Growth Cone Contact Research Articles

Distinct Roles of Different Presynaptic and Postsynaptic NCAM Isoforms in Early Motoneuron-Myotube Interactions Required for Functional Synapse Formation.

The neural cell adhesion molecule (NCAM) is expressed both presynaptically and postsynaptically during neuromuscular junction formation. Genetic deletion in mice of all three isoforms (180, 140, and 120 kDa), or just the 180 isoform, suggested that different isoforms played distinct roles in synaptic maturation. Here we characterized in mice of either sex the earliest adhesive contacts between the growth cones of motoneurons and myotubes and their subsequent maturation into functional synapses in cocultures of motoneurons and myotubes, which expressed their normal complement of NCAM isoforms, or were lacking all isoforms either presynaptically or postsynaptically. Growth cone contact with +/+ mouse myotubes resulted in immediate adhesive contacts and the rapid downregulation of growth cone motility. When contacting NCAM-/- myotubes, growth cones touched and retracted/collapsed multiple times and failed to form stable contacts, even after 10 h. Exogenous expression in myotubes of either the 180 or 140 isoform, but not the 120 kDa isoform, rescued the rapid formation of stable contacts, the accumulation of presynaptic and postsynaptic molecules, and functional transmission. When NCAM was absent only in motoneurons, growth cones did not retract upon myotube contact, but, since their motility was not downregulated, they grew off the ends of the myotubes, failing to form synapses. The agrin receptor Lrp4 was strongly downregulated in NCAM-negative myotubes, and motoneuron growth cones did not make stable contacts with Lrp4-negative myotubes. These studies have identified novel roles for presynaptic and postsynaptic NCAM in mediating early cell-cell interactions required for synapse formation.SIGNIFICANCE STATEMENT Although many molecular signals needed to form the functionally effective neuromuscular synapses required for normal movement have been described, the earliest signals that let motoneuron growth cones make stable adhesive contacts with myotubes and cease motility are not well understood. Using dynamic imaging of motoneuron-myotube cocultures, we show that NCAM is required on both the growth cone and myotube and that different NCAM isoforms mediate initial adhesion and the downregulation of growth cone motility. The agrin receptor Lrp4 was also essential for initial adhesive contacts and was downregulated on NCAM-/- myotubes. Our identification of novel roles for NCAM and Lrp4 and possible interactions between them in transforming motile growth cones into stable contacts opens interesting new avenues for exploration.

Comparison of sensory neuron growth cone and filopodial responses to structurally diverse aggrecan variants, in vitro

