Effects of antibodies to neural cell adhesion molecule (N-CAM) on the differentiation of neuromuscular contacts between ciliary ganglion neurons and myotubes in vitro

Previous experiments have suggested that the neural cell adhesion molecule (N-CAM) may have a role in initial nerve-muscle adhesion. To determine whether N-CAM might be involved in synaptic differentiation, we grew ciliary ganglion neurons and embryonic myotubes together in the presence and absence of monovalent antibodies to N-CAM. In normal cultures, undifferentiated neurites contact myotubes, and the nerve at some of these neurite-myotube contacts acquires concentrations of synaptic vesicle antigens. Most of these vesicle antigen-positive contacts become associated with patches of acetylcholine receptor (AChR) on the surface of the underlying myotube. Contacts without concentrations of vesicle antigens do not become associated with AChR patches. In the presence of antibodies to N-CAM, adhesion between neuronal somata and myotubes was reduced, but neurites contacted myotubes with near-normal frequency. The subsequent differentiation of nerve and muscle at these contacts, as assayed by the localization of vesicle antigens and AChR, proceeded normally in the presence of anti-N-CAM antibodies. The results suggest that N-CAM-mediated adhesion between neurite and myotube is not required for synaptic differentiation.     

Changes in synaptic potential properties during acetylcholine receptor accumulation and neurospecific interactions in Xenopus nerve-muscle cell culture

Acetylcholine receptors in the muscle cell membrane accumulate at the nerve contact area in Xenopus cell cultures. The correlation between spontaneous synaptic potential properties and extent of acetylcholine receptor accumulation was studied. Small and infrequent miniature endplate potentials were measured before acetylcholine receptor accumulation which was observed with fluorescence microscopy using tetramethylrhodamine-conjugated α-bungarotoxin. As acetylcholine receptors accumulate at the nerve contact area, these synaptic potentials become larger and their frequency increases dramatically. In nerve-contacted muscle cells where spontaneous synaptic activity could not be detected, extensive acetylcholine receptor accumulation was not found at sites of nerve contact. Furthermore, muscle cells which exhibited extensive acetylcholine receptor accumulation along the nerve always produced miniature endplate potentials. Thus acetylcholine receptor accumulation and the presence of miniature endplate potentials were strongly correlated. Noncholinergic neurons from dorsal root ganglia did not form functional synaptic contacts with muscle cells nor acetylcholine receptor accumulation along the path of contact. Furthermore, explants from tadpole spinal cord formed functional synaptic contacts with muscle cells but rarely caused AChR localization. These data are discussed in terms of developmental processes during neuromuscular junction formation.     

The involvement of axonin-1/SC2 in mediating notochord-derived chemorepulsive activities for dorsal root ganglion neurites

Previous studies have suggested that the developing notochord secretes diffusible axon guidance molecules that repel dorsal root ganglion (DRG) neurites (R. Keynes et al., 1997, Neuron 18, 889-897; K. Nakamoto and T. Shiga, 1998, Dev. Biol. 202, 304-314). Neither notochord-derived chemorepellents nor their receptors on DRG neurites are, however, known. Here we investigated whether cell adhesion molecules (CAMs) of the immunoglobulin/fibronectin type III subfamily present on DRG neurites, including axonin-1/SC2, N-CAM, Ng-CAM, and Nr-CAM, are required for mediating the notochord-derived chemorepulsion. Using collagen gel cocultures of DRGs and notochord explants, we found that an antibody against axonin-1/SC2 diminished the effects of the chemorepulsive activity from the notochord, whereas antibodies against N-CAM, Ng-CAM, and Nr-CAM had no effect. We further showed that the removal of glycosylphosphatidylinositol-anchored cell surface molecules, including axonin-1/SC2, from DRG neurites diminished the effects of the notochord-derived chemorepulsive activity to an extent similar to that of treatment with the anti-axonin-1/SC2 antibody. These results suggest that axonin-1/SC2 expressed on DRG neurites may be involved in mediating the notochord-derived chemorepulsive activity.     

