Effects of fasciculation on the outgrowth of neurites from spinal ganglia in culture

This report describes the influence of neurite fasciculation on two aspects of nerve growth from chick spinal ganglia in vitro: the inhibition of outgrowth by high concentrations of nerve growth factor (NGF) and the preferential growth of neurites toward a capillary tube containing NGF. These studies involved a comparison of cultures of single cells, cell aggregates, and intact ganglia and the use of antibodies against the nerve cell adhesion molecule (CAM) to perturb fasciculation under a variety of conditions. The inhibition of outgrowth, which was observed with ganglia and aggregates but not with single cells, was correlated with a thickening of neurite fascicles. In accord with this observation, anti-CAM, which diminishes fasciculation by inhibiting side-to-side interactions between individual neurites, also partially reversed the inhibition of neurite outgrowth at high NGF concentrations. On the basis of these and other studies, we consider the possibility that neurite bundling causes an increase in the elastic tension of a fascicle without a compensatory increase in its adhesion to substratum. It is proposed that this imbalance could inhibit neurites from growing out from a ganglion and even result in retraction of preexisting outgrowth. In the analysis of NGF-directed growth, it was found that a capillary source of NGF produced a steep but transient NGF gradient that subsided before most neurites had emerged from the ganglion. Nevertheless, the presence of a single NGF capillary caused a dramatic and persistent asymmetry in the outgrowth of neurites from ganglia or cell aggregates. In contrast, processes of individual cells did not appear to orient themselves toward the capillary. The most revealing finding was that anti-CAM antibodies caused a decrease in the asymmetry of neurite outgrowth. These results suggest that side-to-side interactions among neurites can influence the guidance of nerve bundles by sustaining and amplifying an initial directional signal.     

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.     

Studies on the action of nerve growth factor. III. Role of RNA and protein synthesis in the process of neurite outgrowth.

Chick embryo dorsal root and sympathetic ganglia cultured on untreated tissue culture plates exhibited a dependence upon both RNA and protein synthesis for the expression of nerve growth factor-mediated neurite outgrowth. Neurite outgrowth was no longer dependent upon RNA synthesis, but remained dependent upon continued protein synthesis when ganglia were cultured in plasma clots, or on either collagen or poly-l-lysine coated plates. Nerve growth factor-induced neurite outgrowth was dependent upon the presence of either microexudates, which may play an important role as functional components of the substratum across which neurites migrate, or exogenous substrata such as collagen, fibrin, or poly-l-lysine.     

Promotion of retinal neurite outgrowth by substratum-bound fibronectin

We have examined conditions under which aggregates of embryonic chick neural retina will extend neurities in vitro. Trypsin-dispersed cells from 7-day embryonic chick neural retina were aggregated in rotation culture for 8 hr and maintained in serum-free medium on a variety of standard culture substrate. Aggregates extend few neurites on untreated plastic, glass, or collagen substrata. However, pretreatment of these substrata with human plasma fibronectin enhances their capacity to support retinal neurite outgrowth. Aggregates cultured on fibronectin-treated substrata extend long, radially oriented neurites within 36 hr in vitro. The morphology of these neurites is distinct from that seen when aggregates are cultured on polylysine-treated substrata. In the latter case, neurites are highly branched and grow concentrically around the aggregate perimeter. Addition of fibronectin to polylysine-treated substrata stimulates radial neurite outgrowth. Promotion of neurite outgrowth is dependent on the amount of fibronectin bound to the culture substratum and on the pH at which binding occurs. The requirements for fibronectin-mediated neurite outgrowth are more stringent than those previously reported for fibroblast attachment and spreading.     

Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG).

