Rac1-dependent actin filament organization in growth cones is necessary for β1-integrin–mediated advance but not for growth on poly-D-lysine

The activity of filopodia and lamellipodia determines the advance, motility, adhesion, and sensory capacity of neuronal growth cones. The shape and dynamics of these highly motile structures originate from the continuous reorganization of the actin cytoskeleton in response to extracellular signals. The small GTPases, Rac1, Rho, and CDC42, regulate the organization of actin filament structures in nonneuronal cells; yet, their role in growth cone motility and neurite outgrowth is poorly understood. We investigated in vitro the function of Rac1 in neurite outgrowth and differentiation by introducing purified recombinant mutants of Rac1 into primary chick embryo motor neurons via trituration. Endogenous Rac1 was expressed in growth cone bodies as well as in the tips and shafts of filopodia, where it often colocalized with actin filament structures. The introduction of constitutively active Rac1 resulted in an increase in rhodamine–phalloidin staining, presumably from an accumulation of actin filaments in growth cones, while dominant negative Rac1 caused a decrease in rhodamine–phalloidin staining. Nevertheless, both Rac1 mutants retarded growth cone advance, and hence attenuated neurite outgrowth and inhibited differentiation of neurites into axons and dendrites on laminin and fibronectin. In contrast, on poly-D -lysine, neither Rac1 mutant affected growth cone advance, neurite outgrowth, or neurite differentiation despite inducing similar changes in the amount of rhodamine–phalloidin staining in growth cones. Our data demonstrate that Rac1 regulates actin filament organization in neuronal growth cones and is pivotal for β1 integrin–mediated growth cone advance, but not for growth on poly-D lysine. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 524–540, 1998     

Sprouting and functional regeneration of an identified serotonergic neuron following axotomy

An identified serotonergic neuron (C1) in the cerebral ganglion of Helisoma trivolvis sprouts following axotomy and rapidly (seven to eight days) regenerates to recover its regulation of feeding motor output from neurons of the buccal ganglia. The morphologies of normal and regenerated neurons C1 were compared. Intracellular injection of the fluorescent dye, Lucifer Yellow, into neuron C1 was compared with serotonin immunofluorescent staining of the cerebral and buccal ganglia. The two techniques revealed different and complimentary representations of the morphology of neuron C1. Lucifer Yellow provided optimal staining of the soma, major axon branches, and dendritic arborization. Immunocytochemical staining revealed terminal axon branches on distant targets and showed an extensive plexus of fine fibers in the sheaths of ganglia and nerve trunks. In addition to C1, serotonin-like immunoreactivity was localized in approximately 30 other neurons in each of the paired cerebral ganglia. Only cerebral neurons C1 had axons projecting to the buccal ganglia. No neuronal somata in the buccal ganglia displayed serotonin-like immunoreactivity. Observations of regenerating neurons C1 demonstrated: Actively growing neurites, both in situ and in cell culture, displayed serotonin-like immunoreactivity; severed distal axons of C1 retained serotonin-like immunoreactivity for up to 28 days; axotomized neurons C1 regenerated to restore functional control over the feeding motor program.     

The role of endogenous heparan sulfate proteoglycan in adhesion and neurite outgrowth from dorsal root ganglia

Some phases of dorsal root ganglion (DRG) substratum attachment and growth cone morphology are mediated through endogenous cell surface heparan sulfate proteoglycan. The adhesive behavior of intact embryonic chicken DRG (spinal sensory ganglia) is examined on substrata coated with fibronectin, fibronectin treated with antibody to the cell-binding site (anti-CBS), and the heparan sulfate-binding protein platelet factor four. DRG attach to fibronectin, anti-CBS-treated fibronectin, and platelet factor four. The ganglia extend an extensive halo of unfasciculated neurites on fibronectin and produce fasciculated neurite outgrowth on platelet factor four and anti-CBS antibody-treated FN. Treatment with heparinase, but not chondroitinase, abolishes adhesion to fibronectin and platelet factor four. Growth cones of DRG on fibronectin have well-spread lamellae and microspikes. On platelet factor four, and anti-CBS-treated FN, growth cones exhibit microspikes only. Isolated Schwann cells adhere equally well to fibronectin and platelet factor four, spreading more rapidly on fibronectin. Isolated DRG neurons adhere equally well on both substrata, but only 10% of the neurons extend long neurites on platelet factor four. The majority of the isolated neurons on platelet factor four exhibit persistent microspike production resembling that of the early stages of normal neurite extension. Endogenous heparan sulfate proteoglycan supports the adhesion of whole DRG, isolated DRG neurons, and Schwann cells, as well as extensive microspike activity by DRG neurons, one important part of growth cone activity.     

