Accelerated neurite growth from spiral ganglion neurons exposed to the Rho kinase inhibitor H-1152

Upon the death of their hair cell synaptic partners, bipolar cochlear spiral ganglion neurons either die or retract their peripheral nerve fibers. Efforts to induce the regrowth of the peripheral neurites have had to rely on limited knowledge of the mechanisms underlying spiral ganglion neurite regeneration and have been restricted by the impracticality of undertaking large numbers of manual analyses of neurite growth responses. Here we have used dissociated cultures of postnatal mouse spiral ganglia to assess the effects of the Rho kinase inhibitor H-1152 on neurite growth and to determine the utility of automated high content analysis for evaluating neurite length from spiral ganglion neurons in vitro. In cultures of postnatal mouse spiral ganglion, greater than 95% of the neurons develop bipolar, monopolar or neurite-free morphologies in ratios dependent on whether the initial medium composition contains leukemia inhibitory factor or bone morphogenetic protein 4. Cultures under both conditions were maintained for 24 h, then exposed for 18 h to H-1152. None of the cultures exposed to H-1152 showed decreased neuronal survival or alterations in the ratios of different neuronal morphologies. However, as measured manually, the population of neurite lengths was increased in the presence of H-1152 in both types of cultures. High content analysis using the Arrayscan VTi imager and Cellomics software confirmed the rank order differences in neurite lengths among culture conditions. These data suggest the presence of an inhibitory regulatory mechanism(s) in the signaling pathway of Rho kinase that slows the growth of spiral ganglion neurites. The automated analysis demonstrates the feasibility of using primary cultures of dissociated mouse spiral ganglion for large scale screens of chemicals, genes or other factors that regulate neurite growth.     

Promoting neurite outgrowth from spiral ganglion neuron explants using polypyrrole/BDNF-coated electrodes

Release of neurotrophin-3 (NT3) and brain-derived neurotrophic factor (BDNF) from hair cells in the cochlea is essential for the survival of spiral ganglion neurons (SGNs). Loss of hair cells associated with a sensorineural hearing loss therefore results in degeneration of SGNs, potentially reducing the performance of a cochlear implant. Exogenous replacement of either or both neurotrophins protects SGNs from degeneration after deafness. We previously incorporated NT3 into the conducting polymer polypyrrole (Ppy) synthesized with para-toluene sulfonate (pTS) to investigate whether Ppy/pTS/NT3-coated cochlear implant electrodes could provide both neurotrophic support and electrical stimulation for SGNs. Enhanced and controlled release of NT3 was achieved when Ppy/pTS/NT3-coated electrodes were subjected to electrical stimulation. Here we describe the release dynamics and biological properties of Ppy/pTS with incorporated BDNF. Release studies demonstrated slow passive diffusion of BDNF from Ppy/pTS/BDNF, with electrical stimulation significantly enhancing BDNF release over 7 days. A 3-day SGN explant assay found that neurite outgrowth from explants was 12.3-fold greater when polymers contained BDNF (p < 0.001), although electrical stimulation did not increase neurite outgrowth further. The versatility of Ppy to store and release neurotrophins, conduct electrical charge, and act as a substrate for nerve-electrode interactions is discussed for specialized applications such as cochlear implants.     

Expression of Wnt receptors in adult spiral ganglion neurons: frizzled 9 localization at growth cones of regenerating neurites

