Vasoactive intestinal peptide influences neurite outgrowth in cultured rat spinal cord neurons

Abstract

Vasoactive intestinal peptide (VIP) is a neuropeptide which has been shown to exhibit a wide range of neurotrophic effects both in vivo and in vitro . For the purpose of clarifying the effect of VIP on spinal cord neurons, we studied the effect of VIP on neurite outgrowth of fetal rat ventral and dorsal portions of spinal cord in cultures. VIP-treated ventral spinal cord cultures (VSCC), compared with control VSCC, had a significant neurite outgrowth at 10^-8, 10^-6, and 10^-4 M. The effect was considered to be concentration dependent. Morphological changes of the dorsal spinal cord cultures (DSCC) remained unchanged by VIP treatment. Because of their close sequence homology with VIP, PHI-27 (peptide, histidylisoleucine amide) and secretin were also examined with the same experimental conditions as was VIP. Both PHI-27 and secretin had neurite promoting effects in VSCC at 10^-8 and 10^-6 M, respectively. However, there were no neurite promoting effects in DSCC in both of them at any concentrations. VIP had the most potent effect on neurite outgrowth in VSCC, followed by PHI-27, and secretin in their effectiveness concentrations. Our data showing VIP, PHI-27 and secretin have neurotrophic action on VSCC and suggest that a potential therapeutic use of VIP and its related peptides in treating diseases that involve degeneration and death of spinal motor neurons, such as motor neuropathy and amyotrophic lateral sclerosis. [Neurol Res 2001; 23: 851-854]     

Acidic and basic fibroblast growth factors enhance neurite outgrowth in cultured rat spinal cord neurons

Abstract

We have studied neurotrophic effects of acidic fibroblast growth factor (aFCF) and basic fibroblast growth factor (bFGF) on explanted ventral and dorsal spinal cord cultures from 13- and 14-day-old rat embryos. Cultures treated with aFCF and bFGF significantly enhanced neurite outgrowth with cultures of ventral spinal cord, but not with cultures of dorsal spinal cord. Our data suggest that aFCF and bFGF are potent neurotrophic factors on rat ventral spinal cord neurons in vitro. [Neurol Res 1995; 17: 70-72]     

蛋白激酶C调节脊髓神经元突起的生长(英文)

目的关于蛋白激酶C(PKC)在神经元突起生长和神经再生中的作用,目前仍存有争议。本研究主要观察PKC对离体培养的脊髓神经元生长的调节作用,旨在阐明PKC对突起生长的调节作用。方法分离纯化胎龄14天(E14)的SD胎鼠的脊髓前角神经元,进行原代培养,并检测不同时相点膜/浆PKC活性(m/c-PKCactivity)的比值。结果神经元培养3-11d期间,神经元内m/c-PKC比值以及PKC-βII在突起中的表达水平均与突起生长呈显著相关关系(r=0.95,P<0.01;r=0.73,P<0.01)。此外,PKC激动剂PMA能显著提高m/c-PKC比值,且与神经突起的生长一致(r=0.99,P<0.01)。而PKC抑制剂GF109203X则能显著抑制突起生长,且不被PMA作用所逆转。结论PKC的活性在脊髓神经元突起生长调节中具有重要作用,其中βII亚型可能扮演重要角色。     

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.     

Vasoactive intestinal polypeptide neurons in fetal cortical homografts to adult rat spinal cord

Transplants of central nervous system to adult spinal cords are considered as potential aids in regeneration of the spinal cord and/or recovery of function after injury. The organization and development of the implant are important issues in seeking the potential for a transplant and host to become functionally integrated. This study uses embryonic cerebral cortex transplanted into the spinal cord of adult rats (T6) and examined the development and organization of the transplant with an antibody to vasoactive intestinal polypeptide (VIP). The cell bodies of VIP neurons are in the implants at 30 days postimplantation, but few of the somata have processes. By 45 days postimplantation, VIP neurons in the implant have dendrites and axons and are clearly recognizable as cortical bipolar cells which are not normally present in the thoracic spinal cord. These data show that neurons in embryonic cerebral cortical implants into the spinal cord elaborate the appropriate biochemical and morphological constituents in spite of the ectopic location. However, the cell processes develop at a slower than normal pace. Morphological interaction between the host spinal cord and the implant can be demonstrated possibly as early as 45 days postimplantation and clearly at 6 months following the implant. Thus, further examination of cerebral cortical implants as a potential aid in allevation of paraplegia subsequent to spinal cord injury is warranted.     