The regulation of neurite outgrowth, growth cone motility, and electrical synaptogenesis by serotonin

Identified neurons of the buccal ganglion of the snail Helisoma when isolated from their ganglionic environment and plated in cell culture grow new neurites that are tipped with motile growth cones. Addition of the neurotransmitter serotonin to the culture medium surrounding actively growing neurons causes an immediate, premature cessation of neurite elongation in specific identified neurons. Serotonin selectively inhibits neurite extension of neurons B19 and P5 while having no effect on the extension of neuron B5. Coincident with the serotonin evoked inhibition of neurite elongation is an inhibition of growth cone motile activities and a retraction of growth cone filopodia and lamellipodia. One site of serotonin's growth inhibitory actions is directly at the growth cone rather than at the neurites or cell body. A second area of this study concerns connectivity. In Helisoma neurons the formation of electrical synaptic connections critically relies on both potential partner neurons having a mutual interaction of actively growing neurites. Neurons in a nongrowing state do not form electrical synapses (Hadley et al., 1983). As a result of inhibiting neurite extension, serotonin is able to affect synaptogenesis by preventing certain neurons (neurons B19) from forming electrical synaptic connections with other neurons (neurons B5) that are themselves competent to interconnect. Thus, by inhibiting neurite extension, serotonin is capable of regulating both the development of arborizations and the formation of connectivity.     

The differential regulation of formation of chemical and electrical connections in Helisoma

Novel chemical and electrical connections form between neurons not normally connected in the buccal ganglia of the snail Helisoma. We examined the cellular and environmental conditions required for the formation of each type of connection. Previous work in situ showed that novel electrical connections could form in response to axotomy. We have now found that axotomy can evoke the formation of novel unidirectional chemical connections between neurons B5 and B4 in addition to a novel electrical connection. The novel chemical connections display all of the normal properties of chemical synapses in Helisoma ganglia. These connections, however, are transient in nature and break 4 days following axotomy. Previous work has shown that conjoint outgrowth is required for the formation of electrical connections. In cell culture we have investigated whether conjoint outgrowth is also required for chemical synaptogenesis. Using neurons B5 and B19 we have found that when neuron pairs make contact in cell culture, under conditions of synchronous neurite extension, both electrical and chemical synapses form. However, if one neuron has ceased extension prior to contact by a growing neuron, electrical synapses never form (Hadley et al., 1983, 1985) but chemical synapses do form. Furthermore, the addition of serotonin (10(-6) M) to culture medium to inhibit neurite extension of B19, but not that of B5, selectively prevents the formation of electrical connections while permitting the formation of chemical synapses. Thus, the timing of contact in relation to the state of neurite extension can specify the type of connection a given neuron can form.     

Cell adhesion molecules NgCAM and axonin-1 form heterodimers in the neuronal membrane and cooperate in neurite outgrowth promotion

The axonal surface glycoproteins neuronglia cell adhesion molecule (NgCAM) and axonin-1 promote cell-cell adhesion, neurite outgrowth and fasciculation, and are involved in growth cone guidance. A direct binding between NgCAM and axonin-1 has been demonstrated using isolated molecules conjugated to the surface of fluorescent microspheres. By expressing NgCAM and axonin-1 in myeloma cells and performing cell aggregation assays, we found that NgCAM and axonin-1 cannot bind when present on the surface of different cells. In contrast, the cocapping of axonin-1 upon antibody-induced capping of NgCAM on the surface of CV- 1 cells coexpressing NgCAM and axonin-1 and the selective chemical cross-linking of the two molecules in low density cultures of dorsal root ganglia neurons indicated a specific and direct binding of axonin- 1 and Ng-CAM in the plane of the same membrane. Suppression of the axonin-1 translation by antisense oligonucleotides prevented neurite outgrowth in dissociated dorsal root ganglia neurons cultured on an NgCAM substratum, indicating that neurite outgrowth on NgCAM substratum requires axonin-1. Based on these and previous results, which implicated NgCAM as the neuronal receptor involved in neurite outgrowth on NgCAM substratum, we concluded that neurite outgrowth on an NgCAM substratum depends on two essential interactions of growth cone NgCAM: a trans-interaction with substratum NgCAM and a cis-interaction with axonin-1 residing in the same growth cone membrane.     

