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PubMed1988

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

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

Animals↗
PubMed1987

Adenosine 5'-triphosphate synthesis and metabolism localized in neurites of cultured sympathetic neurons.

Adenosine triphosphate synthesis and metabolism in cultured sympathetic neurons was studied after the incorporation of [2-3H]adenine into intact or microdissected neurites to determine whether ATP is provided locally during neurite outgrowth, when and where it is synthesized and how its levels are regulated at rest and following depolarization. Neurites maintained an independent capability for synthesis of ATP at any stage of growth: [3H]ATP levels increased in neurites in direct proportion to neurite length and equivalent amounts of [3H]ATP were synthesized by intact neurites, by neurites separated from cell bodies or by neurites further segmented into sections. Thus, metabolic labelling of cultured neurons with [3H]adenine provides a simple method to measure relative neurite outgrowth. Neurite ATP was maintained mainly by respiration but also by glycolysis and [3H]ATP levels were stable for at least 14 h after adenine withdrawal when cells were at rest. Depolarization overcame respiratory control, causing a quantitative conversion of ATP to adenosine monophosphate (AMP) and inosine monophosphate (IMP) and the release of nucleosides (adenosine and inosine) and nucleotides [adenosine diphosphate (ADP) and adenosine monophosphate (AMP)]. Release of nucleosides, but not of nucleotides or [3H]noradrenaline, was enhanced by NaN3 or 2-deoxyglucose under nondepolarizing conditions and was prevented by the adenosine transport inhibitor p-nitrobenzyl-6-thioinosine. It is concluded that neurites can use local mechanisms for ATP synthesis that do not depend on a functional connection to the cell body. Any metabolic stress which causes ATP breakdown causes these cells to express a transient purinergic phenotype involving release of adenosine and inosine by facilitated diffusion. To promote the release of purine nucleotides, however, more specific stimuli are required.

Adenine↗
PubMed1990

Inhibition of neurite polarity by tau antisense oligonucleotides in primary cerebellar neurons.

Neurons in culture can have fundamentally distinct morphologies which permit their cytological identification and the recognition of their neurites as axons or dendrites. Microtubules may have a role in determining morphology by the selective stabilization of spatially distinct microtubule subsets. The plasticity of a neurite correlates inversely with the stability of its component microtubules: microtubules in growth cones are very dynamic, and in initial neurites there is continuous incorporation of labelled subunits, whereas in mature neurites, microtubules are highly stabilized. The binding of microtubule-associated proteins to the microtubules very probably contributes to this stability. Cerebellar neurons in dissociated culture initially extend exploratory neurites and, after a relatively constant interval, become polarized. Polarity becomes evident when a single neurite exceeds the others in length. These stable neurites cease to undergo the retractions and extensions characteristic of initial neurites and assume many features of axons and dendrites. We have now studied the role of the neuronal microtubule-associate protein tau in neurite polarization by selectively inhibiting tau expression by the addition of antisense oligonucleotides to the culture media. Although the extension of initial exploratory neurites occurred normally, neurite asymmetry was inhibited by the failure to elaborate an axon.

Animals↗
PubMed1988

Tension and compression in the cytoskeleton of PC-12 neurites. II: Quantitative measurements.

We assessed the mechanical properties of PC-12 neurites by applying a force with calibrated glass needles and measured resulting changes in neurite length and deflection of the needle. We observed a linear relationship between force and length change that was not affected by multiple distensions and were thus able to determine neurite spring constants and initial, nondistended, rest tensions. 81 out of 82 neurites showed positive rest tensions ranging over three orders of magnitude with most values clustering around 30-40 mu dynes. Treatment with cytochalasin D significantly reduced neurite rest tensions to an average compression equal to 14% of the former tension and spring constants to an average of 17% of resting values. Treatment with nocodazole increased neurite rest tensions to an average of 282% of resting values but produced no change in spring constant. These observations suggest a particular type of complementary force interaction underlying axonal shape; the neurite actin network under tension and neurite microtubules under compression. Thermodynamics suggests that microtubule (MT) assembly may be regulated by changes in compressive load. We tested this effect by releasing neurite attachment to a polylysine-coated surface with polyaspartate, thus shifting external compressive support onto internal elements, and measuring the relative change in MT polymerization using quantitative Western blotting. Neurons grown on polylysine or collagen without further treatment had a 1:2 ratio of soluble to polymerized tubulin. When neurites grown on polylysine were treated with 1% polyaspartate for 15-30 min, 80% of neurites retracted, shifting the soluble: polymerized tubulin ratio to 1:1. Polyaspartate treatment of cells grown on collagen, or grown on polylysine but treated with cytochalasin to reduce tension, caused neither retraction nor a change in the soluble:polymerized tubulin ratio. We suggest that the release of adhesion to the dish shifted the compressive load formerly borne by the dish onto Mts causing their partial depolymerization. Our observations are consistent with the possibility that alterations in MT compression during growth cone advance integrates MT assembly with the advance.

