Explant cultures of teleost spinal cord: source of neurite outgrowth

The source of neurite outgrowth in explant cultures of normal adult Apteronotus spinal cord was examined. Explants which contained the central region of spinal cord, including ependyma, showed neurite outgrowth in culture. Explants which did not contain ependyma showed no neurite outgrowth. It is concluded that the ependymal region is necessary for neurite outgrowth in these cultures of adult teleost spinal cord. In addition, our failure to observe axon outgrowth clearly attributable to fluorescently back-labeled electromotor neurons in these cultures suggests that the exuberant neurite outgrowth in vitro is most probably due to cells other than the electromotor neurons. This explant culture system provides a unique opportunity to study neuronal differentiation, regeneration, and neurogenesis in vitro.     

Peripheral nerve extract promotes long-term survival and neurite outgrowth in cultured spinal cord neurons

The hypothesis that peripheral, skeletal muscle tissue contains a trophic factor supporting central neurons has recently been investigated in vitro by supplementing the culture medium of spinal cord neurons with muscle extracts and fractions of extract. We extended these studies asking whether or not a trophic factor is present in peripheral nerves, the connecting link between muscle and central neurons via which factors may be translocated from muscle to neurons by the retrograde transport system. Lumbar, 8-day-old chick spinal cords were dissociated into single cells and then cultured in the presence of peripheral nerve extract. Cytosine arabinoside was added to inhibit proliferation of nonneuronal cells. In the presence of nerve extract, spinal cord neurons survived for more than a month, extended numerous neurites, and showed activity of choline acetyltransferase. In the absence of extract, neurons attached and survived for a few days but then died subsequently in less than 10 days. Neurite outgrowth did not occur in the absence of extract. Withdrawal of extract from the medium of established neuronal cultures caused progressive loss of both cells and neurites. Other tissues also contained neuron supporting activity but less than that found in nerve extract. These studies indicate that peripheral nerves contain relatively high levels of spinal cord neuron-directed trophic activity, suggesting translocation of neurotrophic factor from muscle to central target neurons. The neurotrophic factor has long-term (weeks) effects, whereas short-term (days) survival is factor independent.     

Effects of docosahexaenoic acid on the survival and neurite outgrowth of rat cortical neurons in primary cultures

Effects of docosahexaenoic acid (DHA) on survival and neurite outgrowth were investigated in primary cultures of rat cortical neurons. Cell cultures were prepared from cortex on embryonic day 18 (E-18) for treatment with a series of DHA concentrations (12.5, 25, 50, 75, 100 and 200 microM). Docosahexaenoic acid (25-50 microM) significantly enhanced neuronal viability, but lower concentration of DHA (12.5 microM) did not show an obvious effect. In contrast, higher concentrations of DHA (100-200 microM) exerted the significant opposite effects by decreasing neuronal viability. Furthermore, treatment with 25 microM DHA significantly prevented the neurons from death after different culture days in vitro (DIV). Moreover, measurements from the cultures exposed to 25 microM DHA immediately after plating showed significant increases in the percentage of cells with neurites, the mean number of neurite branches, the total neuritic length per cell and the length of the longest neurite in each cell after 24 and 48 h in vitro (HIV). The DHA-treated neurons had greater growth-associated protein-43 (GAP-43) immunoactivity and higher phosphatidylserine (PS) and phosphatidylethanolamine (PE) contents, but lower phosphatidylcholine (PC) content than control neurons. The significant increased DHA contents were also observed in both PE and PS in the treated neurons. These findings suggest that optimal DHA (25 microM) may have positive effects on the survival and the neurite outgrowth of the cultured fetal rat cortical neurons, and the effects probably are related to DHA-stimulating neuron-specific protein synthesis and its enhancing the discrete phospholipid (PL) content through enrichment of DHA in the PL species.     