Little is known about signaling pathways, besides those of neurotrophic factors, that are operational in adult spiral ganglion neurons. In patients with sensorineural hearing loss, such pathways could eventually be targeted to stimulate and guide neurite outgrowth from the remnants of the spiral ganglion towards a cochlear implant, thereby improving the fidelity of sound transmission. To systematically identify neuronal receptors for guidance cues in the adult cochlea, we conducted a genome-wide cDNA microarray screen with 2-month-old CBA/CaJ mice. A meta-analysis of our data and those from older mice in two other studies revealed the presence of neuronal transmembrane receptors that represent all four established guidance pathways—ephrin, netrin, semaphorin, and slit—in the mature cochlea as late as 15 months. In addition, we observed the expression of all known receptors for the “wingless-related MMTV integration site” (Wnt) morphogens, whose neuronal guidance function has only recently been recognized. In situ hybridizations located the mRNAs of the Wnt receptors frizzled 1, 4, 6, 9, and 10 specifically in adult spiral ganglion neurons. Finally, frizzled 9 protein was found in the growth cones of adult spiral ganglion neurons that were regenerating neurites in culture. We conclude from our results that adult spiral ganglion neurons are poised to respond to neurite damage, owing to the constitutive expression of a large and diverse collection of guidance receptors. Wnt signaling, in particular, emerges as a candidate pathway for guiding neurite outgrowth towards a cochlear implant after sensorineural hearing loss.     

组织外植块对培养的鸡胚背根节神经突起生长的影响

为了解神经元的发育和周围组织的关系,本文分别用了:(1)10天鸡胚背根节与10天鸡胚心脏、皮肤、角膜、骨骼肌、肠、大脑、脊髓外植块联合培养;(2)不同时期的鸡角膜与不同胚龄的鸡胚背根节联合培养;(3)10天鸡胚背根节与雏鸡角膜内、外层联合培养。实验结果表明鸡胚心脏、皮肤和角膜外植块对背根节神经突起生长具较强的促进作用且对生长方向有明显诱导作用;骨骼肌、肠和大脑对神经突起生长也有不同程度促进作用,但脊髓却无明显作用。14、16、18天鸡胚角膜对8、10、12、14天鸡胚背根节神经突起生长有明显促进作用;12、14天鸡胚背根节在各时期角膜作用下神经突起生长都较丰富。含上皮层的雏鸡角膜外植块促进背根节神经突起生长的作用比含内皮层的角膜外植块强。     

L-type calcium channels in the regulation of neurite outgrowth from rat dorsal root ganglion neurons in culture

Dorsal root ganglion neurons from neonatal rats were grown in culture for 12 h and the extent of neurite outgrowth determined by counting the fraction of neurons with neurites. In the presence of high K+, veratridine or bradykinin the extent of neurite outgrowth was reduced by about 60%. The inhibitory effect of depolarisation was reversible and was abolished by nifedipine. gamma-Aminobutyric acid (GABA), baclofen and 2-chloroadenosine had no effect on neurite outgrowth in control cultures but abolished the inhibitory effect of depolarisation.     

Analysis of the action of euxanthone, a plant-derived compound that stimulates neurite outgrowth

We have investigated the neurite growth-stimulating properties of euxanthone, a xanthone derivative isolated from the Chinese medicinal plant Polygala caudata. Euxanthone was shown to exert a marked stimulatory action on neurite outgrowth from chick embryo dorsal root ganglia explanted in collagen gels, in the absence of added neurotrophins. It was also shown to promote cell survival in explanted chick embryo ganglia, and to stimulate neurite outgrowth from isolated adult rat primary sensory neurons in vitro. The further finding that euxanthone stimulates neurite outgrowth from explants of chick embryo retina and ventral spinal cord suggests an action on signaling pathways downstream of neuronal receptors for specific neurotrophic factors. Consistent with this, euxanthone did not promote neurite outgrowth from non-transfected PC12 cells, or from PC12 cells transfected with TrkB or TrkC, under conditions in which these cells extended neurites in response to, respectively, the neurotrophins nerve growth factor, brain-derived neurotrophic factor and neurotrophin 3. Western blot analysis of euxanthone-stimulated dorsal root ganglion explants showed that expression of phospho-mitogen-activated protein (MAP) kinase was up-regulated after 1 h of euxanthone-treatment. Inhibition of the MAP kinase pathway using PD98059, a specific inhibitor of MAP kinase kinase, blocked all euxanthone-stimulated neurite outgrowth. However, analysis of phospho-Akt expression indicated that the phosphatidylinositol-3 kinase-Akt pathway, another major signaling pathway engaged by neurotrophins, is not significantly activated by euxanthone. These results suggest that euxanthone promotes neurite outgrowth by selectively activating the MAP kinase pathway.     

