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.     

Purified proteins acting on cultured chick embryo ciliary ganglion neurons

Chick embryo ciliary ganglion neurons in dissociated monolayer culture have been used to examine molecular requirements for neuronal survival and neurite growth. These neurons will rapidly die in vitro unless supplied with an adequate level of ciliary neuronotrophic factor (CNTF), and even in the presence of CNTF they will not vigorously extend neurites on polyornithine substrata unless supplied with appropriate amounts of polyornithine-binding neurite-promoting factors (PNPFs). Recent work on the purification and partial characterization of embryonic chick eye CNTF and rat schwannoma PNPF is reviewed, and in vitro responses of ciliary ganglion neurons to other purified proteins such as laminin, fibronectin, insulin, and nerve growth factor are mentioned.     

Distinct molecular interactions mediate neuronal process outgrowth on non-neuronal cell surfaces and extracellular matrices

We have compared neurite outgrowth on extracellular matrix (ECM) constituents to outgrowth on glial and muscle cell surfaces. Embryonic chick ciliary ganglion (CG) neurons regenerate neurites rapidly on surfaces coated with laminin (LN), fibronectin (FN), conditioned media (CM) from several non-neuronal cell types that secrete LN, and on intact extracellular matrices. Neurite outgrowth on all of these substrates is blocked by two monoclonal antibodies, CSAT and JG22, that prevent the adhesion of many cells, including neurons, to the ECM constituents LN, FN, and collagen. Neurite outgrowth is inhibited even on mixed LN/poly-D-lysine substrates where neuronal attachment is independent of LN. Therefore, neuronal process outgrowth on extracellular matrices requires the function of neuronal cell surface molecules recognized by these antibodies. The surfaces of cultured astrocytes, Schwann cells, and skeletal myotubes also promote rapid process outgrowth from CG neurons. Neurite outgrowth on these surfaces, though, is not prevented by CSAT or JG22 antibodies. In addition, antibodies to a LN/proteoglycan complex that block neurite outgrowth on several LN-containing CM factors and on an ECM extract failed to inhibit cell surface-stimulated neurite outgrowth. After extraction with a nonionic detergent, Schwann cells and myotubes continue to support rapid neurite outgrowth. However, the activity associated with the detergent insoluble residue is blocked by CSAT and JG22 antibodies. Detergent extraction of astrocytes, in contrast, removes all neurite- promoting activity. These results provide evidence for at least two types of neuronal interactions with cells that promote neurite outgrowth. One involves adhesive proteins present in the ECM and ECM receptors on neurons. The second is mediated through detergent- extractable macromolecules present on non-neuronal cell surfaces and different, uncharacterized receptor(s) on neurons. Schwann cells and skeletal myotubes appear to promote neurite outgrowth by both mechanisms.     

Differential expression and localization of neuronal intermediate filament proteins within newly developing neurites in dissociated cultures of Xenopus laevis embryonic spinal cord

The molecular subunit composition of neurofilaments (NFs) progressively changes during axon development. In developing Xenopus laevis spinal cord, peripherin emerges at the earliest stages of neurite outgrowth. NF-M and XNIF (an alpha-internexin-like protein) appear later, as axons continue to elongate, and NF-L is expressed after axons contact muscle. Because NFs are the most abundant component of the vertebrate axonal cytoskeleton, we must understand why these changes occur before we can fully comprehend how the cytoskeleton regulates axon growth and morphology. Knowing where these proteins are localized within developing neurites and how their expression changes with cell contact is essential for this understanding. Thus, we examined by immunofluorescence the expression and localization of these NF subunits within dissociated cultures of newly differentiating spinal cord neurons. In young neurites, peripherin was most abundant in distal neuritic segments, especially near branch points and extending into the central domain of the growth cone. In contrast, XNIF and NF-M were usually either absent from very young neurites or exhibited a proximal to distal gradient of decreasing intensity. In older neurites, XNIF and NF-M expression increased, whereas that of peripherin declined. All three of these proteins became more evenly distributed along the neurites, with some branches staining more intensely than others. At 24 h, NF-L appeared, and in 48-h cultures, its expression, along with that of NF-M, was greater in neurites contacting muscle cells, arguing that the upregulation of these two subunits is dependent on contact with target cells. Moreover, this contact had no effect on XNIF or peripherin expression. Our findings are consistent with a model in which peripherin plays an important structural role in growth cones, XNIF and NF-M help consolidate the intermediate filament cytoskeleton beginning in the proximal neurite, and increased levels of NF-L and NF-M help further solidify the cytoskeleton of axons that successfully reach their targets.     