Stimulation by melanocortins of neurite outgrowth from spinal and sensory neurons in vitro

Using ELISAs for B-50/GAP43 and neurofilament (NF), we tested ACTH(1–24), -MSH, ACTH(4–10), and an ACTH(4–9) analogue (ORG2766) for their ability to induce sprouting and neuritogenesis from spinal and sensory neurons. Dissociated fetal rat spinal cord neurons or neonatal rat dorsal root ganglion (DRG) cells were cultured with peptide and assayed after 24, 48, or 96 h. In spinal neurons, -MSH and ACTH(1–24) induced the expression of B-50 dose dependently. After 24 h -MSH had a stimulatory effect (from 10 nM onwards), with a maximum at 100 μM (36% increase). After 96 h the maximal effect of 100 μM -MSH on B-50/GAP43 was lower (19%). ACTH(1–24) (100 μM) stimulated B-50/GAP43 by 19%. Neurofilament levels (96 h) were elevated maximally by 64% at 100 μM -MSH. In DRG neurons a bell-shaped dose-response curve was found for -MSH, the maximal effect being observed after 48 h at 100 nM: 54% for B-50/GAP43 and 22% for NF. In both culture systems neither ACTH(4–10) nor ORG2766 was effective. We conclude that -MSH stimulates the expression of B-50/GAP43 (sprouting) and the formation of NF (neurite elongation) and may therefore be considered a neurotrophic factor.     

Effect of protein phosphorylation on neurite outgrowth in cultured embryonic Xenopus spinal neurons

Intracellular signaling pathways involved in neurite outgrowth have been extensively studied in a variety of cell systems. While most of these studies utilized continuous neuronal-like cell lines, fewer studies have been conducted in primary neuronal culture. One primary culture system that has recently been used to dissect the signaling pathways involved in axon guidance consists of spinal neurons derived from embryonic Xenopus laevis. In this study, we used Xenopus to study neurite outgrowth by treating neuronal cultures with pharmacological agents that activate or inhibit various protein kinases or that inhibit protein phosphatases. We found that agents which affected signaling via cAMP-dependent protein kinase, calmodulin, cyclin-dependent kinase 5, or protein phosphatases had effects on Xenopus neurite outgrowth that were similar to those reported in other primary neurons or in neuronal-like cell lines. However, agents which affected protein kinase C signaling had effects on Xenopus neurite outgrowth that were distinct from those reported in neuronal-like cell lines. Although continuous cell lines have several advantages for the dissection of signaling pathways involved in neurodevelopment, these observations underscore the importance of also using primary neurons to examine these pathways.     

Effect of 710 nm visible light irradiation on neurite outgrowth in primary rat cortical neurons following ischemic insult

Stanniocalcin 2 (STC2) is a homolog of stanniocalcin 1, a 56kD glycoprotein hormone that originally was found to confer calcitonin-like activity in fish. Human STC2 is expressed in various tissues such as kidney, spleen, heart, and pancreas. STC2 has been demonstrated to be induced by different kinds of stress and display cytoprotective activity, but the molecular mechanism is poorly understood. Heme oxygenase 1 (HO1) degrades heme to biliverdin, carbon monoxide and free iron, and is a stress-responsive protein. Using yeast two-hybrid screening we identified HO1 as a binding partner of STC2. The interaction was validated by in vivo co-immunoprecipitation and immunofluorescence. The binding site for HO1 was located to amino acids 181-200 of STC2. We also found that STC2 binds hemin via a consensus heme regulatory motif. Moreover, STC2 expression was induced by heat shock in HEK293 cells. Taken together, our findings point to three novel functions of STC2, and suggest that STC2 interacts with HO1 to form a eukaryotic 'stressosome' involved in the degradation of heme.     

