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PubMed1989

Growth cone-growth cone interactions in cultures of rat sympathetic neurons.

Growth cones of sympathetic neurons from the superior cervical ganglia of neonatal rats were studied using video-microscopy to determine events following contact between growth cones and other cell surfaces, including other growth cones and neurites. A variety of behaviors were observed to occur upon contact between growth cones. Most commonly, one growth cone would collapse and subsequently retract upon establishing filopodial contact with the growth cone of another sympathetic neuron. Contacts resulting in collapse and retraction were often accompanied by a rapid and transient burst of lamellipodial activity along the neurite 30-50 microns proximal to the retracting growth cone. In no instances did interactions between growth cones and either fibroblasts or red blood cells result in the growth cone collapsing, suggesting that a specific recognition event was involved. On several occasions, growth cones were seen to track other growth cones, although fasciculation was rare. In some cases, there was no obvious response between contacting growth cones. Growth cone-growth cone contact was almost four times more likely to result in collapse and retraction than was growth cone-neurite contact (45% vs 12%, respectively). These observations suggest that the superior cervical ganglion may be composed of neurons with different cell surface determinants and that these determinants are more concentrated on the surface of growth cones than on neurites. These results further suggest that contact-mediated inhibition of growth cone locomotion may play an important role in growth cone guidance.

Animals↗

PubMed1999

Quantitative estimation of F-actin in single growth cones.

Growth cones at distal ends of elongating neurites are characterized by a bunch of motile filopodia. Filamentous actin (F-actin) is the supporting cytoskeletal structure of growth cone filopodia. Normal growth cone motility requires balanced polymerization and depolymerization rates of F-actin. If this balance is disturbed, growth cone shape is altered and extension may fail. Image acquisition by confocal scanning microscopy was used as a very efficient tool to optically isolate single growth cones from the rest of the cell to study morphological and physiological behavior. The relative F-actin content (r.a.c.) of a single growth cone area was defined as a parameter describing different growth cone states. To estimate r.a.c., a double-labeling technique was applied. F-actin was selectively labeled by fluorescent rhodamine-conjugated phalloidin and total protein was unspecifically labeled by 5-(4, 6-dichlorotriazin-2-yl)aminofluorescein (DTAF). The r.a.c. was calculated by rationing and averaging digitized rhodamine and DTAF fluorescence of single growth cone areas. Subsequently, r.a.c. was used as a numeric descriptor of the variable F-actin underlying morphological structures of growth cones. The method allowed an analysis of local changes in growth cone morphology measured as a change in F-actin due to signaling events. It can be used to quantify ligand-receptor effects at subcellular areas of intact cells.

Actins↗

PubMed1999

Organization of point contacts in neuronal growth cones.

Growth cones from rat dorsal root ganglia plated on laminin contain integrin clusters over the entire growth cone surface, and growth cones make transient adhesions at sites called point contacts. We examined, by immunocytochemistry and confocal microscopy, the composition and distribution of point contacts in neuronal growth cones. Vinculin was concentrated in the central domain of growth cones and at the tips of filopodia. Vinculin was specifically associated with integrin clusters at the membrane-substrate interface and thus marked point contacts. The cytoskeletal proteins paxillin and talin colocalized with beta1 integrin in a subpopulation of clusters restricted to the central domain of the growth cone and to the tips of filopodia. The neuron-specific kinase, FAK+ also distributed with the vinculin-positive clusters. The Rho family proteins RhoA, RhoB, and Cdc42 were present in growth cones, and a few Rho clusters were colocalized with vinculin. Examination of proteins resistant to detergent extraction in PC12 cells confirmed the retention of beta1 integrin, paxillin, talin, and vinculin with the cytoskeleton. Moreover, we detected FAK+ and RhoA in the detergent-resistant cytoskeleton, supporting their distribution to point contacts. Our observations indicate that two types of integrin clusters are present in growth cones: those associated with vinculin at the cell substratum interface, and those not associated with vinculin. Point contacts are mature adhesion sites defined by the presence of both beta1 integrin and vinculin, and they are associated with signaling proteins.

Animals↗

PubMed1985

Analysis of microspike movements on the neuronal growth cone.

