Cell-to-substratum adhesion and guidance of axonal elongation

The behavior of axonal growth cones on surfaces with patterned variations in substratum was observed. Cells from sensory ganglia of 8-day-old chicken embryos were cultured on plastic petri dishes, plastic tissue culture dishes, and polyornithine-coated tissue culture dishes, all of which contained gridlike patterns of palladium (Pd) deposition.The results indicated that growth cones elongated on the Pd-shadowed areas vs areas lacking Pd deposits depending on the relative adhesivity of the growth cones to the substrata. In petri dishes, growth cones stay on the Pd; in tissue culture dishes, they cross from one surface to the other; and in polyornithine-coated dishes, they elongate for great distances on the Pd-free areas. Analyses of time-lapse movies showed that, on Pd-shadowed polyornithine dishes, growth cones often approach the Pd-coated areas and microspikes touch the Pd surface. Yet, the axon tip continues to elongate on the Pd-free polyornithine surface.The conclusion is offered that interactions between microspikes and the substratum adjacent to the growth cone are important determinants of the directions and pathways of axonal elongation.     

A cytomechanical investigation of neurite growth on different culture surfaces

We have examined the relationship between tension, an intrinsic stimulator of axonal elongation, and the culture substrate, an extrinsic regulator of axonal elongation. Chick sensory neurons were cultured on three substrata: (a) plain tissue culture plastic; (b) plastic treated with collagen type IV; and (c) plastic treated with laminin. Calibrated glass needles were used to increase the tension loads on growing neurites. We found that growth cones on all substrata failed to detach when subjected to two to threefold and in some cases 5-10-fold greater tensions than their self-imposed rest tension. We conclude that adhesion to the substrate does not limit the tension exerted by growth cones. These data argue against a "tug-of-war" model for substrate-mediated guidance of growth cones. Neurite elongation was experimentally induced by towing neurites with a force-calibrated glass needle. On all substrata, towed elongation rate was proportional to applied tension above a threshold tension. The proportionality between elongation rate and tension can be regarded as the growth sensitivity of the neurite to tension, i.e., its growth rate per unit tension. On this basis, towed growth on all substrata can be described by the simple linear equation: elongation rate = sensitivity x (applied tension - tension threshold) The numerical values of tension thresholds and neurite sensitivities varied widely among different neurites. On all substrata, thresholds varied from near zero to greater than 200 mudynes, with some tendency for thresholds to cluster between 100 and 150 mudynes. Similarly, the tension sensitivity of neurites varied between 0.5 and 5.0 microns/h/mudyne. The lack of significant differences among sensitivity or threshold values on the various substrata suggest to use that the substratum does not affect the internal "set points" of the neurite for its response to tension. The growth cone of chick sensory neurons is known to pull on its neurite. The simplest cytomechanical model would assume that both growth cone-mediated elongation and towed growth are identical as far as tension input and elongation rate are concerned. We used the equation above and mean values for thresholds and sensitivity from towing experiments to predict the mean growth cone-mediated elongation rate based on mean rest tensions. These predictions are consistent with the observed mean values.     

Effects of lipid-derivatized glycosaminoglycans (GAGs), a novel probe for functional analyses of GAGs, on cell-to-substratum adhesion and neurite elongation in primary cultures of fetal rat hippocampal neurons

The effects of glycosaminoglycans (GAG) on cell-to-substratum adhesion and neurite elongation were examined in primary cultures of fetal rat hippocampal neurons using tissue culture dishes coated with GAGs coupled to dipalmitoylphosphatidylethanolamine (PE), a novel probe for biological functions of GAGs. Both chondroitin sulfate conjugate to PE (CS-PE) and hyaluronic acid conjugate to PE (HA-PE) promoted neurite elongation from neurons in a dose-dependent manner when immobilized onto polylysine-coated dishes at various concentrations up to 1.0 microg/ml. The coating of CS-PE or HA-PE at a concentration higher than 1.0 microg/ml resulted in failure of neurite extension and adhesion of neurons to the substrata. In contrast, heparin conjugate to PE (HP-PE) did not exert any effects on neurite elongation or on cell attachment at these concentrations. These findings suggest that GAGs serve as a modulator for neurite elongation during neuronal network formation in the developing central nervous system.     

