary myotubes. EM analysis of staining with the fast Ca2(+)-ATPase antibody confirmed that it recognizes only secondary myotubes in this region. This study establishes that antibodies to slow myosin heavy chain and fast Ca2(+)-ATPase are suitable markers for selective labeling of primary and secondary myotubes in the iliofibularis; these markers are used in the following article to describe and quantify the effects that chronic blockade of neuromuscular activity or denervation has on these populations of myotubes.

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PubMed1991

A reevaluation of the role of innervation in primary and secondary myogenesis in developing chick muscle.

The neural dependence of primary and secondary myogenesis and its relation to fiber-type differentiation was immunocytochemically investigated in chicken limb muscles. In a previous study, we demonstrated that a novel combination of slow myosin and fast Ca2(+)-ATPase antibodies differentially stained mutually exclusive populations of myotubes, which in the slow region of the iliofibularis allowed us to visualize primary and secondary myotubes and to quantify their development. When these antibodies were used to stain myotubes in muscles that were either chronically paralyzed by d-tubocurarine or denervated, we were surprised to observe by both LM and EM analysis that secondary myotubes formed in both cases, in contrast to the widely held tenet that nerve activity is necessary for secondary myogenesis. Also, an unexpected decrease in the number of primary myotubes occurred before the onset of secondary myotube formation. Although the total quantity of myotubes formed was drastically reduced by curare treatment or denervation, the ratio of fast to slow myotubes increased normally between st 34 and 39 1/2. Paralysis by curare did produce a striking increase in the size of individual myotube clusters, indicating that blocking nerve activity either increases adhesion between myotubes or prevents a normal decrease in adhesion during development which may be necessary for myofiber separation from clusters. Our findings indicate that both slow primary and fast secondary myotube populations are composed of nerve-dependent and independent individuals and that the relative quantities of fast and slow myotubes are regulated independent of innervation.

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PubMed1991

The regulation of synaptogenesis during normal development and following activity blockade.

The mature neuromuscular junction is characterized by the tight spatial colocalization of synaptic vesicles and acetylcholine receptor (AChR) clusters. Although a large body of work exists on the interactions between motoneurons and myotubes leading to synaptogenesis in tissue culture, how the neuromuscular junction acquires its highly specialized structure in vivo is not well understood, particularly during the earliest period of synaptogenesis. In this study, the development of the neuromuscular synapse in chick hindlimb muscles was examined and quantified by simultaneously labeling the pre- and postsynaptic elements from the time the main nerve trunks leave the lumbosacral plexus region to enter the developing limb (St 24) through the end of the motoneuron cell death period (St 36). Based on these results, synaptogenesis can be divided into several distinct stages that are intimately connected to the innervation sequence described in a previous paper (Dahm and Landmesser, 1988). Briefly, as large nerve trunks approach the developing muscles and the first AChR clusters are induced to form on nearby myotubes, none of these initial receptor clusters are in direct contact with a nerve profile. The first appearance of nerve-contacted clusters (synapses) is coincident with the growth of large, unbranched nerve trunks into the muscles. The next step is initiated by the formation of small nerve side branches that grow out from the larger intramuscular nerve trunks to bring most axons and myotubes into contact for the first time. As side branches form, synapses appear around them, and non-nerve-contacted receptor clusters disappear from around the main intramuscular nerve trunks. The next step in synaptogenesis is the restriction of synaptic vesicle antigen to sites of synaptic contact. These early stages of synaptogenesis are also characterized by the growth of the presynaptic terminal to match the length of the postsynaptic receptor cluster. This study showed that AChR cluster formation during early in vivo neuromuscular development does not require close anatomical nerve contact, but that the presence of the nerves is necessary for AChR clusters to form. This suggests that the nerves normally induce AChR clustering via the release of a diffusible substance, a suggestion substantiated by the observation that AChR clusters do not form on aneural myotubes in vivo. In order to assess the role of synapse formation in the regulation of motoneuron number, synaptogenesis was quantitatively examined after chronic neuromuscular blockade, which prevents motoneuron cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1988

The regulation of intramuscular nerve branching during normal development and following activity blockade.