Roles of 5-HT on phase transition of neurite outgrowth in the identified serotoninergic neuron C1, <Emphasis Type="Italic">Helisoma trivolvis</Emphasis>

Neurite outgrowth is a morphological marker of neuronal differentiation and neuroregeneration, and the process includes four essential phases, namely initiation, elongation, guidance and cessation. Intrinsic and extrinsic signaling molecules seem to involve morphological changes of neurite outgrowth via various cellular signaling cascades phase transition. Although mechanisms associated with neurite outgrowth have been studied extensively, little is known about how phase transition is regulated during neurite outgrowth. 5-HT has long been studied with regard to its relationship to neurite outgrowth in invertebrate and vertebrate culture systems, and many studies have suggested 5-HT inhibits neurite elongation and growth cone motility, in particular, at the growing parts of neurite such as growth cones and filopodia. However, the underlying mechanisms need to be investigated. In this study, we investigated roles of 5-HT on neurite outgrowth using single serotonergic neurons C1 isolated from Helisoma trivolvis. We observed that 5-HT delayed phase transitions from initiation to elongation of neurite outgrowth. This study for the first time demonstrated that 5-HT has a critical role in phase-controlling mechanisms of neurite outgrowth in neuronal cell cultures.     

Neuron-specific modulation by serotonin of regenerative outgrowth and intracellular calcium within the CNS of Helisoma trivolvis

We have investigated the cell-specific effect of serotonin (5-HT) on regenerating neurons within the adult central nervous system of the pond snail, Helisoma trivolvis. In culture, 5-HT arrests outgrowth of buccal neurons B19 but not neurons B5 (Haydon, McCobb, and Kater, 1984). After axotomy, neurons within the Helisoma nervous system typically exhibit profuse regenerative outgrowth. This study, on neurons within the CNS, shows that 5-HT selectively inhibits the outgrowth of specific identified neurons, and also causes significant elevations in intracellular calcium concentrations as measured by the calcium indicator dye, Fura-2. The outgrowth of neurons B19 and C1 was selectively inhibited when ganglia were incubated in 5 X 10(-5) M 5-HT. The outgrowth of buccal neurons B5, however, was not affected. Moreover, 5-HT caused significant transient elevations of calcium concentrations in neurons B19 over 30 minutes, but neurons B5 did not show any increases in calcium concentrations with the addition of 5-HT. These results suggest that the effect of 5-HT upon outgrowth of regenerating neurons may be due to an increase in the intracellular calcium concentration.     

Laminin-like immunoreactivity in the snail Helisoma: involvement of approximately 300 kD extracellular matrix protein in promoting outgrowth from identified neurons

Polyclonal antibodies directed against laminin (LM), and against the A and B chains of reduced LM were used to identify antigenically related proteins in the extracellular matrix (ECM) of the snail Helisoma trivolvis. Immunofluorescence of snail central ganglionic rings using either the anti-LM or anti-B chain antibodies labeled the ECM within ganglionic sheaths as well as basal laminae surrounding the ganglia. Both the anti-LM and anti-B chain antibodies recognized a prominent, approximately 300-kD protein on immunoblots of a snail central ganglion preparation enriched in ECM components. The anti-A chain antibody failed to label any structures in sections of snail ganglia or to recognize any proteins on immunoblots of ganglionic ECM. A polyclonal antibody was raised against the approximately 300-kD snail protein. Immunofluorescence of snail ganglia with the anti- approximately 300-kD antibody gave a distribution of labeled structures comparable to that obtained with the anti-LM antibody. Immunofluorescent labeling of sections of snail muscle and salivary gland with the anti- approximately 300-kD antibody revealed a distribution of reactive protein characteristic of an ECM component. Probing immunoblots of ganglionic ECM with the anti- approximately 300-kD antibody revealed the recognition of the same approximately 300-kD protein as identified by the anti-LM antibodies. Media conditioned by Helisoma central ganglionic rings (CM) contains an unidentified neurite outgrowth promoting factor (NOPF). Immunoblots of CM probed with the anti-B chain and anti- approximately 300-kD antibodies reveal the recognition of a soluble approximately 300-kD protein similar to the approximately 300-kD protein identified in snail ECM. The ganglionic ECM preparation containing the approximately 300-kD protein supported outgrowth from cultured snail buccal neurons B5, and addition of anti- approximately 300-kD Fab fragments to CM abolished its outgrowth promoting activity. These results suggest that the approximately 300-kD ECM protein may be the NOPF in CM and/or functions in promoting neurite outgrowth.     