Novel effects of serotonin on neurite outgrowth in neurons cultured from embryos of Helisoma trivolvis

The neurotransmitter serotonin has been shown to inhibit neurite outgrowth in specific identified neurons isolated from adult Helisoma. While in vivo experiments on Helisoma embryos have supported the hypothesis that endogenous serotonin regulates neurite outgrowth during embryonic development, direct effects of serotonin on embryonic neurons have not been measured. In the present study, cultures of dissociated embryonic neurons were used to test the direct actions of serotonin on developing embryonic neurons. Serotonin arrested neurite outgrowth in a significant percentage of elongating neurites in a dose-dependent manner. Furthermore, analysis of neurons with stable, nonelongating neurites revealed a novel response. Serotonin caused the reinitiation of neurite outgrowth in a significant percentage of nonelongating neurites. The arrestment of outgrowth and reinitiation of outgrowth occurred in similar percentages of elongating and nonelongating neurites, respectively. Parallel experiments on cultures of dissociated adult neurons were carried out to determine whether serotonin could also induce both inhibitory and stimulatory responses in adult cells. Serotonin arrested neurite outgrowth in a similar percentage of neurites to that observed in cultures of embryonic neurons. In contrast, serotonin did not reinitiate neurite outgrowth in a significant percentage of adult neurites. These data support the hypothesis that serotonin regulates neurite outgrowth in developing embryonic neurons. Furthermore, only some of these regulatory effects appear to be conserved from embryonic to adult neurons.     

Cortical neurite outgrowth and growth cone behaviors reveal developmentally regulated cues in spinal cord membranes

Corticospinal axon outgrowth in vivo and the ability to sprout or regenerate after injury decline with age. This developmental decline in growth potential has been correlated with an increase in inhibitory myelin‐associated proteins in older spinal cord. However, previous results have shown that sprouting of corticospinal fibers after contralateral lesions begins to diminish prior to myelination, suggesting that a decrease in growth promoting and/or an increase in inhibitory molecules in spinal gray matter may also regulate corticospinal axon outgrowth. To address this possibility, we carried out in vitro experiments to measure neurite outgrowth from explants of 1‐day‐old hamster forelimb sensorimotor cortex that were plated onto membrane carpets or membrane stripe assays prepared from white or gray matter of 1‐to 22‐day‐old cervical spinal cord. On uniform carpets and in the stripe assays cortical neurites grew robustly on young but not older membranes from both white and gray matter. Mixtures of membranes from 1‐ and 15‐day spinal cord inhibited neurite outgrowth, suggesting that the presence of inhibitory molecules in the 15‐day cord overwhelmed permissive or growth promoting molecules in membranes from 1‐day cord. Video microscopic observations of growth cone behaviors on membrane stripe assays transferred to glass coverslips supported this view. Cortical growth cones repeatedly collapsed at borders between permissive substrates (laminin or young membrane stripes) and nonpermissive substrates (older membrane stripes). Growth cones either turned away from the older membranes or reduced their growth rates. These results suggest that molecules in both the gray and white matter of the developing spinal cord can inhibit cortical neurite outgrowth. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 393–406, 1999     

Effects of phenylalanine on the survival and neurite outgrowth of rat cortical neurons in primary cultures: possible involvement of brain-derived neurotrophic factor

Bax inhibitor-1 (BI-1), a newly identified apoptosis inhibitor, has recently been found to be overexpressed in several human carcinomas and its specific down-regulation by RNA interference (RNAi) could lead to cell death. The purpose of this study is to investigate the role of BI-1 in apoptosis-resistance and the underlying mechanisms in human nasopharyngeal carcinoma (NPC) cells. Our results showed that BI-1 was expressed in two different human NPC cell lines, CNE-2Z and CNE-1, and specific inhibition of BI-1 expression by siRNA caused a significant increase in spontaneous apoptosis in both cell lines. Mechanistically, we demonstrated that down-regulation of BI-1 protein expression decreased the ratio of Bcl-X_L/Bcl-2 with Bax protein as determined by Western blot and increased the activity of caspase-3 by colorimetric analysis, thus leading to the activation of the associated cell death pathways. Taken together, these results have provided evidence that BI-1 could serve as an important molecular target gene for the development of new therapeutic strategy against human NPCs.     