Conditioned medium promotes neurite growth from both central and peripheral neurons

Explants or dissociated neurons of spinal cord and other parts from chick embryos and rat fetuses were cultured for 1–4 days. Extensive outgrowth of neurites from the spinal cord was observed in the medium which had been conditioned by skeletal and heart muscle cells. In the fresh medium, the neurites were short and accompanied by the migrating non-neuronal cells. The conditioned medium promoted also the neurite outgrowth from retina, optic tectum, ciliary ganglion, superior cervical ganglion and dorsal root ganglion.     

Functional involvement of synaptotagmin I/II C2A domain in neurite outgrowth of chick dorsal root ganglion neuron

Synaptotagmin I or II (Syt I/II) is involved in Ca²⁺-regulated exocytosis of secretory vesicles, probably serving as a Ca²⁺-sensor via its C2A domain. Synaptotagmin is also known to be expressed in neuronal growth cone vesicles, but its functional involvement in neurite outgrowth remains largely unknown. In this study, we examined the function of Syt I/II in neurite outgrowth in cultured chick dorsal root ganglion neurons using an anti-synaptotagmin I and II C2A domain (anti-STI/II-C2A) antibody that inhibits Ca²⁺-regulated exocytosis. Immunoblots confirmed the high specificity of the anti-STI/II-C2A antibody and showed the expression of synaptotagmin I or II in chick dorsal root ganglion neurons. Immunocytochemistry revealed that synaptotagmin I or II is enriched at the growth cone region of chick dorsal root ganglion neurons, in both lamellipodia and filopodia. Whole or Fab-fragment of the anti-STI/II-C2A antibody loaded into dorsal root ganglion neurons by trituration significantly inhibited neurite outgrowth, whereas preimmune IgG had no effect. These results showed that the C2A domain of synaptotagmin I or II plays a crucial role in neurite outgrowth.     

Extracellular matrix glycoproteins inhibit neurite outgrowth of different types of identified leech neurons in culture

We explored the contribution of inhibitory peanut-binding extracellular matrix glycoproteins to the regeneration of characteristic outgrowth patterns by different types of identified neurons. Adult leech neurons were isolated one by one and plated in culture on a substrate that consisted of the capsules that encase the CNS ganglia. On the inside surface of this substrate, a combination of growth-promoting and -inhibiting extracellular matrix glycoproteins regulates the regeneration of distinctive outgrowth patterns by different neuron types. The role of inhibitory glycoproteins that bind to peanut lectin was studied by perturbation experiments in which peanut lectin was added to the culture medium. The effects of peanut lectin on the outgrowth patterns depended on the specific cell type that was tested. Anterior pagoda neurons, which on capsules produce a bipolar outgrowth pattern, in the presence of the lectin multiplied the number of primary neurites and the total neurite length and also lost their bipolarity. Annulus erector motoneurons, which on capsules grow poorly, in the presence of peanut lectin sprouted 70% more neurites and duplicated their total neurite length. By contrast, Retzius neurons which grow profusely on ganglion capsules, in the presence of peanut lectin increased the number of primary neurites without increasing their total neurite length or branch points. When neurons were plated on plastic, peanut lectin added to the culture medium did not affect the growth of neurons, thus showing that the effects of peanut lectin were induced by blocking the binding of neurons to inhibitory glycoproteins on the capsules. These results show that regeneration of different neuron types has different regulation by inhibitory extracellular matrix molecules.     

