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PubMed2000

Different extrinsic trophic factors regulate neurite outgrowth and synapse formation between identified Lymnaea neurons.

The requirement for trophic factors in neurite outgrowth is well established, though their role in synapse formation is yet to be determined. Moreover, the issue of whether the trophic factors mediating neurite outgrowth are also responsible for synapse specification has not yet been resolved. To test whether trophic factors mediating neurite outgrowth and synapse formation between identified neurons are conserved in two molluscan species and whether these developmental processes are differentially regulated by different trophic factors, we used soma-soma and neurite-neurite synapses between identified Lymnaea neurons. We demonstrate here that the trophic factors present in Aplysia hemolymph, although sufficient to induce neurite outgrowth from Lymnaea neurons, do not promote specific synapse formation between excitatory partners. Specifically, the identified presynaptic neuron visceral dorsal 4 (VD4) and postsynaptic neuron left pedal dorsal 1 (LPeD1) were either paired in a soma-soma configuration or plated individually to allow neuritic contacts. Cells were cultured in either Lymnaea brain-conditioned medium (CM) or on poly-L-lysine dishes that were pretreated with Aplysia hemolymph (ApHM), but contained only Lymnaea defined medium (DM; does not promote neurite outgrowth). In ApHM-coated dishes containing DM, Lymnaea neurons exhibited extensive neurite outgrowth, but appropriate excitatory synapses failed to develop between the cells. Instead, inappropriate reciprocal inhibitory synapses formed between VD4 and LPeD1. Similar inappropriate inhibitory synapses were observed in Aplysia hemolymph-pretreated dishes that contained dialyzed Aplysia hemolymph. These inhibitory synapses were novel and inappropriate, because they do not exist in vivo. A receptor tyrosine kinase inhibitor (Lavendustin A) blocked neurite outgrowth induced by both Lymnaea CM and ApHM. However, it did not affect inappropriate inhibitory synapse formation between the neurons. These data demonstrate that neurite outgrowth but not inappropriate inhibitory synapse formation involves receptor tyrosine kinases. Together, our data provide direct evidence that trophic factors required for neurite outgrowth are conserved among two different molluscan species, and that neurite extension and synapse specification between excitatory partners are likely mediated by different trophic factors.

Animals↗
PubMed1994

Secretion of nerve growth factor from septum stimulates neurite outgrowth and release of the amyloid protein precursor of Alzheimer's disease from hippocampal explants.

Alzheimer's disease (AD) is characterized by the deposition of amyloid in the extracellular and intracellular compartments of the cerebral cortex. The extracellular amyloid consists of a protein (beta A4) which is derived from a larger precursor, the amyloid protein precursor (APP). Several studies have implicated APP in the regulation of neurite outgrowth during development, although the precise function of APP remains unknown. To examine the role of APP in the regulation of neurite outgrowth from hippocampal neurons, an explant culture system was developed. Explants of E18 mouse hippocampus were found to extend neurites when co-cultured with explants of E18 mouse septum. This finding demonstrated that the septum can release a neurite outgrowth-promoting factor (NOPF). As nerve growth factor (NGF) was also able to stimulate neurite outgrowth from the hippocampal explants, this suggested that the NOPF might be NGF. Immunoprecipitation of NGF from septal conditioned medium using a specific monoclonal antibody (27/21) completely blocked the neurite outgrowth-promoting effect, supporting this conclusion. Concomitant with its ability to stimulate neurite outgrowth, NGF stimulated the release of APP from the hippocampal explants. As previous studies have suggested that the binding of APP to heparan sulfate proteoglycans (HSPGs) in the extracellular matrix might be an important step in the regulation of neurite outgrowth by NGF, we examined the effect of APP on neurite outgrowth from dissociated hippocampal cells cultured on various protein substrates. When cells were cultured on a substrate of APP and HSPG, neurite outgrowth was markedly stimulated. No stimulation of neurite outgrowth was seen when neurons were cultured on substrates of either APP or HSPG alone. The results suggest that secreted forms of APP may be involved in stimulating neurite outgrowth from hippocampal neurons and that interactions between APP and HSPG may be important for a neurite outgrowth-promoting function.

