Developmental regulation of chick embryo retina neurite extension by extrinsic factors

Developmental changes in the control of neurite extension by extracellular factors can be examined by utilizing cultures of neurons from various aged embryos. Several conditioned media and tissue extracts were added to cultures of chick embryo retinal neurons on collagen substrates for 1, 3 and 5 days in vitro. Neurite outgrowth, measured as the percentage of neurons with neurites and the length of neurites, was promoted by optic tectal extract and cornea conditioned medium in retina neurons from younger ages (6- to 12-day embryos), but not from older ages (14- and 16-day embryos). The promotion of neurite outgrowth by optic tectal extracts may be mediated by a promotion of glial cell growth. The developmental changes in neurite extension may be due to either an altered sensitivity to the neurite promoting factors or by an altered intrinsic ability of retinal neurons to extend processes.     

Raphe-spinal neurons display an age-dependent differential capacity for neurite outgrowth compared to other brainstem-spinal populations

Functional regeneration of brainstem-spinal pathways occurs in the developing chick when the spinal cord is severed prior to embryonic day (E) 13. Functional spinal cord regeneration is not observed in animals injured after E13. This developmental transition from a permissive to a restrictive repair period may be due to the formation of an extrinsic inhibitory environment preventing axonal growth, and/or an intrinsic inability of mature neurons to regenerate. Here, we investigated the capacity of specific populations of brainstem-spinal projection neurons to regrow neurites in vitro from young (E8) versus mature (E17) brainstem explants. A crystal of carbocyanine dye (DiI) was implanted in ovo into the E5 cervical spinal cord to retrogradely label brainstem-spinal projection neurons. Three or 12 days later, discrete regions of the brainstem containing DiI-labeled neurons were dissected to produce explant cultures grown in serum-free media on laminin substrates. The subsequent redistribution of DiI into regenerating processes permitted the study of in vitro neurite outgrowth from identified brainstem-spinal neurons. When explanted on E8, i.e., an age when brainstem-spinal neurons are normally elongating through the spinal cord and are capable of in vivo functional regeneration, robust neurite outgrowth was observed from all brainstem populations, including rubro-, reticulo-, vestibulo-, and raphe-spinal neurons. In contrast, when explanted on E17, robust neurite outgrowth was seen only from raphe-spinal neurons. Neurite outgrowth from raphe-spinal neurons was 5-hydroxy-tryptamine immunoreactive. This study demonstrates that in growth factor-free environments with permissive growth substrates, neurite outgrowth from brainstem-spinal neurons is dependent on both neuronal age and phenotype.     

Heparan sulfate proteoglycan and laminin mediate two different types of neurite outgrowth

Spinal cord neurons cultured in vitro have been shown to respond to changes in their environment by means of 2 different types of neurite outgrowth: (1) neurite elongation and (2) emergence and branching of newly formed neurites. Culture of spinal cord neurons with heparan sulfate proteoglycan (HSPG) medium resulted in a 3-fold increase in neurite elongation compared to the control. Extensive branching was seen when neurons were cultured in laminin-supplemented culture medium. HSPG-induced elongation and laminin-induced branching of neurites were blocked by specific anti-HSPG and antilaminin sera, respectively. Furthermore, laminin antibodies did not inhibit neurite elongation and HSPG antibodies did not block neurite branching. Conditioned medium from primary embryonic rat muscle cultures (MCM) mimicked the effects of both HSPG and laminin on neurite outgrowth. Immunoprecipitation with anti-HSPG and antilaminin antibodies demonstrated that MCM contains these 2 basal lamina components. Our observations suggest that HSPG and laminin might be highly effective molecules for promoting neurite outgrowth of rat spinal cord neurons in vitro.     

Effect of the substrate on neurofilament phosphorylation in mixed cultures of rat embryo spinal cord and dorsal root ganglia.

The effect of the substrate on neurofilament phosphorylation was studied in primary cultures of spinal cord and dorsal root ganglia dissociated from 15-day-old rat embryos. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites. (Primaria dishes have a modified plastic surface with a net positive charge). On both polylysine and Primaria substrates, spinal cord neurons were stained with neurofilament monoclonal antibodies reacting with phosphorylated epitopes appearing early in rat embryo development, i.e. soon after neurofilament expression. Conversely, immunoreactivity with antibodies recognizing late phosphorylation events was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, only occurred after several days in culture, at a time when non-neuronal cells (mainly astrocytes) had formed a confluent monolayer.     