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.     

In vitro mineralization by mesenchymal stem cells cultured on titanium scaffolds

Titanium has been utilized in the field of orthopaedic and dental reconstructive surgery, but mineralization through osteogenic differentiation of osteogenic cells on titanium surfaces has not been fully investigated. Here we cultured rat mesenchymal stem cells (MSCs) on the surfaces of titanium dishes in osteogenic media containing calcein which is a calcium-binding fluorescence dye. On titanium dishes, MSCs showed high viability to adhere to the surfaces and excellent proliferation. At day 14 of culture, MSCs differentiated into osteoblasts to form mineralized matrices on titanium dishes as well as tissue culture polystyrene (TCPS) dishes which are widely recognized as optimal culture substrates. Calcein was incorporated into the bone minerals fabricated by MSCs cultured on both substrates to show green emission under fluorescence microscopy. The fluorescence intensity was quantified with an image analyser during culture periods. These results indicate that the surfaces of titanium showed a high adhesion/proliferation potential to MSCs and that the titanium effectively supported the osteogenic differentiation of MSCs comparable to TCPS dishes. Therefore, the titanium is an effective scaffold that is applicable in bone reconstruction surgery.     

Bubble Contact Angle Method for Evaluating Substratum Interfacial Characteristics and Its Relevance to Bacterial Attachment

A bubble contact angle method was used to determine interfacial free-energy characteristics of polystyrene substrata in the presence and absence of potential surface-conditioning proteins (bovine glycoprotein, bovine serum albumin, fatty acid-free bovine serum albumin), a bacterial culture supernatant, and a bacterial exopolymer. Clean petri dish substrata gave a contact angle of 90°, but tissue culture dish substrata were more hydrophilic, giving an angle of 29° or less. Bubble contact angles at the surfaces exposed to the macromolecular solutions varied with the composition and concentration of the solution. Modification by pronase enzymes of the conditioning effect of proteins depended on the nature of both the substratum and the protein, as well as the time of addition of the enzyme relative to the conditioning of the substratum. The effects of dissolved and substratum-adsorbed proteins on the attachment of Pseudomonas sp. strain NCMB 2021 to petri dishes and tissue culture dishes were consistent with changes in bubble contact angles (except when proteins were adsorbed to tissue culture dishes before attachment) as were alterations in protein-induced inhibition of bacterial attachment to petri dishes by treatment with pronase. Differences between the attachment of pseudomonads to petri dishes and tissue culture dishes suggested that different mechanisms of adhesion are involved at the surfaces of these two substrata.     

In vitro regulation of neuronal morphogenesis and polarity by astrocyte-derived factors

Mesencephalic neurons were cultured from 2 to 5 days in mesencephalic (CM Gmes) or striatal (CM Gstr) astrocyte conditioned media or in the soluble (S100) and insoluble (P100) fractions prepared from these media by ultracentrifugation. CM Gmes as well as all soluble fractions induced dendritic and axonal elongation, whereas CM Gstr and the insoluble fractions promoted axonal growth only. The study of the shape of the neuronal cell bodies and the measurement of their adhesion to the substratum revealed that axons elongated under low adhesion conditions, but that dendrite growth was highly dependent upon adhesion and spreading of the neuronal soma. This different dependency of axonal and dendritic elongation upon spreading is explained by a model in which we consider the respective viscosities of axons and dendrites. From these observations and speculations we propose that axons and dendrites have different modes of elongation and that the primary effect of the astrocyte-derived factors capable of regulating neuronal polarity is to modify the adhesion of the neurons to their culture substratum.     

