Synaptic plasticity: fastening synapses by adhesion.
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Animals↗
Biomedical subjects
L Landmesser
Publications and source records attributed to L Landmesser.
Polysialic acid in the vertebrate nervous system: a promoter of plasticity in cell-cell interactions.
Polysialic acid (PSA), a homopolymer attached to the neural cell adhesion molecule (NCAM), serves as a modulator of cell interactions. Polysialic acid exhibits a highly regulated expression pattern. During embryonic development its abundant expression is closely correlated with axon pathfinding and targeting, and with certain aspects of muscle formation. Its level also can be altered by synaptic activity. During neonatal development and in the adult brain, PSA expression is more restricted, being primarily associated with regions capable of morphological or physiological plasticity. The ability to perturb PSA in vivo by a specific glycosidase and by the creation of NCAM-deficient mice has led to extensive analysis of its biological function. These studies suggest that the primary role of PSA is to promote changes in cell interactions and thereby facilitate plasticity in the structure and function of the nervous system.
Animals↗
Cell adhesion/recognition molecule-mediated steps during the guidance of commissural and motor axons.
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Animals↗
Contractile activity regulates isoform expression and polysialylation of NCAM in cultured myotubes: involvement of Ca2+ and protein kinase C.
Muscle development involves a series of complex cell-cell interactions that are mediated, at least in part, by several different cell adhesion molecules. Previous work from this lab showed that the different isoforms of NCAM and its level of polysialylation are developmentally regulated during chick myogenesis in vivo and that this regulation is important for normal muscle development. Using developing chick secondary myotubes grown in culture, we show here that both the polysialylation of NCAM and the developmental switch in isoform expression are regulated by activity and that Ca2+ entry through voltage-gated channels and the subsequent activation of protein kinase C are required for the developmental changes in NCAM isoform synthesis. Specifically, PSA expression was shown to be developmentally regulated with high expression being temporally correlated with the onset of spontaneous contractile activity. Furthermore, blocking contractile activity caused a decrease in PSA expression, while increasing activity with electrical stimulation resulted in its up-regulation. Immunoblot and metabolic labeling studies indicated that dividing myoblasts synthesize primarily 145-kD NCAM, newly formed, spontaneously contracting myotubes synthesize 130-, 145-, and 155-kD NCAM isoforms, while older, more mature myotubes primarily synthesize the glycosylphosphatidylinositol-anchored 130-kD isoform which, in contrast to the other three isoforms, had a high rate of turnover. This developmental switch in NCAM isoform expression could be inhibited with Ca2+ channel blockers and inhibitors of protein kinase C. Taken together, these results suggest that Ca2+ ions and protein kinase C are involved in a second messenger cascade coupling membrane depolarization with transcriptional factors that regulate NCAM isoform synthesis and polysialylation.
Alternative Splicing↗
Polysialic acid regulates growth cone behavior during sorting of motor axons in the plexus region.
Removal of polysialic acid (PSA) from N-CAM during the time when chick motoneuron axons are segregating into target-specific fascicles at the base of the limb was previously shown to result in motoneuron projection errors. Here, it is established that these errors are associated with altered growth cone behavior in the plexus. In contrast to control embryos, in which individual axons were observed to exhibit dramatic changes in direction and extensive divergence, axonal trajectories following the removal of PSA were relatively straight. To determine whether enhanced axon-axon fasciculation following PSA removal had prevented growth cones from responding appropriately to guidance cues at the base of the limb, we also examined the role of L1, a major mediator of axon-axon fasciculation in this system. Anti-L1 reversed the effects of PSA removal on both growth cone trajectories and projection errors. These results indicate that PSA plays a permissive role, attenuating axon-axon interactions in the plexus and thereby allowing the axonal reorganization that is essential for the formation of specific motoneuron projections.
Animals↗
Axonal outgrowth and pathfinding.
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Animals↗
Regulation and activity-dependence of N-cadherin, NCAM isoforms, and polysialic acid on chick myotubes during development.