Adrenal Gland Neoplasms↗
PubMed1980

Effects of fasciculation on the outgrowth of neurites from spinal ganglia in culture.

This report describes the influence of neurite fasciculation on two aspects of nerve growth from chick spinal ganglia in vitro: the inhibition of outgrowth by high concentrations of nerve growth factor (NGF) and the preferential growth of neurites toward a capillary tube containing NGF. These studies involved a comparison of cultures of single cells, cell aggregates, and intact ganglia and the use of antibodies against the nerve cell adhesion molecule (CAM) to perturb fasciculation under a variety of conditions. The inhibition of outgrowth, which was observed with ganglia and aggregates but not with single cells, was correlated with a thickening of neurite fascicles. In accord with this observation, anti-CAM, which diminishes fasciculation by inhibiting side-to-side interactions between individual neurites, also partially reversed the inhibition of neurite outgrowth at high NGF concentrations. On the basis of these and other studies, we consider the possibility that neurite bundling causes an increase in the elastic tension of a fascicle without a compensatory increase in its adhesion to substratum. It is proposed that this imbalance could inhibit neurites from growing out from a ganglion and even result in retraction of preexisting outgrowth. In the analysis of NGF-directed growth, it was found that a capillary source of NGF produced a steep but transient NGF gradient that subsided before most neurites had emerged from the ganglion. Nevertheless, the presence of a single NGF capillary caused a dramatic and persistent asymmetry in the outgrowth of neurites from ganglia or cell aggregates. In contrast, processes of individual cells did not appear to orient themselves toward the capillary. The most revealing finding was that anti-CAM antibodies caused a decrease in the asymmetry of neurite outgrowth. These results suggest that side-to-side interactions among neurites can influence the guidance of nerve bundles by sustaining and amplifying an initial directional signal.

Animals↗
PubMed1984

Uptake and transport of herpes simplex virus in neurites of rat dorsal root ganglia cells in culture.

Attachment and neuritic transport of herpes simplex virus (HSV) type 1 (McIntyre) were studied in a cell culture system with dissociated cells of rat dorsal root ganglia. The two-chamber cell culture system containing a diffusion barrier penetrated by neurites of cultured sensory neurons permitted infection of neurites extending outside the diffusion barrier without exposure of the neuronal cell soma. HSV adsorbed to neuritic extensions and reached the neuronal soma within 1.5 h post-inoculation. Neuritic uptake and transport of HSV were inhibited in the presence of cytochalasin B. Internalization of virus in neurites was preceded by attachment of virus to the neurite plasma membrane. Neurites transported viral nucleocapsids (NC) through the diffusion barrier of the cultures. Destruction of the neuritic extensions before or shortly after peripheral virus inoculation blocked spread of infection to the cell soma. No infection was established when neuritic extensions were exposed to viral NC and NC were then not observed inside the neurite plasma membrane. Virus produced in neurons, when HSV was inoculated into the inner culture chamber containing the neuronal cell bodies, was transported as enveloped virus in cytoplasmic vesicles from the neuronal cell body towards the periphery. Schwann cells were infected by viropexis. Shortly after infection virions were observed in vacuoles of the cytoplasm.

Animals↗
PubMed1987

Newly synthesized catalytic and regulatory components of adenylate cyclase are expressed in neurites of cultured sympathetic neurons.