Cdc42 stimulates neurite outgrowth and formation of growth cone filopodia and lamellipodia

To assess the role of cdc42 during neurite development, cmyc-tagged constitutively active (CA) and dominant negative (DN) cdc42 were expressed in dissociated primary chick spinal cord neurons using adenoviral-mediated gene transfer. Three days after infection, >85% of the neurons in infected cultures expressed cdc42 proteins, as detected by indirect immunofluorescence against cmyc. Growth cones of infected neurons displayed 1.83- (CAcdc42) and 1.93-fold (DNcdc42) higher cmyc immunofluorescence per square micrometer than uninfected controls. CAcdc42 expression stimulated growth cones, almost doubling growth cone size and number of filopodia, and increased neurite growth rates by 65-89%. In neurons plated onto fibronectin, the percent of growth cones with both filopodia and lamellipodia increased from 71 to 92%. Total Texas Red-phalloidin staining in these growth cones doubled, and the percent of growth cones with F-actin localized to peripheral regions increased from 52% in controls to 78% after CAcdc42 expression. Expression of DNcdc42 did not significantly alter growth cone morphology or neurite growth rates. Addition of soluble laminin to spinal cord neurons resulted in the identical phenotype as CAcdc42 expression, including changes in growth cone morphology, F-actin localization, and neurite growth rates. Significantly, expression of DNcdc42 blocked the effects of laminin on growth cones. These results show that cdc42 promotes neurite outgrowth and filopodial and lamellipodial formation in growth cones and suggests that cdc42 and laminin share a common signaling pathway during neurite development. Addition of laminin to CAcdc42-expressing neurons is inhibitory to growth cones, indicating that laminin also may activate some other pathways.     

Phenotypic properties of adult mouse intrinsic cardiac neurons maintained in culture

Intrinsic cardiac neurons are core elements of a complex neural network that serves as an important integrative center for regulation of cardiac function. Although mouse models are used frequently in cardiovascular research, very little is known about mouse intrinsic cardiac neurons. Accordingly, we have dissociated neurons from adult mouse heart, maintained these cells in culture, and defined their basic phenotypic properties. Neurons in culture were primarily unipolar, and 89% had prominent neurite outgrowth after 3 days (longest neurite length of 258 +/- 20 microm, n = 140). Many neurites formed close appositions with other neurons and nonneuronal cells. Neurite outgrowth was drastically reduced when neurons were kept in culture with a majority of nonneural cells eliminated. This finding suggests that nonneuronal cells release molecules that support neurite outgrowth. All neurons in coculture showed immunoreactivity for a full complement of cholinergic markers, but about 21% also stained for tyrosine hydroxylase, as observed previously in sections of intrinsic cardiac ganglia from mice and humans. Whole cell patch-clamp recordings demonstrated that these neurons have voltage-activated sodium current that is blocked by tetrodotoxin and that neurons exhibit phasic or accommodating patterns of action potential firing during a depolarizing current pulse. Several neurons exhibited a fast inward current mediated by nicotinic ACh receptors. Collectively, this work shows that neurons from adult mouse heart can be maintained in culture and exhibit appropriate phenotypic properties. Accordingly, these cultures provide a viable model for evaluating the physiology, pharmacology, and trophic factor sensitivity of adult mouse cardiac parasympathetic neurons.     

Influence of ganglion age,nonneuronal cells and substratum on neurite outgrowth in culture