Neuritogenesis on collagen substrates. Involvement of integrin-like matrix receptors in retinal fibre outgrowth on collagen.

Extracellular matrix molecules such as laminin, fibronectin and collagen promote neurite outgrowth in vitro. We have investigated the capacity of hydrated gels of collagen types I-III and monomeric collagen types I-VI on plastic surfaces to support neuritogenesis. The attachment and survival of explants from the day 6 chick embryo were studied and neurite outgrowth measured as mean elongation rate and maximal neurite length. Collagen types I and III, both as three-dimensional gels or as native monomers supported neuritogenesis equal to or better than laminin. Collagen type V also supported neurite out-growth although less effectively. Collagen types II, IV and VI, as well as denatured collagens of all types tested, did not support outgrowth. The monoclonal anti-beta 1 integrin antibody (CSAT), as well as rabbit polyclonal antibodies directed to the integrin beta 1-chain, effectively inhibited neurite outgrowth on permissive collagenous substrata, indicating that collagen-binding integrins were involved in the neuritogenesis. These beta 1-integrins were independent of Arg-Gly-Asp (RGD) since neurite formation proceeded in the presence of synthetic RGD-containing peptides. Fluorescence immunohistochemistry revealed the presence of the integrin beta 1-chain on the outgrowing neurites. The results suggest a possible function of collagen and collagen-binding integrins in the development of the visual system.     

Vimentin-GFAP transition in primary dissociated cultures of rat embryo spinal cord

Primary dissociated cultures derived from 15-day-old rat embryo spinal cord with or without dorsal root ganglia (DRG) were grown on polylysine, Primaria and laminin substrates. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. After 2 days in culture, the number of cells stained with GFAP antibodies progressively increased within the vimentin-positive monolayer surrounding the neuronal aggregates. These astrocytes had the typical appearance of astrocytes in primary dissociated cultures derived from late fetal or early neonatal murine brain, i.e. large flat or stellate cells with thick processes staining equally well with GFAP and vimentin antibodies. Astrocytes found within the neuronal aggregates in 4-5 day cultures were markedly different, i.e. small stellate cells with slender processes forming a delicate mesh throughout the aggregate. These GFAP-positive cells stained only weakly with vimentin antibodies. Spinal cord neurons formed aggregates on laminin substrates but failed to extend neurites and rapidly degenerated. The large flat cells in the surrounding monolayer gradually invaded the aggregates. These cells stained with both GFAP and vimentin antibodies. DRG neurons developed equally well on Primaria and laminin substrates, extending their neurites on the vimentin-positive flat cells forming the monolayer regardless of their reactivity with GFAP antibodies.     

Age-dependent patterns and rates of neurite outgrowth from CNS neurons on Schwann cell-derived basal lamina and laminin substrata

In the present study, we have examined the growth characteristics of CNS neurons on type I collagen, detergent-treated collagen (dColl), Schwann cell-derived basal lamina (SC-BL), and purified laminin substrata. Neurons from the cerebral cortex, septal basal forebrain, and lumbosacral spinal cord were obtained from embryonic age (E) 15 and E18 rats and grown in vitro as explants on the test substrata. Neurons from either embryonic age displayed radial neurite outgrowth on collagen and dColl substrata. However, pretreatment of collagen with detergents slightly diminished its ability to support neurite outgrowth, as evidence by the 20-40% decrease in the rate of neurite growth on dColl versus the rate calculated for neurons on collagen. In contrast to the similar growth characteristics of E15 and E18 neurons on collagen and dColl, the pattern of neurite outgrowth for CNS neurons on SC-BL and laminin substrata was age dependent. Most E15 neurons grown on SC-BL extended neurites that grew identically to those observed on dColl; these 'non-orienting' neurites maintained a radial orientation to their outgrowth despite encountering interposing channels of SC-BL and grew at rates equal to that calculated for neurons on dColl. E15 neurons placed on laminin substrata showed similar growth patterns and rates equal to that calculated for neurons on dColl. E15 neurons placed on laminin substrata showed similar growth patterns and rates to neurons on collagen. In contrast, neurons from E18 rats exhibited neurites that preferentially grew in intimate association with SC-BL channels once contact with the channels was established. These 'orienting' neurites faithfully elongated within the SC-BL and demonstrated a 1.4- to 2.0-fold increase in growth rate compared with the sister cultures of neurons grown on dColl. Furthermore, E18 neurons exhibited a 1.4-fold increase in growth on laminin compared with E18 neurons grown on collagen. A minor population of neurites exhibiting similar characteristics to orienting neurites was also observed in E15 cultures. It is hypothesized that orienting and non-orienting neurites reflect the outgrowth of 'regenerating' and 'developing' neurons, respectively, and may indicate an inherent difference in the ability of regenerating and developing neurons to recognize and respond to the same guidance signals.     