Amyloid beta-Protein Precursor↗
PubMed1993

Neurite outgrowth from N18TG2 neuroblastoma induced by H-7, a protein kinase inhibitor, in the presence of colchicine.

Our previous studies have demonstrated that the protein kinase inhibitor H-7 promotes neurite outgrowth from mouse neuroblastoma N18TG2 cells as well as from primary cerebellar cells, and also that the neurites induced by H-7 were more tolerant of colchicine (COL) than those induced by dibutyryl cAMP (dB-cAMP). In the present study, we tested the effects of H-7 and dB-cAMP on neurite growth from N18TG2 cells in the presence of COL. We found that only H-7 promoted neurite formation in the presence of COL. The percentage of cells with neurites induced by H-7 in the presence of COL (H-7 + COL) was similar to that induced by H-7 alone. The neurites induced by H-7 + COL grew straight. They were very thin (less than 1 micron in diameter) and had round varicosities, as did the neurites induced by H-7 alone. By transmission electron microscopy, the neurites induced by H-7 + COL were found to contain longitudinally arranged intermediate filaments (IF). Microtubules (MT) were not observed within the neurites. We also examined the effect of cytochalasin B (CB) on the neurites induced by H-7 + COL and by H-7 alone. The neurites induced by H-7 + COL were tolerant to CB, but those induced by H-7 were resorbed completely within 24 h after CB was applied. Neurites tolerant to CB contained longitudinally IF. Simultaneous application of CB with H-7 + COL or with H-7 alone did not induce neurite formation.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

A simple procedure for quantification of neurite outgrowth based on stereological principles.

The molecular mechanisms controlling formation and remodelling of neuronal extensions are of considerable interest for the understanding of neuronal development and plasticity. Determination of neurite outgrowth in cell culture is a widely used approach to investigate these phenomena. This is generally done by a time consuming tracing of individual neurites and their branches. We have used stereological principles to determine the length of neurites. The total neuritic length per cell was estimated by counting the number of intersections between neurites and test lines of an unbiased counting frame superimposed on images of cell cultures obtained by conventional computer-assisted microscopy. The absolute length, L, of neurites per cell was subsequently estimated from the number of neurite intersections, I, per cell by means of the equation L=(pid/2)I describing the relationship between the number of neurite intersections and the vertical distance, d, between the test lines used. When measuring neurite outgrowth from PC12 cells and primary hippocampal neurons, data obtained by counting neuritic intersections correlated statistically significantly with data obtained using a conventional tracing technique. However, information was acquired more efficiently using the stereological approach. Thus, using the described set-up, the stereological procedure was approximately five times less time consuming than the conventional method based on neurite tracing. The study shows that stereological estimation of neuritic length provides a precise and efficient method for the study of neurite outgrowth in cultures of primary neurons and cell lines.

Animals↗
PubMed1994

Respective roles of neurofilaments, microtubules, MAP1B, and tau in neurite outgrowth and stabilization.