Schwann cell-conditioned medium supports neurite outgrowth and survival of spinal cord neurons in culture

The effect of Schwann cell-conditioned medium (SCM) on the development in vitro of spinal cord neurons was studied. Spinal cord neurons from 18-day-old rat embryos were cultured in serum-free conditioned medium obtained from confluent rat Schwann cells. In cultures fed SCM, the cells developed typical neuronal morphology and were identified by indirect immunofluorescence using a monoclonal antibody to neurofilament protein. SCM stimulated neurite outgrowth and supported survival of spinal cord neurons. Preliminary characterization suggests that the neurotrophic factor in SCM appears to be a protein with a molecular weight greater than 8000 daltons.     

Alpha-synuclein promotes early neurite outgrowth in cultured primary neurons

We previously showed that alpha-synuclein (α-Syn), a protein implicated in the pathogenesis of several neurodegenerative diseases, is a microtubule-associated protein (MAP), facilitating the polymerization of tubulin into microtubules. Therefore, we hypothesized that α-Syn might promote neurite outgrowth, a process that requires microtubule assembly. To test this hypothesis, recombinant human wild type (WT) and mutant (A30P and A53T) α-Syn proteins were added to cultured primary rat cortical neurons, and their effects on early neurite outgrowth were observed. The WT and mutant α-Syn proteins entered the neurons after 1–4 h of incubation. However, a significant increase in neurite outgrowth was observed only in neurons treated with WT α-Syn. MES23.5 dopaminergic neuronal cells overexpressing WT α-Syn also exhibited enhanced neurite outgrowth, indicating that the ability of α-Syn to promote neurite outgrowth was not due to a direct action on the cell membrane or by the membrane translocation process. Co-immunoprecipitation demonstrated that the recombinant human α-Syn was bound to tubulin. In addition, the α-Syn-treated neurons displayed increased levels of polymerized tubulin. Because α-Syn’s MAP functionality is mediated by specific domains, we generated N-terminal (a.a. 1–65), non-amyloid-β (non-Aβ) component (NAC) (a.a. 61–95) and C-terminal (a.a. 96–140) fragments and added them to the primary neurons. After 1–4 h of incubation, the various α-Syn fragments had entered the neurons. However, only the NAC and C-terminal fragments, which have been previously shown to mediate MAP functionality, promoted neurite outgrowth. These results suggest that α-Syn promotes neurite outgrowth by facilitating the polymerization of tubulin into microtubules.     

Effects of calcium ion on neurite outgrowth of rat spinal cord neurons in vitro: the role of non-neuronal cells in regulating neurite sprouting

The interactions of nerve cells with their environment and other cells are specific to different stages of cellular differentiation. Neurite outgrowth was measured from cultured spinal cord neurons under the influence of different Ca²⁺ concentrations. We used fluorodeoxyuridine (FuDr), an antimitotic agent which reduces significantly the proportion of non-neuronal cells in spinal cord cell cultures, to examine the effects of non-neuronal cells on neurite outgrowth. Spinal cord neurons responded to changes in their environment by means of two types of neurite outgrowth: sprouting and elongation. The concurrent presence of non-neuronal cells led to increased sprouting of neurites in certain ionic environments, thus lending support to the idea that non-neuronal cells release diffusible factors which influence sprouting and guide neurite outgrowth.     

Small Nogo-66-binding peptide promotes neurite outgrowth through RhoA inhibition after spinal cord injury

Abortive regeneration in the adult mammalian central nervous system (CNS) is partially mediated through CNS myelin proteins, among which Nogo-A plays an important role. Nogo-66, which is located at the C-terminus of Nogo-A, inhibits axonal regrowth through the Nogo-66/NgR signalling pathway. In this study, two small peptides were tested in a neurite outgrowth assay and spinal cord injury (SCI) model to examine the effects of these molecules on the inhibition of Nogo-66/NgR signalling. PepIV was selected from a phage display peptide library as a Nogo-66 binding molecule. And PepII was synthesized as a potential NgR antagonist. The results indicated that PepIV and PepII decrease the mRNA levels of the small GTPase RhoA and partially neutralize CNS myelin inhibition to cultured cerebellar granule cells (CGCs). Moreover, treatment with both peptides was propitious to maintaining residual axons after SCI, thereby promoting regeneration and locomotion recovery. Because RhoA plays a role in stabilizing the cytoskeleton in growth cones and axons, enhanced neurite outgrowth might reflect a decrease in RhoA expression through PepIV and PepII treatment. Moreover, PepIV induced lower RhoA mRNA expression compared with PepII. Therefore, PepIV could block Nogo-66/NgR signalling and reduce RhoA mRNA level, and then contribute to neuronal survival and axonal regrowth after SCI, showing its ability to reverse CNS myelin inhibition to regeneration. Furthermore, selected small peptide might cover some unknown active sites on CNS myelin proteins, which could be potential targets for improving neurite outgrowth after injury.     