Growth cones of chick sensory ganglion neurons in tissue culture were photographed at 60-sec intervals as they advanced over the substratum. Numbers of microspikes (or "filopodia") were recorded together with the time and position of their appearance, their rate of elongation, their lateral movements, their lifespan, and the position and manner of their disappearance. All microspikes go through cycles of extension, lateral movement, and shortening. These are irregular and unpredictable but show systematic differences depending on where on the growth cone they occur. At the leading edge of the growth cone microspike extension occurs at highest frequency and microspike shortening occurs at the lowest frequency; when the latter occurs in this region it often involves the advance of the margin of the cell in the form of a lamellipodium. Microspike loss occurs most often at the base of the growth cone, usually by the retraction of the microspike into the cell. Calculations of the gain and loss of microspikes at different regions of the growth cone show that they undergo a net retrograde flow, the rate of which is correlated with the forward advance of the growth cone. Individual microspikes can also move backward from the growth cone onto the axon (or "neurite"), an event that occurs most often on adhesive substrata. Our observations support a direct role of microspike movement in the advance of the growth cone. The primary force for axonal elongation appears to be the contraction of microspikes pulling the leading margin of the growth cone forward. At more proximal and peripheral regions of the growth cone, microspikes undergo a retrograde sweeping motion, followed by retraction into the cell, which may also contribute to the forward movement of the growth cone. We interpret these movements as arising from a flow of actin filaments and associated proteins which are incorporated into microspikes and lamellipodia at the leading edge of the growth cone, passing backward, and being deposited into the actin-rich membrane-associated cortex of the axonal cylinder.

Actins↗

PubMed1996

L1, N-cadherin, and laminin induce distinct distribution patterns of cytoskeletal elements in growth cones.

Growth cones are highly motile extensions of growing neuronal processes that have a well-characterized cytoskeleton, which is necessary for motility and directed growth. In culture, neurons have been observed to extend processes on a variety of substrates made of cell adhesion molecules (CAMs) or extracellular matrix molecules (ECM molecules). We have previously shown that retinal ganglion cell (RGC) growth cones exhibit characteristic morphologies dependent on the substrate on which they are grown (Payne et al., 1992: Cell Motil. Cytoskel. 21:65-73). Upon contact with a sharp border between two substrates, the growth cones display rapid changes in morphology (Burden-Gulley et al., 1995: J. Neurosci. 15:4370-4381) that may result from extensive restructuring of the cytoskeleton. In the present study, immunocytochemical methods were used to examine the distribution of three cytoskeletal elements in RGC growth cones growing on L1, N-cadherin, or laminin as well as on dishes coated with alternating lanes of these substrates. Distinct distribution patterns of f-actin, microtubules (MTs), and neurofilaments (NFs) were observed in growth cones growing on individual substrates. At border regions between two substrates, growth cones with extensive lamellipodial contact with the second substrate were observed to have f-actin and MT distribution patterns appropriate for the new substrate encountered. Contact via filopodia alone did not evoke this change. Redistribution of NFs was observed only after the majority of the growth cone had crossed onto the second substrate. These results suggest that actin and MTs, but probably not NFs, are directly influenced by CAMs and ECM molecules to produce changes in growth cone morphology. The distribution of two members of the protein tyrosine kinase family, pp60arc and p59fyn, and phosphorylated tyrosine residues was also examined. No differences were observed in the distribution patterns of the kinases and phosphorylated tyrosine residues in growth cones on any of the substrate molecules tested. In addition, the distribution patterns were unchanged in growth cones that contacted and crossed borders between two substrates. These results suggest that redistribution of pp60arc or p59fyn is not required to produce alterations in growth cone morphology induced by contact with L1, N-cadherin or laminin.

Actins↗

PubMed1991

Control of microtubule assembly in growth cones.