The respective roles of neurofilaments (NFs), microtubules (MTs), and the microtubule-associated proteins (MAPs) MAP 1B and tau on neurite outgrowth and stabilization were probed by the intracellular delivery of specific antisera into transiently permeabilized NB2a/d1 cells during treatment with dbcAMP. Intracellular delivery of antisera specific for the low (NF-L), middle (NF-M), or extensively phosphorylated high (NF-H) molecular weight subunits did not prevent initial neurite elaboration, nor did it induce retraction of existing neurites elaborated by cells that had been previously treated for 1 d with dbcAMP. By contrast, intracellular delivery of antisera directed against tubulin reduced the percentage of cells with neurites at both these time points. Intracellular delivery of anti-NF-L and anti-NF-M antisera did not induce retraction in cells treated with dbcAMP for 3 d. However, intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, tau, or tubulin induced similar levels of neurite retraction at this time. Intracellular delivery of monoclonal antibodies (RT97 or SMI-31) directed against phosphorylated NF-H induced neurite retraction in cell treated with dbcAMP for 3 d; a monoclonal antibody (SMI-32) directed against nonphosphorylated NF-H did not induce neurite retraction at this time. By contrast, none of the above antisera induced retraction of neurites in cells treated with dbcAMP for 7 d. Neurites develop resistance to retraction by colchicine, first detectable in some neurites after 3 d and in the majority of neurites after 7 d of dbcAMP treatment. We therefore examined whether or not colchicine resistance was compromised by intracellular delivery of the above antisera. Colchicine treatment resulted in rapid neurite retraction after intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, or tau into cells that had previously been treated with dbcAMP for 7 d. By contrast, colchicine resistance was not compromised by the intracellular delivery of antisera directed against NF-L, NF-M, or tubulin. These findings support previous studies indicating that MT polymerization mediates certain aspects of axonal neurite outgrowth and suggest that NFs do not directly participate in these events. These findings further suggest that NFs function in stabilization of the axonal cytoskeleton, apparently by interactions among NFs and MTs that are mediated by NF-H and MAPs.

Animals↗
PubMed2005

Neurite outgrowth involves adenomatous polyposis coli protein and beta-catenin.

Neuronal morphogenesis involves the initial formation of neurites and then differentiation of neurites into axons and dendrites. The mechanisms underlying neurite formation are poorly understood. A candidate protein for controlling neurite extension is the adenomatous polyposis coli (APC) protein, which regulates membrane extensions, microtubules and beta-catenin-mediated transcription downstream of Wnt signaling. APC is enriched at the tip of several neurites of unpolarized hippocampal neurons and the tip of only the long axon in polarized hippocampal neurons. Significantly, APC localized to the tip of only one neurite, marked by dephospho-tau as the future axon, before that neurite had grown considerably longer than other neurites. To determine whether neurite outgrowth was affected by beta-catenin accumulation and signaling, a stabilized beta-catenin mutant was expressed in PC12 cells, and neurite formation was measured. Stabilized beta-catenin mutants accumulated in APC clusters and inhibited neurite formation and growth. Importantly, these effects were also observed was independently of the gene transcriptional activity of beta-catenin. These results indicate that APC is involved in both early neurite outgrowth and increased growth of the future axon, and that beta-catenin has a structural role in inhibiting APC function in neurite growth.

Adenomatous Polyposis Coli Protein↗
PubMed1991

Retinal neurite growth on astrocytes is not modified by extracellular matrix, anti-L1 antibody, or oligodendrocytes.

Two factors that may influence the course of axonal regeneration in the central nervous system (CNS) are extracellular matrix (ECM) and cell surface molecules that may enhance or inhibit neurite outgrowth. Whereas cultured astrocytes have been reported to be a good substratum for neurite outgrowth, there is recent evidence that cultured oligodendrocytes are inhibitory. To test the influences of 1) ECM components, 2) the L1 adhesion molecule, and 3) the inhibitory potential of mature oligodendrocytes in the astrocytic environment, we have utilized a culture system in which neurites from embryonic rat retina grow vigorously on astrocyte monolayers. The major ECM components were assembled in neonatal rat cortical astrocyte-retina co-cultures only when the medium contained serum. In electron microscopic studies of serum containing cultures, retinal neurites were seen to be related to astrocyte surfaces but rarely were found in contact with ECM; in serum-free medium the association between neurites and astrocytes was similar. In addition, the growth of neurites was vigorous whether ECM was present or absent. Presence of antibodies against the cell surface adhesion molecule L1 did not inhibit retinal neurite elongation on glial fibrillary acidic protein-positive astrocytes. When oligodendrocytes from adult rat spinal cord were combined with the astrocytes, retinal neurites grew as well on the mixed glial population as on astrocytes alone. Immunostaining for galactocerebroside showed many oligodendrocyte processes to be aligned in the direction of neurite growth, suggesting association between the two cell types. This association was verified by electron microscopy. Furthermore, retinal explants extended neurites among myelin basic protein-positive oligodendrocytes cultured without astrocytes. Thus, the astrocyte surface is a strong promoter of neurite growth from embryonic rat retina. This growth did not depend upon either ECM or the L1 adhesion molecule. Because neurites grew on astrocytes in the presence of mature oligodendrocytes or among oligodendrocytes alone, we conclude that oligodendrocytes do not inhibit neurite growth under certain conditions.