Regulation of retinal neurite growth by alterations in MAPK/ERK kinase (MEK) activity

Activation of the extracellular-signal regulated kinase (ERK) cascade may be involved in the promotion of neurite outgrowth by a variety of stimuli. For example, we have previously shown that laminin (LN) and N-cadherin activate ERK2 in chick retinal neurons, and that pharmacological inhibition of MAPK/ERK kinase (MEK), the major upstream ERK2 activator, severely impairs neurite growth induced by these proteins. We have therefore hypothesized that ERK activation through MEK is required for optimal induction of neurite growth by these proteins. Here we show that expression of mutant MEK in transfected retinal neurons alters neuronal responses to LN in a manner consistent with this hypothesis. Neurons expressing a constitutively active MEK construct extended longer neurites on LN than controls, while neurons transfected with a dominant negative construct extended shorter neurites. Further, experiments in which transfected neurons were replated onto polylysine substrates suggest that activation of MEK is sufficient for neurite promotion on a non-inducing substrate, and neurons replated onto LN confirm the pharmacological data that inhibition of MEK activation inhibits LN-induced neurite growth. We conclude that ERK activation plays a direct role in the promotion of neurite outgrowth from retinal neurons by LN.     

Choroid coat extract and ciliary neurotrophic factor strongly promote neurite outgrowth in the embryonic chick retina

Previous studies have shown that extracts from the target optic tectum stimulate neurite outgrowth from retinal explants. The present study indicates that the choroid coat is an even richer source of retinotrophic activity. We thus studied the effects of recombinant rat ciliary neurotrophic factor (CNTF) on primary cultures of dissociated chick ciliary ganglion neurons and retinal explants for a comparison with choroid coat extract from the E 18 chick. For our assays, E9 ciliary neurons were incubated in collagen gels and retinal explants were cultured on collagen gels with the addition of the trophic factors and maintained for two or four days. Survival of ciliary neurons per area as well as maximal neurite length in retinal cultures were determined. Growth responses occurred in a dose-dependent manner both to CNTF and choroid extract. Immunofluorescence examination of cells and developing processes showed 200 kdal neurofilament positivity demonstrating that the cells studied were neurons with neurites. It is concluded that a trophic activity of the choroid as well as the recombinant CNTF stimulate retinal neuron survival and neurite extension. The results suggest that CNTF may have developmental functions in the establishment of the visual pathways.     

Schwann cell surfaces but not extracellular matrix organized by Schwann cells support neurite outgrowth from embryonic rat retina

Despite evidence that glial cell surfaces and components of the extracellular matrix (ECM) support neurite outgrowth in many culture systems, the relative contributions of these factors have rarely been compared directly. Specifically, it remains to be determined which components of peripheral nerve support growth of central nerve fibers. We have directly compared neurite outgrowth from embryonic day 15 rat retinal explants placed onto beds of (1) Schwann cells without ECM, (2) Schwann cells expressing ECM (including a basal lamina), (3) cell-free ECM prepared from neuron-Schwann cell cultures, (4) nonglial cells (fibroblasts), and (5) 2 isolated ECM components, laminin and type I collagen. From the first day in culture, retinal explants extended neurites when placed on Schwann cells without ECM. Outgrowth on Schwann cells expressing ECM was also extensive, but not obviously different form that on Schwann cells alone. Ultrastructural study revealed that 95% of retinal neurites in ECM-containing cultures contacted other neurites and Schwann cell surfaces exclusively. On cell-free ECM prepared from neuron-Schwann cell cultures, neurite extension was poor to nonexistent. No neurite outgrowth occurred on fibroblasts. Retinal explants also failed to extend neurites onto purified laminin and ammoniated type I collagen substrata; however, growth was rapid and extensive on air-dried type I collagen. In cultures containing islands of air-dried type I collagen on a laminin-coated coverslip, retinal explants attached and extended neurites on collagen, but these neurites did not extend off the island onto the laminin substratum. We conclude from these experiments that neurite extension from embryonic rat retina is supported by a factor found on the surface of Schwann cells and that neither organized nor isolated ECM components provide this neurite promotion. These findings are discussed in relation to possible species differences in growth requirements for retinal ganglion cell neurites and to the specificity of response of different CNS neurites to ECM substrata.     