In vertebrates, approximately 50% of the lumbosacral motoneurons die during a short period of development that coincides with synaptogenesis in the limb. Although it has been postulated that these motoneurons die because they fail to obtain adequate trophic support from the muscles, it is not clear how this factor is supplied. The mechanism by which activity blockade prevents motoneurons cell death is also unknown. In order to begin to understand the nature of these proposed trophic interactions, we have examined the temporal sequence of axonal invasion and ramification within two muscles of the chick hindlimb, the predominantly slow iliofibularis and the fast posterior iliotibialis, during the cell death period. We found striking differences in intramuscular nerve ingrowth and branching between fast and slow muscle. We also observed differences in the molecular composition of fast and slow myotubes that may contribute to the nerve pattern differences. In addition, we observed a progressive increase in the degree of intramuscular nerve fasciculation as well as a precise temporal sequence of nerve branching. The earliest detectable response to chronic curarization was a dramatic decrease in the degree of intramuscular nerve fasciculation. Activity blockade also greatly enhanced nerve branching within the muscles from the time that nerve branches normally formed, and, additionally, interfered with the normal cessation of axon growth. Our results support the idea that nerve endings are the sites of trophic uptake. Furthermore, although our results do not allow us to exclude other activity-dependent influences on motoneuron survival, they suggest the following testable hypotheses: (1) the normal regulation of motoneuron survival may result from the precise control of intramuscular nerve branching, (2) activity blockade may increase motoneuron survival by enhancing intramuscular nerve branching, and (3) anything which affects this complex process of nerve branching may also alter motoneuron survival.

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PubMed1986

Activation patterns of embryonic chick hind-limb muscles following blockade of activity and motoneurone cell death.

Motoneurone cell death and spontaneous embryonic motility were blocked in chick embryos by daily in ovo injections of d-tubocurarine from stage 28-36 (E5-10). Isolated spinal cord-hind-limb preparations were prepared from these embryos and movement sequences in response to electrical stimulation of the thoracic cord were assessed, after drug wash-out, by electromyogram (e.m.g.) or muscle-nerve recordings. In embryos in which complete blockade of lumbar motoneurone cell death was later confirmed histologically, flexor and extensor motoneurone pools were found to be activated in alternating bursts as occurs in control embryos. Thus the development of the basic cord circuits responsible for these patterns of motoneurone activation does not require motoneurone cell death. Partial blockade of motoneurone cell death by guanosine 3',5'-phosphate (cyclic GMP) was also without effect on muscle activation patterns. In ovo injection of d-tubocurarine or alpha-bungarotoxin in doses sufficient to block embryonic motility was found to have a direct effect on the spinal cord, preventing the patterned activation of motoneurone pools in alternating bursts. Cords removed from treated embryos behaved similarly to cords in which these drugs were applied acutely in the bath. Minor changes in muscle activation patterns that occurred with chronic drug treatment were also observed in acutely treated cords and appear to be a direct and persistent effect of the drugs on cord circuits. It is possible to conclude that cholinergic circuits within the chick lumbar cord play a role in the normal patterned activation of flexor and extensor motoneurone pools. Systemically applied drugs can have access to these circuits, indicating a need for caution when interpreting the results of drugs applied in this manner to developing embryos. We also conclude that neither the activation of motoneurones in patterned bursts, nor the afferent feed-back from the movements that result, are required to form the basic spinal cord circuits responsible for the activation of extensor and flexor motoneurone pools in alternating bursts.

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PubMed1986

Neurites and growth cones in the chick embryo. Enhanced tissue preservation and visualization of HRP-labeled subpopulations in serial 25-microns plastic sections cut on a rotary microtome.

Study of axonal guidance in developing vertebrates has been hindered by an inability to readily visualize individual growth cones, determine the neuronal population from which they originate, trace their trajectories, and discern their interactions with their embryonic environment. We report a method that combines plastic embedding and serial sectioning with horseradish peroxidase labeling of subpopulations of neurons in the chick embryo. This method labels individual neurites from the soma to the tip of the growth cones, allowing their trajectory to be inferred and their identity to be determined by the position of the somata. As sections are up to 25 micron thick, entire growth cones can often be visualized without laborious reconstruction. Tissue preservation is much better than with similar material embedded in paraffin. Sections are cut relatively quickly using a steel knife on a standard rotary microtome and are suitable for subsequent electron microscopy.