Animals↗
PubMed1990

Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells requires thrombin inhibition.

Protease nexin-1 (PN-1) is a protein proteinase inhibitor recently shown to be identical with the glial-derived neurite-promoting factor or glial-derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN-1:thrombin complexes might mediate the neurite outgrowth activity of PN-1. However, the present studies showed that such complexes, unlike free PN-1, did not promote neurite outgrowth. The neurite outgrowth activity of PN-1 was only detected in the presence of thrombin or serum (which contains thrombin). PN-1 did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum-free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in the presence or absence of PN-1. The neurite-promoting activity of PN-1 was inhibited by an anti-PN-1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin III, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin III and PN-1 corresponded to their inhibition of thrombin. Together, these observations show that PN-1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites.

Amyloid beta-Protein Precursor↗
PubMed1991

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.

Aging↗
PubMed1987

A second cell-binding domain on fibronectin (RGDS-independent) for neurite extension of human neuroblastoma cells.

Human neuroblastoma cells (Platt and La-N1) have previously been shown to adhere and extend neurites on tissue-culture substrata coated with a 120K chymotryptic cell-binding fragment (CBF) of plasma fibronectin (pFN), a fragment which lacks heparan sulfate- and collagen-binding activities, and to adhere to--but not extend neurites on--substrata coated with the heparan sulfate (HS)-binding protein, platelet factor-4 (PF4) (Tobey et al., Exp Cell Res 158 (1985) 395 [3]). The mechanisms of these processes on CBF, on the intact pFN molecule, or on heparin-binding fragments of pFN have been tested using a heptapeptide (peptide A) containing the Arg-Gly-Asp-Ser (RGDS) sequence which recognizes a specific 'receptor' on the surface of a variety of cells or a control peptide with a single amino acid substitution. Adherence and neurite extension were completely inhibited on the 120K CBF by peptide A but not by control peptide; these results indicate that the RGDS-dependent 'receptor' is solely responsible for adhesive responses to the 120K CBF-containing region of the pFN molecule. When peptide A was added to cells on CBF which had already formed neurites to test reversibility, retraction of all neurite processes was induced by 1 h and cells eventually detached. In contrast, on intact pFN, peptide A had very limited effects on either initial adherence or neurite extension, revealing a second 'cell-binding' domain on the fibronectin molecule outside of the 120K region competent for neurite differentiation; addition of peptide A at later times to pFN-adherent, neurite-containing cells could induce only a small subset of neurites to retract, thus supporting evidence for the presence of this second domain. A second 'cell-binding' domain was further confirmed by quantitation of neurite outgrowth on these substrata and by analyses of cells on substrata coated with mixtures of CBF/PF4. When substrata coated with chymotrypsin-liberated HBF were tested in a similar fashion, adherence was rapid but neurite outgrowth required much longer times and was completely sensitive to RGDS peptides; supplementation of cells with the complex ganglioside GT1b could not induce RGDS-resistant neurites on heparin-binding fragments (HBF). These latter results indicate that neurite extension on HBF is a consequence of a low concentration of RGDS-dependent activity in HBF (but not to HS-binding activity as characterized by Tobey et al. [3]) and that the second 'cell-binding' domain is sensitive to chymotrypsin digestion of pFN during the liberation of HBF.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Lymphoid tissues induce NGF-dependent and NGF-independent neurite outgrowth from rat superior cervical ganglia explants in culture.