Adult rat olfactory nerve ensheathing cells are effective promoters of adult central nervous system neurite outgrowth in coculture

A coculture method is described for ensheathing glial cells from adult rat olfactory nerve, serving as a substrate for the regrowth of neurites from adult rat retinal ganglion cells. Immunocytochemically identified phenotypes present in primary cultures of olfactory nerve cells are described, and their ability to promote neurite outgrowth is compared with neonatal astrocytes and Schwann cells, with other nonglial cells, and with laminin. Ensheathing cell cultures were more effective than any other substrate tested and also directed the orientation of regrowing neurites. In comparison with cultured Schwann cells, which released neurotrophic factors into the culture medium, there was no evidence of a similar activity in ensheathing cell cultures. Combinations of ensheathing cell–conditioned medium and substrates of laminin, merosin, or 3T3 cells also failed to show the release of factors enhancing either survival or neurite outgrowth from retinal ganglion cells. Evidence is presented for a partial inhibition of neurite outgrowth in the presence of calcium channel antagonists or an intracellular calcium‐chelating reagent. This provides evidence for a contribution from an intracellular calcium signaling mechanism, possibly implicating ensheathing cell adhesion molecules in promoting neurite outgrowth. GLIA 25:256–269, 1999. © 1999 Wiley‐Liss, Inc.     

Ciliary neurotrophic factor stimulates neurite outgrowth from spinal cord neurons

Ciliary neurotrophic factor (CNTF) has been shown to promote the survival of motoneurons, but its effects on axonal outgrowth have not been examined in detail. Since nerve growth factor (NGF) promotes the outgrowth of neurites within the same populations of neurons that depend on NGF for survival, we investigated whether CNTF would stimulate neurite outgrowth from motoneurons in addition to enhancing their survival. We found that CNTF is a powerful promoter of neurite outgrowth from cultured chick embryo ventral spinal cord neurons. An effect of CNTF on neurite outgrowth was detectable within 7 hours, and at a concentration of 10 ng/ml, CNTF enhanced neurite length by about 3- to 4-fold within 48 hours. The neurite growth-promoting effect of CNTF does not appear to be a consequence of its survival-promoting effect. To determine whether the effect of CNTF on spinal cord neurons was specific for motoneurons, we analyzed cell survival and neurite outgrowth for motoneurons labeled with diI, as well as for neurons taken from the dorsal half of the spinal cord, which lacks motoneurons. We found that the effect of CNTF was about the same for motoneurons as it was for neurons from the dorsal spinal cord. The responsiveness of a variety of spinal cord neurons to CNTF may broaden the appeal of CNTF as a candidate for the treatment of spinal cord injury or disease. © 1996 Wiley-Liss, Inc.     

Polyornithine-attached neurite-promoting factors (PNPFs). Culture sources and responsive neurons

We have recently reported the existence within chick embryo heart cell conditioned medium (HCM) of two distinct and independently assayable factors. One agent, ciliary neuronotrophic factor (CNTF), supports the in vitro survival of 8-day chick embryo ciliary ganglionic (CG) neurons. The other factor, polyornithine-attachable neurite promoting factor (PNPF) is required for extensive neuritic growth from these same CNTF-supported CG neurons.In the present study we have examined the occurrence of PNPF activity within nearly 100 different conditioned media using our previously described chick CG bioassay system. From this screening we conclude that: (1) PNPF production is a rather widespread property of cultured neural as well as non-neural cells; and (2) the chick bioassay is sensitive to PNPF activity from all the species examined, including mouse, rat, human and chick cells.We next examined the effects of 3 representative PNPF-containing conditioned media (from chick heart, mouse Schwann and rat Schwannoma) on neurite production from 3 other peripheral ganglionic neuronal cultures (8-day chick dorsal root, 11-day chick sympathetic, and neonatal mouse dorsal root ganglia) as well as 4 central neuronal cultures (8-day chick embryo telencephalon, optic lobe and spinal cord and neonatal mouse cerebellum). The results of these studies indicate: (1) that the peripheral neurons exhibit a dramatic increase in neurite production in response to PNPF which can be easily recognized both qualitatively and quantitatively; whereas (2) the CNS neurons showed essentially no PNPF-induced increase in neurite production. The sole exception to the latter was the appearance within the chick spinal cord cultures of a neuronal population which extended very long neurites in response to PNPF.     