Growth cones are transient structures present at the tips of growing axons and dendrites (neurites). They are crucial to neuronal development because of their pathfinding ability and their role in synaptogenesis (reviewed by Dodd and Jessell, 1988). In the last few years it has become apparent that growth cones are also involved in the assembly of the cytoskeleton of the elongating neurite (reviewed by Gordon-Weeks, 1989, 1991). We are particularly interested in the assembly of microtubules in the growth cone and its importance for neurite advance. The microtubules in the neurite are bundled into fascicles, presumably by the cross-linking action of microtubule-associated proteins (MAPs), and on entering the growth cone they splay out like the ribs of a fan with their 'plus' ends, the ends at which assembly takes place preferentially, oriented distally. Within the growth cone there is a large pool of assembly competent tubulin that provides subunits for microtubule elongation. Several observations point to the existence of precise mechanisms controlling assembly of this soluble tubulin pool. If the control mechanisms are disrupted the ability of the neurite to advance is severely compromised. We have examined the possibility that MAPs are important components in this control mechanism. Many of the known MAPs are present within growth cones, including MAP 1B, MAP 2 and tau. Experiments with neuronal cultures and growth cones isolated as a subcellular fraction from developing rat brain point particularly toward the phosphorylated form of MAP 1B as an essential component in the concerted assembly of microtubules at the growth cone and in particular in the bundling of microtubules in the neurite.

Animals↗

PubMed1987

Recycling of plasmalemma in chick tectal growth cones.

Growth cones from freeze-substituted intact chick optic tectum were analyzed in serial reconstructions of thin-section electron micrographs in order to determine which internal organelles might contribute membrane for plasmalemmal expansion. These growth cones contain numerous stacked and single lumenless membrane-limited disks; the stacks are arrays of single disks interconnected, and possibly organized, by intervening electron-dense cross-links. The single and stacked disks together account for 80% of the total intracellular membrane in the growth cones. Single disks frequently lie close to and occasionally contact the filopodial plasmalemma; regularly spaced electron-dense cross-links also occur at these juxtapositions between single disks and the plasmalemma. Some of the juxtaposed disk membranes contact the growth cone plasmalemma, and images of some of these contacts appear to indicate fusion of the disk membrane with the plasmalemma. When excised optic tecta are exposed to cationized ferritin for various times, ferritin micelles appear sequentially in coated pits, coated vesicles, smooth vesicles, vacuoles, and then in stacked and single disks, including some of those contacting the plasmalemma. Because the cytoplasmic disks filled only at the longest times after exposure to ferritin, the membrane continuities between the disks and the plasmalemma are thought to be indicative of exocytosis rather than endocytosis. We propose, therefore, that components of the plasma membrane are recycled through the stacks of lumenless disks in the chick tectal growth cones; the disks therefore represent a pool of internal membrane waiting to be added to the growth cone plasmalemma that could be used for filopodial extension or neuritic extension.

Animals↗

PubMed1996

Clustering and functional cooperation of Ng-CAM and axonin-1 in the substratum-contact area of growth cones.

Growth cones and neurites of chicken dorsal root ganglia neurons cultured on laminin, Ng-CAM, or axonin-1 exhibit substratum-dependent morphology and growth patterns which are accompanied by distinctive distributions of axonin-1 and Ng-CAM in the growth cone membrane. On either Ng-CAM or axonin-1 substratum, both Ng-CAM and axonin-1 were depleted from some areas of the apical growth cone membrane. In contrast, on laminin, both axonin-1 and Ng-CAM remained randomly distributed. Removal of axonin-1 from growth cones resulted in a blockage of neurite outgrowth on both Ng-CAM and axonin-1 substrata, indicating that in these neurons axonin-1 cooperates with Ng-CAM in the activation of axon growth. Based on these results possible molecular models for cooperation between axonin-1 and Ng-CAM on the growth cone are discussed.

Animals↗

PubMed1987

Calcium current in growth balls from isolated Helix aspersa neuronal growth cones.

Growth cones were severed from their neurites in primary cultures of Helix aspersa neurons. Following isolation, growth cones rolled up into 5-10-micron-diameter spheres, which remained attached to a poly-L-lysine or lectin-coated glass coverslip. Whole-cell-configuration patch-clamp recordings from isolated growth cones revealed inward calcium currents upon block of outward currents with internally perfused CsCl. Up to 50 microM tetrodotoxin did not affect this current. In 20-micron-diameter spheres, a peak current of 1.2 nA was reached within 3 ms under voltage-clamp conditions for a 60-mV pulse from a holding potential of -50 mV. Channel density calculations averaged to approximately one channel per square micrometer. A two-phase inactivation was evident under voltage-clamp steps from -50 mV to +15 mV. The growth balls described can be internally perfused and voltage clamped to measure ionic currents involved in growth cone function.