Culture conditions determine the prevalence of bipolar and monopolar neurons in cultures of dissociated spiral ganglion

To gain insight into the mechanisms that control the generation or maintenance of the characteristic bipolar morphology of cochlear spiral ganglion neurons, we have taken advantage of our recently developed procedure for culture of dissociated newborn mouse spiral ganglion. In these cultures, inclusion of the cytokine leukemia inhibitory factor (LIF) in the medium increases neuronal survival and the number of bipolar neurons. Here we tested effects of two other LIF-type cytokines (ciliary neurotrophic factor, CNTF; and human recombinant oncostatin M, hOSM) and of bone morphogenetic protein 4 (BMP4) on survival, morphology and neurite lengths of neurons in cultures of dissociated spiral ganglion. Like LIF, CNTF and hOSM increased neuronal survival and the number of surviving bipolar neurons. BMP4 also increased neuronal survival, but unlike LIF, CNTF and hOSM, increased the number of monopolar neurons and neurons with no neurites. In addition, population histograms demonstrate that the population lengths of the longer and shorter neurites of bipolar neurons were shorter in BMP4 containing cultures than in control or LIF cultures. When LIF and BMP4 were simultaneously added to the cultures, the BMP4 effects predominated. These experiments demonstrate that exposure to different environmental conditions can result in different morphologies in the surviving population of spiral ganglion neurons in culture.     

Distinct roles for Sema3A, Sema3F, and an unidentified trophic factor in controlling the advance of geniculate axons to gustatory lingual epithelium

Geniculate ganglion axons arrive in the lingual mesenchyme on embryonic day 13 (E13), 3–4 days before penetrating fungiform papilla epithelium (E17). This latency may result from chemorepulsion by epithelial Sema3A (Dillon et al. (2004) Journal of Comparative Neurology 470, 13–24), or Sema3F, which we report is also expressed in this epithelium. Sema3A and Sema3F repelled or suppressed geniculate neurite outgrowth, respectively, and these effects were stage and neurotrophic factor dependent. BDNF-stimulated outgrowth is repelled by Sema3A until E17, but insensitive to Sema3F from E16. NT-4-stimulated neurite outgrowth is sensitive to Sema3A and Sema3F through E18, but NT-4 has not been detected in E15–18 tongue. E15–18 tongue explants did not exhibit net chemorepulsion of geniculate neurites, but the ability of tongue explants to support geniculate neurite outgrowth fluctuates: E12–13 (Rochlin et al. (2000), Journal of Comparative Neurology, 422, 579–593) and E17–18 explants promote and may attract geniculate neurites, but stages corresponding to intralingual arborization do not. The E18 trophic and tropic effects were evident even in the presence of BDNF or NT-4, suggesting that some other factor is responsible. Intrinsic neurite outgrowth capability (without exogenous neurotrophic factors) fluctuated similarly: ganglia deteriorated at E15, but exhibited moderate outgrowth at E18. The chemorepulsion studies are consistent with a role for Sema3A, not Sema3F, in restricting geniculate axons from the epithelium until E17, when axons penetrate the epithelium. The transient inability of tongue explants to promote geniculate neurite outgrowth may signify an alternative mechanism for restricting geniculate axons from the epithelium: limiting trophic factor access. This work was supported by a research grant from the NIDCD, 1 R03 DC04965-01 to MWR.     

Brain derived neurotrophic factor and neurotrophic factor 3 modulate neurotransmitter receptor expressions on developing spiral ganglion neurons

Cochlear spiral ganglion neurons (SGN) provide the only pathway for transmitting sound evoked activity from the hair cells to the central auditory system. Neurotrophic factor 3 (NT-3) and brain derived neurotrophic factor (BDNF) released from hair cells and supporting cells exert a profound effect on SGN survival and neural firing patterns; however, it is unclear what the effects NT-3 and BDNF have on the type of neurotransmitter receptors expressed on SGN. To address this question, the whole-cell patch clamp recording technique was used to determine what effect NT-3 and BDNF had on the function and expression of glutamate, GABA and glycine receptors (GlyR) on SGN of cochlea from postnatal C57 mouse. Receptor currents induced by the agonist of each receptor were recorded from SGN cultured with or without BDNF or NT-3. NT-3 and BDNF exerted different effects. NT-3, and to a lesser extent BDNF, enhanced the expression of GABA receptors and had comparatively little effect on glutamate receptors. Absence of BDNF and NT-3 resulted in the emergence of glycine-induced currents; however, GlyR currents were absent from the short term cultured SGN. In contrast, NT-3 and BDNF suppressed GlyR expression on SGN. These results indicate that NT-3 and BDNF exert a profound effect on the types of neurotransmitter receptors expressed on postnatal SGN, results that may have important implications for neural development and plasticity.     