Neuronal models for studying lipid metabolism and transport

New methods have been developed for studying lipid metabolism and transport in primary cultures of neurons. Sympathetic neurons from rats and mice, as well as retinal ganglion neurons from rats, can be cultured in three-compartmented culture dishes in which the cell bodies reside in a compartment separate from that housing the distal axons. In addition, the three compartments contain completely independent fluid environments. Consequently, these neuronal cultures represent an excellent model for studying the intra-neuronal transport of lipids and proteins between cell bodies and distal axons. In addition, compartmented neuron cultures are particularly appropriate for investigating factors that regulate axonal growth and neuronal survival. The application of the compartmented culture model for use with murine neurons has opened up many new possibilities for studying lipid metabolism in neurons derived from genetically modified mice. Examples are given in which compartmented cultures of primary neurons have been used in studies on (i) lipid analysis of distal axons and cell bodies/proximal axons, (ii) immunoblotting of neuronal proteins involved in lipid metabolism, (iii) the compartmentalization of lipid metabolism, (iv) the role of lipids in axonal growth and survival, and (v) intracellular lipid transport.     

Relationship between neuronal migration and cell-substratum adhesion: laminin and merosin promote olfactory neuronal migration but are anti- adhesive

Regulation by the extracellular matrix (ECM) of migration, motility, and adhesion of olfactory neurons and their precursors was studied in vitro. Neuronal cells of the embryonic olfactory epithelium (OE), which undergo extensive migration in the central nervous system during normal development, were shown to be highly migratory in culture as well. Migration of OE neuronal cells was strongly dependent on substratum- bound ECM molecules, being specifically stimulated and guided by laminin (or the laminin-related molecule merosin) in preference to fibronectin, type I collagen, or type IV collagen. Motility of OE neuronal cells, examined by time-lapse video microscopy, was high on laminin-containing substrata, but negligible on fibronectin substrata. Quantitative assays of adhesion of OE neuronal cells to substrata treated with different ECM molecules demonstrated no correlation, either positive or negative, between the migratory preferences of cells and the strength of cell-substratum adhesion. Moreover, measurements of cell adhesion to substrata containing combinations of ECM proteins revealed that laminin and merosin are anti-adhesive for OE neuronal cells, i.e., cause these cells to adhere poorly to substrata that would otherwise be strongly adhesive. The evidence suggests that the anti- adhesive effect of laminin is not the result of interactions between laminin and other ECM molecules, but rather an effect of laminin on cells, which alters the way in which cells adhere. Consistent with this view, laminin was found to interfere strongly with the formation of focal contacts by OE neuronal cells.     

PC12 adhesion and neurite formation on selected substrates are inhibited by some glycosaminoglycans and a fibronectin-derived tetrapeptide

The effects of added soluble glycosaminoglycans (GAGs) on adhesion and neurite formation by cultured PC12 pheochromocytoma cells on several substrates were tested. PC12 cells adhere more rapidly to Petri plastic coated with fibronectin, laminin, poly-L-lysine, or conA, than to either uncoated Petri plastic or tissue culture plastic. Adhesion to poly-L-lysine, fibronectin- and laminin-coated dishes was significantly inhibited by added dextran sulfate and to a lesser extent heparin--but not by chondroitin sulfate. PC12 adhesion to fibronectin could also be totally inhibited by the putative fibronectin cell binding tetrapeptide L-arginyl-glycyl-L-aspartyl-L-serine (Pierschbacher, MD & Ruoslahti, E, Nature 309 (1984) 30). The inhibitory effects of combinations of this tetrapeptide and heparin or dextran sulfate (but not chondroitin sulfate or hyaluronic acid) were additive. Nerve growth factor (NGF) pretreatment increased the percentage of PC12 cells adherent to all substrates and reduced the GAG inhibition of adhesion. PC12 cells previously treated with NGF to induce morphologic differentiation will rapidly re-extend neurites when plated on all four substrates. On fibronectin and poly-L-lysine-coated dishes this neurite growth is inhibited by added heparin and dextran sulfate, while on laminin it is not. Neurite formation on fibronectin-coated dishes was also inhibited by low concentrations of fibronectin tetrapeptide. In summary, PC12 adhesion and neurite formation can be inhibited by sulfated GAGs on some substrates, including fibronectin, but not other substrates, suggesting that these cells have at least two independent molecular adhesion mechanisms.     