Sulfated proteoglycans (PGs) may play a significant role in the regulation of neurite outgrowth. They are present in axon-free regions of the developing nervous system and repel elongating neurites in a concentration-dependent manner in vitro. The addition of growth-promoting molecules, such as laminin, can modify the inhibitory effect of PGs on neurite outgrowth (Snow, Steindler, and Silver, 1990b). Substrata containing a high-PG/low-laminin ratio completely inhibit neurite outgrowth, while normal, unimpeded outgrowth is observed on low-PG/high-laminin substrata. Therefore, different patterns of neurite outgrowth may result from regulation of the ratio of growth-promoting molecules to growth-inhibiting molecules. Using video microscopy, embryonic chicken dorsal root ganglia neurons (DRG), chicken retinal ganglia neurons (RGC), and rat forebrain neurons (FB) were analyzed as they extended processes from a substratum consisting of laminin alone onto a step gradient of increasing concentrations of chondroitin sulfate proteoglycan (CS-PG) bound to laminin. In contrast to neurite outgrowth inhibition that occurs at the border of a single stripe of high concentration of CS-PG (Snow et al., 1990b and this study), growth cones grew onto and up CS-PG presented in a step-wise graded distribution. Although the behavior of the different cell types was unique, a common behavior of each cell type was a decrease in the rate of neurite outgrowth with increasing CS-PG concentration. These data suggest that appropriate concentrations of growth-promoting molecules combined with growth-inhibiting molecules may regulate the direction and possibly the timing of neurite outgrowth in vivo. The different responses of different neuronal types suggest that the presence of sulfated PG may have varying effects on different aspects of neuronal development.     

The axonally secreted protein axonin-1 is a potent substratum for neurite growth

Axonin-1 is a neuronal glycoprotein occurring both as a membrane-bound and a secreted form. Membrane-bound axonin-1 is predominantly located in membranes of developing nerve fiber tracts and has recently been characterized as a cell adhesion molecule; the soluble form is secreted from axons and accumulates in the cerebrospinal fluid and the vitreous fluid of the eye. In the present study, we addressed the question as to whether secreted axonin-1 was released in a functionally competent form and we found that it strongly promotes neurite outgrowth when presented to neurons as an immobilized substratum. Neurite lengths elaborated by embryonic dorsal root ganglia neurons on axonin-1 were similar to those on the established neurite-promoting substrata L1 and laminin. Fab fragments of axonin-1 antibodies completely inhibited neurite growth on axonin-1, but not on other substrata. In soluble form, axonin-1 had an anti-adhesive effect, as revealed by perturbation of neurite fasciculation. In view of their structural similarity, we conclude that secreted and membrane-bound axonin-1 interact with the same growth-promoting neuritic receptor. The fact that secreted axonin-1 is functionally active, together with our previous findings that it is secreted from an internal cellular pool, suggests a functional dualism between membrane-bound and secreted axonin-1 at the site of secretion, which is most likely the growth cone. The secretion of adhesion molecules could represent a powerful and rapidly acting regulatory element of growth cone-neurite interactions in the control of neurite elongation, pathway selection, and possibly target recognition.     

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.     

Distinct roles for Sema3A,Sema3F,and an unidentified trophic factor in controlling the advance of geniculate axons to gustatory lingual epithelium

Geniculate ganglion axons arrive in the lingual mesenchyme on embryonic day 13 (E13), 3–4 days before penetrating fungiform papilla epithelium (E17). This latency may result from chemorepulsion by epithelial Sema3A (Dillon et al. (2004) Journal of Comparative Neurology470, 13–24), or Sema3F, which we report is also expressed in this epithelium. Sema3A and Sema3F repelled or suppressed geniculate neurite outgrowth, respectively, and these effects were stage and neurotrophic factor dependent. BDNF-stimulated outgrowth is repelled by Sema3A until E17, but insensitive to Sema3F from E16. NT-4-stimulated neurite outgrowth is sensitive to Sema3A and Sema3F through E18, but NT-4 has not been detected in E15–18 tongue. E15–18 tongue explants did not exhibit net chemorepulsion of geniculate neurites, but the ability of tongue explants to support geniculate neurite outgrowth fluctuates: E12–13 (Rochlin et al. (2000), Journal of Comparative Neurology, 422, 579–593) and E17–18 explants promote and may attract geniculate neurites, but stages corresponding to intralingual arborization do not. The E18 trophic and tropic effects were evident even in the presence of BDNF or NT-4, suggesting that some other factor is responsible. Intrinsic neurite outgrowth capability (without exogenous neurotrophic factors) fluctuated similarly: ganglia deteriorated at E15, but exhibited moderate outgrowth at E18.The chemorepulsion studies are consistent with a role for Sema3A, not Sema3F, in restricting geniculate axons from the epithelium until E17, when axons penetrate the epithelium. The transient inability of tongue explants to promote geniculate neurite outgrowth may signify an alternative mechanism for restricting geniculate axons from the epithelium: limiting trophic factor access.     