Membrane properties of type II spiral ganglion neurones identified in a neonatal rat cochlear slice

Neuro-anatomical studies in the mammalian cochlea have previously identified a subpopulation of approximately 5% of primary auditory neurones, designated type II spiral ganglion neurones (sgnII). These neurones project to outer hair cells and their supporting cells, within the 'cochlear amplifier' region. Physiological characterization of sgnII has proven elusive. Whole-cell patch clamp of spiral ganglion neurones in P7-P10 rat cochlear slices provided functional characterization of sgnII, identified by biocytin or Lucifer yellow labelling of their peripheral neurite projections (outer spiral fibres) subsequent to electrophysiological characterisation. SgnII terminal fields comprised multiple outer hair cells and supporting cells, located up to 370 μm basal to their soma. SgnII firing properties were defined by rapidly inactivating A-type-like potassium currents that suppress burst firing of action potentials. Type I spiral ganglion neurones (sgnI), had shorter radial projections to single inner hair cells and exhibited larger potassium currents with faster activation and slower inactivation kinetics, compatible with the high temporal firing fidelity seen in auditory nerve coding. Based on these findings, sgnII may be identified in future by the A-type current. Glutamate-gated somatic currents in sgnII were more potentiated by cyclothiazide than those in sgnI, suggesting differential AMPA receptor expression. ATP-activated desensitising inward currents were comparable in sgn II and sgnI. These data support a role for sgnII in providing integrated afferent feedback from the cochlear amplifier.     

An aligned 3D neuronal-glial co-culture model for peripheral nerve studies

In this study we report on the development of a 3D in vitro peripheral nerve model using aligned electrospun polycaprolactone fibre scaffolds manufactured under tightly controlled and reproducible conditions with uniform diameters of 1 μm, 5 μm and 8 μm. Fibres were characterized by SEM for diameter, density and alignment properties and formed in to scaffolds for 3D in vitro culture. Three different approaches were adopted using: i) neuronal or primary Schwann cell cultures alone; ii) neuronal and primary Schwann cells in co-culture and iii) isolated dorsal root ganglion cultures, containing both neuronal and Schwann cells, with immunohistochemical and 3D confocal microscopy analysis. Neurite guidance was evident on all fibres diameters with the longest neurites detected on 8 μm fibres when cultured alone. However, co-culture with primary Schwann cells was found to enable neurite formation on all scaffolds. Dorsal root ganglion explants when grown on scaffolds showed both organised aligned neurite guidance and notably the co-localization of Schwann cells with neurites. Neurite lengths of up to 2.50 mm were routinely observed using 1 μm diameter fibres after 10 days and all cultures demonstrated a migrating Schwann cell 'front' of up to 2.70 mm (1 μm diameter fibres). Thus, a direct relationship was found between fibre diameter, neurite outgrowth and Schwann cell morphology. This work therefore supports the use of aligned electrospun PCL microfibres for the development of 3D peripheral nerve models in vitro. We envisage such models having future value in a number of areas including developmental biology, disease studies and the design of devices and scaffolds for peripheral nerve repair.     