Vasoactive intestinal peptide immunoreactive neurons in the rat suprachiasmatic nucleus demonstrate diurnal variation

The suprachiasmatic nucleus (SCN) of mammals is considered to be a circadian oscillator and it also demonstrates circadian rhythmicity of its multiple unit activity. A number of neuropeptides have been found in the SCN. Vasoactive intestinal peptide (VIP)- and vasopressin-containing neurons comprise large populations of these cells and have a distinct distribution within the nucleus. Therefore we attempted to examine whether the VIP neurons show a diurnal alteration of their immunoreactivity by combined immunocytochemistry and color image analysis. Our results demonstrate that VIP-like immunoreactive neurons show a diurnal change in the amount of immunoreactivity. Immunoreactivity was most intense in the sections from rats maintained in the cyclic photoperiod and sacrificed at 02.00 h and weakest in the SCN from animals sacrificed at 14.00 h. We considered that VIP-like immunoreactive neurons showed diurnal variation of VIP synthesis depending strongly on the light from the retina.     

A glia-derived nexin promotes neurite outgrowth in cultured chick sympathetic neurons

A glia-derived neurite-promoting factor has been purified from medium conditioned by C6 rat glioma cells. It induces neurite outgrowth in cultured mouse neuroblastoma cells and inhibits granule cell migration in explants of mouse cerebellum. This factor is a potent serine protease inhibitor which has recently been shown to belong to the protease nexin family. It has therefore been called glia-derived nexin (GDN). We report here that GDN also promotes neurite outgrowth in dissociated chick superior cervical ganglion neurons grown in serum-free medium. In these neurons, the presence of nerve growth factor is not required for the stimulatory effect of GDN in the initial phase of neurite outgrowth. These experiments demonstrate that a glia-derived protein with protease inhibitory activity can modulate neurite outgrowth in cultured chick sympathetic neurons.     

Grafting of cultured microglial cells into the lesioned spinal cord of adult rats enhances neurite outgrowth

There is contrasting in vitro and in vivo evidence regarding glial cell involvement in central nervous system (CNS) regeneration. This study has investigated the histological events that follow implantation of either microglia, mixed microglia/astrocytes, or astrocytes into the injured adult rat spinal cord. We have conducted an immunohistochemical characterization of the cellular profiles within and neuritic extension into various grafts consisting of gelfoam (GF) matrices impregnated with cultured microglia and/or astrocytes. After 2–5 weeks, prominent neuritic growth was observed into OX-42-immunoreactive (IR) microglial implants. These grafts were infiltrated by numerous host cellular elements including microvasculature and Schwann cells, and they demonstrated conspicuous laminin IR. Often, the patterns for laminin and OX-42 IR in microglial grafts were overlapping, suggesting partial expression of laminin on transplanted microglial cells. Mixed grafts of microglia and astrocytes demonstrated presence of neurites and laminin-IR elements with similar intensity as microglial grafts, while astroglial implants showed the least amount of neurite ingrowth. Some control implants consisting of cell-free GF showed marginal in-growth of neurites in areas of infiltrating OX-42-IR host cells. Collectively, our findings support a neurite growth-promoting role of activated microglia and suggest that microglia may counteract mechanisms that inhibit CNS regeneration. It remains to be determined whether the observed neurite growth-promoting effects are mediated directly by grafted and/or endogenous microglia, or whether this occurs via the recruitment of host Schwann cells. J. Neurosci. Res. 47:34–48, 1997. © 1997 Wiley-Liss, Inc.     

Vasoactive intestinal peptide fibers innervate neuroendocrine dopaminergic neurons

Hypothalamic neuroendocrine dopaminergic neurons exhibit a diurnal rhythm. Higher level input to these neurons has not been described. In the present study, we identified fibers known to originate in the suprachiasmatic nucleus (SCN), which were associated with neuroendocrine dopaminergic neurons. Hypothalamic sections were obtained from either ovariectomized (OVX) female rats or OVX female rats implanted with estrogen and progesterone (E+P). Confocal microscopic images were acquired from the periventricular nucleus, as well as the rostral, dorsomedial, ventrolateral, and caudal regions of the arcuate nucleus. Using antibodies directed against vasoactive intestinal peptide (VIP) and tyrosine hydroxylase (TH) the rate-limiting enzyme in dopamine synthesis, fine VIP fibers in close apposition to TH-immunoreactive (IR) soma and proximal dendrites were revealed. Of the antibodies for the two VIP receptor subtypes (VIP1R and VIP2R), only VIP2R was found on TH-IR neurons. E+P significantly increased the incidence and density of neuroendocrine dopaminergic neurons expressing VIP2R, when compared to OVX animals. E+P did not affect the percent of neuroendocrine dopaminergic neurons associated with VIP fibers. No VIP fibers or VIP2R were found on dopaminergic neurons in the zona incerta. Brain sections triple labeled for Synapsin (a protein localized in synaptic vesicles) VIP, and TH demonstrated that Synapsin was colocalized with VIP fibers that were associated with TH-IR neurons in the arcuate nucleus. Double-label immuno-electron microscopy of hypothalamic sections labeled with antibodies for VIP and TH revealed VIP boutons associated with TH-IR soma and proximal dendrites. These results suggest VIPergic neurons may directly regulate neuroendocrine dopaminergic neuron activity, and ovarian steroids may play a modulatory role.     

