Ultrastructural differences during embryonic cell death in normal and peripherally deprived ciliary ganglia.

Normally occurring neuron death and that brought about by prior removal of the peripheral target organ was studied ultrastructurally in embryonic chick ciliary ganglion in order to better understand the mechanism of cell death in this system. Before the period of cell death, all neurons in the normal ganglion developed a well-organized rough endoplasmic reticulum (RER) which coincided with peripheral synapse formation. None of the peripherally deprived neurons underwent this change, suggesting that some interaction with the periphery, possibly synapse formation, triggered them into the secretory state. Cell death in peripherally deprived neurons was signalled by nuclear changes followed by freeing of ribosomes from polysomes and RER and presumably cessation of protein synthesis. In contrast, normal cell death was brought about by dilation of the RER with eventual cytoplasmic disruption, nuclear changes appearing only secondarily. It is suggested that failure to form or maintain peripheral synapses could result in the accumulation of transmission-related proteins with consequent cisternal dilation, and eventual cell death.

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.

Fate of ganglionic synapses and ganglion cell axons during normal and induced cell death.

In order to understand the significance of cell death in the formation of neural circuits, it is necessary to determine whether before cell death neurons have (a) sent axons to the periphery; (b) reached the proper target organs; and (c) have established synaptic connections with them. Axon counts demonstrated that, after sending out initial axons, ciliary cells sprouted numerous collaterals at the time of peripheral synapse formation. Subsequently, large numbers of axons were lost from the nerves, slightly later than the onset of ganglion cell death. A secondary loss of collaterals later occurred unaccompanied by cell death. Measurements of conduction velocity and axon diameters indicated that all ganglion cell axons grew down the proper pathways from the start, but it was not possible to determine whether all axons had actually formed proper synapses. This was ascertained, however, in the ganglion itself where preganglionic fibres were shown to synapse selectively with all ganglion cells before cell death. During this period, degenerating preganglionic synapses were observed on normal cells. It can therefore be inferred that at least some preganglionics established proper synapses before dying and that a single synapse is not sufficient to prevent cell death. In this system neither preganglionic nor ganglionic cell death seems designed to remove improper connections but rather to remove cells that have not competed effectively for a sufficient number of synapses, resulting in a quantitative matching up of neuron numbers.

Size and shape of transverse tubule openings in frog twitch muscle fibers.

The openings of transverse tubules in frog twitch fibers are described. The tubules open to the extracellular space by a narrow neck, with an inner diameter of 20 nm. The most peripheral portion of the tubules is tortuous and has a variable diameter. The similarity in size of the openings of T tubules and caveolae and the meandering path of the tubules are sufficient to account for the paucity of observed openings.

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.

Axotomy mimicked by localized colchicine application.

Comparable depression of synaptic transmission in the avian ciliary ganglion resulted from either section or localized colchicine treatment of the ciliary nerves. Both colchicine treatment and axotomny produced similar changes in RNA distribution in the cell bodies as well. Colchicine did not directly affect transmission, and action potential propagation along the ciliary nerves was normal. Interference with axoplasmic transport of material in both cases is postulated to signal the observed chromatolytic changes.

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.

Axonin-1, Nr-CAM, and Ng-CAM play different roles in the in vivo guidance of chick commissural neurons.

Immunoglobulin/fibronectin type III-like cell adhesion molecules have been implicated in axon pathfinding based on their expression pattern in the developing nervous system and on their complex interactions described in vitro. The present in vivo study demonstrates that interactions by two of these molecules, axonin-1 on commissural growth cones and Nr-CAM on floor plate cells, are required for accurate pathfinding at the midline. When axonin-1 or Nr-CAM interactions were perturbed, many commissural axons failed to cross the midline and turned instead along the ipsilateral floor plate border. In contrast, though perturbation of Ng-CAM produced a defasciculation of the commissural neurites, it did not affect their guidance across the floor plate.

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.

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.

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.

Alterations in transmission, vesicle dynamics, and transmitter release machinery at NCAM-deficient neuromuscular junctions.