Induction of neurite outgrowth from superior cervical ganglia (SCG) by rat lymphoid tissues was studied using a tissue culture model. Neonatal rat SCG were cultured with 6-12-week-old rat thymus, spleen, or mesenteric lymph node (MLN) explants in a Matrigel layer, in defined culture medium without exogenous nerve growth factor (NGF). SCG were also co-cultured with neonatal rat heart (as positive control) or spinal cord (SC; as negative control). To determine whether inflammation affects the ability of lymphoid tissues to induce neurite outgrowth, we also examined MLN at various times after infecting rats with Nip-postrongylus brasiliensis (Nb-MLN). In one series of experiments, a single lymphoid tissue explant was surrounded by four SCG at a distance of 1 mm. The extent of neurite outgrowth was determined by counting the number of neurites 0.5 mm away from each ganglion at several time points. Adult thymus and, to a lesser extent, spleen had strong stimulatory effects on neurite outgrowth from SCG after 12 hr or more in culture. For thymus tissue, this was similar to the positive control heart explants. MLN from normal rats had minimal effect on neurite outgrowth; however, Nb-MLN showed a time-dependent enhancement of the neurite outgrowth, maximal at 3 weeks after infection. The relative efficacy of neurite outgrowth induction (heart > or = thymus > or = Nb-MLN > or spleen > or = MLN > or = SC) was confirmed in a second series of experiments where one SCG was surrounded by three different tissue explants. We then examined the role of 2.5S NGF, a well-known trophic factor for sympathetic nerves, in the lymphoid tissue-induced neurite outgrowth. Anti-NGF treatment of co-cultures of SCG and heart almost completely blocked the neurite outgrowth. Anti-NGF also significantly inhibited thymus- and spleen-induced neurite outgrowth, but not as effectively as heart-induced neuritogenesis (93, 80, and 77% inhibition at 24 hr; 86, 70, and 68% inhibition at 48 hr for heart, thymus, and spleen, respectively). On the other hand, anti-NGF inhibited only 8% of neurite outgrowth induced by 3-week post-infection Nb-MLN at 24 hr, and 41% at 48 hr. These data show that several adult rat lymphoid tissues exert neurotrophic/tropic effects. The predominant growth factor in thymus and spleen is NGF, while Nb-MLN produces factor(s) which is (are) immunologically distinguishable from NGF.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

Neurochemical diversity of dystrophic neurites in the early and late stages of Alzheimer's disease.

We examined the neurochemical and morphological diversity of abnormal neurites associated with beta-amyloid plaque formation in the early and late stages of Alzheimer's disease. Preclinical Alzheimer's disease was characterised by the presence of abnormal neurites containing either neurofilament or chromogranin A immunoreactivity. All clustered dystrophic neurites in these cases were associated with beta-amyloid plaques. Neurofilament immunoreactive dystrophic neurites in preclinical Alzheimer's disease could be further subclassified into bulb- and ring-like structures, and these abnormal neurites contained both phosphorylated and dephosphorylated neurofilament epitopes. Dystrophic neurites in Alzheimer's disease could be subdivided into predominantly neurofilament, tau, or chromogranin A immunolabeled forms. Some neurofilament immunoreactive neurites had a core region labeled for tau. The neurofilaments of the dystrophic neurites in Alzheimer's disease had the same complement of phosphorylation- and dephosphorylation-dependent epitopes as observed in preclinical cases. Therefore, an abnormal accumulation of variably phosphorylated neurofilaments represent the earliest cytoskeletal alteration associated with dystrophic neurite formation. Furthermore, these data indicate that dystrophic neurites may "mature" through neurofilament-abundant forms to the neurites containing the profoundly altered filaments labeled for tau. The precise morphological and neurochemical changes associated with dystrophic neurite formation suggests that beta-amyloid plaques are causing physical damage to surrounding axons. The resultant axonal sprouting and profound cytoskeletal alterations would follow the chronic stimulation of the stereotypical reaction to such physical trauma.