Developmental decrease in neurite extension in cultured chick embryo retina and spinal cord neurons

Retina and spinal cord neurons from chick embryos attach to culture substrates and extend neurites. There is a statistically significant age-related decrease in the percentage and average length of neurites formed in 24-hr cultures of chick retina and spinal cord neurons between 6 and 16 days of embryonic age. The developmental decrease of neurite extension may be important for synaptogenesis in the developing nervous system.     

Nerve fibers in culture and their interactions with non-neural cells visualized by immunofluorescence

Cultures of embryonic mouse spinal cord explants, alone or in combination with rat myotubes, were stained by indirect immunofluorescence using antibodies against three structural proteins to: (a) reveal the distribution of these proteins among different cell types, and (b) test the usefulness of antibody staining to reveal the gross morphology of the neurite network in complex cultures. Affinity column purified antibodies were used against chicken gizzard actin, porcine brain tubulin, and skeletal muscle alpha-actinin. Neurites were stained intensely by anti-actin as was the stress fiber pattern of underlying fibroblasts. With anti-tubulin, the staining of neurites was an order of magnitude more intense than the staining of the microtubule pattern of background fibroblasts. Neurite cell bodies and astrocyte-like glia cells were stained with anti-tubulin and their nuclei remained unstained. Anti-tubulin could thus be used to trace even the finest extensions of nerve processes in spinal cord and spinal cord-muscle cultures. Furthermore, it could be combined with the histochemical reaction for acetylcholinesterase (AChE, EC 3.1.1.7) to demonstrate AChE-positive neurons and specialized nerve-muscle contact sites. The staining of neural elements with anti-alpha-actinin was generally much weaker than with anti-actin and anti-tubulin. Neurites were stained only moderately in comparison to myotube Z lines in the same culture. However, a distinct staining of the periphery of dorsal root ganglion cells was observed. Thus, a protein immunologically related to muscle alpha-actinin is present in the nervous system. In myotubes, Z lines were stained intensely with anti-alpha-actinin while I bands were only faintly stained with anti-actin. In isolated myofibrils, both structures were stained intensely with the same antibody preparations.     

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.     

Motoneurons purified by cell sorting respond to two distinct activities in myotube-conditioned medium

Spinal motoneurons from chick embryos were purified by retrograde transport and fluorescence-activated cell sorting. Growth conditions for motoneurons were studied, with experiments focused on the effects of conditioned media from chick myotubes, fibroblasts, and spinal cord dividing cells. Motoneurons rapidly extended neurites when plated onto polylysine-coated dishes that had been exposed to these conditioned media. Enzymatic analysis of the substratum-binding, neurite outgrowth-promoting activity from myotube-conditioned medium indicated that it contained heparan sulfate and protein. The neurite outgrowth-promoting activity sedimented as a peak centered at a density of 1.34 in associative cesium chloride gradients, and eluted near the void volume of a Sepharose CL-6B column. Inclusion of myotube conditioned medium in the culture medium of motoneurons also enhanced their survival over periods greater than 2 days in culture. This enhancement of survival could not be explained by myotube-conditioned medium providing motoneurons with a continuous supply of the neurite outgrowth-promoting activity. Media conditioned by spinal cord dividing cells and fibroblasts supported motoneuron survival to some extent, but this effect was not as great as that of myotube-conditioned medium.     

ras p21 protein promotes survival and fiber outgrowth of cultured embryonic neurons