Action Potentials↗

PubMed2000

Identification of an invariant response: stable contact with schwann cells induces veil extension in sensory growth cones.

Growth cones sense cues by filopodial contact, but how their motility is altered by contact remains unclear. Although contact could alter motile dynamics in complex ways, our analysis shows that stable contact with Schwann cells induces motility changes that are remarkably discrete and invariant. Filopodial contact invariably induces local veil extension. Even when contacts are brief, veils always extend before the filopodia retract. Contact at filopodial tips suffices for induction. Moreover, veils extend significantly sooner than on filopodia contacting laminin, which often detach without extending veils. The overall behavioral responses of the growth cone, such as increased area and turning, result from integrating multiple discrete responses. Cycles of veil induction enlarge the growth cone and often lead it onto the cell. Invariant veil induction is abolished by blocking N-cadherin signaling. We propose an axonal guidance model in which different guidance cues act by inducing different but discrete and invariant responses.

Animals↗

PubMed1998

Distribution of phosphorylated GAP-43 (neuromodulin) in growth cones directly reflects growth cone behavior.

Phosphorylation of GAP-43 (neuromodulin) by protein kinase C (PKC) occurs at a single site, serine41. In vivo, phosphorylation is induced after initiation of axonogenesis and is confined to distal axons and growth cones. Within individual growth cones, phosphorylation is nonuniformly distributed. Here, we have used high-resolution video-enhanced microscopy of cultured dorsal root ganglia neurons together with immunocytochemistry with a monoclonal antibody that recognizes PKC-phosphorylated GAP-43 to correlate the distribution of phosphorylated GAP-43 with growth cone behavior. In "quiescent," nontranslocating growth cones, phosphorylated GAP-43 was confined to the proximal neurite and the central organelle-rich region, and was low in organelle-poor lamellae. However, levels in lamellae were elevated when they became motile. Conversely, levels of phosphorylated GAP-43 were low in either lamellae that were actively retracting or in the central organelle-rich region and proximal neurite of growth cones that had totally collapsed. The results suggest a mechanism whereby phosphorylation of GAP-43 by PKC, potentially in response to extracellular signals, could direct the functional behavior of the growth cone.

Animals↗

PubMed1989

Developmental changes in the calcium dependency of gamma-aminobutyric acid release from isolated growth cones: correlation with growth cone morphology.

We have investigated the development of Ca2+-dependent gamma-[3H]aminobutyric acid [( 3H]GABA) release in superfused growth cone fractions isolated from rats between the postnatal ages of 1 and 11 days. We have compared this release with the overall morphology of the subcellular fractions, and identified those structures taking up [3H]GABA by electron microscopical autoradiography. In fractions isolated from rats between 1 and 5 days, K+-evoked [3H]GABA release was completely independent of extracellular Ca2+. After 5 days a Ca2+ dependency appeared, which increased with age, such that by 10 days approximately 50% of the K+-evoked release was Ca2+ dependent. Electron microscopical analysis showed that, at all ages, large numbers of GABAergic growth cones were present in the subcellular fractions. Up to postnatal day 5, the growth cones were synaptic vesicle sparse but, after this age, increasing numbers of synaptic vesicle-containing growth cones were seen. These results suggest that during maturation of GABAergic growth cones into synapses there is, initially, a mechanism for release that is independent of extracellular Ca2+ and that the appearance of a Ca2+-dependent [3H]GABA release from growth cones correlates with the appearance of synaptic vesicles.

Aging↗

PubMed1992

Biochemical characterization of nerve growth cones isolated from both fetal and neonatal rat forebrains: the growth cone particle fraction mainly consists of axonal growth cones in both stages.

