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.

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.

The acquisition of motoneuron subtype identity and motor circuit formation.

Experiments in chick embryos using classical transplantation techniques introduced by Viktor Hamburger are reviewed; these demonstrated that chick-limb innervating motoneurons become specified by extrinsic signals prior to axon outgrowth and that they selectively grow to appropriate muscles by actively responding to guidance cues within the limb. More recent experiments reveal that fast/slow and flexor/extensor subclasses of motoneurons are distinct by E4-5 and that they exhibit patterned spontaneous activity while still growing to their targets. These observations are then related to the combinatorial code of LIM transcription factor expression, which has been hypothesized to specify motoneuron subtypes.

Structural and functional alterations of neuromuscular junctions in NCAM-deficient mice.

The role of neural cell adhesion molecule (NCAM) in the development and maturation of the neuromuscular junction (NMJ) was explored by characterizing structurally and functionally NMJs from postnatal day 11 (P11) to P30 +/+, +/-, and -/- NCAM null mutant mice. Differences in NCAM levels resulted in alterations in the size and shape of NMJs, with -/- NMJs being smaller. Additionally both the withdrawal of polyneuronal innervation and the selective accumulation of synaptic vesicle protein in the presynaptic terminal were delayed. These observations suggest that the bidirectional signaling responsible for these events is impaired at -/- NMJs. Functionally, miniature end plate potential size, end plate potential size, and quantal content did not differ from that of wild type under either normal or low release conditions. However at normal release conditions, -/- NMJs, unlike +/+ NMJs, lacked paired-pulse facilitation. The most striking abnormality was the inability of NCAM null junctions to maintain transmitter output with repetitive stimuli. Combined electrophysiological and FM1-43-labeling studies suggest that NCAM null junctions are unable either to dock or to mobilize a sufficient number of vesicles at high but physiological rates of transmitter release. Taken together our observations show that many aspects of transmission are normal and, thus, that many presynaptic and postsynaptic molecules have assembled properly in the absence of NCAM. However, the fact that NCAM was required for specific aspects of transmission, including paired-pulse facilitation and reliable transmission with repetitive stimuli, suggests that NCAM either is directly involved in these processes or is required for the proper organization and/or function of other molecules underlying these processes.

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.

Cholinergic and GABAergic inputs drive patterned spontaneous motoneuron activity before target contact.

Patterned spontaneous electrical activity has been demonstrated in a number of developing neural circuits and has been proposed to play a role in refining connectivity once axons reach their targets. Using an isolated spinal cord preparation, we have found that chick lumbosacral motor axons exhibit highly regular bursts of activity from embryonic day 4 (E4) (stage 24-25), shortly after they exit the spinal cord and while still en route toward their target muscles. Similar bursts could be evoked by stimulating descending pathways at cervical or thoracic levels. Unlike older embryonic cord circuits, the major excitatory transmitter driving activity was not glutamate but acetylcholine, acting primarily though nicotinic non-alpha7 receptors. The circuit driving bursting was surprisingly robust and plastic, because bursting was only transiently blocked by cholinergic antagonists, and following recovery, was now driven by GABAergic inputs. Permanent blockade of spontaneous activity was only achieved by a combination of cholinergic antagonists and bicuculline, a GABAA antagonist. The early occurrence of patterned motor activity suggests that it could be playing a role in either peripheral pathfinding or spinal cord circuit formation and maturation. Finally, the characteristic differences in burst parameters already evident between different motoneuron pools at E4 would require that the combination of transcription factors responsible for specifying pool identity to have acted even earlier.

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.

Selective fasciculation and divergent pathfinding decisions of embryonic chick motor axons projecting to fast and slow muscle regions.

Proper motor function requires the precise matching of motoneuron and muscle fiber properties. The lack of distinguishing markers for early motoneurons has made it difficult to determine whether this matching is established by selective innervation during development or later via motoneuron-muscle fiber interactions. To examine whether chick motoneurons selectively innervate regions of their target containing either fast or slow muscle fibers, we backlabeled neurons from each of these regions with lipophilic dyes. We found that motor axons projecting to fast and slow muscle regions sorted into separate but adjacent fascicles proximally in the limb, long before they reached the muscle. More distally, these fascicles made divergent pathfinding decisions to course directly to the appropriate muscle fiber region. In contrast, axons projecting to different areas of an all-fast muscle did not fasciculate separately and became more intermingled as they coursed through the limb. Selective fasciculation of fast- and slow-projecting motoneurons was similar both before and after motoneuron cell death, suggesting that motoneurons specifically recognized and fasciculated with axons growing to muscle regions containing the appropriate muscle fiber type. Taken together, these results strongly support the hypothesis that "fast" and "slow" motoneurons are molecularly distinct before target innervation and that they use these differences to selectively fasciculate, pathfind to, and branch within the correct muscle fiber region from the outset of neuromuscular development.

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).

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.

Selective innervation of fast and slow muscle regions during early chick neuromuscular development.

The electrical properties of adult motoneurons are well matched to the contractile properties of the fast or slow muscle fibers that they innervate. How this precise matching occurs developmentally is not known. To investigate whether motoneurons exhibit selectivity in innervating discrete muscle regions, containing either fast or slow muscle fibers during early neuromuscular development, we caused embryonic chick hindlimb muscles to become innervated by segmentally inappropriate motoneurons. We used the in vitro spinal cord-hindlimb preparation to identify electrophysiologically the pools of foreign motoneurons innervating the posterior iliotibialis (pITIB), an all-fast muscle, and the iliofibularis (IFIB), a partitioned muscle containing discrete fast and slow regions. The results showed that the pITIB and the fast region of the IFIB were exclusively innervated by motoneurons that normally supply fast muscles. In contrast, the slow region of the IFIB was always innervated by motoneuron pools that normally supply slow muscles. Some experimental IFIB muscles lacked a fast region and were innervated solely by "slow" motoneurons. In addition, the intramuscular nerve branching patterns were always appropriate to the fast-slow nature of the muscle (region) innervated. The selective innervation was found early in the motoneuron death period, and we found no evidence that motoneurons grew into appropriate muscle regions, but failed to form functional contacts. Together, these results support the hypothesis that different classes of motoneurons exhibit molecular differences that allow them to project selectively to, and innervate, muscle fibers of the appropriate type during early neuromuscular development.

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.

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.

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.