Autonomous and non-autonomous regulation of mammalian neurite development by Notch1 and Delta1

BACKGROUND: On the basis of experiments suggesting that Notch and Delta have a role in axonal development in Drosophila neurons, we studied the ability of components of the Notch signaling pathway to modulate neurite formation in mammalian neuroblastoma cells in vitro. RESULTS: We observed that N2a neuroblastoma cells expressing an activated form of Notch, Notch1(IC), produced shorter neurites compared with controls, whereas N2a cell lines expressing a dominant-negative Notch1 or a dominant-negative Delta1 construct extended longer neurites with a greater number of primary neurites. We then compared the effects on neurites of contacting Delta1 on another cell and of overexpression of Delta1 in the neurite-extending cell itself. We found that N2a cells co-cultured with Delta1-expressing quail cells produced fewer and shorter neuritic processes. On the other hand, high levels of Delta1 expressed in the N2a cells themselves stimulated neurite extension, increased numbers of primary neurites and induced expression of Jagged1 and Notch1. CONCLUSIONS: These studies show that Notch signals can antagonize neurite outgrowth and that repressing endogenous Notch signals enhances neurite outgrowth in neuroblastoma cells. Notch signals therefore act as regulators of neuritic extension in neuroblastoma cells. The response of neuritic processes to Delta1 expressed in the neurite was opposite to that to Delta1 contacted on another cell, however. These results suggest a model in which developing neurons determine their extent of process outgrowth on the basis of the opposing influences on Notch signals of ligands contacted on another cell and ligands expressed in the same cell.     

Galactosyl transferase-dependence of neurite outgrowth on substratum-bound laminin

The cell surface enzyme beta 1-4 galactosyl transferase (galtase) has been implicated in a number of cellular events involving adhesion and recognition, among them migration of neural crest and mesenchymal cells as well as initiation and elongation of neurites from PC12 cells. Results presented here demonstrate that reagents that specifically alter galtase activity modulate the rate of neurite outgrowth from chick dorsal root ganglia on substrata coated with the large extracellular matrix glycoprotein, laminin (LN), a known substrate for galtase activity. Not all neurites responded equally to reagent addition, and in every experiment a subset of neurites was ostensibly unaffected by reagent, even at the highest concentration tested. Those neurites that were affected demonstrated an ability to adapt to the continued presence of reagent and resume normal elongation. These results support the hypothesis that cell surface galtase activity plays an important role in mediating neurite elongation and suggest further that differential expression of galtase at the nerve growth cone might contribute to axonal guidance through glycoconjugate-rich environments in vivo.     

Factors affecting neurite outgrowth of occipital cortical explants

The effects of various substrata including laminin, collagen gel, collagen I, and human amniotic basement membrane on neurite outgrowth of occipital cortical and diencephalic explants were studied. The results showed that the extent and pattern of growing neurites of cortical explants varied considerably depending on the substrata used. While an elaborated network of growing neurites was observed when cortical explants were plated on laminin, the most extensive neurite outgrowth was observed when collagen gel was used as the substratum. In contrast, diencephalic explants did not grow on most of the substrata. The significance of the findings are discussed.     

