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.     

The culture of chick embryo dorsal root ganglionic cells on polylysine-coated plastic

Polylysine-coated culture surfaces are strongly adhesive for neural cells, restrict locomotion on nonneuronal elements, but do not inhibit neurite elongation. In the present study, culture dishes were pre-treated with poly-d-lysine (PDL) at various concentrations, seeded with dissociates from 8-day chick embryo dorsal root ganglia, and incubated under conditions that normally support both neuronal survival and nonneuronal proliferation. Pretreatment with low (0.1 mg/ml) PDL concentrations had no effect on neuronal survival and neuritic growth, but entirely prevented an increase in ganglionic nonneurons, yielding a numerically stable culture greatly enriched in neurons. Higher PDL concentrations caused increasing losses in both cell classes. The 50% levels of cell loss were achieved at about the same PDL dose, but earlier for neurons than nonneurons and still with no impairment of neuritic growth from the surviving neurons. A procedure was developed to compare acid-soluble and acid-precipitable accumulation of radioactivity under 1-hr pulses of [³H]uridine, which was applicable even to poorly attached cells. The cytotoxic effects of higher PDL pretreatments was revealed as early as 6 hr after seeding by 2- to 4-fold lower radioaccumulation. The data are discussed in terms of possible regulations of cell permeability and metabolism by adhesive interactions between cells and their substratum, or other cells.     

Neurite-Promoting Factor in Conditioned Medium from RN22 Schwannoma Cultures: Bioassay, Fractionation, and Properties

Abstract: On polyornithine (PORN) substrata dissociated 8-day chick embryo ciliary ganglionic neurons will survive if the culture medium is supplemented with Ciliary Neuronotrophic Factor. However, neuritic growth will not occur unless the substratum is derivatized with a PORN-bindable Neurite Promoting Factor (PNPF). In this preliminary study we report that soluble PNPF can be (1) assayed by a convenient in vitro system; (2) obtained in relatively large amounts from serum-free media conditioned over RN22 Schwannoma cultures; (3) concentrated by using Amicon XM100 ultrafiltration; and (4) separated from nearly all of the non-active protein by using ion-exchange chromatography. The partially purified PNPF can be concentrated using XM100 and is heat- and protease-sensitive. In the course of these fractionation studies we observed in some cases a concentration-dependent interference with the expression of PNPF activity in the bioassay; we propose graphical methods to permit the simultaneous determination of PNPF and the extent of such interference. Different treatments that affected the interference property did not always affect PNPF activity in a reciprocal manner, leaving open the possibility that the interference with PNPF activity results from reversible alteration of the PNPF molecule, or that there exists a separate interfering agent.     

Stage-dependent stimulation of neurite outgrowth exerted by nerve growth factor and chick heart in cultured embryonic ganglia

The ability of embryonic chick heart to elicit neuritic outgrowth in different ganglia was tested to examine (1) whether stimulative activity is possessed by the heart only at specific stages and (2) whether the ability of the ganglionic neurons to respond is limited to certain periods of development. As an assay, ganglia were explanted into thin collagen gels with ventricular tissue placed at a distance of about 1 mm. Neuritic outgrowth was measured after 2 days. Control ganglia and ganglia cultured with added nerve growth factor (NGF) were also scored. Four types of tested ganglia, including the ciliary ganglion, showed a peak in neuritic outgrowth when cultured with heart of embryonic Day 18, at about which age the heart becomes sympathetically innervated in ovo. No age-related size differences that could account for this temporal pattern were found among the heart explants when measuring their protein content. A peak in neuronal susceptibility to heart tissue was evident in the 6-day ciliary ganglion and in the 8-day paravertebral, Remak, and spinal ganglia, roughly coinciding with the onset of fibre outgrowth in ovo. Neurite extension is concluded to have been triggered by a factor spread from the heart explants and being distinct from the mouse type of NGF since anti-NGF did not at any stage block the events and since added NGF at all stages failed to evoke neurite formation in the ciliary ganglia. A testable hypothesis is that this factor regulates the growth of sympathetic and possibly parasympathetic and sensory fibres in the developing chick heart.     