Aged↗
PubMed1996

GTP and guanosine synergistically enhance NGF-induced neurite outgrowth from PC12 cells.

Six per cent of rat pheochromocytoma (PC12) cells extended neurites (processes greater than one cell diameter in length) in the presence of 300 microM extracellular GTP or 300 microM guanosine for 48 hr, compared to only 2.5% of cells in control cultures. In the presence of 40 ng/ml of 2.5S NGF, about 20-35% of PC12 cells had neurites after 48 hr, and the addition of 300 microM guanosine or GTP together with NGF synergistically increased the proportion of cells with neurites to 40-65%. GTP and guanosine also increased the average number of branches per neurite, from 0.6 in NGF-treated cultures to 1.2 (guanosine) or 1.5 (GTP). Neurites formed after exposure to NGF alone had axonal characteristics as determined by immunocytochemistry with antibody, SMI-31, against axonal-specific polyphosphorylated neurofilament epitopes. Neurites generated with the addition of both guanosine or GTP had the same characteristics. GTP probably did not exert its effects via the P2X or P2Y purinoceptors because the adenine nucleotides ATP, ATP gamma S, ADP beta S, and ADP, which are all agonists of these receptors, inhibited rather than enhanced, NGF-induced neurite outgrowth. UTP also enhanced the proportion of cells with neurites, although not to the same degree as did GTP. This may indicate activity through a P2U-like nucleotide receptor. However, the response profile obtained, GTP > UTP >> ATP, does not fit the profile of any known P2Y, P2X or P2U receptor. The poorly hydrolyzable GTP analogues, GTP gamma S and GDP beta s were also unable to enhance the proportion of cells with neurites. This implied that GTP may produce its effects through a GTP-specific ectoenzyme or kinase. This idea was supported by results showing that another poorly hydrolyzable analogue, GMP-PCP, competitively inhibited the effects of GTP on neurite outgrowth. GTP did not exert its effects after hydrolysis to guanosine since the metabolic intermediates GDP and GMP were also ineffective in enhancing the proportion of cells with neurites. Moreover, the effects of GTP and guanosine were mutually additive, implying that these two purines utilized different signal transduction mechanisms. The effects of guanosine were not affected by the nucleoside uptake inhibitors nitrobenzylthioinosine (NBTI) and dipyridamole, indicating that a transport mechanism was not involved. Guanosine also did not activate the purinergic P1 receptors, because the A2 receptor antagonists, 1,3-dipropyl-7-methylxanthine (DPMX) or CGS15943, and the A1 receptor antagonist, 1,3-dipropyl-8-(2-amino-4-chloro)xanthine (PACPX) did not inhibit its reaction. Therefore guanosine enhanced neurite outgrowth by a signal transduction mechanism that does not include the activation of the P1 purinoceptors. The enhancement of the neuritogenic effects of NGF by GTP and guanosine may have physiological implications in sprouting and functional recovery after neuronal injury in the CNS, due to the high levels of nucleosides and nucleotides released from dead or injured cells.

Adenosine Triphosphate↗
PubMed1999

Cation channel control of neurite morphology.

The development of neuronal polarity and morphology is essential for a functioning nervous system. The present study was undertaken to explore whether blockade of specific channels alter neuronal morphology. Retinal ganglion cells were cultured in the presence of antagonists to NMDA, AMPA/kainate, L-, N-, P-, and Q-type voltage-dependent calcium channels (VDCCs). Five parameters were measured under these conditions: the number of neurites at the cell body, total neurite length, the length of the longest neurite, the number of branch points per neurite, and the diameter of the cell soma. Antagonists to NMDA and L-type VDCCs reduce the number of neurites at the cell body; antagonists to P- and Q-type VDCCs increase the number of neurites. Antagonists to the N-type VDCCs increase total neurite outgrowth, while antagonists to the NMDA and P-type channels reduce total neurite length. Antagonists to the NMDA and L-type channels increase the length of a single neurite, while decreasing the number of branch points; antagonists to the P- and Q-type VDCCs do essentially the opposite-increase the number of neurites, while decreasing the length of each. Blockade of one or more cation channels in developing retinal ganglion cells significantly perturbs neurite morphology. This study may help elucidate part of the role that cation channel signaling plays in neuritic development.