This study characterizes the outgrowth patterns of superior cervical ganglia (SCG) obtained from embryonic (E15), perinatal (E20–21), and adult (P35) rats when placed in culture on various substrata. Outgrowth morphology, degree of fasciculation, and outgrowth length were examined on collagen (COL), polyornithine (PO), polylysine (PL), fibronectin (FN), and nonneuronal cells (NNCs) from the ganglion. COL and FN supported extensive neuritic outgrowth; PO and PL provided poor support. Outgrowth pattern, degree of fasciculation, neurite growth rate, and the number of NNCs in the outgrowth varied considerably depending upon the COL configuration. When undiluted COL (～5 mg/ml) was air dried, a three-dimensional loose fibrillar network was formed. Upon COL dilution or gelling undiluted COL by ammoniation, an essentially two-dimensional layer was formed. On two-dimensional COL, NNCs were able to proliferate and migrate extensively from ganglia of all ages; their presence influenced the form and extent of neurite growth. E15, E20, and P35 neurites responded differently to their endogenous NNCs. E15 neurites extended in relation to NNC surfaces and were predominantly nonfasciculated. E20 neurites became more fasciculated in the presence of NNCs that exhibited morphological and behavioral differences from those migrating from E15 ganglia. E20 neurite bundles became defasciculated when they extended into E15 outgrowth. Far fewer neurites grew from P35 explants in the presence of their NNCs. Three-dimensional COL greatly slowed NNC migration and thus allowed investigation of neurite outgrowth from ganglia of differing age in the absence of NNCs. We conclude that neuritic outgrowth patterns on varying substrata reflect not only neurite differences depending upon ganglion age but also variation in the behavior of accompanying NNCs.     

Novel effects of serotonin on neurite outgrowth in neurons cultured from embryos of Helisoma trivolvis

The neurotransmitter serotonin has been shown to inhibit neurite outgrowth in specific identified neurons isolated from adult Helisoma. While in vivo experiments on Helisoma embryos have supported the hypothesis that endogenous serotonin regulates neurite outgrowth during embryonic development, direct effects of serotonin on embryonic neurons have not been measured. In the present study, cultures of dissociated embryonic neurons were used to test the direct actions of serotonin on developing embryonic neurons. Serotonin arrested neurite outgrowth in a significant percentage of elongating neurites in a dose-dependent manner. Furthermore, analysis of neurons with stable, nonelongating neurites revealed a novel response. Serotonin caused the reinitiation of neurite outgrowth in a significant percentage of nonelongating neurites. The arrestment of outgrowth and reinitiation of outgrowth occurred in similar percentages of elongating and nonelongating neurites, respectively. Parallel experiments on cultures of dissociated adult neurons were carried out to determine whether serotonin could also induce both inhibitory and stimulatory responses in adult cells. Serotonin arrested neurite outgrowth in a similar percentage of neurites to that observed in cultures of embryonic neurons. In contrast, serotonin did not reinitiate neurite outgrowth in a significant percentage of adult neurites. These data support the hypothesis that serotonin regulates neurite outgrowth in developing embryonic neurons. Furthermore, only some of these regulatory effects appear to be conserved from embryonic to adult neurons.     

Cultured sympathetic neurons: Effects of cell-derived and synthetic substrata on survival and development

The long-term culture of dissociated rat sympathetic neurons requires strong adhesion of the neuronal processes to the culture substratum. A variety of artificial and cell-derived substrata was examined for their effects on the survival, neurite outgrowth, and neurotransmitter development of these neurons. Compared to dried collagen films, both three-dimensional hydrated collagen gels and surfaces coated with basic polymers provided a substratum highly adherent for developing neurons. Polylysine and polyornithine were most suitable for long-term culture when covalently linked with glutaraldehyde to an underlying layer of dried gelatin. Dissociated neurons also attached strongly to a substratum of killed nonneuronal cells fixed by paraformaldehyde, heat, ethanol, or trichloroacetic acid. In addition, an extracellular, substrate-associated material apparently produced by nonneuronal cells (rat cardiac myocytes and associated fibroblasts) promoted the long-term adhesion of growing neurites. The adhesive property of this microexudate was sensitive to trypsin, periodate, and alkali, but resistant to hyaluronidase, chondroitinase, 8 M urea, and 0.5 M acetic acid. Similar characteristics have been reported for fibronectin, an extracellular glycoprotein produced by many cells and cell lines. This protein, or one with similar features, may function in vivo in the extension and guidance of neuronal fibers. The choice and development of neurotransmitter function were unaffected by the various substrata tested, with one exception. Nonneuronal cells fixed with paraformaldehyde caused a significant induction of cholinergic properties similar to that seen with nonneuronal conditioned medium.     