Neuronal survival in intact ciliary ganglia in vivo and in vitro: Ciliary neuronotrophic factor as a target surrogate

Ciliary neuronotrophic factor (CNTF) requirements for neuronal survival in the intact ciliary ganglion (CG) have been investigated in organ culture. Exogenous CNTF was not essential for neuronal survival until embryonic Day 8. Three-day cultures from 5-day ganglia were similar with or without CNTF, showing numerous neurons and extensive neuritic development. In 3-day cultures from 8-day-old ganglia, however, no neurons survived without CNTF, and the ganglia contained only nonneuronal cells and cell debris. Similar ganglia cultured with CNTF contained many neurons, surrounded by nonneuronal cells, and abundant neuritic processes. Morphologic maturation of the neurons was less advanced in CNTF-supported ganglia than in their in vivo counterparts.     

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.     

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.     

Cholinergic neuronotrophic factors: VI. Age-dependent requirements by chick embryo ciliary ganglionic neurons

During development, ciliary ganglionic neurons become postmitotic and extend neurites in apparent independence of the presence of their future intraocular innervation targets. After reaching their peripheral innervation territory, however, these neurons become target dependent and about half of them die. We have previously reported that chick embryo intraocular target tissues contain a ciliary neuronotrophic factor (CNTF), which can be extracted and partially purified in a soluble form and which ensures near-total survival of 8-day chick embryo ciliary ganglionic neurons in monolayer cultures. In this study we have dissociated and cultured ciliary ganglia from embryonic Day (ED) 5 through 14, and examined dependence and responsiveness of their neurons to exogenously added CNTF. Two cell classes (dark and bright) could be distinguished by phase microscopy and differentially counted in cell dissociates from ED7–14, but not in ED5–6 ones. Dark cell number per ganglion increased from 6000 to 78,000 over this developmental time period. In contrast, bright cells (putative neurons) declined from a maximum of about 10,000 to 6000, suggesting a correlation with the expected neuronal cell death in vivo. Dissociated cells from ED5–14 ganglia were seeded on a polyornithine substratum coated with neurite promoting factor, cultured for 24 hr with or without added CNTF, and numerically examined for survival and neuritic development. Cultures from ED7–14 ganglia showed two cell categories: (i) flat nonneuronal elements dramatically increased in number with ganglionic age (thereby correlating with the increasing number of dark cells in the dissociates) and (ii) large, bright cells (often displaying neurite outgrowth) decreased in number in parallel with bright cell number in the dissociate. The survival of these neuronal elements was strictly dependent on exogenously added CNTF between ED7 and 10, but became progressively independent with older ages. ED14 neurons (fully capable of surviving for 24 hr without added CNTF) continued to require CNTF for neurite extension, thus displaying retained sensitivity to this factor. Although the ED5–6 cultures contained well-recognizable flat cells, the dominant category comprised cells with variable morphology, practically all of which exhibited neurite-like processes. Both the survival and neurite extension of these cells, which we tentatively interpret as immature neurons were independent of the presence of added CNTF.     

Antiserum Against Neurite Outgrowth Factor in Chick Gizzard Extract and Its Inhibitory Effect on Neuritic Response in Cultured Ciliary Neurons

Abstract: Antiserum against a neurite outgrowth factor (NOF) of gizzard extract that promotes neurite outgrowth from dissociated ciliary ganglionic neurons (CG neurons) of 8-day-old chick embryo was prepared to determine whether or not the antiserum inhibits neurite outgrowth from cultured neurons or explants of chick and murine tissues. When CG neurons were cultured on a polyornithine-coated well exposed to NOF (NOF-bound POR well), marked neurite outgrowth was observed. When NOF-bound POR wells were exposed to antiserum, neurite outgrowth from CG neurons was gradually inhibited with increasing amounts of antiserum, while exposure to preimmune serum did not prevent neurite outgrowth. Antiserum had no effect on neuronal survival during a 48-h incubation. The diluted antiserum, which produced nearly 100% inhibition of the NOF activity, was almost equally active in suppressing the activity of NOFs in conditioned media (CM) of various chick embryo tissues, but showed much less inhibitory effects on NOFs in CM of murine tissues. The appearance of neurites from explants of spinal cord, dorsal root ganglion, or retina of chick embryo was also inhibited by the antiserum. These results indicate that antiserum against NOF from gizzard extract suppressed the activity of NOFs from various sources, and that there are species differences in NOFs, at least between chick and murine.     