Following spinal cord injury, a regenerating neurite encounters a glial scar enriched in chondroitin sulfate proteoglycans (CSPGs), which presents a major barrier. There are two points at which a neurite makes contact with glial scar CSPGs: initially, filopodia surrounding the growth cone extend and make contact with CSPGs, then the peripheral domain of the entire growth cone makes CSPG contact. Aggrecan is a CSPG commonly used to model the effect CSPGs have on elongating or regenerating neurites. In this study, we investigated filopodia and growth cone responses to contact with structurally diverse aggrecan variants using the common stripe assay. Using time-lapse imaging with 15-s intervals, we measured growth cone area, growth cone width, growth cone length, filopodia number, total filopodia length, and the length of the longest filopodia following contact with aggrecan. Responses were measured after both filopodia and growth cone contact with five different preparations of aggrecan: two forms of aggrecan derived from bovine articular cartilage (purified and prepared using different techniques), recombinant aggrecan lacking chondroitin sulfate side chains (produced in CHO-745 cells) and two additional recombinant aggrecan preparations with varying lengths of chondroitin sulfate side chains (produced in CHO-K1 and COS-7 cells). Responses in filopodia and growth cone behavior differed between the structurally diverse aggrecan variants. Mutant CHO-745 aggrecan (lacking chondroitin sulfate chains) permitted extensive growth across the PG stripe. Filopodia contact with the CHO-745 aggrecan caused a significant increase in growth cone width and filopodia length (112.7%±4.9 and 150.9%±7.2 respectively, p<0.05), and subsequently upon growth cone contact, growth cone width remained elevated along with a reduction in filopodia number (121.9%±4.2; 72.39%±6.4, p<0.05). COS-7 derived aggrecan inhibited neurite outgrowth following growth cone contact. Filopodia contact produced an increase in growth cone area and width (126.5%±8.1; 150.3%±13.31, p<0.001), and while these parameters returned to baseline upon growth cone contact, a reduction in filopodia number and length was observed (73.94%±5.8, 75.3%±6.2, p<0.05). CHO-K1 derived aggrecan inhibited neurite outgrowth following filopodia contact, and caused an increase in growth cone area and length (157.6%±6.2; 117.0%±2.8, p<0.001). Interestingly, the two bovine articular cartilage aggrecan preparations differed in their effects on neurite outgrowth. The proprietary aggrecan (BA I, Sigma-Aldrich) inhibited neurites at the point of growth cone contact, while our chemically purified aggrecan (BA II) inhibited neurite outgrowth at the point of filopodia contact. BA I caused a reduction in growth cone width following filopodia contact (91.7%±2.5, p<0.05). Upon growth cone contact, there was a further reduction in growth cone width and area (66.4%±2.2; 75.6%±2.9; p<0.05), as well as reductions in filopodia number, total length, and max length (75.9%±5.7, p<0.05; 68.8%±6.0; 69.6%±3.5, p<0.001). Upon filopodia contact, BA II caused a significant increase in growth cone area, and reductions in filopodia number and total filopodia length (115.9%±5.4, p<0.05; 72.5%±2.7; 77.7%±3.2, p<0.001). In addition, filopodia contact with BA I caused a significant reduction in growth cone velocity (38.6nm/s±1.3 before contact, 17.1nm/s±3.6 after contact). These data showed that neuron morphology and behavior are differentially dependent upon aggrecan structure. Furthermore, the behavioral changes associated with the approaching growth cone may be predictive of inhibition or growth.

Lateral assembly of the immunoglobulin protein SynCAM 1 controls its adhesive function and instructs synapse formation

Synapses are specialized adhesion sites between neurons that are connected by protein complexes spanning the synaptic cleft. These trans-synaptic interactions can organize synapse formation, but their macromolecular properties and effects on synaptic morphology remain incompletely understood. Here, we demonstrate that the synaptic cell adhesion molecule SynCAM 1 self-assembles laterally via its extracellular, membrane-proximal immunoglobulin (Ig) domains 2 and 3. This cis oligomerization generates SynCAM oligomers with increased adhesive capacity and instructs the interactions of this molecule across the nascent and mature synaptic cleft. In immature neurons, cis assembly promotes the adhesive clustering of SynCAM 1 at new axo-dendritic contacts. Interfering with the lateral self-assembly of SynCAM 1 in differentiating neurons strongly impairs its synaptogenic activity. At later stages, the lateral oligomerization of SynCAM 1 restricts synaptic size, indicating that this adhesion molecule contributes to the structural organization of synapses. These results support that lateral interactions assemble SynCAM complexes within the synaptic cleft to promote synapse induction and modulate their structure. These findings provide novel insights into synapse development and the adhesive mechanisms of Ig superfamily members.

Microtubule and Cell Contact Dependency of ER-bound PTP1B Localization in Growth Cones

PTP1B is an ER-bound protein tyrosine phosphatase implied in the regulation of cell adhesion. Here we investigated mechanisms involved in the positioning and dynamics of PTP1B in axonal growth cones and evaluated the role of this enzyme in axons. In growth cones, PTP1B consistently localizes in the central domain, and occasionally at the peripheral region and filopodia. Live imaging of GFP-PTP1B reveals dynamic excursions of fingerlike processes within the peripheral region and filopodia. PTP1B and GFP-PTP1B colocalize with ER markers and coalign with microtubules at the peripheral region and redistribute to the base of the growth cone after treatment with nocodazole, a condition that is reversible. Growth cone contact with cellular targets is accompanied by invasion of PTP1B and stable microtubules in the peripheral region aligned with the contact axis. Functional impairment of PTP1B causes retardation of axon elongation, as well as reduction of growth cone filopodia lifetime and Src activity. Our results highlight the role of microtubules and cell contacts in the positioning of ER-bound PTP1B to the peripheral region of growth cones, which may be required for the positive role of PTP1B in axon elongation, filopodia stabilization, and Src activity.