In vitro studies on the control of nerve fiber growth by the extracellular matrix of the nervous system

1. Cultured neurons from embryonic chick sympathetic ganglia or dorsal root ganglia grow nerve fibers extensively on simple substrata containing fibronectin, collagens (types I, III, IV), and especially laminin. 2. The same neurons cultured on substrata containing glycosaminoglycans grow poorly. Glycosaminoglycans (heparin) inhibit nerve fiber growth on fibronectin substrata. 3. Proteolytic fragments of fibronectin support nerve fiber growth only when the cell attachment region is intact. For example, a 105 kD fragment, encompassing the cell attachment region, supports growth when immobilized in a substratum, but a 93 kD subfragment, lacking the cell attachment region, is unable to support fiber growth. When it is added to the culture medium, the 105 kD fragment inhibits fiber growth on substrata containing native fibronectin. 4. In culture medium lacking NGF, DRG neurons extend nerve fibers only on laminin and not on fibronectin, collagen or polylysine. Studies with radioiodinated laminin indicate that laminin binds with a relatively high affinity (kd approximately equal to 10(-9) M) to DRG neurons, and to a variety of other neural cells (NG108 cells, PC12 cells, rat astrocytes, chick optic lobe cells). We have isolated a membrane protein (67 kD) by affinity chromatography on laminin columns and are characterizing this putative laminin receptor. 5. Dissociated DRG neurons or ganglionic explants cultured on complex substrata consisting of tissue sections of CNS or PNS tissues extend nerve fibers onto the PNS (adult rat sciatic nerve) but not CNS (adult rat optic nerve) substrata. Other tissue substrata which support fiber growth in vivo (embryonic rat spinal cord, goldfish optic nerve) support growth in culture. While substrata from adult CNS, which support meager regeneration in vivo (adult rat spinal cord) support little fiber growth in culture. 6. Ganglionic explants cultured in a narrow space between a section of rat sciatic nerve and optic nerve grow preferentially onto the sciatic nerve suggesting that diffusible growth factors are not responsible for the differential growth on the two types of tissues. 7. Dissociated neurons adhere better to sections of sciatic nerve than optic nerve. Laminin, rather than fibronectin or heparan sulfate proteoglycan, is most consistently identifiable by immunocytochemistry in tissues (sciatic nerve, embryonic spinal cord, goldfish optic nerve) which support nerve fiber growth. Taken together, these data suggest that ECM adhesive proteins are important determinants of nerve regeneration.     

Responses of cultured neural retinal cells to substratum-bound laminin and other extracellular matrix molecules

The responses of cultured chick embryo retinal neurons to several extracellular matrix molecules are described. Retinal cell suspensions in serum-free medium containing the "N1" supplement (J. E. Bottenstein, S. D. Skaper, S. Varon, and J. Sato, 1980, Exp. Cell Res. 125, 183-190) were seeded on tissue culture plastic surfaces pretreated with polyornithine (PORN) and with one of the factors to be tested. Substantial cell survival could be observed after 72 hr in vitro on PORN pretreated with serum or laminin, whereas most cells appeared to be degenerating on untreated PORN, PORN-fibronectin, and PORN-chondronectin. Cell attachment, although quantitatively similar for all these substrata, was temperature-dependent on serum and laminin but not on fibronectin or untreated PORN. In a short-term bioassay, neurite development was abundant on laminin, scarce on serum and fibronectin, and absent on PORN. No positive correlation between cell spreading and neurite production could be seen: cell spreading was more extensive on PORN and fibronectin than on laminin or serum, while on laminin-treated dishes, spreading was similar for neurite-bearing and non-neurite-bearing cells. Laminin effects on retinal neurons were clearly substratum dependent. When bound to tissue culture plastic, laminin showed a dose-dependent inhibitory effect on cell attachment and did not stimulate neurite development. PORN-bound laminin, on the other hand, did not affect cell attachment but caused marked stimulation of neurite development, suggesting that laminin conformation and/or the spatial distribution of active sites play an important role in the neurite-promoting function of this extracellular matrix molecule. Investigation of the embryonic retina with ELISA and immunocytochemical methods showed that laminin is present in this organ during development. Therefore, in vivo and in vitro observations are consistent with the possibility that laminin might influence neuronal development in the retina.     