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.     

Tissue explants affecting extension and orientation of axons in cultured chick embryo ganglia

Various ganglia from 10-day-old chick embryos were cultured for 3 days in substrata of hydrated collagen lattices. Each ganglion was surrounded at a distance of about 1 mm by three tissue expiants which were identical in one series of cultures and taken from different organs in another. The extent of axon outgrowth towards the different explants was estimated by counting intersections between the axons and test lines arranged perpendicularly to the radial outgrowth direction. The various organs stimulated axon formation to distinctly different extents. Spinal cord, skeletal muscle, skin, liver, colon, kidney and heart had, in that order, increasingly stimulative influence on sympathetic chain ganglia. Colon, followed by heart and liver, had the strongest stimulative effect on Remak's colon ganglion. Spinal and trigeminal ganglia showed dense outgrowth of fibroblast-like cells and were not included in the calculations. However, they appeared to be stimulated to extend axons by exposure to heart explants. The results imply that the tissue explants release various amounts of stimulative factors that reach the ganglia by diffusion. When presented to different tissue explants, the same ganglion showed different extents of outgrowth towards the various tissues. Also, ganglia showed dense outgrowth of axons directed towards inserted capillary tubes containing nerve growth factor. The courses taken by the axons as revealed in silver impregnated whole mounted ganglia suggest that chemotaxis can account for the directed axon outgrowth.     

Fibronectin-like immunoreactivity in Helisoma buccal ganglia: evidence that an endogenous fibronectin-like molecule promotes neurite outgrowth

We examined the distribution of fibronectin-like (FNL) immunoreactivity associated with intact buccal ganglia, cell-cultured buccal ganglia neurons and nonneuronal cells, and brain-conditioned medium from the snail Helisoma. In addition, the possible roles of fibronectin in the regulation of neurite outgrowth were studied. Immunofluorescent staining for FNL antigens revealed intense staining in patches and fibrous arrays over the connective tissue sheaths of buccal ganglia and nerve trunks. Within the ganglia, heavy staining was seen surrounding neurons and in track-like arrangements. In cell cultures, specific staining was associated with nonneuronal cell surfaces and to a lesser degree with the surface of identified neurons. In addition, a noncellular, substrate-bound component of brain-conditioned medium displayed FNL immunoreactivity. Since cultured Helisoma neurons require a substrate-associated, brain-derived conditioning factor (CF) in order to elaborate neurites with motile growth cones, we tested whether the FNL immunoreactive substance might act as a neuritotropic agent. Fibronectin antiserum suppressed, in a dose-dependent manner, the CF-induced sprouting of identified neurons in isolated cell culture. When added at increasing concentrations to neurons already growing in response to CF, fibronectin antiserum exerted a biphasic effect on neurite elongation; outgrowth was accelerated at low, but inhibited at high, antiserum concentrations. In contrast, growth cone structures associated with motility (filopodia and lamellipodia) were progressively reduced by increasing levels of antiserum. A short peptide derived from fibronectin's cell-binding domain (Arg-Gly-Asp-Ser) also greatly reduced neurite outgrowth. The combined results of this study indicate an abundance of FNL immunoreactive molecules within the CNS of Helisoma, their probable production by nonneuronal cells, and their function as a substrate-associated component of CF which promotes growth cone filopodial and lamellipodial activity.     

Regulation of vasoactive intestinal peptide expression in sympathetic neurons in culture and after axotomy: The role of cholinergic differentiation factor/leukemia inhibitory factor

Vasoactive intestinal peptide (VIP) expression increases in sympathetic neurons when they are grown in dissociated cell or explant cultures and when they are axotomized in vivo. In dissociated cell culture, the magnitude of the VIP increase was reduced when nonneuronal cells were removed and medium conditioned by ganglionic nonneuronal cells increased VIP in neuron-enriched cultures. Antiserum Against cholinergic differentiation factor (also leukemia inhibitory factor; CDF/LIF), but not against ciliary neurotrophic factor, immunoprecipitated this activity. Medium conditioned by sympathetic ganglion explants also contained a VIP-stimulatory molecule that was immunoprecipitated by CDF/LIF antiserum, and CDF/LIF antiserum partially blocked VIP induction in explants. CDF/LIF mRNA was increased in dissociated cell cultures, in ganglion explants and in vivo after axotomy. Our results suggest that CDF/LIF released from ganglionic nonneuronal cells plays an important role in regulating VIP after axotomy. 1994 John Wiley & Sons, Inc.     