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PubMed1986

Interspecies selective motoneuron projection patterns in chick-quail chimeras.

During normal development chick motoneurons have been shown to project selectively to appropriate muscles by responding to a series of cues, both specific and nonspecific, within the limb. We tested the ability of motoneurons from another avian species, the Japanese quail, to respond to these cues by transplanting chick limb buds onto quail embryos and quail limb buds onto chick embryos between stages 17 1/2 and 19. Feulgen staining, which distinguishes chick from quail cells on the basis of nuclear chromatin, revealed that all limb tissue, including muscle, was of donor origin, indicating that the migration of somite-derived muscle precursor cells had been completed by the time of transplantation. Normal quail motoneuron pools for most muscles were located in the same relative positions as homologous chick pools. In chick-quail chimeras we found that the motoneuron pools of one species selectively innervated the homologous muscles in the limb of opposite species with considerable precision. This was determined by defining the segmental innervation pattern of the muscles electrophysiologically and by retrogradely labeling motoneuron pools with HRP. Selective innervation was confirmed by using the functional activation patterns of the motoneuron pools as an additional means of identifying motoneurons. We conclude that any limb-derived cues required by motoneurons to project to their appropriate muscles must be similar in chick and quail and that the growth cones of both species must have similar detector systems for responding to these cues. Only 7 spinal segments were found to innervate the quail limb (versus 8 for the chick), resulting in an anterior shift in the spinal segments innervating several posterior quail muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1986

Cell death of lumbosacral motoneurons in chick, quail, and chick-quail chimera embryos: a test of the quantitative matching hypothesis of neuronal cell death.

The quantitative matching hypothesis of neuronal cell death was tested for the chick hindlimb by determining the relationship between myotube number at the onset of motoneuron cell death and the number of motoneurons that survive in chicks, quail, and chick-quail chimeras. Hindlimb buds, which differ in size between the 2 species, were exchanged at stages 16 1/2-19, myosin ATPase-stained myotubes in selected thigh muscles were counted during the cell death period (stages 30-34), and lumbosacral motoneurons were counted following the cell death period (stage 38). No quail motoneurons were rescued when quail cords innervated chick limbs. When chick cords innervated quail limbs, the number of surviving motoneurons was significantly decreased but not to quail values. We consider that this occurred because chicks develop more slowly than quail, and we found that transplanted chick limbs were developmentally younger than the contralateral quail limb at the onset of motoneuron cell death and contained fewer myotubes. Similarly, transplanted quail limbs contained more myotubes at the onset of cell death than normal stage 30 quail limbs. An excellent correlation was obtained during normal development of both species between the number of myotube clusters at the onset of cell death and the number of surviving motoneurons. This correlation was also observed for chick-quail chimeras, and when the data points were plotted for control chick, control quail, chick host-quail limb, and quail host-chick limb, the correlation coefficient was 0.996. This strongly suggests that some parameter closely related to myotube number limits the number of motoneurons that will survive. A proposal consistent with our observations is that motoneuron survival is dependent on the uptake of a myotube-derived trophic factor that can only be taken up at synaptic sites and that the number of such sites is limited and directly related to myotube number. In conclusion, our observations strongly support a quantitative-matching component in the process of neuronal cell death. However, since we were unable to rescue any neurons, we cannot exclude the possibility that some proportion of neurons normally dies for reasons other than peripheral competition.

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PubMed1985

Development of the major pathways for neurite outgrowth in the chick hindlimb.