Calcium transients in growth cones and axons of cultured Helisoma neurons in response to conditioning factors

Accumulating evidence indicates that cytosolic calcium levels regulate growth cone motility and neurite extension. The purpose of this study was to determine if intracellular calcium levels also influence the initiation of neurite extension induced by growth-promoting factors. An in vitro preparation of axotomized neurons that can be maintained in the absence of growth-promoting factors was utilized. The distal axons of cultured Helisoma neurons plated into defined medium do not extend neurites until they are exposed to Helisoma brain-conditioned medium. This provided the opportunity to study the intracellular changes associated with neurite extension. Cytosolic calcium levels were monitored with the calcium-sensitive dye fura 2 at the distal axon. In control medium calcium levels in the distal axon were constant. However, transient elevations in cytosolic calcium in the axonal growth cone occurred after addition of conditioned medium and coincident with the initiation of neurite extension. Application of calcium channel blockers showed that the transients resulted from calcium influx across the neuronal membrane. The transients, however, were not required for neurite extension, although they did influence the rate and extent of neurite outgrowth. Simultaneous extracellular patch recordings demonstrated that the calcium transients were correlated temporally with an increase in rhythmic spontaneous electrical activity of cells, suggesting that conditioned medium influences ionic membrane properties of these neurons. © 1995 John Wiley & Sons, Inc.     

Ouabain binding to preimplantation rabbit blastocysts

Ciliary ganglia (CG) from 8-day-old chick embryos were cultured as explants on a highly adhesive collagen substratum in the presence of the ciliary neuronotrophic factor (CNTF). A remarkable correlation was found between the formation of an outgrowth of ganglionic nonneuronal cells and the timing and extent of neuritic development outside the ganglion. Neurites were not seen to emerge from the ganglion before the onset (24 hr after explantation) of a nonneuronal cell outgrowth. After nonneurons began to migrate over the collagen substratum, neurites could be seen to extend up to, but not beyond the distal limit of the nonneuronal outgrowth. Time-lapse analysis showed that neuritic growth cones could move in synchrony with a nonneuron with which they were in contact as well as over the nonneuronal cell surface, but not on the collagen located distally to the external edge of the nonneuronal outgrowth.Freshly dissected CGs were also grown as secondary explants on preformed host monolayers of ganglionic nonneurons. These secondary explants showed considerable neuritic development within 24 hr, while control ganglia explanted on collagen had not produced neurites. Autoradiographic experiments indicated that this neuritic outgrowth occurred on nonneuronal cells emerging precociously from the secondary explant, rather than on the preexisting host nonneurons. Electron microscopy of 24-hr explants demonstrated that, inside the ganglion, neurites were also very closely associated with the surface of nonneuronal cells.Neuritic behavior in this nonneuron/collagen terrain is compared with previously described observations of CG explants on polyornithine (PORN) or dissociated CG neurons on PORN or collagen. These observations led to the identification of a PORN-bindable neurite promoting factor (PNPF) which does not bind to, and is not active on, collagen. The hypothesis is discussed that PNPF molecules are present on the surface of nonneuronal cells and that the cells owe to those molecules their competence as a suitable terrain for the elongation of neuritic processes.     

GAP-43 phosphorylation is dynamically regulated in individual growth cones

In vivo, kinase C phosphorylation of the growth-associated protein GAP-43 is spatially and temproally associated with the proximity of growing axons to their targets. Here we have used dissociated dorsal root ganglia (DRG)s and an antibody specific for the phosphorylated form of GAP-43 to demonstrate that neurite regeneration in culture also begins in the absence of detectable levels of phosphorylated GAP-43. Since the β isoform of kinase C was found to be enriched in growth cones before stably phosphorylated GAP-43 was detected, it may normally be inactive during initial neurite outgrowth; however, premature phosphorylation of GAP-43 could be stimulated in newly dissociated DRGs by plating them on cultures in which phosphorylation had already been initiated; media conditioned by such cultures caused no response suggesting an effect of either cell-cell or cell-substrate contact. Increased GAP-43 phosphorylation correlated with a reduced extent of neurite outgrowth but not with the rate at which individual growth cones translocated so that motile growth cones contained very low levels of phosphorylated GAP-43, whereas stationary growth cones showed much more immunoreactivity. Downregulation of kinase C by phorbol ester prevented increased GAP-43 phosphorylation and led to growth cone collapse. Finally, phosphorylated GAP-43 was found to be differently distributed within growth cones. Increased immunoreactivity was frequently observed in the neck of the growth cone and was heterogeneously distributed in lamellae and filopodia. These results, which demonstrate the dynamic regulation of GAP-43 phosphorylation in individual growth cones, are discussed with reference to the association between changes in growth cone shape and the ability to translocate and change direction. © 1992 John Wiley & Sons, Inc.     