CLP36 interacts with palladin in dorsal root ganglion neurons

CLP36, a member of the α-actinin-associated LIM protein (ALP)/enigma protein family, plays a role in neurite outgrowth in the peripheral nervous system. However, the underlying molecular mechanisms are not known. In this study, we performed yeast two-hybrid screening of an E18 mouse whole-body cDNA library with CLP36 as the bait and isolated palladin as a CLP36-binding protein. Palladin is an actin-binding protein and it was shown to have a role in the extension of cortical neurons. A coimmunoprecipitation study showed that CLP36 and palladin formed a complex in the dorsal root ganglion (DRG). In addition, CLP36 and palladin were colocalized in the neurites and cell bodies of primary DRG neurons. Furthermore, sciatic nerve transection increased the expression of both CLP36 and palladin mRNAs in DRG neurons, with the increase in CLP36 mRNA being more prominent. This implies that CLP36 has a more specific role in nerve regeneration than palladin. Our results suggest that CLP36 may interact with palladin to influence neurite outgrowth during sciatic nerve regeneration.     

Metallothionein-IIA promotes neurite growth via the megalin receptor

Metallothionein (MT)-I/II has been shown to be neuroprotective and neuroregenerative in a model of rat cortical brain injury. Here we examine expression patterns of MT-I/II and its putative receptor megalin in rat retina. At neonatal stages, MT-I/II was present in retinal ganglion cells (RGCs) but not glial or amacrine cells; megalin was present throughout the retina. Whilst MT-I/II was absent from adult RGC in normal animals and after optic nerve transection, the constitutive megalin expression in RGCs was lost following optic nerve transection. In vitro MT-IIA treatment stimulated neuritic growth: more RGCs grew neurites longer than 25 μm (P < 0.05) in dissociated retinal cultures and neurite extension increased in retinal explants (P < 0.05). MT-IIA treatment of mixed retinal cultures increased megalin expression in RGCs, and pre-treating cells with anti-megalin antibodies prevented MT-IIA-stimulated neurite extension. Our results indicate that MT-IIA stimulates neurite outgrowth in RGCs and may do so via the megalin receptor; we propose that neurite extension is triggered via signal transduction pathways activated by the NPxY motifs of megalin’s cytoplasmic tail.     

Influence of an endogenous lectin substrate on cultured dorsal root ganglion cells

Summary Dorsal root ganglion neurons normally contain a 14 500 molecular weight lactose-binding lectin, designated L-14. Although this lectin is developmentally regulated, and is localized to specific neurons as well as to specific areas of the rat spinal cord, its function in the nervous system is not known. In an effort to study the possible role of this lectin on peripheral neurons, they were dissociated and grown on substrates consisting of either L-14 or laminin, a molecule known to support neurite outgrowth. In contrast to the random distribution and fine neurites displayed by neurons on laminin, those growing on L-14 formed large aggregates with highly fasciculated neurite bundles. Experiments using plant lectins with sugar-binding specificity similar to that of L-14, as well as another endogenous rat lectin not present in neurons, resulted in essentially no neuronal attachment or neurite outgrowth. In addition, the effects induced by L-14 were not blocked by high concentrations of competing sugars, suggesting that it interacts with neurons by a domain distinct from its carbohydrate-binding site.     

Regulation of neurite outgrowth mediated by neuronal calcium sensor-1 and inositol 1,4,5-trisphosphate receptor in nerve growth cones

Calcium acts as an important second messenger in the intracellular signal pathways in a variety of cell functions. Strictly controlled intracellular calcium is required for proper neurite outgrowth of developing neurons. However, the molecular mechanisms of this process are still largely unknown. Neuronal calcium sensor-1 (NCS-1) is a high-affinity and low-capacity calcium binding protein, which is specifically expressed in the nervous system. NCS-1 was distributed throughout the entire region of growth cones located at a distal tip of neurite in cultured chick dorsal root ganglion neurons. In the central domain of the growth cone, however, NCS-1 was distributed in a clustered specific pattern and co-localized with the type 1 inositol 1,4,5-trisphosphate receptor (InsP₃R1). The pharmacological inhibition of InsP₃ receptors decreased the clustered specific distribution of NCS-1 in the growth cones and inhibited neurite outgrowth but did not change the growth cone morphology. The acute and localized loss of NCS-1 function in the growth cone induced by chromophore-assisted laser inactivation (CALI) resulted in the growth arrest of neurites and lamellipodial and filopodial retractions. These findings suggest that NCS-1 is involved in the regulation of both neurite outgrowth and growth cone morphology. In addition, NCS-1 is functionally linked to InsP₃R1, which may play an important role in the regulation of neurite outgrowth.     