Reduced sympathetic neurite outgrowth on uterine tissue sections from rats treated with estrogen

In order to evaluate the contribution of substrate-bound factors to the extent and patterning of the sympathetic innervation of rat uterus following estrogen treatment, superior cervical ganglion explants from neonatal and adult ovariectomized rats were cultured on tissue sections of fresh frozen uterus from adult ovariectomized rats treated with estrogen or a vehicle. The main findings were: (1) neurite growth was greatly influenced by histological features of the underlying section; (2) on myometrial sections, neurites followed the orientation of the main axis of the longitudinally sectioned muscle cells; (3) neurites showed limited growth on transversally sectioned smooth muscle; (4) neuritic patterning was unaffected by a reduction in migrating ganglionic non-neuronal cells; (5) neurite outgrowth, but not non-neural cell migration, was markedly reduced on myometrial sections from rats treated with estrogen. These results suggest that adult myometrium continues to provide signals allowing the organotypic patterning and growth of sympathetic axons, that estrogen treatment modifies myometrial substrate properties so that it is less supportive for sympathetic neurite growth, and that adult sympathetic neurons retain their ability to recognize substrate-bound cues present in the myometrium. On endometrial sections, neurites formed radially symmetric halos, which were reduced in size on estrogen-treated endometrial substrates. Thus, changes in the neuritogenic capacity of the uterus underlie plasticity in uterine sympathetic nerves, and alterations in substrate-bound factors contribute to the diminished receptivity of the estrogenized uterus to its sympathetic innervation.     

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.     

Conditioned medium enhances neuritic outgrowth from rat spinal cord explants

Medium conditioned by primary cultures of fetal or neonatal rat skeletal muscle, fibroblasts, or lung cells dramatically increases the neuritic outgrowth from spinal cord explants. After 7 days in vitro, the outgrowth of neurites from 15- to 16-day fetal rat spinal cord slices grown in conditioned medium (CM) covers a 3- to 4-fold greater area than that from slices grown in fresh, nonconditioned (control) medium. Moreover, the pattern of neuritic outgrowth is markedly different in CM-treated slices. In control slices, the neurites form a tangled, dense network of neurites which usually extend only a small distance from the slice edge, while in CM-treated slices, the neurites form a more open network, with the majority of neurites extending radially for long distances (up to several millimeters) from the slice edge. The effect of CM on neuritic outgrowth is not due to a detoxification or modification of the serum in the medium, because increased neuritic outgrowth was observed in slices grown in medium conditioned in the presence or absence of 10% fetal calf serum. The outgrowth-enhancing factor(s) in CM has a high molecular weight, since all outgrowth-enhancing activity is retained by membrane filters with a nominal molecular weight cutoff of 10⁵ daltons. This factor(s) is stable at 58°C for 30 min, and does not appear to be βNGF or fibronectin.     

Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF, lack NGF receptors but are supported by a liver-derived neurotrophic factor