2-Amino-5-phosphonovalerate↗
PubMed2001

Calcium influx is necessary for optimal regrowth of transected neurites of rat sympathetic ganglion neurons in vitro.

The calcium ion serves many major roles in cellular processes, and therefore, its specific role in the regrowth of neurites after transection has been difficult to define. In the present study the effect of calcium on regrowth of transected rat superior cervical ganglion neurons in culture independent of its effects on neuronal survival was examined. Superior cervical ganglion neurons in culture for 10-14 days were preloaded with the calcium chelator 1,2 bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), and then the neurites were transected. The chelation of calcium inhibited the initiation of regrowth, and also decreased the number of branches in the regrowing neurite. In a second experiment, neurites were transected in zero calcium medium to eliminate calcium influx, and no regrowth was observed for up to 6h after transection. At this time the medium was changed to 1.8mM calcium, and the neurites showed regrowth 2h afterwards. After neurite transection in 1.8mM calcium there was a rise in calcium in the soma demonstrated with the calcium indicator dye Fluo-3 AM. This rise was attenuated with BAPTA-AM preloading or transection in zero calcium medium. Cultures were also grown in compartmented chambers which allowed isolation of the cell body from the neurite environment, and then the neurites were transected with the neurites in 1.8mM calcium medium, and the cell bodies immersed in zero calcium medium. Under these conditions the majority of neurons showed a marked delay of initiation of regrowth. From these data we suggest the following about mammalian neurite regrowth after transection. (1) Calcium is necessary for the initiation of regeneration. (2) Calcium entry at the cell body is necessary for optimal initiation of neurite regrowth. (3) Intracellular calcium is necessary for the branching of regrowing neurites.

Animals↗
PubMed1993

Re-evaluation of the structural organization of neuritic plaques in Alzheimer's disease.

We re-examined the relationship among synaptic pathology, subcellular abnormalities within neurites in the plaques and beta-amyloid deposits of Alzheimer's disease (AD) using laser confocal imaging and computer-aided serial section reconstruction techniques. Analysis of serial optical sections of neuritic plaques double-immunolabeled for anti-beta-amyloid/anti-tau-2 revealed that 35% of this type of plaque contained a dense amyloid core with clusters of peripheral abnormal neurites. The other 65% were without a dense core and were mainly composed of abundant abnormal neuritic clusters with bundles of amyloid distributed throughout the neuritic plaque. While two-dimensional (2-D) analysis of the plaques has suggested that neurites are distributed in the plaque periphery with beta-amyloid localized in its center, serial section analysis showed the opposite arrangement can also be true. Three-dimensional (3-D) reconstructions of serial optical sections showed that the tau-positive tortuous axons clustered in the neuritic plaques were often continuous with synaptophysin-positive distended terminals. Analysis of electron micrographs from serial sections showed continuity among the different segments of the neurites. Further analysis of the computer generated 3-D reconstructed neuritic plaques (both from serial electron micrographs and serial optical sections), viewed as continuous rotating loops, confirmed that a great majority of the plaque volume was occupied by the clustered and continuous abnormal neurites, while the amyloid fibrils were compressed and displaced to the periphery of the plaque. The 3-D imaging of the neuritic plaques in AD suggests a more widespread and active neuritic damage than that predicted from 2-D observations.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMedDate not supplied

Neurite growth capability of rat fetal neuronal cells against matured CNS myelin in vitro.