Invariant mantling of growth cones by Schwann cell precursors characterize growing peripheral nerve fronts

Little is known about the cytoarchitecture of growth fronts in developing mammalian nerves. We report here the first quantitative, ultrastructural analysis of growth cones (GCs) and their immediate cellular and tissue environment at tips of growing nerves that are nearing their targets in fore limbs of E14 rat embryos. Schwann cell precursor (SCP) marker, p75 neurotrophin receptor, and growth cone marker, SCG10, were used to identify nerve fronts, respectively. Using confocal 3D reconstructions and immunoelectron microscopy, we found that growth cone and Schwann cell precursor migrate together at the nerve front, where growth cone contact adjacent growth cone and Schwann cell precursor with similar frequency. Schwann cell precursor are extensively connected by adherens junctions and form elaborate scaffolds that enmantle growth cone at nerve fronts, so that 80% of the nerve front surface is covered by Schwann cell precursor. Although they interdigitate in complex ways among growth cone, the total contact area between growth cone and glial membranes is remarkably constant among the 100 growth fronts analyzed. In contrast to this consistency, other growth cone contacts varied markedly from front to front such that the frequencies of GC-GC contacts are increasing proportional to their decreasing contacts with mesenchymal tissue. Thus, at the nerve front, it is the Schwann cell precursor that are most exposed to extracellular environment while forming a surprisingly invariant substrate for advancing growth cone. This study shows for the first time that Schwann cell precursor are close and consistent cellular companions of growth cone in their approach to their final targets in the developing limb and suggests a previously unappreciated role for Schwann cell precursor in growth cone advance through the limb mesenchyme.

Membrane-bound CSPG mediates growth cone outgrowth and substrate specificity by Schwann cell contact with the DRG neuron cell body and not via growth cone contact

The central nervous system and peripheral nervous system (CNS/PNS) contain factors that inhibit axon regeneration, including myelin-associated glycoprotein (MAG), the Nogo protein, and chondroitin sulfate proteoglycan (CSPG). They also contain factors that promote axon regeneration, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Axon regeneration into and within the CNS fails because the balance of factor favors inhibiting regeneration, while in the PNS, the balance of factor favors promoting regeneration. The balance of influences in the CNS can be shifted toward promoting axon regeneration by eliminating the regeneration-inhibiting factors, overwhelming them with regeneration-promoting factors, or making axon growth cones non-receptive to regeneration-inhibiting factors. The present in vitro experiments, using adult rat dorsal root ganglion (DRG) neurons, were designed to determine whether the regeneration-inhibiting influences of Schwann cell CSPG are mediated via Schwann cell membrane contact with the DRG neuron cell body or their growth cones. The average longest neurite of neurons in cell body contact with Schwann cells was 7.4-fold shorter than those of neurons without Schwann cell-neuron cell body contact (naked neurons), and the neurites showed substrate specificity, growing only on the Schwann cell membranes and not extending onto the laminin substrate. The neurites of naked neurons showed no substrate specificity and extended over the laminin substrate, as well as onto and off the Schwann cells. After digesting the Schwann cell CSPG with the enzyme C-ABC, neurons in cell body contact with Schwann cells extended neurites the same length as those of naked neurons, and their neurites showed no substrate selectivity. Further, the neurites of naked neurons were not longer than those of naked neurons not exposed to C-ABC. These data indicate that the extent of neurite outgrowth from adult rat DRG neurons and substrate specificity of their growth cone is mediated via contact between the Schwann cell membrane-bound CSPG and the DRG neuron cell body and not with their growth cones. Further, there was no apparent influence of diffusible or substrate-bound CSPG on neurite outgrowth. These results show that eliminating the CSPG of Schwann cells in contact with the cell body of DRG neurons eliminates the sensitivity of their growth cones to the CSPG-induced outgrowth inhibition. This may in turn allow the axons of these neurons to regenerate through the dorsal roots and into the spinal cord.

Axon guidance

Axon guidance

Fluorescence microscopy of calcium and synaptic vesicle dynamics during synapse formation in tissue culture.