Muscle development in vivo involves a complex sequence of cell-cell interactions in which secondary myotubes first form in association with primary myotubes and subsequently separate from them. We show here that during this process N-cadherin and the different structural forms of NCAM are regulated in a pattern that involves both temporal changes in expression and localization to particular regions of the muscle cell surface. In particular, levels of N-cadherin on maturing myotubes are decreased, and the form of NCAM synthesized by the muscle changes from a transmembrane non-polysialylated to a lipid-linked polysialylated membrane protein. Moreover, while NCAM was distributed on all myotube surfaces, the polysialyated form of NCAM was restricted to regions of the myotube surface that had recently separated from neighboring cells. We previously found that blockade of nerve-induced activity by d-Tubocurarine perturbed muscle cell interactions, resulting in a failure of myotubes to separate. We now show that this activity blockade also alters adhesion molecule expression. First, N-cadherin was no longer down-regulated in maturing myotubes, and its persistence on the surfaces of mature myotubes may partly explain their failure to separate. Secondly, the developmental switch from transmembrane to lipid-linked NCAM did not occur, and polysialylated NCAM was no longer formed. As the unusual physical properties of PSA have been proposed to impede cell-cell interactions, this alteration would also be expected to compromise cell separation. Together, these results suggest that the regulated expression of both N-cadherin and NCAM isoforms including their polysialylation, is an essential mechanism for the normal separation of secondary myotubes from primary myotubes.
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Reduction of intramuscular nerve branching and synaptogenesis is correlated with decreased motoneuron survival.
Blockade of neuromuscular activity during the period of naturally occurring cell death increases intramuscular nerve branching, synaptogenesis, and survival of embryonic chicken motoneurons. These results suggested that the enhanced motoneuron survival observed might result not from increased production of a trophic factor by the target, as often suggested, but rather from the enhanced ability of motoneurons to take up this factor via additional branches and/or synapses. Since removal of polysialic acid (PSA) by PSA-specific endoneuraminidase (endo N) during the period of nerve ingrowth and motoneuron cell death had been shown to reduce intramuscular nerve branching by altering adhesive interactions between axons and myotubes, we wished to determine, first, if this resulted in a reduction of synapse formation, and second, if this was correlated with decreased motoneuron survival. When PSA was removed from embryonic day 6 (E6) to E9 by injecting endo N into the limb, the number of synapses in the posterior iliotibialis muscle was greatly reduced both at E6.5, the onset of the cell death period, and at E7.5-E8, the middle of the cell death period. Total motoneuron counts at both E7.5-E8 and E10 (close to the end of cell death period) were significantly decreased compared to controls. These results indicate that a treatment that reduces intramuscular nerve branching and synapse formation also affects motoneuron survival. Furthermore, since removal of PSA in ovo via endo N altered neither the spontaneous embryonic motility nor the activation pattern of motoneurons or muscles, these experiments avoid the complication of activity blockade that may alter motoneuron survival by affecting events other than, or in addition to, intramuscular nerve branching.
Activity Cycles↗
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.
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Polysialic acid influences specific pathfinding by avian motoneurons.
The influence of polysialic acid (PSA) on the neural cell adhesion molecule on motoneuron outgrowth and pathway formation was investigated by determining its temporal and spatial pattern of expression and by the effect that its removal had on motoneuron projection patterns. Motoneurons first expressed PSA as their growth cones began to segregate into motoneuron pool-specific groups in the plexus region; furthermore, PSA levels differed between motoneurons projecting to different targets. When PSA was removed during the period of axonal segregation in the plexus region projection errors were common. However, later removal during the process of muscle nerve formation did not result in projection errors. These results suggest that PSA modulates interactions between motoneuron axons and guidance molecules in the plexus region during axonal pathfinding.
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Temporal and spatial modulation of a cytoskeletal antigen during peripheral axonal pathfinding.