Growth cone interactions with a glial cell line from embryonic Xenopus retina

We have isolated a nonneuronal cell line from Xenopus retinal neuroepithelium (XR1 cell line). On the basis of immunocytochemical characterization using monoclonal antibodies generated in our laboratory as well as several other glial-specific antibodies, we have established that the XR1 cells are derived from embryonic astroglia. A monolayer of XR1 cells serves as an excellent substrate upon which embryonic retinal explants attach and elaborate neurites. This neurite outgrowth promoting activity appears not to be secreted into the medium, as medium conditioned by XR1 cells is ineffective in promoting outgrowth. Cell-free substrates were prepared to examine whether outgrowth promoting activity is also associated with the XR1 extracellular matrix (ECM). Substrates derived from XR1 cells grown on collagen are still capable of promoting outgrowth following osmotic shock and chemical extraction. This activity does not appear to be associated with laminin or fibronectin. Scanning electron microscopy was used to examine growth cones of retinal axons on XR1 cells and other substrates that supported neurite outgrowth. Growth cones and neurites growing on a monolayer of XR1 cells, or on collagen conditioned by XR1 cells, closely resemble the growth cones of retinal ganglion cells in vivo. A polyclonal antiserum (NOB1) generated against XR1 cells effectively and specifically inhibits neurite outgrowth on XR1-conditioned collagen. We therefore propose that neurite outgrowth promoting factors produced by these cells are associated with the extracellular matrix and may be glial specific.     

Capzb2 Interacts with β-Tubulin to Regulate Growth Cone Morphology and Neurite Outgrowth

Capping protein (CP) is a heterodimer that regulates actin assembly by binding to the barbed end of F-actin. In cultured nonneuronal cells, each CP subunit plays a critical role in the organization and dynamics of lamellipodia and filopodia. Mutations in either α or β CP subunit result in retinal degeneration in Drosophila. However, the function of CP subunits in mammalian neurons remains unclear. Here, we investigate the role of the β CP subunit expressed in the brain, Capzb2, in growth cone morphology and neurite outgrowth. We found that silencing Capzb2 in hippocampal neurons resulted in short neurites and misshapen growth cones in which microtubules overgrew into the periphery and completely overlapped with F-actin. In searching for the mechanisms underlying these cytoskeletal abnormalities, we identified β-tubulin as a novel binding partner of Capzb2 and demonstrated that Capzb2 decreases the rate and the extent of tubulin polymerization in vitro. We mapped the region of Capzb2 that was required for the subunit to interact with β-tubulin and inhibit microtubule polymerization. A mutant Capzb2 lacking this region was able to bind F-actin and form a CP heterodimer with α2-subunit. However, this mutant was unable to rescue the growth cone and neurite outgrowth phenotypes caused by Capzb2 knockdown. Together, these data suggest that Capzb2 plays an important role in growth cone formation and neurite outgrowth and that the underlying mechanism may involve direct interaction between Capzb2 and microtubules.     

Absence of persistent spreading, branching, and adhesion in GAP-43- depleted growth cones

The growth-associated protein GAP-43 is a major protein kinase C substrate of growth cones and developing nerve terminals. In the growth cone, it accumulates near the plasma membrane, where it associates with the cortical cytoskeleton and membranes. The role of GAP-43 in neurite outgrowth is not yet clear, but recent findings suggest that it may be a crucial competence factor in this process. To define the role of GAP- 43 in growth cone activity, we have analyzed neurite outgrowth and growth cone activity in primary sensory neurons depleted of GAP-43 by a specific antisense oligonucleotide procedure. Under optimal culture conditions, but in the absence of GAP-43, growth cones adhered poorly, displayed highly dynamic but unstable lamellar extensions, and were strikingly devoid of local f-actin concentrations. Upon stimulation, they failed to produce NGF-induced spreading or insulin-like growth factor-1-induced branching, whereas growth factor-induced phosphotyrosine immunoreactivity and acceleration of neurite elongation were not impaired. Unlike their GAP-43-expressing counterparts, they readily retracted when exposed to inhibitory central nervous system myelin-derived liposomes. Frequency and extent of induced retraction were attenuated by NGF. Our results indicate that GAP-43 can promote f- actin accumulation, evoked morphogenic activity, and resistance to retraction of the growth cone, suggesting that it may promote regulated neurite outgrowth during development and regeneration.