Nerve growth cones are responsible for the exact pathway finding, and for the establishment of neurocytoarchitecture. To elucidate the developmental changes of biochemical characteristics of nerve growth cones, growth cone particle (GCP) fractions were isolated biochemically from embryonal day 17 (E17) rat forebrain and from postnatal day 5 (P5). There were no significant differences in protein phosphorylation pattern in a Ca(2+)-dependent manner between E17-GCP fraction and that of P5. As for the membrane lipid composition, molar ratios of cholesterol to total phospholipids were well conserved during these ages. The immunoreactivity to anti-synaptophysin monoclonal antibody as a marker of mature synaptic elements could not be detected either in E17-GCP or P5-GCP fractions. To exclude the possibility of the contamination of dendritic elements, RNA contents and immunoreactivity to anti-high molecular weight microtubule-associated protein 2 (MAP2) monoclonal antibody were examined. RNA contents of the GCP fractions were extremely low compared to those of other subcellular fractions both in E17 and P5. No immunoreactivities to anti-MAP2 antibody were observed in either GCP fraction. Our results suggest that the GCP fractions, isolated from forebrains of E17 to P5 rat, are free from the contamination of the synaptic elements, and that the GCP fractions are mainly composed of axonal growth cones.

Aging↗

PubMed1995

Characterization of spontaneous calcium transients in nerve growth cones and their effect on growth cone migration.

This study examines the mechanisms of spontaneous and induced [Ca2+]i spiking in nerve growth cones and the effect of spikes on growth cone migration. Over a 10-20 min observation period, 29% of DRG growth cones undergo spontaneous and transient elevations in physiological extracellular Ca2+ ((Ca2+)o; 2 mM), whereas 67% of growth cones exposed to 20 mM (Ca2+)o exhibit similar [Ca2+]i spikes. Spontaneous [Ca2+]i spiking was not observed in neuronal cell bodies or nonneuronal cells. Ca2+ influx through non-voltage-gated Ca2+ channels was required for spontaneous [Ca2+]i spikes in growth cones, since removal of (Ca2+)o, or addition of the general Ca2+ channel blockers La3+ or Ni2+, reversibly blocked [Ca2+]i spiking, while blockers of the voltage-gated Ca2+ channels did not. Experiments using agents that influence intracellular Ca2+ stores suggest that Ca2+ stores may buffer and release Ca2+ during growth cone [Ca2+]i spikes. Growth cone migration was immediately and transiently inhibited by [Ca2+]i spikes, but eventually returned to prespike rates.

Animals↗

PubMed1999

En passant synaptic varicosities form directly from growth cones by transient cessation of growth cone advance but not of actin-based motility.

Formation of terminal synapses at sites such as the neuromuscular junction involves transformation of the motile growth cone into the nonmotile synaptic terminal. However, transformation does not need to be the mechanism when a neurite forms multiple widely spaced synaptic varicosities along a target in an en passant configuration. Synaptic varicosities could form here by specialization of the neurite after the growth cone has advanced past the site. We examined this issue by using cocultures of identified sensory (SN) and motor (L7) neurons from Aplysia. Living SNs were labeled with fluorescent dye and their neurites were observed at high resolution every few minutes growing along the axon of L7, allowing a fine-grained analysis of the behavior of the growth cone at the sites of synapse formation. All varicosities whose formation was observed indeed developed from the growth cone. Sensory varicosities were shown by electron microscopy to contain features characteristic of active zones for transmitter release within a day of their formation on the motor axon. Growth cone advance slowed or stopped transiently during varicosity formation, but the motile activity of the peripheral region of the growth cone (veils and filopodia) was maintained. These results suggest that target "stop signals" involved in the formation of synapses, at least of the en passant variety, may be of a different type from the growth inhibitory molecules, such as the collapsins, which guide axons to their targets.

Actins↗

PubMed1997

Selective changes in cell bodies and growth cones of nerve growth factor-differentiated PC12 cells induced by chemical hypoxia.