Although evidence obtained with the PC12 cell line has suggested a role for the ras oncogene proteins in the signal transduction of nerve growth factor-mediated fiber outgrowth, little is known about the signal transduction mechanisms involved in the neuronal response to neurotrophic factors in nontransformed cells. We report here that the oncogene protein T24-ras, when introduced into the cytoplasm of freshly dissociated chick embryonic neurons, promotes the in vitro survival and neurite outgrowth of nerve growth factor-responsive dorsal root ganglion neurons, brain-derived neurotrophic factor-responsive nodose ganglion neurons, and ciliary neuronotrophic factor-responsive ciliary ganglion neurons. The proto-oncogene product c-Ha-ras also promotes neuronal survival, albeit less strongly. No effect could be observed with truncated counterparts of T24-ras and c-Ha-ras lacking the 23 C-terminal amino acids including the membrane-anchoring, palmityl-accepting cysteine. These results suggest a generalized involvement of ras or ras-like proteins in the intracellular signal transduction pathway for neurotrophic factors.     

N-cadherin and integrins: two receptor systems that mediate neuronal process outgrowth on astrocyte surfaces

Receptor-mediated interactions between neurons and astroglia are likely to play a crucial role in the growth and guidance of CNS axons. Using antibodies to neuronal cell surface proteins, we identified two receptor systems mediating neurite outgrowth on cultured astrocytes. N-cadherin, a Ca2(+)-dependent cell adhesion molecule, functions prominently in the outgrowth of neurites on astrocytes by E8 and E14 chick ciliary ganglion (CG) neurons. beta 1-class integrin ECM receptor heterodimers function less prominently in E8 and not at all in E14 neurite outgrowth on astrocytes. The lack of effect of integrin beta 1 antibodies on E14 neurite outgrowth reflects an apparent loss of integrin function, as assayed by E14 neuronal attachment and process outgrowth on laminin. N-CAM appeared not to be required for neurite outgrowth by either E8 or E14 neurons. Since N-cadherin and integrin beta 1 antibodies together virtually eliminated E8 CG neurite outgrowth on cultured astrocytes, these two neuronal receptors are probably important in regulating axon growth on astroglia in vivo.     

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.     

Purification and characterization of a neurite outgrowth factor from chicken gizzard smooth muscle

Chicken gizzard extract contains a macromolecular glycoprotein that promotes neurite outgrowth of dissociated neurons from the ciliary ganglia of chick embryos. Using conventional purification procedures, the factor responsible for the neurite outgrowth (neurite outgrowth factor (NOF)) was purified about 2000-fold to an apparent single protein band (as judged by agarose-polyacrylamide gel electrophoresis). Twenty fmol/cm2 of the purified NOF bound to the culture well was sufficient to exert maximal neuritic response of cultured ciliary ganglia neurons from 8-day-old chick embryos. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that NOF migrated as a single polypeptide of 700 and 210 kDa under nonreducing and reducing conditions, respectively. NOF stained with periodic acid-Schiff reagent and had a sedimentation coefficient of 12 s, a Stokes radius of 114 A, and an isoelectric point of about 5.1. Gizzard NOF was trypsin-sensitive, but resistant to treatment with heparinase, beta-galactosidase, and neuraminidase. Antibody prepared against the purified NOF blocked NOF activity in a dose-dependent manner. The antibody did not inhibit the biological activity of mouse laminin, although it cross-reacted weakly with laminin. Immunohistochemical analysis showed that the antibody against NOF strongly stained the extracellular matrix of cells in thin sections of gizzard, skeletal muscle, heart, liver, and ciliary ganglion, and also the membrane and the cytoplasm of cultured gizzard muscle cells. The present data suggest that gizzard NOF is a novel extracellular matrix glycoprotein which has a role in neurite outgrowth promotion from peripheral neurons in vivo. Although unlikely, the possibility that the NOF is a chick laminin could not be excluded.     