Forskolin- and guanine nucleotide-stimulated adenylate cyclase activities were measured in microdissected sections of neurites from small explants and in dispersed cell cultures of sympathetic ganglion neurons to determine whether a competent system for regulated formation of cAMP, consisting of both catalytic units of adenylate cyclase and regulatory GTP binding proteins, is synthesized during neurite outgrowth and where it is distributed in the neuron. An increase in both guanine nucleotide- and forskolin-dependent activity of adenylate cyclase occurred concomitantly with neurite outgrowth and was directly proportional to neurite length. Separate analysis of adenylate cyclase activity in explant cell bodies or neurites showed that the increased activity was localized entirely in the neurites, while activity in the cell bodies remained virtually constant during growth. Concentric sections of neurites of approximately 500 microns width, which contained similar volumes of neurites as determined with the indicator BCECF (Rink et al., 1982), produced similar levels of cAMP, indicating an even distribution of adenylate cyclase in the neurites. Cell bodies, when stimulated by GTP gamma S, produced 236 +/- 46 attomol cAMP/min (30 degrees C)/cell body and an additional 52.6 +/- 20 attomol cAMP/min (30 degrees C)/neuron were produced with each day of neurite growth (approximately 400 microns). Assuming a turnover number of 2000 min-1, cell bodies and neurites were calculated to contain similar densities of catalytic unit molecules on their surface (9-28 molecules/micron 2). An abundant GTP binding protein, detected by ADP-ribosylation with pertussis toxin, was also widely distributed in the neuron.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1988

Suppression of nerve growth factor-directed neurite outgrowth in PC12 cells by sphingosine, an inhibitor of protein kinase C.

The formation of vertebrate neural circuitry is regulated in part by neurotrophic agents, such as nerve growth factor (NGF); however, the biochemical mechanisms involved in neurite outgrowth have yet to be completely resolved. Phorbol ester tumor promoters are known to influence the extension of neurites in a variety of neurodevelopmental systems, and protein kinase C, the major phorbol ester receptor, has been implicated in this process. In the present study, sphingosine, a specific pharmacological inhibitor of protein kinase C, was employed to investigate the role of this enzyme in the elaboration of neurites in PC12 pheochromocytoma cells. Normally, PC12 cells respond to NGF by morphologically differentiating into sympathetic neuron-like cells, exhibiting a marked hypertrophy, and extending slender neurites piloted by well defined growth cones. The elaboration of NGF-induced neurites was found to be reversibly inhibited by sphingosine in a dose-dependent manner (IC50 = 2.5-5 microM), while similar concentrations of several structural analogs were inactive. The suppression of neurite outgrowth by sphingosine was antagonized by the addition of 12-O-tetradecanoylphorbol 13-acetate (TPA), which binds to and directly activates protein kinase C. In the presence of NGF, TPA treatment increased the incidence of neurite outgrowth, and this increase, in turn, was antagonized by sphingosine. The binding of [3H]phorbol 12,13-dibutyrate to specific phorbol ester binding sites in PC12 cells was inhibited by sphingosine at concentrations similar to those which inhibited neurite outgrowth. The effects of sphingosine on TPA-directed protein phosphorylation were examined in situ, revealing inhibition of [32P]phosphate incorporation into cellular proteins. The specific TPA-directed phosphorylation of tyrosine hydroxylase was inhibited by sphingosine, as was the resulting increase in enzyme activity. The effects of sphingosine on the levels of alpha- and beta-tubulin mRNAs were also examined in an effort to delimit the locus of protein kinase C action. Concentrations of sphingosine which suppressed neurite outgrowth did not inhibit the NGF-directed elevation of tubulin transcript levels. Taken together, these results reveal the presence of a sphingosine-sensitive pathway in neurite outgrowth and indicate that protein kinase C plays a role in mediating the neuritogenic effects of NGF. Furthermore, the results suggest that protein kinase C acts at a distal segment of the neurite growth pathway.

Adrenal Gland Neoplasms↗
PubMed1986

The selective inhibition of growth cone extension by specific neurites in culture.

We have found that neurites from embryonic chick retinal and sympathetic explants do not mix in culture, while retinal neurites mix with retinal neurites, and sympathetic neurites mix with sympathetic neurites. These results confirm those obtained with embryonic rat tissues by Bray et al. (1980). We have also used video time-lapse techniques to examine the behavior of individual retinal and sympathetic growth cones as they attempt to cross retinal and sympathetic neurites. We have found that retinal growth cones cross retinal neurites without delay, retinal growth cones usually retract from sympathetic neurites but often cross them on a second advance, sympathetic growth cones repeatedly retract from retinal neurites, rarely managing to cross them at all, and sympathetic growth cones cross sympathetic neurites but are sometimes delayed. Our results indicate that retinal and sympathetic growth cones can distinguish between specific labels associated with retinal and sympathetic neurites. They also suggest that active avoidance could play an important role in growth cone navigation.