Geometry of isolated sensory neurons in culture. Effects of embryonic age and culture substratum

Sensory neurons were dissociated from lumbar dorsal root ganglia of embryonic chick and put into culture, either directly or after removing non-neuronal cells by density gradient centrifugation. The cells were grown on culture substrata of various kinds in medium containing nerve growth factor (NGF). After 24 h the cultures were fixed, mounted and analysed. Lengths of neurites were measured, and the numbers of primary processes formed at the cell body and of growth cones were counted. From these values, the rates of growth cone advance and frequency of growth cone branching were calculated. Neuronal outgrowths increased strikingly in length and complexity with embryonic age; there was a 3.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones when neurons from 15-day embryos (E15) were compared with those from 8-day embryos (E8) grown on the same substratum (glass). Growth was markedly greater on surfaces prepared with laminin or conditioned medium compared with plain glass or air-dried collagen. When E15 neurons grown on glass were compared with those grown on laminin, for example, a 2.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones was observed. Calculations showed that a major factor in these changes was an increase in the frequency of growth cone branching. The number of initial processes emanating from the cell body changed with age, but not with the different substrata tested. Non-neuronal cells when present in low numbers and in contact with neurons did not appear to influence neuronal geometry in a systematic way. Our results document the fact that both external factors (in this case, the nature of the culture substratum) and intrinsic factors (stage of development of the neuron) can influence the geometry of neurite outgrowth.     

Quantitative effects of laminin concentration on neurite outgrowth in vitro

Recent studies indicate that mediation of neurite outgrowth by the glycoprotein laminin may be a significant factor in the outgrowth of neurites to their targets during embryogenesis. To further characterize the possible role of this extracellular matrix molecule during development, we have systematically measured several features of outgrowth by neonatal rat sympathetic neurons on different concentrations of laminin. Individual neurons, obtained by mechanical dissociation of superior cervical ganglia (SCG), were cultured at low density on laminin substrates ranging from 0.01 to 1.0 microgram/cm2. Outgrowth characteristics were subsequently analyzed for noninteracting cells in both fixed and live cultures. Data obtained from neurons fixed after 11 hr of culture showed approximately twofold increases in neurite initiation and outgrowth, and a twofold decrease in branching for a corresponding 100-fold increase in adsorbed laminin concentration. In time-lapse videomicroscopy observations, the root-mean square speed of growth cone movement increased from 60 to 90 microns/hr over the same range in concentration, while the persistence time remained constant at 0.10 hr. In general, neurite outgrowth parameters were relatively insensitive to changes in laminin concentration, supporting the idea that laminin is a permissive rather than an "instructive" substrate during development. Data obtained from fixed cultures were examined in terms of probability models to suggest possible mechanisms contributing to the dose-dependent effects observed.     

Tenascin is accumulated along developing peripheral nerves and allows neurite outgrowth in vitro.

The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Here we studied the expression of tenascin along developing peripheral nerves in chick embryos and tested its activity as a substrate for cultured neurons. Motor axons grow out through the tenascin-rich, anterior part of the sclerotome. Shortly after, tenascin surrounds axon fascicles of ventral roots. At the limb levels, outgrowing axons accumulate in the tenascin-containing girdle region forming a plexus. In the limb, tenascin first appears in bracket-like structures that surround the precartilage cell condensations of the femur and humerus, respectively. These regions coincide with the channels along which axons first grow in from the girdle plexus to form the limb nerves. Later, the major tenascin staining is associated with the cartilage and tendon primordia, and not with the limb nerves. We used tenascin as a substrate for cultured neural explants and single cells in order to test for its function in neurite outgrowth. Dissociated embryonic neurons of various types attached to mixed polylysine/tenascin substrates and sprouted rapidly after a lag of several hours. Outgrowth was inhibited and neurites were detached by anti-tenascin antibodies. On substrates coated with tenascin alone, neurite outgrowth was achieved from 3 day spinal cord explants. Whereas growth cones were well spread and rapidly moving, the neurites were poorly attached, straight and rarely branched. We speculate that in vivo tenascin allows axonal outgrowth, but inhibits branching and supports fasciculation of newly formed axons.     