脊髓提取液对培养脊髓神经细胞中GABA能及DYN能神经元突起生长的影响

本文以体外培养的小鼠脊髓神经元为模型研究了人胚脊髓提取液对Ｅ１２－１５小鼠脊髓中ＧＡＢＡ能神经元和ＤＮＹ能神经元的突起生长的营养作用,结果发现人胚脊髓提取液在蛋白浓度为２５０μｇ／ｍｌ时对ＧＡＢＡ能神经元的突起生长无营养作用,但对ＤＮＹ能神经元的突起生长有显著的促进作用。提示了人胚脊髓提取液中有促进神经元突起生长的营养物质,且对特定胎龄的不同的神经元有不同的作用。     

Cortical neurite outgrowth and growth cone behaviors reveal developmentally regulated cues in spinal cord membranes.

Corticospinal axon outgrowth in vivo and the ability to sprout or regenerate after injury decline with age. This developmental decline in growth potential has been correlated with an increase in inhibitory myelin-associated proteins in older spinal cord. However, previous results have shown that sprouting of corticospinal fibers after contralateral lesions begins to diminish prior to myelination, suggesting that a decrease in growth promoting and/or an increase in inhibitory molecules in spinal gray matter may also regulate corticospinal axon outgrowth. To address this possibility, we carried out in vitro experiments to measure neurite outgrowth from explants of 1-day-old hamster forelimb sensorimotor cortex that were plated onto membrane carpets or membrane stripe assays prepared from white or gray matter of 1-to 22-day-old cervical spinal cord. On uniform carpets and in the stripe assays cortical neurites grew robustly on young but not older membranes from both white and gray matter. Mixtures of membranes from 1- and 15-day spinal cord inhibited neurite outgrowth, suggesting that the presence of inhibitory molecules in the 15-day cord overwhelmed permissive or growth promoting molecules in membranes from 1-day cord. Video microscopic observations of growth cone behaviors on membrane stripe assays transferred to glass coverslips supported this view. Cortical growth cones repeatedly collapsed at borders between permissive substrates (laminin or young membrane stripes) and nonpermissive substrates (older membrane stripes). Growth cones either turned away from the older membranes or reduced their growth rates. These results suggest that molecules in both the gray and white matter of the developing spinal cord can inhibit cortical neurite outgrowth.     

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.     

Protein trafficking mechanisms associated with neurite outgrowth and polarized sorting in neurons.

Neuronal differentiation in vitro and in vivo involves coordinated changes in the cellular cytoskeleton and protein trafficking processes. I review here recent progress in our understanding of the membrane trafficking aspects of neurite outgrowth of neurons in culture and selective microtubule-based polarized sorting in fully polarized neurons, focusing on the involvement of some key molecules. Early neurite outgrowth appears to involve the protein trafficking machineries that are responsible for constitutive trans-Golgi network (TGN) to plasma membrane exocytosis, utilizing transport carrier generation mechanisms, SNARE proteins, Rab proteins and tethering mechanisms that are also found in non-neuronal cells. This vectorial TGN-plasma membrane traffic is directed towards several neurites, but can be switch to concentrate on the growth of a single axon. In a mature neuron, polarized targeting to the specific axonal and dendritic domains appears to involve selective microtubule-based mechanisms, utilizing motor proteins capable of distinguishing microtubule tracks to different destinations. The apparent gaps in our knowledge of these related protein transport processes will be highlighted.     