A neuron-specific cytoskeletal antigen (5E10), whose expression pattern during initial motoneuron outgrowth into the chick limb suggests that it is playing a role in axon guidance, is described. This antigen, which was shown to be a phosphorylated epitope, probably of the intermediate weight neurofilament protein (NF-M), exhibits a highly stereotyped and spatially heterogeneous pattern of expression. The point of onset of expression, which was abrupt and occurred in the distal axon and base of the growth cone, differed between groups of neurons that projected to different targets. Specifically, expression occurred from positions where previous perturbation experiments suggested that the axons in question would begin responding to specific guidance cues, and it remained high along the axon from this point to the target. Expression of this antigen could also be induced in cultured motoneurons by activating several second messenger systems.
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Polysialic acid on the surface of axons regulates patterns of normal and activity-dependent innervation.
Studies of the cell-cell adhesion molecules NCAM and L1 have indicated that their combined action is an important determinant in establishing normal patterns of muscle innervation. Moreover, they participate in activity-dependent changes in axonal sprouting. Recent findings in vivo, however, suggest that the central variable in both events is not altered NCAM or L1 expression, but rather changes in the amount of polysialic acid (PSA) at the cell surface. This finding is consistent with the proposed role of PSA as a regulator of cell-cell interactions. Because these molecular entities are present in most of the nervous system, it is likely that this mechanism can influence many aspects of axonal behavior during development and regeneration.
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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)
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Polysialic acid as a regulator of intramuscular nerve branching during embryonic development.
The role of polysialic acid (PSA) during initial innervation of chick muscle was examined. Previously, the adhesion molecules L1 and N-CAM were shown to be important in balancing axon-axon and axon-muscle adhesion during this process. Here we demonstrate developmental changes in the pattern of innervation that are not correlated with levels of L1 or N-CAM expression, but rather with the amount of PSA at the axon surface. Removal of PSA by a specific endoneuraminidase (Endo-N) increased axon fasciculation and reduced nerve branching. In contrast, the nerve trunk defasciculation and increased branching produced by neuromuscular activity blockade were associated with an increase in axonal PSA levels. Furthermore, Endo-N prevented these inactivity-induced effects on branching. Together these results illustrate the potential of PSA as a regulator of cell-cell interactions and provide a direct example of a molecular link between the morphogenic effects of adhesion-mediated and synaptic activity-dependent processes.
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Distinct roles for adhesion molecules during innervation of embryonic chick muscle.
In vitro studies have suggested that the cell adhesion molecules NCAM and G4/L1 contribute to a variety of events during neural development. We have directly tested the role played by these molecules in the process of initial nerve ingrowth and ramification in the embryonic chick iliofibularis muscle by in ovo injections of specific adhesion-blocking antibodies and analysis of the resultant nerve branching pattern in muscle whole mounts. Antibodies against both molecules produced axonal defasciculation, which resulted in an enhanced transverse projection to the fast region of the muscle. In the case of anti-G4/L1, we also observed a large increase in the number of side branches that form from nerve trunks in the slow region and an enhancement of nerve branching in the fast region. Conversely, anti-NCAM produced a striking decrease in both the number and length of side branches in the slow region, and a reduction in nerve branching in the fast region. A similar reduction of nerve branching was obtained following injection of an endosialidase, which removes sialic acid from NCAM, and which was observed to enhance fiber-fiber apposition, presumably by increasing cell adhesion. Based on their biochemical properties in vitro and their in vivo distribution, both NCAM and G4/L1 are in a position to contribute to axon-axon adhesive interactions, whereas NCAM would be expected to also promote axon-myotube interactions. Our observations in fact indicate that these two adhesion molecules play different but complementary roles during muscle innervation and, specifically, that axon-axon fasciculation is influenced by both NCAM and G4/L1 in an anatomically distinct manner to regulate the overall pattern of nerve branching and that NCAM-mediated axon-myotube interactions are necessary for the attainment of the normal stereotyped pattern of nerve branching in both fast and slow regions of this muscle.
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Distribution of fiber types in embryonic chick limb muscles innervated by foreign motoneurons.