Identification of HGF-like protein as a novel neurotrophic factor for avian dorsal root ganglion sensory neurons

HGF-like protein (HLP) is a member of the hepatocyte growth factor (HGF) family. Although HGF is shown to have neurotrophic activities on many of CNS and PNS neurons, the role of HLP in the nervous system is poorly understood despite the knowledge that Ron/HLP receptor is expressed in embryonic neurons. Here we show that HGF but not HLP promotes neurite extension and migration emanating from chick embryonic day 9 (E9) dorsal root ganglia (DRG) explants in the presence of low levels of NGF, however, HLP does promote neurite extension and cellular migration from E15 chick DRG explants with low levels of NGF. Ron-Fc, a chimeric molecule composed of the extracellular domain of Ron fused with immunoglobulin Fc, eliminated activities of HLP, such as cellular migration and long neurite extension emanating from E15 DRG explants in the presence of NGF, but did not eliminate short neurites. These results suggested that promotion of long neurite extension and migration depends on activities of HLP through its receptor/Ron. Taken together, we propose that HLP may play an important role in chick sensory ganglia at relatively late stages of development. This is the first evidence that HLP functions as a neurotrophic factor.     

The development of functional neuromuscular junctionsin vitro: An ultrastructural and physiological study

Neuromuscular junctions were formedin vitro between rat spinal cord explants and myotubes. At various intervals after the spinal cord explants were added to the myotube culture (7 hr to 15 days of coculture), the presence of functional neuromuscular junctions was determined by recording miniature endplate potentials (mepps) from the myotubes contacted by a few neurites. Electron microscopical studies were conducted on identified myotubes in which mepps were recorded. Mepps were already found as early as 7 hr after coculture. The fine structure of these newly formed neuromuscular junctions was simple. No synaptic specializations were observed except the presence of a small number of synaptic vesicles in the nerve. The neuromuscular junctions differentiated during the coculture period. Synaptic vesicles formed a cluster at the prejunctional membrane with a localized density in the middle. Basal lamina started to form in 4-day-old cocultures and became continuous in cocultures of 10 days or longer. Clear postjunctional foldings were observed in 15-day-old cocultures. Higher mepp frequencies were correlated with more advanced ultrastructure.     

The polymeric immunoglobulin receptor accumulates in specialized endosomes but not synaptic vesicles within the neurites of transfected neuroendocrine PC12 cells

We have expressed in neuroendocrine PC12 cells the polymeric immunoglobulin receptor (pIgR), which is normally targeted from the basolateral to the apical surface of epithelial cells. In the presence of nerve growth factor, PC12 cells extend neurites which contain synaptic vesicle-like structures and regulated secretory granules. By immunofluorescence microscopy, pIgR, like the synaptic vesicle protein synaptophysin, accumulates in both the cell body and the neurites. On the other hand, the transferrin receptor, which normally recycles at the basolateral surface in epithelial cells, and the cation-independent mannose 6-phosphate receptor, a marker of late endosomes, are largely restricted to the cell body. pIgR internalizes ligand into endosomes within the cell body and the neurites, while uptake of ligand by the low density lipoprotein receptor occurs primarily into endosomes within the cell body. We conclude that transport of membrane proteins to PC12 neurites as well as to specialized endosomes within these processes is selective and appears to be governed by similar mechanisms that dictate sorting in epithelial cells. Additionally, two types of endosomes can be identified in polarized PC12 cells by the differential uptake of ligand, a housekeeping type in the cell bodies and a specialized endosome in the neurites. Recent findings suggest that specialized axonal endosomes in neurons are likely to give rise to synaptic vesicles (Mundigl, O., M. Matteoli, L. Daniell, A. Thomas-Reetz, A. Metcalf, R. Jahn, and P. De Camilli. 1993. J. Cell Biol. 122:1207- 1221). Although pIgR reaches the specialized endosomes in the neurites of PC12 cells, we find by subcellular fractionation that under a variety of conditions it is efficiently excluded from synaptic vesicle- like structures as well as from secretory granules.     