Reconstruction of neurocircuits by transplanted cells is expected to become an effective therapy for brain damage. In order to establish the transplantation therapy, it is necessary to find transplantable cells capable of reconstructing the lesioned neurocircuitry. We have reported that the younger neuronal cells such as neural stem cells are useful transplant materials because of their vigorous capacity for forming abundant neurites. On the other hand, it was reported that myelin-associated neurite growth inhibitor prevents neurite regeneration. In this study, we used rat fetal neuronal cells to examine the neurite growth capacity in the presence of mature CNS myelin. Crude CNS myelin was prepared from the brains of adult Wistar rats using previously described procedures. Testing wells were precoated with poly-L-lysine and additionally by over-night drying of a suspension containing 0, 5, 10, 15, or 20 microg/cm2 of the crude myelin protein. On embryonic days 10, 12, 15, and 17 (E10, E12, E15, and E17) embryos were surgically removed, mesencephalic neural plates were dissected out from the E10 embryos, and midbrain cells were taken from the E12, E15, and E17 embryos. The neural plates and midbrain cells were placed on the myelin-coated wells. After 24 h of culture (72 h in the case of neural plates), the number of surviving cells and the length of the neurites were examined immunocytochemically using anti-neurofilament (NF) antibody. Neurite length was measured by image analyzer Luzex-F. The mesencephalic neural plate was able to grow neurites even on 20 microg/cm2 central myelin. Almost the same number of midbrain cells attached themselves to the wells without myelin in every culture obtained from various stages of embryos. The number of cells attached on the myelin-coated wells decreased with the concentration of myelin. The number of NF-positive cells was higher in cultures of materials obtained from older embryos than in cultures obtained from younger embryos. The younger cells grew longer neurites than the older cells in the myelin noncoated wells. Neurite growth was inhibited strongly when the concentration of the central myelin was 10 microg/cm2 or greater, but on the 5 microg/cm2 myelin, the younger the cells were, the longer neurites they had. When the length of the longest neurites in one field of the image analyzer was further examined in the same way, the younger the cells were, the longer their axons grew on 0 and 5 microg/cm2 myelin. Thus, CNS myelin was seen to be a significant inhibitor of the recovery of injured neural tissue of the adult CNS. Younger cells grew longer neurites than older cells on CNS myelin, and so it was suggested that neural stem cells or younger neurons may serve as tissue for transplantation therapy.

Animals↗
PubMed2001

Potentiation of nerve growth factor-induced elongation of neurites by gelsemiol and 9-hydroxysemperoside aglucone in PC12D cells.

In PC12D cells, nerve growth factor (NGF) increased the proportion of neurite-bearing cells and made neurites longer. A methanol extract of Verbena littoralis H. B. K. collected in Paraguay only slightly potentiated the proportion of PC12D cells with neurites but markedly increased the length of neurites in the presence of NGF (2 ng mL(-1)). The methanol extract was partitioned between ethyl acetate and water followed by further extraction of water fraction with n-butanol. The potentiating activity of NGF-action was observed in the ethyl acetate and n-butanol fractions. The n-butanol fraction was separated by silica gel chromatography, monitoring the NGF-potentiating activity to give gelsemiol and 9-hydroxysemperoside aglucone (9-OHSA). Neither compound (30-300 microM) exhibited neurite-inducing activity alone. Gelsemiol (100-300 microM) markedly enhanced an increase in the proportion of neurite-bearing cells and an extension of the neurite length in the presence of NGF (2 ng mL(-1)). Interestingly, in the presence of NGF (2 ng mL(-1)), 9-OHSA (100-300 microM) enhanced the elongation of neurites without affecting the increase in the proportion of cells with neurites. These results suggested that gelsemiol and 9-OHSA were major active components of V. littoralis in the NGF-potentiating action. It was possible that the mechanism of neurite elongation by NGF was different from that of the increase in the proportion of neurite-bearing cells, and that 9-OHSA selectively affected the neurite elongation mechanism.

Cell Culture Techniques↗