The signal transduction process involved in the development of the nerve terminal is an intriguing question in developmental neurobiology. During the formation of the neuromuscular junction, presynaptic development is induced by growth cone's contact with the target muscle cell. Fluorescence microscopy with specific markers has made it possible to follow signalling events during this process. By using fluorescent calcium indicators, such as fura-2 and fluo-3, we found that a rise in intracellular calcium is elicited in the growth cone upon its contact with a target, and this calcium signal can also be elicited by local application of basic fibroblast growth factor. To monitor the clustering of synaptic vesicles in response to target contact, the fluorescent vesicular probe FMl-43 was used. With this probe, we observed that packets of synaptic vesicle are already present along the length of naïve neurite, which has not encountered its synaptic target. The activity-dependent loading of FMl-43 indicates that these packets can undergo exocytosis and endocytosis upon depolarization. Time-lapse recording showed that these packets are quite mobile. Upon target contact, synaptic vesicles become clustered and immobilized at the contact site. The methodology and instrumentation used in these studies are described in this article.

Guidance of CNS growth cones by substratum grooves and ridges: effects of inhibitors of the cytoskeleton, calcium channels and signal transduction pathways.

We exploited our observation that embryonic Xenopus spinal neurites align parallel to grooves in a quartz surface and that embryonic rat hippocampal neurites align perpendicular to shallow, narrow grooves (see companion paper: A. M. Rajnicek, S. Britland and C. D. McCaig, 1997) (J. Cell Sci. 110, 2905-2913) to investigate the mechanism of growth cone contact guidance. Substratum topography affected the pattern of growth cone filopodia and microtubules but parallel orientation of Xenopus neurites and perpendicular orientation of hippocampal neurites were unperturbed by cytochalasin B, which virtually eliminated filopodia. Hippocampal growth cone orientation and turning in response to grooves was unaffected by disruption of microtubules using taxol or nocodazole. Gross cytoskeletal reorganization on grooved substrata was therefore not required for growth cone steering. Inhibitors were used to identify the signal transduction pathway for perpendicular alignment of hippocampal neurites. Alignment persisted in the presence of gadolinium chloride, a blocker of stretch-activated calcium channels, the G protein inhibitor pertussis toxin, the protein tyrosine kinase inhibitor genistein, the protein kinase A and G inhibitor HA1004, the protein kinase A inhibitor KT5720and the protein kinase G inhibitor KT5823. Low concentrations of the protein kinase C inhibitors stauro-sporine, bisindolylmaleimide or H-7 did not affect perpendicular orientation but higher concentrations inhibited it. The calcium channel blockers flunarizine, nifedipine and diltiazem also inhibited perpendicular orientation. Influx of calcium and protein kinase C activity therefore appear to be involved in perpendicular contact guidance.

Role of laminin and integrin interactions in growth cone guidance.

Laminin is well known to promote neuronal adhesion and axonal growth, but recent experiments suggest laminin has a wider role in guiding axons, both in development and regeneration. In vitro experiments demonstrate that laminin can alter the rate and direction of axonal growth, even when growth cone contact with laminin is transient. Investigations focused on a single neuronal type, such as retinal ganglion cells (RGCs), strongly implicate laminin as an important guidance molecule in development and suggest the involvement of integrins. Integrins are receptors for laminin, and neurons express multiple types of integrins that bind laminin. Morphologically, integrins cluster in point contacts, specialized regions of the growth cone that may coordinately regulate adhesion and motility. Recent evidence suggests that the structure and regulation of point contacts may differ from that of their nonneuronal counterpart, focal contacts. In part, this may be because the interaction of the cytoplasmic domain of integrin with the cytoskeleton is different in point contacts and focal contracts. Mutational studies where the cytoplasmic domain is truncated or altered are leading to a better understanding of the role of the alpha and beta subunit in regulating integrin clustering and binding to the cytoskeleton. In addition, whereas integrins may regulate motility through direct physical linkages to the growth cone cytoskeleton, an equally important role is their ability to elicit signaling, both through protein tyrosine phosphorylation and modulating calcium levels. Through such mechanisms integrins likely regulate the dynamic attachment and detachment of the growth cone as it moves on laminin substrates.