Factors released by ciliary neurons and spinal cord explants induce acetylcholine receptor mRNA expression in cultured muscle cells

The nuclei of cultured noninnervated muscle cells are heterogeneous with respect to production of mRNA for the nicotinic acetylcholine receptor (AChR). Some nuclei actively express AChR mRNA while others have a low level of activity or are inactive. To determine if innervation, or a factor released by neurons, influences nuclear expression of AChR mRNA, we examined mRNA at a single cell level via in situ hybridization and autoradiography with an alpha-subunit AChR genomic probe. Four days after plating, we co-cultured chicken primary muscle cells with spinal cord explants, ciliary neurons, or dorsal root ganglia (DRG) cells. In situ hybridization of the spinal-cord and muscle-cell co-cultures with the AChR alpha-subunit probe revealed a high density of silver grains on muscle cells, which were within two explant diameters of the spinal cord explant, and a graded decrease in silver grain density as the distance from the explant increased, as well as the appearance of a strikingly nonhomogenous distribution of active and inactive muscle cell nuclei. When ciliary neurons were uniformly distributed over the muscle cells, a high level of AChR mRNA was induced, but no gradients appeared. Neither an increased mRNA level nor a gradient was observed when DRG cells were co-cultured with muscle cells. When ciliary neurons are cultured within Costar permeable inserts, which prevent any contact between the neurons and the underlying muscle cells, AChR messenger RNA is still induced, showing that diffusible factors are responsible. Our results indicate that molecules released by cholinergic neurons regulate the expression of AChR mRNA in the myotubes and raise the possibility that AChR expression depends on both neuronal signals and on intracellular information from the muscle cell.     

Segmentation of sensory and sympathetic ganglia: interactions between neural crest and somite cells.

The segmental pattern of peripheral ganglia in higher vertebrates is generated by interactions between neural crest and somite cells. Each mesodermal somite is subdivided into at least two distinct domains represented by its rostral and caudal halves. Most migratory pathways taken by neural crest cells in trunk regions of the axis, as well as the outgrowth of motoneuron fibers are restricted to the rostral domain of each somite. Experimental modification of the somites, achieved by constructing a mesoderm composed of multiple rostral half-somites, results in the formation of continuous and unsegmented nerves, dorsal root ganglia (DRG) and sympathetic ganglia (SG). In contrast, both neurites and crest cells are absent from a mesoderm composed of multiple-caudal half somites. However, the mechanisms responsible for gangliogenesis within the rostral half of the somite, appear to be different for DRG and SG. Vertebral development from the somites is also segmental. In implants of either multiple rostral or caudal somite-halves, the grafted mesoderm dissociates normally into sclerotome and dermomyotome. However, the morphogenetic capabilities of each somitic half differ. The lateral vertebral arch is continuous in the presence of caudal half-somite grafts and is virtually absent in rostral half-somite implants. Therefore, the rostrocaudal subdivision of the sclerotome determines the segmental pattern of neural development and is also important for the proper metameric development of the vertebrae.     

Neurite outgrowth on cryostat sections of innervated and denervated skeletal muscle

To localize factors that guide axons reinnervating skeletal muscle, we cultured ciliary ganglion neurons on cryostat sections of innervated and denervated adult muscle. Neurons extended neurites on sections of muscle (and several other tissues), generally in close apposition to sectioned cell surfaces. Average neurite length was greater on sections of denervated than on sections of innervated muscle, supporting the existence of functionally important differences between innervated and denervated muscle fiber surfaces. Furthermore, outgrowth was greater on sections of denervated muscle cut from endplate-rich regions than on sections from endplate-free regions, suggesting that a neurite outgrowth-promoting factor is concentrated near synapses. Finally, 80% of the neurites that contacted original synaptic sites (which are known to be preferentially reinnervated by regenerating axons in vivo) terminated precisely at those contacts, thereby demonstrating a specific response to components concentrated at endplates. Together, these results support the hypothesis that denervated muscles use cell surface (membrane and matrix) molecules to inform regenerating axons of their state of innervation and proximity to synaptic sites.     

Synapses with a different synaptic contact structure in the cerebellar cortex

The ultrastructure of cerebellar axosomatic (inhibitory) and axo-dendritic (excitatory) synapses were studied on the Purkinje cells and in the lower molecular layer of guinea-pigs and rats, respectively. It was shown that synaptic contacts of excitatory and inhibitory synapses differed in the existence of desmosome-like structures near the active zones. The classification of synaptic functions according to the ultrastructure of specialized contacts, earlier developed to identify neurons of lower vertebrates, is supposed to be applicable to the nervous system of higher vertebrates.     