Animals↗
PubMed1992

Analysis of neuritic outgrowth from severed giant axons in Lumbricus terrestris.

This study analyzes the detailed morphometric pattern at various postoperative times of neuritic outgrowths from the proximal and distal stumps of two uniquely identifiable axons. Morphological patterns of neuritic outgrowths from stumps of severed axons were compared for medial and lateral giant axons in the central nervous system of the earthworm Lumbricus terrestris. Outgrowths from proximal and distal stumps were labeled by injection of fluorescent dye into axonal stumps and assessed according to morphometric parameters. Outgrowths from axonal stumps of severed giant axons were statistically indistinguishable for most morphometric measures of neuritic quantity, shape, direction, and location. There were two exceptions to this general rule: 1) proximal stumps of medial giant axons produced significantly more neurites than distal stumps of medial giant axons, and 2) proximal stumps of lateral giant axons produced significantly longer neurites than proximal stumps of medial giant axons. No measure of neuritic outgrowth showed a significant change from the second through seventh postoperative week, suggesting that most outgrowth occurred in the first two postoperative weeks and that neuritic morphology remained stable through the seventh postoperative week. Neurites grew across the lesion site in relatively straight trajectories parallel to the longitudinal axis of the ventral nerve cord and often grew alongside the appropriate axonal stump across the lesion site. The length of neurites growing in close apposition to appropriate axonal stumps or giant axons was much greater than expected, had outgrowth been randomly directed. These data provide a basis for future investigations of the mechanisms that regulate neuritic outgrowth.

Animals↗
PubMed1993

Evidence that transmitter-containing dystrophic neurites precede paired helical filament and Alz-50 formation within senile plaques in the amygdala of nondemented elderly and patients with Alzheimer's disease.

Immunocytochemical techniques were employed to examine the temporal ordering whereby amyloid beta-protein (A beta P) and neuronal elements collectively come together to form senile plaques in Alzheimer's disease (AD). Specifically, we addressed three questions: (1) whether A beta P deposition precedes or follows neuritic changes; (2) whether paired helical filament (PHF) formation is an early or late event in the genesis of the dystrophic neurites which participate in plaque formation; and (3) whether the density of senile plaques displays any relationship with the prevalence of PHF or Alz-50 containing neurons. To address these questions we studied the amygdala from a group of patients with AD, a group of nondemented age-matched individuals exhibiting a sufficient number of senile plaques to be classified by neuropathological criteria as AD, and a group of age-matched controls without AD pathology. Amyloid-bearing plaques were demonstrated by A beta P immunolabeling and thioflavine-S staining. Neuritic changes in the form of dystrophic neurites were observed with the aid of antibodies against PHF, Alz-50, as well as antibodies against several neuropeptides (i.e., substance P, somatostatin, and neurotensin) and the acetylcholine biosynthetic enzyme, choline acetyltransferase. By using a graded range of pathologic changes both within and across the patient population to provide us with a means of evaluating plaque deposition from its earliest to most advanced stages of development, we observed in patients and/or regions of the amygdala displaying a mild degree of pathologic change A beta P deposition in the absence of any neuritic changes. With increasing density of A beta P, however, we began to observe dystrophic neurites within plaques. In regions of relatively few plaques, the dystrophic neurites were immunolabeled only with antibodies against the various neurotransmitters and they lacked evidence of cytoskeletal pathology (i.e., Alz-50 or PHF). Only as the density of A beta P increased further within a region, were dystrophic neurites observed that exhibited Alz-50 or PHF. In no instance did we observe a relationship between the density of A beta P deposition and the density of Alz-50 or PHF-immunoreactive neurons. Collectively, our data suggest that the deposition of A beta P is an early pathologic event in senile plaque formation. Thereafter, swollen neurites can be seen in the vicinity of A beta P. This early neuritic response, which can first be visualized by immunolabeling for one or another transmitter substance, is followed by alterations in the cytoskeleton as recognized initially by antibodies to Alz-50 and subsequently by the presence of PHF.

Adult↗
PubMed2006

Automated neurite labeling and analysis in fluorescence microscopy images.