Developmental patterns of neurite outgrowth from chick embryo spinal cord and retinal neurons on laminin substrates

It has been previously found that neurite outgrowth on collagen substrates decreases with increasing gestational age of chick embryo spinal cord and retinal neurons in tissue culture. In the current study, laminin, polylysine and collagen were compared in their efficacy in promoting neurite extension from chick embryo spinal cord neurons aged 6-16 days or retinal neurons aged 8-16 days in ovo. The percentage of neurons with neurites and the length of the neurites were determined at 1 and 3 days in culture. There was a significant increase in neuritogenesis by laminin and polylysine compared to collagen for both spinal cord and retinal neurons. Further, in spinal cord cultures grown on a laminin substrate, there was no decline in neurite outgrowth with increasing developmental age of the neurons as was seen on collagen and polylysine. Neurite length measurements also demonstrated a significant stimulation of neuritogenesis for spinal cord, but not retinal, neurons by laminin compared to polylysine or collagen in 1-day cultures. The results demonstrate tissue-specific differences in the developmental patterns of neurite outgrowth. Retinal neurons appear to have intrinsic changes in their ability to respond to extracellular promoting factors or substrates, while spinal cord neurite outgrowth can be regulated by these extrinsic factors.     

Effects of calcium ion on neurite outgrowth of rat spinal cord neurons in vitro: The role of non-neuronal cells in regulating neurite sprouting

The interactions of nerve cells with their environment and other cells are specific to different stages of cellular differentiation. Neurite outgrowth was measured from cultured spinal cord neurons under the influence of different Ca²⁺ concentrations. We used fluorodeoxyuridine (FuDr), an antimitotic agent which reduces significantly the proportion of non-neuronal cells in spinal cord cell cultures, to examine the effects of non-neuronal cells on neurite outgrowth. Spinal cord neurons responded to changes in their environment by means of two types of neurite outgrowth: sprouting and elongation. The concurrent presence of non-neuronal cells led to increased sprouting of neurites in certain ionic environments, thus lending support to the idea that non-neuronal cells release diffusible factors which influence sprouting and guide neurite outgrowth.     

Methyl 3,4-dihydroxybenzoate promotes neurite outgrowth of cortical neurons cultured in vitro

Cerebral cortical neurons from neonatal rats were cultured in the presence of methyl 3,4-dihydroxybenzoate (MDHB;2,4,and 8 μM).Results showed that MDHB significantly promoted neurite outgrowth and microtubule-associated protein 2 mRNA expression,and increased neuronal survival in a dose-dependent manner.Moreover,MDHB induced brain-derived neurotrophic factor expression.These findings suggest that MDHB has a neurotrophic effect,which may be due to its ability to increase brain-derived neurotrophic factor expression.     

Vasoactive intestinal peptide and nitric oxide promote survival of adult rat myenteric neurons in culture

Several motility disorders originate in the enteric nervous system (ENS). Our knowledge of factors governing survival of the ENS is poor. Changes in the expression of vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS) in enteric neurons occur after neuronal injury and in intestinal adaptation. The aim of this study was to evaluate whether VIP and nitric oxide (NO) influence survival of cultured, dissociated myenteric neurons. Neuronal survival was evaluated after 0, 4, and 8 days in culture. Influence of VIP and NO on neuronal survival was examined after culturing in the presence of VIP, NO donor, VIP antiserum, or NOS inhibitor. A marked loss of neurons was noted during culturing. VIP and NO significantly promoted neuronal survival. Corroborating this was the finding of an enhanced neuronal cell loss when cultures were grown in the presence of VIP antiserum or NOS inhibitor.     

Carbamazepine blocks NMDA-activated currents in cultured spinal cord neurons.

The antiepileptic agents, carbamazepine and phenytoin, suppress seizures in man and convulsant-induced hyperactivity in spinal cord nerve cell cultures. In the present study, we have shown by whole cell recording that carbamazepine, in contrast to phenytoin, blocks N-methyl-D-aspartate (NMDA)-activated membrane currents in cultured neurons in a dose-dependent fashion. The NMDA receptor-activated channel, which is blocked at physiological concentrations of Mg2+ at resting membrane potential, can be activated by glutamate in depolarized neurons and thus be involved in epileptogenesis. Therefore, the block of NMDA-evoked membrane currents in cultured neurons may contribute to the clinical effectiveness of carbamazepine.