Distribution and density of α-bungarotoxin binding sites on innervated and noninnervated Xenopus muscle cells in culture

The distribution and density of α-bungarotoxin (α-BT) binding sites on Xenopus muscle cells in culture by autoradiography using ¹²⁵I-α-BT were examined. In muscle cells grown alone α-BT binding sites were fairly uniformly distributed over the entire surface with a mean density of 104/μm² (background density). Occasionally, spots of higher density were observed (“hot spots”) where the mean density was 890/μm². The addition of neural tube cells did not change the background density. Similarly in the majority of cases medium contained with neural tube cells did not affect the density of α-BT binding sites. Previous findings that the background acetylcholine sensitivity of muscle cells increased in the presence of neural tube cells (by approximately 50%) or in conditioned medium (by approximately 70%), therefore, are not likely due primarily to an increase in the acetylcholine receptor (AChR) density. In cocultures of nerve and muscle cells regions of high α-BT binding sites were occasionally associated with the path of neurites. In such regions the density of α-BT binding sites was estimated to be approximately 1000/μm². However, even in these cells the density at non-nerve contacted regions was not different from that in muscle cells cultured alone. Whether the increase in AChR density at the junctional area is sufficient to explain a previous observation of a fivefold increase in the amplitude of spontaneous synaptic potentials during the process of AChR accumulation is discussed.     

Membrane junctions in Xenopus eggs: their distribution suggests a role in calcium regulation

We have observed the presence of membrane junctions formed between the plasma membrane and cortical endoplasmic reticulum of mature, unactivated eggs of xenopus laevis. The parallel, paired membranes of the junction are separated by a 10-mn gap within which electron-dense material is present. This material occurs in patches with an average center-to-center distance of approximately 30 nm. These junctions are rare in immature (but fully grown) oocytes (approximately 2 percent of the plasma membrane is associated with junctions) and increase dramatically during progesterone-induced maturation. Junctions in the mature, unactivated egg are two to three times more abundant in the animal hemisphere (25-30 percent of the plasma membrane associated with junction) as compared with the vegetal hemisphere (10-15 percent). Junction density decreases rapidly to values characteristic of immature oocytes in response to egg activation. The plasma membrane-ER junctions of xenopus eggs are strikingly similar in structure to membrane junctions in muscle cells thought to be essential in the triggering of intracellular calcium release from the sarcoplasmic reticulum. In addition, the junctions’ distinctive, animal-vegetal polarity of distribution, their dramatic appearance during maturation, and their disapperance during activation are correlated with previously documented patterns of calcium-mediated events in anuran eggs. We discuss several lines of evidence supporting the hypothesis that these junctions in xenopus eggs are sites that transduce extracellular events into intracellular calcium release during fertilization and activation of development.     

Active zones and receptor surfaces of freeze-fractured crayfish phasic and tonic motor synapses

Deep and superficial flexor muscles in the crayfish abdomen are innervated respectively by small populations of physiologically distinct phasic and tonic motoneurons. Phasic motoneurons typically produce large EPSP's, releasing 100 to 1000 times more transmitter per synapse than their tonic counterparts, and exhibiting more rapid synaptic depression with maintained stimulation. Freeze-fracturing the abdominal flexor muscles yielded images of phasic and tonic synapse-bearing terminals. The two types of synapse are qualitatively similar in ultrastructure, displaying on the presynaptic membrane's P-face synaptic contacts recognized by relatively particle-free oval plaques which are often framed by the muscle fiber's E-face leaflet with its associated receptor particles. Situated within these presynaptic plaques are discrete clusters of large intramembrane particles, forming active zone (AZ) sites specialized for transmitter release. AZs of phasic and tonic synapses are similar: 80% had a range of 15–40 large particles distributed in either paired spherical clusters or in linear form, with a few depressions denoting sites of synaptic vesicle fusion or retrieval around their perimeters. The packing density of particles is similar for phasic and tonic AZs. The E-face of the muscle membrane displays oval-shaped receptor-containing sites made up of tightly packed intramembranous particles. Phasic and tonic receptor particles are packed at similar densities and the measured values resemble those of several other crustacean and insect neuromuscular junctions. Overall, the similarity between phasic and tonic synapses in the packing density of particles at their presynaptic AZs and postsynaptic receptor surfaces suggests similar regulatory mechanisms for channel insertion and spacing. Furthermore, the findings suggest that morphological differences in active zones or receptor surfaces cannot account for large differences in transmitter release per synapse.     