BACKGROUND: To investigate the intricate nervous processes involved in many biological activities by computerized image analysis, accurate and reproducible labeling and measurement of neurites are prerequisite. We have developed an automated neurite analysis method to assist this task. METHODS: Our approach can be considered as automated with certain user interaction in setting initial parameters. Single and connected centerlines along neurites are extracted. The computerized method can also generate branching and end points. Owing to its multi-scale flexibility, both thick and thin neurites are simultaneously detected. RESULTS: We employ the relative neurite length difference (defined as the difference between the lengths obtained by automated and manual analysis divided by the total length of the latter) and neurite centerline deviation (defined as the area of the regions enclosed by different paths between automated and manual analysis divided by the total length of the former) to evaluate the performance of our algorithm, which is of great interest in neurite analysis. The average of the relative length difference is about 0.02, while the average of the centerline deviation is about 2.8 pixels. The probabilities of the distributions being the same from the Kolmogorov-Smirnov (KS) test of the automatic and manual results are 99.79%. The KS test also shows no significant bias between different observers based on the proposed new validation scheme. CONCLUSIONS: With the accurate and automated extraction of neurite centerlines and measurement of neurite lengths, the proposed method, which greatly reduces human labor and improves efficiency, can serve as a candidate tool for large-scale neurite analysis beyond the capability of manual tracing methods.

Algorithms↗
PubMed1992

Sequential effects of astroglial-derived factors on neurite outgrowth: initiation by protease inhibitors and potentiation by extracellular matrix components.

Astroglial-conditioned medium (GCM) induced two distinct, but intimately related, phases of neuritogenesis in NB2a/d1 neuroblastoma cells--a "rapid-outgrowth," unstable phase, and a delayed, relatively stable phase, which are apparently regulated by glial-derived protease inhibitors and laminin, respectively. The initial rapid outgrowth (less than 4 hr) may be mediated by inhibition of a thrombin-like protease, present as a serum component and/or adsorbed to the outer neuronal surface, since (1) a similar effect was obtained by serum removal or by adding the specific thrombin inhibitor, hirudin; (2) exogenous thrombin inhibited the rapid outgrowth of neurites by GCM; and (3) cell-free enzyme assays confirmed the presence of thrombin-inhibitory activity in GCM. Although neurites induced by removal of serum removal or hirudin addition are rapidly resorbed following serum replenishment or hirudin depletion, GCM-induced neurites continued to elongate after GCM removal, indicating that GCM contained additional neurite-promoting factors. Anti-laminin antiserum did not inhibit the initial elaboration of neurites by GCM but prevented their continued elongation. Anti-laminin antiserum had no affect on neurite outgrowth induced by serum deprivation. The more protracted, second phase of neurite outgrowth could also be achieved by the addition of soluble purified laminin to undifferentiated cells. Unlike neurites at 4 hr, neurites at 24 hr were no longer dependent on the protease inhibitors in GCM, since exogenous thrombin no longer caused them to retract. Simultaneous addition of thrombin and anti-laminin antiserum with GCM had identical inhibitory effects on continued neurite elaboration at 24 hr as did anti-laminin antiserum without thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Developmental regulation of plasticity along neurite shafts.

Although it is becoming increasingly clear that structural dynamics on neurite shafts play important roles in establishing neuronal architecture, the underlying mechanisms are unknown. The present study investigates local induction of filopodia along the shafts of neurites, a process that, by analogy to the growth cone, can represent the first stage in the generation of a new neuronal process. We show that filopodia can be induced reliably along the neurite shaft in response to a localized electric field stimulus that evokes large local intracellular calcium increases. Neither induction of filopodia nor a local rise in intracellular calcium occurred in calcium-free medium. Although calcium induction of neurite filopodia is highly reliable, forming in response to more than 90% of attempts, it is developmental state-dependent, since neurite filopodia could not be induced in neurons previously defined as "stable state." We have found two distinct changes in stable state neurons that can decrease the ability to induce new neurites. The first is a reduced calcium response: Field stimulation produced large local rises (280 nM) in intracellular calcium in growing neurons, whereas the identical stimulus produced smaller changes (148 nM) in stable state neurons. Second, stable state neurons change so that even when the stimulus intensity was increased to elicit a calcium response that would have been sufficient to induce filopodia in growing neurites, neurite filopodia were still not induced. Thus, intracellular calcium plays a key role in structural changes along the shafts of neurites. Furthermore, developmental changes in both calcium homeostatic components and in calcium responsiveness (i.e., the sensitivity of cellular components that modulate neurite morphology) underlie shifts from plasticity to stability of neuronal architecture in this system.