Although functional neuromuscular junctions (NMJs) form in NCAM-deficient mice, they exhibit multiple alterations in presynaptic organization and function. Profound depression and unusual periodic total transmission failures with repetitive stimulation point to a defect in vesicle mobilization/cycling, and these defects were mimicked in (+/+) NMJs by inhibitors of myosin light chain kinase, known to affect vesicle mobilization. Two separate release mechanisms, utilizing different endocytic machinery and Ca(2+) channels, were shown to coexist in (-/-) terminals, with the mature process targeted to presynaptic membrane opposed to muscle, and an abnormally retained immature process targeted to the remainder of the presynaptic terminal and axon. Thus, NCAM plays a critical and heretofore unsuspected role in the molecular organization of the presynaptic NMJ.

Interference with axonin-1 and NrCAM interactions unmasks a floor-plate activity inhibitory for commissural axons.

Axonin-1 and NrCAM were previously shown to be involved in the in vivo guidance of commissural growth cones across the floor plate of the embryonic chicken spinal cord. To further characterize their role in axon pathfinding, we developed a two-dimensional coculture system of commissural and floor-plate explants in which it was possible to study the behavior of growth cones upon floor-plate contact. Although commissural axons readily entered the floor plate under control conditions, perturbations of either axonin-1 or NrCAM interactions prevented the growth cones from entering the floor-plate explants. The presence of antiaxonin-1 resulted in the collapse of commissural growth cones upon contact with the floor plate. The perturbation of NrCAM interactions also resulted in an avoidance of the floor plate, but without inducing growth-cone collapse. Therefore, axonin-1 and NrCAM are crucial for the contact-mediated interaction between commissural growth cones and the floor plate, which in turn is required for the proper guidance of the axons across the ventral midline and their subsequent rostral turn into the longitudinal axis.

Synaptic plasticity: keeping synapses under control.

Recent studies have confirmed that a retrograde signal is produced at the neuromuscular junction that can adjust the efficacy of transmission to meet long-term changing needs. Genetic manipulations in Drosophila have begun to define the circumstances in which such signals are generated and how they act.

Site-directed antibodies to low-voltage-activated calcium channel CaV3.3 (alpha1I) subunit also target neural cell adhesion molecule-180.

Synthetic peptides of defined amino acid sequence are commonly used as unique antigens for production of antibodies to more complex target proteins. We previously showed that an affinity-purified, site-directed polyclonal antibody (CW90) raised against a peptide antigen (CNGRMPNIAKDVFTKM) anticipated to be specific to a T-type voltage-dependent Ca(2+) channel subunit identified recombinant rat alpha1I/Ca(V)3.3 and two endogenous mouse proteins distinct in their developmental expression and apparent molecular mass (neonatal form 260 kDa, mature form 190 kDa) [Yunker AM, Sharp AH, Sundarraj S, Ranganathan V, Copeland TD, McEnery MW (2003) Immunological characterization of T-type voltage-dependent calcium channel Ca(V)3.1 (alpha 1G) and Ca(V)3.3 (alpha 1I) isoforms reveal differences in their localization, expression, and neural development. Neuroscience 117:321-335]. In the present study, we further characterize the biochemical properties of the CW90 antigens. We show for the first time that recombinant alpha1I/Ca(V)3.3 is modified by N-glycosylation. Using peptide:N-glycosidase F (PNGase F), an enzyme that removes polysaccharides attached at Asn residues, and endoneuraminidase-N (Endo-N), which specifically removes polysialic acid modifications, we reveal that differential glycosylation fully accounts for the large difference in apparent molecular mass between neonatal and adult CW90 antigens and that the neonatal form is polysialylated. As very few proteins are substrates for Endo-N, we carried out extensive analyses and herein present evidence that CW90 reacts with recombinant alpha1I/Ca(V)3.3 as well as endogenous neural cell adhesion molecule-180 (NCAM-180). We demonstrate the basis for CW90 cross-reactivity is a five amino acid epitope (AKDVF) present in both alpha1I/Ca(V)3.3 and NCAM-180. To extend these findings, we introduce a novel polyclonal anti-peptide antibody (CW678) that uniquely recognizes NCAM-180 and a new antibody (CW109) against alpha1I/Ca(V)3.3. Western blot analyses obtained with CW678, CW109 and CW90 on a variety of samples confirm that the endogenous CW90 signals are fully attributed to the two developmental forms of NCAM-180. Using CW678, we present novel data on differentiation-dependent NCAM-180 expression in human neuroblastoma IMR32 cells. These results strongly suggest the need for careful analyses to validate anti-peptide antibodies when targeting membrane proteins of low abundance.