The effect of cyclic AMP on neuritic outgrowth in explant cultures of developing chick olfactory epithelium

The effect of cyclic AMP (cAMP) analogs and phosphodiesterase (PDE) inhibitors on neurite outgrowth was studied in explant cultures of olfactory neurons. Nasal pits from 5- or 6-day-old chick embryos were minced, explanted into culture dishes, and grown in a serum-free medium. One of the cyclic AMP analogs, dibutyryl cyclic AMP (dbcAMP) or 8-bromo-cyclic AMP (8-Br-cAMP), or one of the PDE inhibitors, theophylline or isobutylmethylxanthine (IBMX), was added to the culture medium. The explants were examined for neurite outgrowth after 2 days in vitro. Db-cAMP increased the number of explants expressing neurites by 25-35% over control cultures, whereas 8-Br-cAMP had essentially no effect at the same concentrations. Addition of dibutyryl cyclic GMP (dbcGMP) gave no increase in neurite outgrowth, thus indicating that the effect of enhancing neuritic growth is specific to cAMP and not cyclic nucleotides in general. The resulting increase in neurite outgrowth is due to the cyclic nucleotide component of dbcAMP, since both IBMX and theophylline, which elevate intracellular cAMP, also increased neurite outgrowth significantly. When forskolin was added to the culture medium, there was a trend to increased neurite outgrowth; this was significantly enhanced when a subthreshold concentration of theophylline was added in addition to the forskolin.     

Staurosporine-induced morphological differentiation of human neuroblastoma cells

SH-SY-5Y human neuroblastoma cells rapidly elaborated an extensive network of neuritic processes following treatment with staurosporine, an inhibitor of protein kinase C. These neurites were retracted within 24hr following removal of inhibitor. Another inhibitor of protein kinase C, H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], also induced rapid, reversible neurite outgrowth. However, neurites induced by these two inhibitors were morphologically distinct: staurosporine-treated cells elaborated a branching neuritic network adjacent to cell bodies, with some longer, unbranching neurites extending out of this network, while H7-treated cells elaborated only long, unbranching neurites. HA-1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide], which inhibits of cAMP- and cGMP-dependent protein kinases but not protein kinase C, did not induce neuritogenesis. Staurosporine-induced neurite outgrowth did not require protein synthesis but did require microtubule assembly, suggesting that cells contained the necessary components for neuritogenesis, and that alterations in protein phosphorylation alone was sufficient to initiate neurite outgrowth by rearrangement of existing structures or cytoskeletal precursors. These results implicate phosphorylation in the regulation of neuronal differentiation and neuritogenesis.     

The role of cell adhesion molecules in neurite outgrowth on Müller cells

The roles of neural cell adhesion molecule (NCAM), L1, N-cadherin, and integrin in neurite outgrowth on various substrates were studied. Antibodies against these cell surface molecules were added to explants of chick retina and the neurites from retinal ganglion cells were examined for effects of the antibodies on neurite length and fasciculation. On laminin, an anti-integrin antibody completely inhibited neurite outgrowth. The same antibody did not inhibit neurite outgrowth on polylysine or Müller cells. Antibodies to NCAM, L1, and N-cadherin did not significantly inhibit neurite outgrowth on laminin but produced significant inhibition on Müller cells. The inhibition of neurite outgrowth on glia by anti-L1 antibodies supports the hypothesis that L1 is capable of acting in a heterophilic binding mechanism. On laminin, both anti-N-cadherin and anti-L1 caused defasciculation of neurites from retinal ganglion cells, while anti-NCAM did not. None of these antibodies produced defasciculation on Müller cells. The results indicate that these three cell adhesion molecules may be very important in interactions with glia as axons grow from the retina to the tectum and may be less important in axon-axon interactions along this pathway. No evidence was found supporting the role of integrins in axon growth on Müller cells.     

Analysis of high PSA N-CAM expression during mammalian spinal cord and peripheral nervous system development.