Explant and dissociated neuron-enriched cultures of nodose ganglia (inferior or distal sensory ganglion of the Xth cranial nerve) were established from chick embryos taken between embryonic Day 4 (E4) and Day 16 (E16). The response of each type of culture to nerve growth factor (NGF) was examined over this developmental range. At the earliest ages taken (E4-E6), NGF elicited modest neurite outgrowth from ganglion explants cultured in collagen gel for 24 hr, although the effect of NGF on ganglia taken from E4 chicks was only marginally greater than spontaneous neurite extension from control ganglia of the same developmental age. The response of nodose explants to NGF was maximal at E6-E7, but declined to a negligible level in ganglia taken from E9-E10 or older chick embryos. In dissociated neuron-enriched cultures, nodose ganglion neurons were unresponsive to NGF throughtout the entire developmental age range between E5 and E12. In contrast to the lack of effect of NGF, up to 50% of nodose ganglion neurons survived and produced extensive neurites in dissociated cultures, on either collagen- or polylysine-coated substrates, in the presence of extracts of late embryonic or early posthatched chick liver (E18-P7). Antiserum to mouse NGF did not block the neurotrophic activity of chick (or rat or bovine) liver extracts. Whether cultured with chick liver extract alone or with chick liver extract plus NGF, nodose ganglion neurons taken from E6-E12 chick embryos and maintained in culture for 2 days were devoid of NGF receptors, as assessed by autoradiography of cultures incubated with 125I-NGF. Under similar conditions 70-95% of spinal sensory neurons (dorsal root ganglion--DRG) were heavily labeled. 2+     

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.     

The NC1 domain of type IV collagen promotes axonal growth in sympathetic neurons through interaction with the alpha 1 beta 1 integrin

We have examined the effects of collagen IV on the morphological development of embryonic rat sympathetic neurons in vitro. In short-term (less than or equal to 24 h) culture, collagen IV accelerated process outgrowth, causing increases in the number of neurites and total neuritic length. Analysis of proteolytic fragments of collagen IV indicated that the NC1 domain was nearly as active as the intact molecule in stimulating process outgrowth; in contrast, the 7S domain and triple helix-rich fragments of collagen IV were inactive. Moreover, anti-NC1 antiserum inhibited neuritic outgrowth on collagen IV by 79%. In long-term (up to 28 d) cultures, neurons chronically exposed to collagen IV maintained a single axon but failed to form dendrites. Thus, the NC1 domain of collagen IV can alter neuronal development by selectively stimulating axonal growth. Comparison of collagen IV's effects to those of laminin revealed that these molecules exert quantitatively different effects on the rate of initial axon growth and the number of axons extended by sympathetic neurons. Moreover, neuritic outgrowth on collagen IV, but not laminin, was blocked by cycloheximide. We also observed differences in the receptors mediating the neurite-promoting activity of these proteins. Two different antisera that recognize beta 1 integrins each blocked neuritic outgrowth on both collagen IV and laminin; however, an mAb (3A3) specific for the alpha 1 beta 1 integrin inhibited collagen IV but not laminin-induced process growth in cultures of both sympathetic and dorsal root neurons. These data suggest that immunologically distinct integrins mediate the response of peripheral neurons to collagen IV and laminin.     

Developmental potentialities in the nonneuronal population of quail sensory ganglia

Sensory ganglia taken from quail embryos at E4 to E7 were back-transplanted into the vagal neural crest migration pathway (i.e., at the level of somites 1 to 6) of 8- to 10-somite stage chick embryos. Three types of sensory ganglia were used: (i) proximal ganglia of cranial sensory nerves IX and X forming the jugular-superior ganglionic complex, whose neurons and nonneuronal cells both arise from the neural crest; (ii) distal ganglia of the same nerves, i.e., the petrosal and nodose ganglia in which the neurons originate from epibranchial placodes and the nonneuronal cells from the neural crest; (iii) dorsal root ganglia taken in the truncal region between the fore- and hindlimb levels. The question raised was whether cells from the graft would be able to yield the neural crest derivatives normally arising from the hindbrain and vagal crest, such as carotid body type I and II cells, enteric ganglia, Schwann cells located along the local nerves, and the nonneuronal contingent of cells in the host nodose ganglion. All the grafted cephalic ganglia provided the host with the complete array of these cell types. In contrast, grafted dorsal root ganglion cells gave rise only to carotid body type I and II cells, to the nonneuronal cells of the nodose ganglion, and to Schwann cells; the ganglion-derived cells did not invade the gut and therefore failed to contribute to the host's enteric neuronal system. Coculture on the chorioallantoic membrane of aneural chick gut directly associated with quail sensory ganglia essentially reinforced these results. These data demonstrate that the capacity of peripheral ganglia to provide enteric plexuses varies according to the level of the neuraxis from which they originate.