Cytosolic free Ca2+ concentration ([Ca2+]i) was measured in differentiated PC12 cells to test whether chemical hypoxia selectively alters intracellular Ca2+ in growth cones and cell bodies. Hypoxia increased [Ca2+]i and exaggerated its response to K+ depolarization in both parts of the cells. [Ca2+]i in the cell bodies was greater than that in the growth cones under resting conditions and in response to K+ or hypoxia. Ca2+-channel blockers selectively altered these responses. The L-channel blocker nifedipine reduced [Ca2+]i following K+ depolarization by 67% in the cell bodies but only 25% in the growth cones. In contrast, the N-channel blocker omega-conotoxin GVIA (omega-CgTX) diminished K+-induced changes in [Ca2+]i only in the growth cones. During hypoxia, nifedipine was more effective in the cell bodies than in the growth cones. During hypoxia, omega-CgTX diminished K+-induced changes by 50-75% in both parts of the cell, but only immediately after depolarization. The combination of nifedipine and omega-CgTX diminished the [Ca2+]i response to K+ with or without hypoxia by >90% in the cell body and 70% in the growth cones. Thus, the increased Ca2+ entry with K+ during hypoxia is primarily through L channels in the cell bodies, whereas in growth cones influx through L and N channels is about equal. The results show that chemical hypoxia selectively alters Ca2+ regulation in the growth cone and cell body of the same cell.

Animals↗

PubMed1989

The role of cytoskeleton in organizing growth cones: a microfilament-associated growth cone component depends upon microtubules for its localization.

We are interested in the relationship between the cytoskeleton and the organization of polarized cell morphology. We show here that the growth cones of hippocampal neurons in culture are specifically stained by a monoclonal antibody called 13H9. In other systems, the antigen recognized by 13H9 is associated with marginal bands of chicken erythrocytes and shows properties of both microtubule-and microfilament-associated proteins (Birgbauer, E., and F. Solomon. 1989 J. Cell Biol. 109:1609-1620). This dual nature is manifest in hippocampal neurons as well. At early stages after plating, the antibody stains the circumferential lamellipodia that mediate initial cell spreading. As processes emerge, 13H9 staining is heavily concentrated in the distal regions of growth cones, particularly in lamellipodial fans. In these cells, the 13H9 staining is complementary to the localization of assembled microtubules. It colocalizes partially, but not entirely, with phalloidin staining of assembled actin. Incubation with nocodazole rapidly induces microtubule depolymerization, which proceeds in the distal-to-proximal direction in the processes. At the same time, a rapid and dramatic redistribution of the 13H9 staining occurs; it delocalizes along the axon shaft, becoming clearly distinct from the phalloidin staining and always remaining distal to the receding front of assembled microtubules. After longer times without assembled microtubules, no staining of 13H9 can be detected. Removal of the nocodazole allows the microtubules to reform, in an ordered proximal-to-distal fashion. The 13H9 immunoreactivity also reappears, but only in the growth cones, not in any intermediate positions along the axon, and only after the reformation of microtubules is complete. The results indicate that the antigen recognized by 13H9 is highly concentrated in growth cones, closely associated with polymerized actin, and that its proper localization depends upon intact microtubules.

Actin Cytoskeleton↗

PubMed1999

Effects of roundabout on growth cone dynamics, filopodial length, and growth cone morphology at the midline and throughout the neuropile.

roundabout (robo) encodes an axon guidance receptor that controls midline crossing in the Drosophila CNS. In robo mutants, axons that normally project ipsilaterally can cross and recross the midline. Growth cones expressing Robo are believed to be repelled from the midline by the interaction of Robo and its ligand Slit, an extracellular protein expressed by the midline glia. To help understand the cellular basis for the midline repulsion mediated by Robo, we used time-lapse observations to compare the growth cone behavior of the ipsilaterally projecting motorneuron RP2 in robo and wild-type embyros. In wild-type embryos, filopodia can project across the midline but are quickly retracted. In robo mutants, medial filopodia can remain extended for longer periods and can develop into contralateral branches. In many cases RP2 produces both ipsilateral and contralateral branches, both of which can extend into the periphery. The growth cone also exhibits longer filopodia and more extensive branching both at the midline and throughout the neuropile. Cell injections in fixed stage 13 embryos confirmed and quantified these results for both RP2 and the interneuron pCC. The results suggest that Robo both repels growth cones at the midline and inhibits branching throughout the neuropile by promoting filopodial retraction.

Animals↗