Studies of cardiac ganglia in pre- and postnatal rabbits

Summary This investigation was undertaken to describe the ultrastructure of cardiac ganglia in rabbits from day 18 of gestation to day 35 postpartum. Special attention was directed to the types of synaptic contacts made with the principal neurons and with the small granule-containing cells. The cardiac ganglia in all animals consisted mainly of parasympathetic postganglionic neurons, supporting cells, and small granule-containing (small intensely fluorescent) cells. The neurons received afferent synaptic terminals of two types. One type contained mainly small clear vesicles typical of most cholinergic terminals. The second type contained mainly small dense-core vesicles (these were most prominent after treatment of the animal with 5-hydroxydopamine), and were considered to be adrenergic terminals. These adrenergic terminals are probably part of an inhibitory system in the ganglia. The small granule-containing cells received typical afferent synaptic terminals of the cholinergic type, and also formed specialized contacts with certain axonal terminals. These latter specializations are considered to be reciprocal synapses which probably have a role in modulating ganglionic transmission.Supported by the Kentucky Heart Association and the Heart Association of Louisville and Jefferson County     

Normal segmentation and size of the primary sympathetic ganglia depend upon the alternation of rostrocaudal properties of the somites.

Metameric organization of the dorsal root ganglia (DRG) and ventral roots depends on the alternation of rostrocaudal properties within the somites. In addition, the size of DRG is likely to be regulated by the adjacent mesoderm, because unilateral creation of a paraxial mesoderm with only rostral somitic (RS) halves, leads to the development of non-segmented DRG that are larger and contain more cells than the sum of the contralateral, control DRG. We have now extended our studies of the role of the paraxial mesoderm in the morphogenesis of the peripheral nervous system (PNS) to another metameric PNS component, the sympathetic ganglia (SG). The development of the primary sympathetic chain was studied in chick-quail chimeras with multiple half-somite grafts using quantitative morphometric analysis. In the presence of an exclusively rostral or caudal somitic mesoderm, segmentation of the initially homogeneous primary sympathetic chain into ganglia is prevented. Therefore, the SG, like the DRG and ventral roots, require the normal rostrocaudal alternation of the somitic mesoderm for segmental morphogenesis. On embryonic day 4 (E4), there is a 38% average decrease in the volume of the primary sympathetic chain opposite a RS mesoderm, compared with the primary chain on the unoperated side. This is in contrast to the average increase of 27% in the volume of the DRG opposite the grafted mesoderm in the same embryos. Our results, and classical observations, have led us to propose a model in which the mesoderm controls DRG and SG size by modulating the partition of migrating NC precursors between the anlage of these two ganglion types. According to this model, the reduction in SG volume and concomitant increase in DRG volume observed opposite RS grafts, results from the arrest in the DRG anlage of neural crest cells that normally migrate to the SG.     

Ultrastructure of BHK-21 cells treated with dopamine

The ultrastructure of a suspension or of adhered BHK-21 cells exposed to dopamine has been studied. It was found that ultrastructural modifications were observed mainly in cellular regions enriched in actin, including in intercellular desmosome-like contacts, microvilli, and under the membrane cortical layer. Desmosome-like contacts are hypertrophied with increased electron density and fibrillar bridges that appear in specialized contacts. Microvilli fused with each other and with plasma membrane of neighboring cells or were split up and penetrated by fibrils. The cytoplasm is filled with randomly arranged F-actin filaments and has a blotchy appearance. The effect of dopamine is more pronounced in nonattached cells with undifferentiated actin cytoskeletons than in spread cells with stress fibers. The blockage of the D2 receptor with haloperidol does not affect the ultrastructure of BHK-21 or dopamine induced modifications. The data obtained suggest direct interactions between dopamine and the actin cytoskeleton.     