To elucidate mechanisms that may control development of the gross anatomical nerve pattern, motoneuron outgrowth into the chick hindlimb was examined using orthograde labeling, scanning and transmission electron microscopy, and Alcian blue staining. Results show that growth cones are not guided by contact with oriented extracellular fibrils, aligned mesenchyme cells, the myotome, or the vasculature. Pathways are not delineated by cell-free space or channels of lower cell density; however, densely packed mesenchyme may form barriers that channel outgrowth. In addition, abundant mesenchymal cell death was seen at the nerve front. This cell death may provide space that encourages growth cone advancement. Pathways often lie along interfaces between areas that stain darkly and lightly with Alcian blue, which specifically stains glycosaminoglycans, and growth cones never penetrate areas that stain intensely, such as the pelvic girdle, which is known to be a barrier to outgrowth. Leading growth cones form specialized contacts with mesenchyme cells, but the predominant contacts are interneuronal. It is proposed that the anatomical pattern of outgrowth is determined by the distribution of preferred substrata, the most preferred substratum being other neurites. Further, neurites tend to prefer loose mesenchyme to dense mesenchyme or areas rich in glycosaminoglycans.

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PubMed1985

Specificity of early motoneuron growth cone outgrowth in the chick embryo.

During development, chick lumbosacral motoneurons have been reported to form precise topographic projections within the limb from the time of initial outgrowth. This observation implies, first, that motoneurons select the appropriate muscle nerve pathway and, second, that they restrict their ramification within the primary uncleaved muscle masses to appropriate regions. Several reports based on electrophysiology and orthograde horseradish peroxidase (HRP) labeling have shown muscle nerve pathway selection to be fairly precise. However, studies based on retrograde labeling with HRP have produced conflicting reports on the extent to which vertebrate motoneurons make projection errors. Since it is difficult to distinguish between true projection errors and HRP leakage when using retrograde labeling, we decided to assess the distribution of labeled growth cones in 25-micron serial plastic sections, following orthograde labeling of identifiable subpopulations of motoneurons during the period of initial axon outgrowth. Examination of a large number of muscle nerves revealed no segmentally inappropriate axons, confirming earlier reports that muscle nerve pathway selection is very accurate. In addition, we observed that growth cones take widely divergent trajectories into the same muscle nerve, suggesting that growth cones are responding independently to some specific environmental cue rather than being passively channeled at this point. The distribution of labeled growth cones within the muscle masses provided direct evidence that motoneurons did not at any time project to obviously inappropriate muscle regions.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1985

Growth cone morphology and trajectory in the lumbosacral region of the chick embryo.

We quantitatively analyzed several features of orthogradely labeled peripheral growth cones in the lumbosacral region of the chick embryo. We compared motoneuron growth cones in regions where they appear to express specific directional preferences (the plexus region and regions where muscle nerves diverge from main nerve trunks), which we operationally defined as "decision regions," to motoneuron growth cones in other pathway regions (the spinal nerve, nerve trunk, and muscle nerve pathways) which we termed, for contrast, "non-decision region." We found that motoneuron growth cones are larger, more lamellepodial, and have more complex trajectories in decision regions. Sensory growth cone populations, which are thought to be dependent upon motoneurons for outgrowth (Landmesser, L., and M. Honig (1982) Soc. Neurosci. Abstr. 8: 929), do not enlarge or become more lamellepodial in motoneuron decision regions, suggesting that this local environment does not affect all species of growth cones equally and that the alterations in motoneuron growth cones in these regions may be relevant to their specific guidance. In addition, the resemblance between the sensory population and other closely fasciculating growth cones lends support to the suggestion that sensory neurons utilize motoneuron neurites as a substratum. We suggest that the convoluted trajectories, enlarged size, and more lamellepodial morphology of motoneuron growth cones in decision regions is either related directly to the presence of specific cues that guide motoneurons or to some aspect of this environment that allows them to respond to specific cues.

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PubMed1984

Activation patterns of embryonic chick hind limb muscles recorded in ovo and in an isolated spinal cord preparation.