Early ablation of target muscles modulates the arborisation pattern of an identified embryonic Drosophila motor axon

The Drosophila RP3 motor axon establishes a stereotypic arborisation along the adjoining edges of muscles 6 and 7 by the end of embryogenesis. The present study has examined the role of the target muscles in determining this axonal arborisation pattern. Target muscles were surgically ablated prior to the arrival of the RP3 axon. Following further development of the embryo in culture medium, the morphology of target-deprived RP3 motor axons was assayed by intracellular injection with the dye Lucifer Yellow. Axonal arborisations were formed on a variety of non-target muscles when muscles 6 and 7 were removed and these contacts were maintained into stage 16. The pattern of axonal arborisations over non-target muscles varied between preparations in terms of the number of muscles contacted, and the distribution of arborisations on individual muscles. Following removal of muscle 6, the RP3 motor axon frequently contacted muscle 7, and axonal arborisations were present along the distal edge of the muscle. In the absence of muscle 7, the RP3 axon often did not contact muscle 6 and when muscle 6 was contacted, the arborisation of RP3 was poorly developed. Axonal processes were retained on non-target muscles when only one target muscle was present. Therefore, the establishment of a stereotypic arborisation by the RP3 motor axon is apparently dependent on growth cone contact with both target muscles.

Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium‐dependent adhesion molecule, N‐cadherin

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2 mM Ca++ or 0.2 mM Ca++, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca++, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca++ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca++ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca++ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca++ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca++, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca++. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca++ levels had similar effects as lowering the Ca++ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.

Formation of electrical connections between cultured identified neurons and muscle fibers of the snail Helisoma

We studied the formation of connections between identified neurons removed from the buccal ganglion of the snail Helisoma and muscle fibers dissociated from the buccal mass. Three types of identified neurons — B19, B5, and B4 — were placed into cell culture and muscle fibers fom the supralateral tensor muscle (SLT), normally innervated by B19, were subsequently plated adjacent to the neuronal cell bodies. Growth cones from the neurons contacted the muscle fibers within 6–12 h after isolation. Simultaneous intracellular recordings from the neuronal cell bodies and muscle fibers after 4 days in culture indicated that the neurons had formed electrical connections with the fibers. All 3 types of neurons coupled to the muscle fibers but displayed differing probabilities and strengths of connections. The role of growth cone contact in the formation of these connections was tested by plating muscle fibers onto fields of neurites after neuronal growth had stopped. Under these conditions, neurons still became electrically coupled to the muscle fibers, but the strength of these connections differed from those formed by neurons and fibers that were plated simultaneously. Thus, quantitative characteristics of electrical connections formed between cultured Helisoma neurons and dissociated muscle fibers are influenced by neuronal identity and the timing of neuronal contacts.

Effect of the weaver ( wv) mutation on cerebellar neuron differentiation : I. Qualitative observations of neuron behavior in culture

A mutant gene dose-dependent inhibition of cerebellar granule cell neuron survival and neurite growth in dissociated cultures of cerebellum from 7-day-old heterozygous ( + wv ) and homozygous ( wv wv ) weaver mutant mice (M. Willinger, D. M. Margolis, and R. L. Sidman. (1981), J. Supramol. Struc. 17, 79–86) has previously been observed. In the present phase-contrast study time-lapse microcinematography was performed between 10 and 80 hr in culture to determine which properties of neurite growth and neuron migration are affected by weaver gene expression. Neurite growth in +/+ cultures is rapid and discontinuous. Neurites are thin and cylindrical. Membrane movement occurs only at the growth cone. Growth cone contact with cell aggregates or glial somas results in the cessation of cone advancement and the induction of translocation of the neuronal soma toward the astrocyte. In cultures of + wv and wv wv cerebellar cells, abnormal neurite growth is characterized by frequent neurite retractions and reinitiations. Neuronal somas and neurite shafts are motile during elongation. Homozygous neurites and cones are pleomorphic. Normal, + wv , and wv wv neurons undergo nuclear translocation. Like +/+ neurons, + wv neurons migrate in response to growth cone-cell soma contact. In contrast, homozygous soma frequently reverse direction and migrate independently of cell contact. Granule cell death occurs with increasing frequency with increasing gene dosage. Neurons are unusually active prior to the rapid onset of cell death. In summary, the weaver mutation impairs granule cell differentiation by affecting neurite maintenance, membrane motility, and neuron morphology. The loss of viability appears to be independent of, or secondary to, these targets of gene action.