Feedback interactions between cell-cell adherens junctions and cytoskeletal dynamics in newt lung epithelial cells

To test how cell-cell contacts regulate microtubule (MT) and actin cytoskeletal dynamics, we examined dynamics in cells that were contacted on all sides with neighboring cells in an epithelial cell sheet that was undergoing migration as a wound-healing response. Dynamics were recorded using time-lapse digital fluorescence microscopy of microinjected, labeled tubulin and actin. In fully contacted cells, most MT plus ends were quiescent; exhibiting only brief excursions of growth and shortening and spending 87.4% of their time in pause. This contrasts MTs in the lamella of migrating cells at the noncontacted leading edge of the sheet in which MTs exhibit dynamic instability. In the contacted rear and side edges of these migrating cells, a majority of MTs were also quiescent, indicating that cell-cell contacts may locally regulate MT dynamics. Using photoactivation of fluorescence techniques to mark MTs, we found that MTs in fully contacted cells did not undergo retrograde flow toward the cell center, such as occurs at the leading edge of motile cells. Time-lapse fluorescent speckle microscopy of fluorescently labeled actin in fully contacted cells revealed that actin did not flow rearward as occurs in the leading edge lamella of migrating cells. To determine if MTs were required for the maintenance of cell-cell contacts, cells were treated with nocodazole to inhibit MTs. After 1-2 h in either 10 microM or 100 nM nocodazole, breakage of cell-cell contacts occurred, indicating that MT growth is required for maintenance of cell-cell contacts. Analysis of fixed cells indicated that during nocodazole treatment, actin became reduced in adherens junctions, and junction proteins alpha- and beta-catenin were lost from adherens junctions as cell-cell contacts were broken. These results indicate that a MT plus end capping protein is regulated by cell-cell contact, and in turn, that MT growth regulates the maintenance of adherens junctions contacts in epithelia.     

Studies on the organization and localization of actin and myosin in neurons

The organization of actin in mouse neuroblastoma and chicken dorsal root ganglion (DRG) nerve cells was investigated by means of a variety of electron microscope techniques. Microspikes of neuroblastoma cells contained bundles of 7- to 8-nm actin filaments which originated in the interior of the neurite. In the presence of high concentrations of Mg++ ion, filaments in these bundles became highly ordered to form paracrystals. Actin filaments, but not bundles, were observed in growth cones of DRG cells. Actin was localized in the cell body, neurites, and microspikes of both DRG and neuroblastoma nerve cells by fluorescein-labeled S1. Myosin was localized primarily in the neurites of chick DRG nerve cells with fluorescein-labeled anti-brain myosin antibody. This antibody also stained stress fibers in fibroblasts and myoblasts but did not stain muscle myofibrils.     

Potassium inward rectifier and acetylcholine receptor channels in embryonic Xenopus muscle cells in culture

Embryonic muscle cells of the frog Xenopus laevis were isolated and grown in culture and single-channel recordings of potassium inward rectifier and acetylcholine (ACh) receptor currents were obtained from cell-attached membrane patches. Two classes of inward rectifier channels, which differed in conductance, were apparent. With 140 mM potassium chloride in the electrode, one channel class had a conductance of 28.8 ± 3.4 pS (n = 21), and, much more infrequently, a smaller channel class with a conductance of 8.6 ± 3.6 pS (n = 7) was recorded. Both channel classes had relatively long mean channel open times, which decreased with membrane hyperpolarization. The probability of finding a patch of membrane with an inward rectifier channel was high (66%) and many membrane patches contained more than one inward rectifier channel. The open state probability (with no applied potential) was high for both inward rectifier channel classes so that 70% of the time there was a channel open. Seventy-three percent of the membrane patches with ACh receptor channels (n = 11) also had at least one inward rectifier channel present when the patch electrode contained 0.1 μM ACh. Inward rectifier channels were also found at 71% of the sites of high ACh receptor density (n = 14), which were identified with rhodamine-conjugated α-bungarotoxin. The results indicate that the density of inward rectifier channels in this embryonic skeletal muscle membrane was relatively high and includes sites of membrane that have synaptic specializations. © 1996 John Wiley & Sons, Inc.     

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.