Animals↗
PubMed1997

Truncated trkB receptors on nonneuronal cells inhibit BDNF-induced neurite outgrowth in vitro.

The function of truncated trkB receptors during nervous system plasticity and regeneration is currently unknown. The extensive nonneuronal localization of truncated trkB-T1 receptors, coupled with their up-regulation by CNS glial cells in response to injury, has led to the speculation that these receptors may sequester BDNF and NT-4/5 to reduce their local availability and, thus, limit axonal sprouting. Conversely, trkB-T1 receptors could bind and present neurotrophins to injured axons and facilitate their regeneration in a manor analogous to that proposed for p75(NTR) receptors on Schwann cells. To address this issue, we used an in vitro coculture paradigm in which wild-type 3T3 NIH fibroblasts or two different 3T3 cell clones stably expressing trkB-T1 receptors served as monolayer substrates upon which to evaluate the effect of trkB-T1 receptors on nonneuronal cells to influence neurotrophin (NGF, BDNF, NT-3, and NT-4/5)-induced neurite outgrowth from retinoic acid (RA)-treated SY5Y neuroblastoma cells. In these experiments, BDNF and NT-4/5 produce a strong phosphorylation of trk receptors on the RA-SY5Y cells and induce differentiation of the SY5Y cells (as measured by the development of neurofilament-positive neuritic processes). This ability of the trkB ligands to stimulate neurite outgrowth is dose dependent since increasing concentrations of BDNF (5, 25, and 100 ng/ml) result in an increased percentage of SY5Y cells developing neurites and in progressively longer neurites from SY5Y cells on the control 3T3 monolayers. In these experiments, BDNF and NT-4/5 induce the strongest neurite outgrowth, followed by NT-3 and then NGF. When trkB-T1 receptors are present on the 3T3 cell substratum both BDNF- and NT-4/5-induced neurite extension from the SY5Y cells are strongly inhibited. In contrast, NGF-induced neurite growth is unaffected and NT-3-associated growth is somewhat reduced. These results suggest that the inhibitory effect of the trkB-T1 receptors on the nonneuronal cell substrates is selective for neurite outgrowth that is mediated via the trkB-kinase receptors on the neuroblastoma cells. This ability of trkB-T1 receptors on the nonneuronal substratum to inhibit BDNF-induced neurite outgrowth can be overcome by the addition of high concentrations of BDNF (1 microg/ml). Binding assays using 125I-BDNF suggest that this inhibitory effect could be mediated via binding and internalization of BDNF by the trkB-T1 receptors on the 3T3 cells. These results provide strong support for the hypothesis that the up-regulation of trkB-T1 receptors on astrocytes following CNS lesions enhances the sequestration of the trkB ligands, BDNF and NT- 4/5, at the site of reactive gliosis and, thus, contributes to the inhibition of CNS axonal regeneration from neurons expressing trkB-kinase receptors by removing their ligands from the extracellular environment.

3T3 Cells↗
PubMed1996

Detection of a trigger zone of bradykinin-induced fast calcium waves in PC12 neurites.

Bradykinin and caffeine were used as two different agonists to study inositol 1,4,5-trisphosphate (IP3)-sensitive and caffeine/ryanodine-sensitive intracellular Ca2+ release in the outgrowing neurites of nerve-growth-factor (NGF)-treated rat phaeochromocytoma cells (PC12). Changes in neuritic intracellular free Ca2+ ([Ca2+]i) in single cells were measured after loading with a 1:1 mixture of the acetoxymethyl (AM) ester of the Ca2+-sensitive dyes Fura-red and Fluo-3, in combination with confocal microscopy. Bradykinin-induced Ca2+ release was blocked by U73211, a specific phospholipase C inhibitor. Caffeine-induced Ca2+ release was very low in neurites at rest. It increased after the cells were preloaded with Ca2+. The Ca2+ signal evoked at high concentrations of bradykinin (>500 nM) arose from a trigger zone in the proximal part of the neurite, as a bi-directional wave towards the growth cone and cell body. The speed of neuritic Ca2+ waves was reduced in cells loaded with the Ca2+ chelator 1, 2-bis(2-aminophenoxy)ethane-tetraacetic acid/AM. Preloading of Ca2+ stores led to increased bradykinin-induced Ca2+ release, as seen for caffeine, and faster Ca2+ wave speeds. Caffeine evoked a simultaneous [Ca2+]i rise along the neurites of Ca2+ preloaded cells. Higher Ca2+ signal amplitudes and faster Ca2+ wave speeds, but no longer-lasting IP3-induced [Ca2+]i signals, correlated with increased caffeine-induced Ca2+ release in the neurites. At low concentrations of bradykinin (<1.0 nM), the Ca2+ signals ceased to propagate as complete Ca2+ waves. Instead, repetitive stochastic Ca2+ release events (neuritic Ca2+ puffs) were observed. Neuritic Ca2+ puffs spread across only a few microns, at a slower speed than neuritic Ca2+ waves. These Ca2+ puffs represent elementary Ca2+ release units, whereby the released Ca2+ ions form these elementary events into the shape of a Ca2+ wave.