The growth of spinal ganglion neurons in serum-free medium

The presence of serum in the culture medium affects the morphology of spinal ganglion neurons. With serum present, neuronal cell bodies are rounded and axons are predominantly straight. In serum-free medium axons curve all along their lengths, while both cell bodies and growth cones are spread on the substratum. Such “curved” axons straighten if serum is added to the culture dish.Serum-free medium may increase the adhesion of neurons to the substratum. This can account for the curved morphology of axons in serum-free medium, since such axons may represent a “history” of the movement of the growth cone during axon elongation.     

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 nature of substrate-attached materials in human fibroblast cultures: localization of cell and fetal calf serum components.

Human fibroblasts have been used as an in vitro model to examine the morphology and origin of substrate-attached materials. In cultures of subconfluent cells, no ‘tracks’ or ‘pools’ of material could be detected on substrata by anodic oxide interferometry or electron microscopy. However, a continuous layer of densely staining material was present on Falcon plastic tissue culture dishes never exposed to cells or culture medium. Exposure of substrata to culture medium caused the adsorption of fetal calf serum (FCS) components onto the substratum within a few minutes. Although antigenic FCS components remained on the substrata for several days, they were seldom adsorbed to the cells. The hypothesis was formulated that adhesion was mediated by FCS components on the substrata, but not by cellular materials deposited extracellularly. Support for this hypothesis was obtained by studying serum-dependent differences in cell adhesion. Fibroblasts subcultured in the presence of FCS components were usually separated from the substratum by a distance of at least 30 Å. In the absence of FCS components, the cells were more closely adherent, in the range at which the near van der Walls forces were effective. Fibroblasts subcultured in the absence of serum components could be removed readily from the substratum, leaving lsfootprints’ of cell surface material behind. Although this material has been prepared similarly to ‘microexudates’ from other types of cultured cells, its relationship to those microexudates has not been determined.     

Aplysia hemolymph promotes neurite outgrowth and synaptogenesis of identifiedHelix neurons in cell culture

Hemolymph of adultAplysia californica significantly affects neurite outgrowth of identified neurons of the land snailHelix pomatia. The metacerebral giant cell (MGC) and the motoneuron C3 from the cerebral ganglion and the neuron B2 from the buccal ganglion ofH. pomatia were isolated by enzymatic and mechanical dissociation and plated onto poly-l-lysine-coated dishes either containing culture medium conditioned byHelix ganglia, or pre-treated withAplysia hemolymph. To determine the extent of neuronal growth we measured the neurite elongation and the neuritic field of cultured neurons at different time points.Aplysia hemolymph enhances the extent and rate of linear outgrowth and the branching domain ofHelix neurons. With the hemolymph treatment the MGC neuron more consistently forms specific chemical synapses with its follower cell B2, and these connections are more effective than those established in the presence of the conditioned medium.     

Adhesion site composition of murine fibroblasts cultured on gelatin-coated substrata

Fibroblasts in vivo adhere to a collagenous extracellular matrix. We present here a combined morphological and biochemical analysis of the adhesion sites of fibroblast-like cells cultured in vitro on gelatin-coated plastic, for comparison with earlier model studies using serum (plasma-fibronectin [pFn])-coated plastic. Scanning electron microscopy shows that cell adhesion to the gelatin is quite similar to that on plastic, but with some morphological differences reminiscent of those caused by higher concentrations of fibronectin adsorbed to the substratum. Measurement using 125I-radiolabeled pFn shows the level of substratum-bound pFn adsorbed from serum in the growth medium is, however, comparable on gelatin or plastic; thus, differences due to pFn must be attributed to the quality of the adsorbed protein; not its absolute quantity. Gel electrophoretic analysis of cellular adhesion sites formed on the two substrata shows their compositions to be qualitatively similar, suggesting again that the same fundamental adhesion processes are involved. However, three protein bands do change; notably, cellular fibronectin is increased on gelatin. These three proteins are also the most resistant to saline extraction, suggesting their intrinsic importance in the adhesion sites. The nature of the growth substratum thus appears to modulate a fundamentally unvarying morphology and adhesion site composition of the cells that adhere to it.