An NCAM-derived FGF-receptor agonist, the FGL-peptide, induces neurite outgrowth and neuronal survival in primary rat neurons

The Neural Cell Adhesion Molecule (NCAM) plays a crucial role in development of the central nervous system regulating cell migration, differentiation and synaptogenesis. NCAM mediates cell-cell adhesion through homophilic NCAM binding, subsequently resulting in activation of the fibroblast growth factor receptor (FGFR). NCAM-mediated adhesion leads to activation of various intracellular signal transduction pathways, including the Ras-mitogen activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI3K)-Akt pathways. A synthetic peptide derived from the second fibronectin type III module of NCAM, the FGL peptide, binds to and induces phosphorylation of FGFR without prior homophilic NCAM binding. We here present evidence that this peptide is able to mimic NCAM heterophilic binding to the FGFR by inducing neuronal differentiation as reflected by neurite outgrowth through a direct interaction with FGFR in primary cultures of three different neuronal cell types all expressing FGFR subtype 1: dopaminergic, hippocampal and cerebellar granule neurons. Moreover, we show that the FGL peptide promotes neuronal survival upon induction of cell death in the same three cell types. The effects of the FGL peptide are shown to depend on activation of FGFR and the MAPK and PI3K intracellular signalling pathways, all three kinases being necessary for the effects of FGL on neurite outgrowth and neuronal survival.     

Maturation of neurites in mixed cultures of spinal cord neurons and muscle cells from Xenopus laevis embryos followed with antibodies to neurofilament proteins

Dissociated cell cultures of Xeopus laevis embryonic spinal cord have proved useful for studying the differentiation of neuronal ionic channel and membrane properties and for examining the dynamics of microtubules in developing neurons. To examine their usefulness for studying neurofilaments in developing neurites, we prepared similar cultures from stage 22 embryos. Between 3 and 55 h after plating, these cultures were fixed and immunostained with antibodies directed against various epitopes of neurofilament proteins from X. Laevis. These antibodies were specific for nonphosphorylated epitopes of the two low molecular weight Xenopus neurofilament proteins (Xenopus NF-L and the Xenopus neuronal intermediate filament protein, XNIF), both phosphorylated and nonphosphorylated epitopes of the Xenopus middle molecular weight neurofilament protein (NF-M), and a nonphosphorylated epitope of the Xenopus high molecular weight neurofilament protein (NF-H). The emergence of these neurofilament proteins in culture was compared to the time course previously reported for them in Xenopus spinal cord neurons in situ. To facilitate the comparison of times in culture to developmental stages, the age of cultured neurons was converted to an equivalent Nieuwkoop and Faber normal stage using data presented here on the effect of changing temperature on developmental rates of X. laevis. With the exception of the nonphosphorylated epitope of NF-H, which is indicative of the most mature axons found in situ. the emergence of the other neurofilament protein antibody epitopes closely paralleled that previously reported for these antibodies in situ. Thus, with respect to XNIF, NF-M, and NF-L, the neurities of cultured neurons were typical of young embryonic Xenopus laevis spinal cord axons. This system should prove useful for studying both the function of these neurofilament proteins during the early stages of axonal development and the dynamics of their transport. 1994 John Wiley & Sons, Inc.     

The neurite growth stimulating effect of terrilytin on sensory neurons and the spinal cord in tissue culture

Terrilytin (a protease) was found to promote neurite extension in chick embryo dorsal root ganglia and spinal cord in vitro. This protease was active at the concentration 10-50 ng/ml, which provided extensive neurite outgrowth in the bioassay, compared to the control. Terrilytin may be used for stimulating regenerating processes in nervous tissue.     

Electrophysiologic and morphologic properties of neurons in dissociated chick spinal cord cell cultures

Neurons dissociated from embryonic chick spinal cords mature in relatively sparse cell culture and survive in vitro for several weeks. They generate action potentials and form both excitatory and inhibitory chemical synapses with one another. By electrophysiologic and morphologic criteria, it appears that the neuronal population (after 2–3 weeks) is made up of a variety of different cell types; few, if any, are motoneurons. Neuron cell bodies are not covered by glia or satellite cells and nerve processes are not myelinated. Thus, the cultures should permit more direct microelectrode and pharmacologic analysis of differentiation of cell specific properties and of synapse formation than is possible in the intact central nervous system.