Muscle activation patterns of embryonic chick hind limb muscles were determined from electromyographic (e.m.g.) recordings in an isolated spinal cord/hind limb preparation of stage 34-36 embryos, and were compared with in ovo e.m.g. activity from similarly staged embryos. Muscle activity in ovo consisted of periodically recurring sequences of bursts during which antagonistic muscles often alternated and synergistic muscles were co-active, as compatible with their mature function. However, more variable behaviour was also observed. Burst sequences in ovo were often initiated by a short-duration, high-amplitude discharge that occurred synchronously in all muscles studied, and which was followed by a period of electrical silence that was longest in the flexor muscles. This type of activity has not been described previously in mature animals. In ovo movement sequences were generally initiated by extensor activity which progressively declined in duration and intensity throughout the sequence, while flexor activity progressively intensified. The onset of activity in extensor muscles was accompanied by an abrupt decrease in flexor activity, whereas the converse was not observed. Spontaneous movement sequences also occurred when the spinal cord and hind limb were isolated and maintained in oxygenated Tyrode solution for several hours. Deafferentation experiments indicated that the motor pattern in this preparation was generated centrally by circuits within the spinal cord. Activity from the isolated cord was less variable than that occurring in ovo, consisting of sequences of highly regular recurring bursts. Each burst began with a brief high-amplitude discharge that occurred synchronously in all muscles and which was similar to that observed in ovo. This was followed by a silent period, which was longest in the flexors, and then by a more prolonged burst. Although its behaviour differs from that in ovo in some respects, it is concluded that the isolated cord maintained in vitro produces a spontaneous and patterned motor output.

Action Potentials↗
PubMed1984

The activation patterns of embryonic chick motoneurones projecting to inappropriate muscles.

Chick lumbosacral motoneurones were caused to innervate foreign muscles by surgically rotating or shifting the limb bud about the anterior-posterior axis in stage 17-18 embryos. The activation pattern of such wrongly projecting motoneurones was assessed at stages 35-38 by recording electromyographic activity from muscles in an isolated spinal cord/hind limb preparation. Muscle activity was classed as flexor- or extensor-like according to the characteristics of the patterned sequence of bursts elicited by a single shock to the thoracic cord. Wrongly projecting motoneurones did not have their activation pattern altered to one appropriate for the muscle innervated; therefore in some cases a particular muscle was activated with a pattern similar to its original one, and in other cases in an opposite manner. Mixed flexor-extensor-like activation of a single muscle was, however, rare. The identity of motoneurones projecting to a muscle was determined by their cord location following retrograde labelling with horseradish peroxidase. This allowed us to conclude that motoneurones could develop their normal pattern of activation even when projecting to foreign muscles. It is concluded that the cord circuits (presumably composed of local interneurones responsible for the activation of motoneurones in the isolated cord preparation are not altered by retrograde influences from the muscle. Wrongly projecting motoneurones, which were maintained throughout the normal cell death period, were activated during spontaneous embryonic movements, and in many cases were found to have a behaviourally inappropriate activation pattern. These observations are discussed in relation to proposed mechanisms by which developmental errors in connectivity are corrected.

Action Potentials↗
PubMed1984

Pattern and specificity of axonal outgrowth following varying degrees of chick limb bud ablation.

Motoneurons grow into the chick hindlimb via consistent pathways, within which they make specific decisions leading to their correct targets. To determine which axonal guidance features dictate the position of the pathways and to examine the distribution of specific cues, we totally or partially ablated the early hindlimb bud and determined how the subsequent pattern of nerve outgrowth related to the distribution of tissue remnants. Our results suggest that local elements determine the gross anatomical pattern of outgrowth. First, determinants of individual pathways could be selectively removed without altering the pattern in other regions. Second, neurites were restricted to the plexus region at the base of the leg (within which, for unknown reasons, they proceeded posteriorly) unless distal permissive pathways or nearby target remnants were present. Finally, we found that the central region of the pelvic girdle, adjacent to the plexus region, determines the position where the major nerve trunks enter the leg. When gaps were introduced in this region of the girdle, nerves traversed the gaps and directly innervated adjacent muscle. The developing girdle is probably a nonpermissive environment for axon elongation, and axons enter the leg only where it is locally absent. Our results also support the concept that the specific cues that neurites use to reach their appropriate muscles are local. We find that neurites could make correct and specific decisions in the plexus region in the absence of all tissues distal to the pelvic girdle. This shows that the cues for these decisions are independent of the target and must reside in the local mesenchyme. In addition, when muscle remnants were present they were correctly innervated.(ABSTRACT TRUNCATED AT 250 WORDS)

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