Aniline Compounds↗
PubMed1995

The interaction of vascular endothelial cells and dorsal root ganglion neurites is mediated by vitronectin and heparan sulfate proteoglycans.

The interaction of peripheral nerve and blood vessels during development was studied by using DRG explant culture plated on confluent monolayer of vascular endothelial cells (VEC). The comparison of neurite length on various substrates showed a preference of DRG neurites in the following order; thrombospondin > laminin, vitronectin > fibronectin, VEC monolayer > collagen I, rat astrocyte monolayer. On layers of fibroblasts (3T3) or gliomas (C6), neurite extension was not observed. To identify the neurite outgrowth promoting adhesion molecules on VEC surface, several antibodies and synthetic peptides were added to the culture medium of DRG. With vitronectin antibody or with peptides containing the Arg-Gly-Asp (RGD) sequence, 30-40% of neurite outgrowth was inhibited and these two effects were not additive. Therefore, a part of neurite outgrowth in this system is mediated by vitronectin in RGD dependent manner. Another molecule which promotes neurite outgrowth on VEC was identified by a new monoclonal antibody (MAb) EC1. In the Western blot analysis, the immunoreactive band which was over 400 kDa was intensified by guanidine HCl extraction. EC1 immunoreactive band disappeared after the treatment of heparitinase but not with other glycolyases, indicating that EC1 antigen is heparan sulfate proteoglycan(s). The DRG neurite outgrowth was inhibited by MAb EC1 by about 30-40%. By the combination of MAb EC1 and RGD peptide, the neurite outgrowth in explant culture was inhibited by about 50%, and in DRG dissociated culture nearly 100% inhibition was observed. Thus, for the DRG neurite elongation on VEC, vitronectin and heparan sulfate proteoglycan(s) are playing crucial roles.

Animals↗
PubMed2004

N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) suppresses neuritic degeneration caused by different experimental paradigms including in vitro Wallerian degeneration.

Accumulating evidence indicates that neurite degeneration occurs via a distinct mechanism from somal death programs. We have previously shown that neuritic ATP level in sympathetic neurons decreases, whereas somal ATP level remains unaltered during degeneration caused by the microtubule-disrupting agent, vinblastine. Moreover, caspase activation occurs only in cell soma, supporting the view of somal apoptosis and neuritic necrosis. Therefore, the ATP level of neurites is crucial for their degeneration; it appears to correlate with membrane blebbing or beading which precedes late whole fragmentation of neurites under these conditions. Based on these metabolic and morphological criteria, we have tested the effects of various protease inhibitors on vinblastine-induced neurite degeneration in superior cervical ganglia from neonatal mice. Among agents tested, N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), the trypsin-like serine protease inhibitor, but not N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), the chymotrypsin-like serine protease inhibitor, protected sympathetic neurites from beading formation, neuritic fragmentation and a decrease in their ATP level. The commitment time for the saving effect of TLCK occurred around 7 h following treatment with vinblastine, at a time point after microtubule degradation (2 h) and before massive beading formation (later than 12 h). Moreover, TLCK was also capable of suppressing Wallerian degeneration in culture and neuritic degeneration following withdrawal of NGF in a dose-dependent manner. These results strongly suggest that TLCK intervenes in a common step in the cascade of neuritic degeneration caused by these different experimental paradigms and provides a helpful clue for identifying such a molecular step.

Adenosine Triphosphate↗