Degradation of Chondroitin Sulfate Proteoglycan Enhances the Neurite-Promoting Potential of Spinal Cord Tissue

The contribution of chondroitin sulfate proteoglycan (CSPG) in the suppression of axonal growth in rat spinal cord has been examined by means of anin vitrobioassay in which regenerating neurons are grown on tissue section substrata. Dissociated embryonic chick dorsal root ganglionic neurons were grown on normal and injured adult spinal cord tissue sections treated with chondroitinases. Neuritic growth on normal spinal cord tissue was meager. However, both the percentage of neurons with neurites and the average neurite length were substantially greater on sections treated with chondroitinase ABC. Enzymes that specifically degraded dermatan sulfate or hyaluronan were ineffective. Neuritic growth was significantly greater on injured (compared to normal) spinal cord and a further dramatic increase resulted from chondroitinase ABC treatment. Neurites grew equally within white and gray matter regions after chondroitinase treatment. Observed increases in neurite outgrowth on chondroitinase-treated tissues were largely inhibited in the presence of function-blocking laminin antibodies. These findings indicate that inhibitory CSPG is widely distributed and predominant in both normal and injured spinal cord tissues. Additionally, inhibitory CSPG is implicated in negating the potential stimulatory effects of laminin that might otherwise support spinal cord regeneration.     

A subpopulation of embryonic telencephalic neurons survive and develop in vitro in response to factors derived from the periphery

Denervated chick muscle contains factors that enhance neurite outgrowth in cultures of embryonic chicken spinal neurons. Chromatography of muscle extract on a column of DEAE-Sepharose yielded a fraction which retained most of the starting neurite-promoting activity. This DEAE fraction was tested for its activity on neurons from other regions of the central nervous system of 5-day-old chicken embryos. Both neurite outgrowth and survival of telencephalic neurons in vitro were greatly enhanced when the DEAE fraction was added at protein concentrations around 1 microgram/ml. When cultures were prepared from embryos later than 6 days in ovo, the effects of the DEAE fraction progressively diminished with age. Neurons from the embryonic diencephalon, mesencephalon and rhombencephalon were not responsive to the DEAE fraction, although they all developed neurites on a laminin substratum. Similar neurite-promoting activities for telencephalic neurons were found in extracts of neonatal brain, liver and heart, but not lung.     

Ganglioside potentiation of NGF-independent conditioned medium enhancement of neuritic outgrowth from spinal cord and ciliary ganglia explants

Culture medium conditioned (CM) by embryonic chick skeletal muscle or RN22 Schwannoma cells enhanced dramatically the neuritic development of chick embryonic spinal cord slices explanted onto a collagen substratum. The addition of a mixture of bovine brain gangliosides (BBG) or the monosialoganglioside GM1 to this medium potentiated the nerve growth factor (NGF)-independent CM-mediated neuritogenesis. A 3-4 fold increase in spinal cord outgrowth was due to increased neurite number, length and branching. The ability of the gangliosides to potentiate the positive neuritogenic action of CM was not limited solely to spinal cord cultures since similar results were obtained in parallel studies employing organized cultures of embryonic chick ciliary ganglia. These studies demonstrate the ability of gangliosides to enhance the trophic action of factor(s) present in CM. They suggest further that gangliosides may play a modulatory role in the development of the nervous system.     

Peripheral motoneuron interactions with laminin and Schwann cell-derived neurite-promoting molecules: developmental regulation of laminin receptor function

Schwann cells synthesize several neurite outgrowth-promoting molecules and localize them in either the extracellular matrix (ECM; e.g., laminin) or on the plasma membrane (e.g., L1/NgCAM and N-cadherin). Neurite outgrowth by embryonic chick ciliary ganglion (CG) neurons in response to these Schwann cell molecules largely depends on several specific neuronal cell surface receptors: integrin beta 1-class ECM receptors, L1/NgCAM, and N-cadherin (Bixby et al.: Journal of Cell Biology 107:353-361 1988). To address whether neuronal ECM receptors are regulated independently of cell surface adhesion molecules, we studied the ability of dissociated CG neurons from different developmental ages to extend neurites rapidly on 1) substrates coated with the ECM glycoprotein laminin (either from Schwann cell-conditioned medium or purified from the Engelbreth-Holm-Swarm sarcoma) or 2) the surfaces of Schwann cells or Schwannoma (RN22) cells. CG neurons gradually lost the ability between embryonic day 8 (E8) and E14 to attach to and extend neurites in an integrin-dependent fashion on purified laminin or Schwann cell-derived laminin. The inability of E14 CG neurons to respond to laminin was partially reversed after explantation for 2.5 days in vitro, which increased the percentage of responsive neurons approximately ten-fold. E14 neurons remained capable of extending neurites rapidly on the surfaces of Schwann and Schwannoma cells. Thus, the inability of E14 neurons to respond to laminin reflects a specific loss of laminin receptor function, while other receptors, most likely N-cadherin and L1/NgCAM, remain capable of promoting neurite outgrowth on Schwann cell surfaces. Since integrin beta 1-class heterodimers have been shown to function directly as receptors mediating neuronal attachment and process outgrowth on laminin, our results imply that the expression or function of laminin-binding integrin heterodimers is regulated during the development of CG neurons. The apparent loss of integrin receptor function occurs during the period when the axons of CG neurons innervate their targets. Substantial integrin receptor function is recovered when target contact is disrupted by explantation. Thus, the functions of integrin-class receptors in CG neurons may be regulated by target contact.     