坍塌反应调节蛋白5促进海马神经元突起生长

目的 探讨坍塌反应调节蛋白5(CRMP5)对神经元突起生长的作用。方法 构建CRMP5真核表达载体,采用基因转染、实时定量PCR和免疫印迹技术评估CRMP5基因表达;以空载体为对照组,设3个复孔,用时差成像技术和突起提取技术观察和检测原代培养海马神经元突起的生长。结果 成功构建携带增强型绿色荧光蛋白（EGFP）标签蛋白的CRMP5的真核表达载体。脂质体转染技术可成功把CRMP5基因导入细胞,转染的细胞CRMP5表达高于空载体对照组;CRMP5蛋白表达于神经元的胞体和突起,尤其是胞体、突起起始处和突起末端高表达。过表达CRMP5可明显促进突起生长,主要表现为突起的生长,并形成丰富的侧枝;定量结果显示,CRMP5过表达的细胞突起的长度逐渐延长,而且较空载体转染细胞增多,差异显著(P<0.01)。导入CRMP5的细胞突起提取液的吸光度较对照细胞明显升高(P.<0.01)。 结论 CRMP5能促进神经元突起及其分支的生长。     

Comparison of the Schwann cell surface and Schwann cell extracellular matrix as promoters of neurite growth

Summary The ability of Schwann cells to influence the direction and rate of neurite growth was investigated in a tissue culture model of the bands of Büngner of injured peripheral nerve. The arrangement of this culture system allowed testing of the growth-promoting properties of the Schwann cell surface and extracellular matrix (ECM) assembled by Schwann cells rather than soluble substances secreted into conditioned medium. Various components of peripheral nerve were examined separately as substrata for regenerating neuntes: (i) Schwann cells and their ECM; (ii) Schwann cells alone; (iii) Schwann cell ECM alone; (iv) Schwann cells, fibroblasts, and their assembled ECM; (v) Schwann cells, their ECM and neurites; and (vi) purified laminin. Regenerating peripheral neurites were from expiants of foetal rat dorsal root ganglia, which had been cultured for several weeks to rid them of accompanying non-neuronal cells, or from expiants of foetal rat superior cervical ganglia, which contained non-neuronal cells. CNS neurites from the somatosensory cortex of embryonic rats were also studied; these neurites may be either first growing or regenerating.Neurites from all types of expiants studied grew longer and were guided on a substratum of Schwann cells or Schwann cell ECM compared with a collagen substratum. The presence of fibroblasts during ECM assembly did not enhance the neurite growth-promoting activity. The design of the experiments suggested that the factors by which the Schwann cells or their ECM promoted and guided neurite outgrowth were surface-bound rather than medium-borne. Electron microscopic examination showed that neurites grew on either Schwann cell surfaces or basal lamina material. Attempts to define the chemical nature of the neurite growth-promoting effect of ECM by partial enzymatic digestion did not identify any single component as essential.Purified laminin was a more effective promoter of outgrowth of peripheral neurites than were Schwann cells or Schwann cell ECM. Cortical expiants also grew on laminin, but neurites were accompanied on this substratum by a massive migration of non-neuronal cells; the neurites appeared to extend primarily on the non-neuronal cells rather than by direct attachment to the laminin substratum. This characteristic outgrowth of cortical non-neuronal cells on laminin was not consistently seen on Schwann cell ECM.In conclusion, either the Schwann cell surface or the ECM produced and assembled by Schwann cells promotes neurite outgrowth and guides that outgrowth from the several types of peripheral and CNS neurons studied in this report.