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.

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.

Axon guidance at choice points.

The common theme in many recent axonal pathfinding studies, both in vertebrates and invertebrates, is the demonstration of the importance of a balance between positive and negative cues. The integration of multiple and often opposing molecular interactions at each site along the axon's trajectory, especially at choice points, helps to fine tune the directional response of its growth cone, which continuously samples its environment for guidance cues. The dynamic regulation of the receptors for such cues, in response to extrinsic signals, also enhances the behavioral repertoire of growth cones at different points along their trajectory. Some of the molecules identified as being important for axon guidance at choice points are conserved between invertebrates and vertebrates (e.g. Robo and netrin), whereas other molecules have been identified, so far, only in invertebrates (e.g. Comm) or vertebrates (e.g. axonin-1 and NrCAM).

The pattern of avian intramuscular nerve branching is determined by the innervating motoneuron and its level of polysialic acid.

Most skeletal muscles are composed of a heterogeneous population of fast and slow muscle fibers that are selectively innervated during development by fast and slow motoneurons, respectively. It is well recognized that, in both birds and mammals, fast and slow motoneurons have substantially different intramuscular branching patterns, a difference critical for proper motor function. However, the cellular mechanisms regulating these differences in motoneuron branching are unknown. In a previous study, we showed that the fast and slow pattern of intramuscular branching, in a chick muscle containing distinct fast and slow muscle regions, was remarkably similar to normal when formed by foreign motoneurons. Whether this was attributable to some property of the innervating "fast" or "slow" motoneurons or to some property of the developing fast-slow muscle fibers was not determined. To distinguish between these two possibilities, we performed chick-quail hindlimb chimeras to force slow chick plantaris motoneurons to innervate a fast quail plantaris muscle. The pattern of intramuscular nerve branching in the fast plantaris of these chimeras closely resembled the slow branching pattern normally observed in chick slow plantaris muscles. Enzymatic removal of polysialic acid (PSA) from nerve and muscle during normal quail plantaris development dramatically changed the normal fast pattern to more closely resemble a slow pattern. In contrast, removal of PSA from chick plantaris motoneurons and muscle fibers had little effect on the pattern of nerve branching. Together, these results indicate that the pattern of intramuscular nerve branching is determined by the level of PSA on the innervating motoneurons.

Neuromuscular activity blockade induced by muscimol and d-tubocurarine differentially affects the survival of embryonic chick motoneurons.

To understand better how spontaneous motoneuron activity and intramuscular nerve branching influence motoneuron survival, we chronically treated chicken embryos in ovo with either d-tubocurarine (dTC) or muscimol during the naturally occurring cell death period, assessing their effects on activity by in ovo motility measurement and muscle nerve recordings from isolated spinal cord preparations. Because muscimol, a GABA(A) agonist, blocked both spontaneous motoneuron bursting and that elicited by descending input but did not rescue motoneurons, we conclude that spontaneous bursting activity is not required for the process of normal motoneuron cell death. dTC, which rescues motoneurons and blocks neuromuscular transmission, blocked neither spontaneous nor descending input-elicited bursting and early in the cell death period actually increased burst amplitude. These changes in motoneuron activation could alter the uptake of trophic molecules or gene transcription via altered patterns of [Ca(2+)](i), which in turn could affect motoneuron survival directly or indirectly by altering intramuscular nerve branching. A good correlation was found between nerve branching and motoneuron survival under various experimental conditions: (1) dTC, but not muscimol, greatly increased branching; (2) the removal of PSA from NCAM partially reversed the effects of dTC on both branching and survival, indicating that branching is a critical variable influencing motoneuron survival; (3) muscimol, applied with dTC, prevented the effect of dTC on survival and motoneuron bursting and, to a large extent, its effect on branching. However, the central effects of dTC also appear to be important, because muscimol, which prevented motoneuron activity in the presence of dTC, also prevented the dTC-induced rescue of motoneurons.