L1 substrate enhances outgrowth of tyrosine hydroxylase-immunoreactive neurites in mesencephalic cell culture.

We have evaluated neurite outgrowth from mesencephalic tyrosine hydroxylase-positive neurons grown in vitro on different substrates. Cultures of ventral mesencephalon from rat embryos (E13) were plated on plastic dishes coated with the following substrates: L1, L2/HNK-1 "residual" (mainly J1/160 but also tenascin), MAG antigens from mouse brains, laminin, fibronectin, poly-L-lysine, RGD peptide, and plastic alone. After 3, 4, and 6 days in vitro, the cultures were stained using an antibody against tyrosine hydroxylase (TH), and the length of TH-positive neurites was measured by computer-assisted image analysis in a double-blind fashion. L1 antigen had a significant positive effect on neurite outgrowth compared to the other substrates studied. Laminin and fibronectin were also favorable substrates. In cultures treated with cytosine arabinoside to prevent mitoses and glial proliferation, the positive effect of L1 was abolished, but laminin still had a stimulatory effect. These data indicate that L1 may be indirectly involved in differentiation or axonal elongation of substantia nigra dopaminergic neurons and suggest a complex effect involving both neurons and glia on dopaminergic neurite development.     

Cooperation between nerve growth factor and laminin or fibronectin in promoting sensory neuron survival and neurite outgrowth

Chick embryo dorsal root ganglion (DRG) neurons were purified by differential adhesion to plastic. The purified neurons were used to study the cooperation between nerve growth factor (NGF) and laminin or fibronectin in promoting neuron survival and neurite outgrowth. NGF alone supported the survival of only 20% embryonic day 10 (E10) cells, of which only 40-50% had neurites. Treatment of the substrate with fibronectin or laminin increased survival in the presence of NGF up to 80% of the seeded neurons, all of which showed extensive neurite outgrowth. Survival and neurite outgrowth were also enhanced by the combined effects of elevated potassium and laminin. In contrast to E8-10 cells, 85% of E16 neurons survived in the basal culture conditions, i.e. without additional NGF, fibronectin or laminin, although neurite outgrowth was enhanced by all 3 proteins. Antisera to NGF, laminin and fibronectin, each independently decreased survival and neurite outgrowth of DRG neurons, totally with E9 and partially with E16 cells. The results suggest that the cooperative actions of extracellular matrix proteins and NGF are essential for survival and neurite outgrowth of embryonic DRG neurons and that these neuronal requirements change during development.     

In vitro evidence for a neurite growth-promoting activity in Trembler mouse serum

Basal lamina components, such as heparan sulfate proteoglycan (HSPG) and laminin play an important role in neuritic outgrowth for CNS and PNS neurons in culture. The mutant mouse 'Trembler' is characterized by hypomyelinization and production of an excess of basal lamina layers around Schwann cells in peripheral nerves. In order to analyse whether or not the serum of the mutant animals contains neurite outgrowth-promoting factors, we cultured rat spinal cord neurons in the presence of Trembler serum. Under these conditions, the outgrowth of neurites was increased approx. 2 times as compared to control serum. Trembler serum induces neuritic outgrowth characterized both by an increase in number of primary neurites emerging from the nerve cell body as well as by an increase in peripheral branching of neurites. To characterize the factors implicated in this increase we added antibodies directed against HSPG or laminin to the mutant serum. As a result, the increase in neuritic outgrowth was reduced or abolished in both cases. Trembler effect on neurite growth disappeared when the number of the non-neuronal cells was reduced, suggesting that the mutant serum did not act directly on neurons but by the intermediary action of non-neuronal cells.