Using a monoclonal antibody that recognizes specifically a high polysialylated form of N-CAM (high PSA N-CAM), the temporal and spatial expression of this molecule was studied in developing spinal cord and neural crest derivatives of mouse truncal region. Temporal expression was analyzed on immunoblots of spinal cord and dorsal root ganglia (DRGs) extracts microdissected at different developmental stages. Analysis of the ratio of high PSA N-CAM to total N-CAM indicated that sialylation and desialylation are independently regulated from the expression of polypeptide chains of N-CAM. Motoneurons, dorsal root ganglia cells and commissural neurons present a homogeneous distribution of high PSA N-CAMs on both their cell bodies and their neurites. Sialylation of N-CAM can occur in neurons after their aggregation in peripheral ganglia as demonstrated for dorsal root ganglia at E12. Furthermore, peripheral ganglia express different levels of high PSA N-CAM. With in vitro models using mouse neural crest cells, we found that expression of high PSA N-CAM was restricted to cells presenting an early neuronal phenotype, suggesting a common regulation for the expression of high PSA N-CAM molecules, neurofilament proteins and sodium channels. Using perturbation experiments with endoneuraminidase, we confirmed that high PSA N-CAM molecules are involved in fasciculation and neuritic growth when neurons derived from neural crest grow on collagen substrata. However, we demonstrated that these two parameters do not appear to depend on high PSA N-CAM molecules when cells were grown on a fibronectin substratum, indicating the existence of a hierarchy among adhesion molecules.     

Neurite extension by neuroblastoma cells on substratum-bound fibronectin''s cell-binding fragment but not on the heparan sulfate-binding protein, platelet factor-4

Human and rat neuroblastoma cells extend neurites over plasma fibronectin (pFN)-coated substrata. For resolution of which fibronectin binding activities (the cell-binding domain (CBD), the heparan sulfate-binding domains, or a combination of the two) are responsible for neurite outgrowth, CBD was prepared free of heparan sulfate-binding activity as described by Pierschbacher et al. (Cell 26 (1981) 259-267). Neuroblastoma cells attached and extended neurites as stably and as effectively on CBD-coated substrata as on intact pFN, while cytoplasmic spreading was more extensive on pFN-coated substrata. The structures of growth cones on CBD or pFN were virtually identical. On substrata coated with the model heparan sulfate-binding protein, platelet factor 4 (PF4), cells attached and spread somewhat but never extended neurites. When cells were challenged with substrata coated with various ratios of CBD and PF4, PF4 was found to be an effective inhibitor of CBD-mediated neurite extension. Similarly, cells grown on substrata coated at different locations with CBD or PF4 in order to evaluate topographical dependence of growth cone formation extended neurites only onto the CBD-coated region or along the interface between these two proteins, but never onto the PF4 side of cells that bridged the interface. These studies indicate that (a) the CBD activity of pFN, and not its heparan sulfate-binding activity, is the critical determinant in neurite extension of these neural tumor cells from the central nervous system; (b) under some circumstances, heparan sulfate-binding activity can be antagonistic to neurite extension; (c) the chemical nature of the substratum controls the direction of neurite extension; (d) these neuroblastoma cells respond to these binding proteins very differently than fibroblasts or neurons from the peripheral nervous system.     

Enhanced neurite outgrowth of rat neural cortical cells on surface-modified films of poly(lactic-<Emphasis Type="Italic">co</Emphasis>-glycolic acid)

Neural cortical cells, isolated from prenatal rat cerebra, were grown on surface-modified poly(lactic-co-glycolic acid, 65:35) (PLGA) films coated with poly-D-lysine (PDL) with either laminin (LN), fibronectin (FN) or collagen (CN). Immunocytochemistry showed that the isolated cells were highly immunopositive for both neurofilament and MAP-2 with well-organized neurites and somatodendritic localization. The presence of PDL with LN or FN on the PLGA films was essential for increased neural cell growth. Also, PLGA films coated with either PDL/LN or PDL/FN mixtures had higher neurite outgrowth and regular differentiation.Revisions requested 30 September 2004; Revisions received 10 November 2004