Changes in the number of chick ciliary ganglion neuron processes with time in cell culture

The purpose of this study was to describe the shape of chick ciliary ganglion neurons dissociated from embryonic day 8 or 9 ganglia and maintained in vitro. Most of the neurons were multipolar during the first three days after plating, with an average of 6.0 processes extending directly from the cell body. The neurons became unipolar with time. The remaining primary process accounted for greater than 90% of the total neuritic arbor. This striking change in morphology was not due to the selective loss of multipolar cells, or to an obvious decline in the health of apparently intact cells. The retraction of processes was neither prevented nor promoted by the presence of embryonic muscle cells. Process pruning occurred to the same extent and over the same time course whether the cells were plated on a monolayer of embryonic myotubes or on a layer of lysed fibroblasts. Process retraction is not an inevitable consequence of our culture conditions. Motoneurons dissociated from embryonic spinal cords remained multipolar over the same period of time. We conclude that ciliary ganglion neurons breed true in dissociated cell culture in that the multipolar-unipolar transition reflects their normal, in vivo, developmental program.     

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.     

Laminin promotes neuritic regeneration from cultured peripheral and central neurons

The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co- purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin.     

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.     

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+     

Clusters of intramembrane particles associated with binding sites for alpha-bungarotoxin in cultured chick myotubes

Developing chick myotubes in tissue culture were freeze-fractured to yield complementary replicas of large areas of membrane. Regions of muscle fibers with high concentrations of acetylcholine receptors were identified by binding of fluorescent-labeled alpha-bungarotoxin. Membranes in such regions contained clusters of large (100 A Diam) angular particles, similar in appearance to particles found in postsynaptic membranes of cholinergic synapses. Particles appeared in apposing areas of cytoplasmic and external leaflets but were more prevalent in the cytoplasmic leaflet. The areas of high particle concentration were coextensive with the fluorescence due to bound toxin. Treatment of cultures with tetrodotoxin increased the size of fluorescent spots and areas of high concentration of particles relative to those found in control cultures. In muscle cultures grown in the presence of spinal cord explants, some neurites contacted and innervated nearby myotubes. Intense fluorescence due to binding or alpha-bungarotoxin was present at portions of such neurite-myotube contacts. At these same portions, a high concentration of large angular particles was present in the sarcolemma adjacent to the neurite. In addition, an ordered arrangement of large particles was seen in the cytoplasmic leaflet of the neuronal plasmalemma directly apposing the muscle. The possible significance of these arrangements is discussed. Clusters on myotubes tended to be larger (contain more particles) when they occurred in groups, defined as three or more clusters with an intercluster distance of less than 0.5 micrometers. Clusters were also larger in myotubes treated with tetrodotoxin and in myotubes adjacent to some neurites in nerve-muscle cocultures. Several depressions containing particles similar to those in the clusters were found in the sarcolemma. The implications of these depressions are discussed in light of current theories of incorporation of proteins into cell membranes.     

The cytomechanics of axonal elongation and retraction

Neurites of PC12 and chick dorsal root ganglion neurons behave as viscoelastic solids in response to applied forces. This passive behavior can be modeled with three mechanical elements; a relatively stiff, undamped spring in series with a Voight element composed of a less stiff spring in parallel with a dashpot. In response to applied tensions greater than 100 microdynes, PC12 cells show lengthening behavior distinct from and in addition to the passive viscoelastic response. We interpret this as "towed growth" (Bray, D. 1984. Dev. Biol. 102:379-389) because the neurites can become twice as long without obvious thinning of the neurite and because in two cases neurite tensions fell below original rest tensions, a result that cannot be obtained with passive viscoelastic elements. The rate of towed growth showed a linear dependence of growth rate with applied tensions in 8 of 12 PC12 neurites exposed to applied tension greater than 100 microdynes. Both PC12 and chick sensory neurons showed evidence of retraction when neurite tensions were suddenly diminished. This response was measured as tension recovery after slackening in chick sensory neurites. In 62% of the cases, tension recovery exceeded and sometimes doubled the preexperimental steady-state tension. Our data indicate that this response is active tension generation by the neurite shaft. We conclude that neurite length is regulated by axial tension in both elongation and retraction. Our data suggest a three-way controller: above some tension set point, the neurite is stimulated to elongate. Below some different, lower tension threshold the neurite is stimulated to retract. Between these two tension thresholds, the neurite responds passively as a viscoelastic solid.     

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues¹ and survival factors² located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons³. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons⁴. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro^5,6.Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol⁷) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival⁸.