The differentiation of distinct myotube fiber types in chick limb muscle development is coincident with innervation. The role of motoneurons in influencing fiber type differentiation was analyzed by causing chick hind limb muscles to be innervated by inappropriate motoneurons and then examining experimental muscles for changes in the distribution of myosin ATPase fiber types. Motoneuron innervation of limb muscles was altered by performing either limb shifts, limb reversals, or large spinal cord reversals on early neural tube or limb bud stage chick embryos. The distribution of fiber types was then analyzed in muscles from stage 36 (E10) to stage 45 (E20) embryos after processing hind limb sections for myosin ATPase histochemistry. In the majority of experimental muscles examined (267/312), the distribution of myosin ATPase fiber types was unaltered. In the remaining experimental muscles (14%), alterations in the distribution of myosin ATPase fiber types occurred, indicating that in some cases, foreign innervation may alter the developmental program of differentiating myotubes. The results suggest that myotubes differentiate myosin ATPase staining characteristics according to an intrinsic program and that these differentiating myotubes are selectively innervated by motoneurons of the appropriate type under most conditions including normal development. Under exceptional circumstances of motoneuron-muscle fiber type mismatch, embryonic motoneurons can alter fiber type expression.
Adenosine Triphosphatases↗
The development of hindlimb motor activity studied in the isolated spinal cord of the chick embryo.
The development of hindlimb motor activity was studied in an isolated preparation of the chick spinal cord. The motor output from lumbosacral segments was characterized by recording the pattern of ventral root and muscle nerve discharge in 6-14-d-old embryos. In addition, the synaptic drive underlying motoneuron activity was monitored electrotonically from the ventral roots. Spontaneous motor activity consisted of recurring episodes of cyclical motoneuron discharge. During development, both the number of cycles in each episode and the intensity of discharge in each cycle progressively increased. Monophasic, positive ventral root potentials accompanied each cycle of motoneuron discharge. Prior to the innervation of hindlimb muscles at stage 26, ventral root discharge was barely detectable despite the presence of large ventral root potentials. Following hindlimb muscle innervation, each cycle of activity was initiated by a brief, intense discharge that coincided with the rising phase of the ventral root potential. In embryos older than stage 30, the initial discharge was followed, after a delay, by a more prolonged discharge. The duration of ventral root potentials was shortest in the stage 26 embryos, but was similar in embryos at stage 29 and older. The developmental changes in the coordination of antagonist activity were documented by recording the pattern of discharge in sartorius (flexor) and caudilioflexorius (extensor) muscle nerves between stage 30 and stage 36. At stage 30 both sets of motoneurons were coactivated during the brief discharge that initiated each cycle. By stage 31 a second discharge occurred in each cycle. The second discharge was delayed in flexor, but not in extensor, motoneurons, which led to an alternating pattern of activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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Altered sensory projections in the chick hind limb following the early removal of motoneurons.
Chick sensory neurons grow to their correct targets in the hindlimb from the outset during normal development and following various experimental manipulations. This may result not because sensory neurons respond to specific limb-derived cues, but because they interact in some way with motoneurons which are responsive to such cues. To test this possibility, we removed the ventral part of the neural tube, which contains motoneurons and their precursors, at stages 16 1/2-20 1/2 and later examined the pathways sensory neurons had taken within the limb. Muscle nerves generally were missing or were reduced in diameter beyond the extent expected simply from the absence of motoneuron axons. In many cases, cutaneous nerves were enlarged, presumably due to the addition of other sensory axons. This result suggests that, in the absence of motoneurons, sensory neurons that normally project to muscles are unable to do so and may instead project along cutaneous pathways. Sensory axons from different segments also crossed less extensively in the plexus region than they did in control embryos, suggesting that alterations in their trajectories may normally be facilitated by similar changes in motoneuron pathways. Thus, motoneurons greatly enhance sensory neuron growth to muscles and contribute significantly toward the achievement of the normal sensory projection pattern. Sensory axons may fasciculate with motoneuron axons, or motoneuron axons may provide an aligned substrate for sensory neurons to grow along. Alternatively, motoneuron axons may alter the environment, thereby making certain pathways in the limb permissive for sensory neuron growth.
Afferent Pathways↗