Binding characteristics of chondroitin sulfate proteoglycans and laminin-1, and correlative neurite outgrowth behaviors in a standard tissue culture choice assay

Neuronal growth cones are capable of sophisticated discrimination of environmental cues, on cell surfaces and in the extracellular matrix, to accomplish navigation during development (generation) and following nervous system injury (regeneration). Choices made by growth cones are commonly examined using tissue culture paradigms in which molecules of interest are purified and substratum-bound. From observations of growth cone behaviors using these paradigms, assertions are made about choices neuronal growth cones may make in vivo. However, in many cases, the binding, interactions, and conformations of these molecules have not been determined. In the present study, we investigated the binding characteristics of two commonly studied outgrowth regulatory molecules: chondroitin sulfate proteoglycans (CSPGs), which are typically inhibitory to neurite outgrowth during development and following nervous system injury, and laminin, which is typically outgrowth promoting for many neuronal types. Using a novel combination of radiolabeling and quantitative fluorescence, we determined the precise concentrations of CSPGs and laminin-1 that were bound separately and together in a variety of choice assays. For identically prepared cultures, we correlated neurite outgrowth behaviors with binding characteristics. The data support-our working hypothesis that neuronal growth cones are guided by the ratio of outgrowth-promoting to outgrowth-inhibiting influences in their environment, i.e., they summate local molecular cues. The response of growth cones to these molecular combinations is most likely mediated by integrins and subsequent activation of signal transduction cascades in growth cones.     

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

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

The heparan sulfate proteoglycan agrin modulates neurite outgrowth mediated by FGF‐2

Although the role of agrin in the formation of the neuromuscular junction is well established, other functions for agrin have remained elusive. The present study was undertaken to assess the role of agrin in neurite outgrowth mediated by the heparin‐binding growth factor basic fibroblast growth factor (FGF‐2), which we have shown previously to bind to agrin with high affinity and that has been shown to mediate neurite outgrowth from a number of neuronal cell types. Using both an established neuronal cell line, PC12 cells, and primary chick retina neuronal cultures, we find that agrin potentiates the ability of FGF‐2 to stimulate neurite outgrowth. In PC12 cells and retinal neurons agrin increases the efficacy of FGF‐2 stimulation of neurite outgrowth mediated by the FGF receptor, as an inhibitor of the FGF receptor abolished neurite outgrowth in the presence of agrin and FGF‐2. We also examined possible mechanisms by which agrin may modulate neurite outgrowth, analyzing ERK phosphorylation and c‐fos phosphorylation. These studies indicate that agrin augments a transient early phosphorylation of ERK in the presence of FGF‐2, and augments and sustains FGF‐2 mediated increases in c‐fos phosphorylation. These data are consistent with established mechanisms where heparan sulfate proteoglycans such as agrin may increase the affinity between FGF‐2 and the FGF receptor. In summary, our studies suggest that neural agrin contributes to the establishment of axon pathways by modulating the function of neurite promoting molecules such as FGF‐2. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 261–277, 2003     

Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG).

Sulfated proteoglycans (PGs) may play a significant role in the regulation of neurite outgrowth. They are present in axon-free regions of the developing nervous system and repel elongating neurites in a concentration-dependent manner in vitro. The addition of growth-promoting molecules, such as laminin, can modify the inhibitory effect of PGs on neurite outgrowth (Snow, Steindler, and Silver, 1990b). Substrata containing a high-PG/low-laminin ratio completely inhibit neurite outgrowth, while normal, unimpeded outgrowth is observed on low-PG/high-laminin substrata. Therefore, different patterns of neurite outgrowth may result from regulation of the ratio of growth-promoting molecules to growth-inhibiting molecules. Using video microscopy, embryonic chicken dorsal root ganglia neurons (DRG), chicken retinal ganglia neurons (RGC), and rat forebrain neurons (FB) were analyzed as they extended processes from a substratum consisting of laminin alone onto a step gradient of increasing concentrations of chondroitin sulfate proteoglycan (CS-PG) bound to laminin. In contrast to neurite outgrowth inhibition that occurs at the border of a single stripe of high concentration of CS-PG (Snow et al., 1990b and this study), growth cones grew onto and up CS-PG presented in a step-wise graded distribution. Although the behavior of the different cell types was unique, a common behavior of each cell type was a decrease in the rate of neurite outgrowth with increasing CS-PG concentration. These data suggest that appropriate concentrations of growth-promoting molecules combined with growth-inhibiting molecules may regulate the direction and possibly the timing of neurite outgrowth in vivo. The different responses of different neuronal types suggest that the presence of sulfated PG may have varying effects on different aspects of neuronal development.     

Localization of ganglioside 9‐O‐acetyl GD3 in point contacts of neuronal growth cones

Gangliosides are a large group of sialylated glycosphingolipids widely expressed in mammalian tissues. We have shown previously that the expression of 9‐O‐acetyl GD3 is highly correlated with periods of neurite outgrowth in the developing nervous system, and that the advance of dorsal root ganglia growth cones on laminin was halted in presence of an antibody specific for 9‐O‐acetyl GD3. In this work, we examined by immunocytochemistry and confocal microscopy whether this ganglioside is localized in point contacts in neuronal growth cones. We identified point contacts by immunoreactions with proteins, such as vinculin and β1 integrin, known to be associated with these structures in growth cones. Our observations indicate that 9‐O‐acetyl GD3 is specifically associated with vinculin and β1 integrin in point contacts of growth cones, suggesting a possible role for this particular ganglioside in the modulation of these contacts during neurite outgrowth. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 31–37, 2003     

A neuronal inhibitory domain in the N‐terminal half of agrin

Agrin is required for appropriate pre‐ and postsynaptic differentiation of neuromuscular junctions. While agrin's ability to orchestrate postsynaptic differentiation is well documented, more recent experiments have suggested that agrin is also a “stop signal” for the presynaptic neuron, and that agrin has actions on neurons in the CNS. To elucidate the neuronal activities of agrin and to define the receptor(s) responsible for these functions, we have examined adhesions of neurons and their neurite‐outgrowth responses to purified agrin in vitro. We find that both full‐length agrin and the C‐terminal 95 kDa of agrin (agrin c95), which is sufficient to induce postsynaptic differentiation, are adhesive for chick ciliary ganglion (CG) and forebrain neurons. Consistent with previous findings, our results show that N‐CAM binds to full‐length agrin, and suggest that α‐dystroglycan is a neuronal receptor for agrin c95. In neurite outgrowth assays, full‐length agrin inhibited both laminin‐ and N‐cadherin–induced neurite growth from CG neurons. The N‐terminal 150 kDa fragment of agrin, but not agrin c95, inhibited neurite outgrowth, indicating that domains in the N‐terminal portion of agrin are sufficient for this function. Adhesion assays using protein‐coated beads and agrin‐expressing cells revealed differential interactions of agrin with members of the immunoglobulin superfamily of cell adhesion molecules. However, none of these, including N‐CAM, appeared to be critical for neuronal adhesion. In summary, our results suggest that the N‐terminal half of agrin is involved in agrin's ability to inhibit neurite outgrowth. Our results further suggest that neither α‐dystroglycan nor N‐CAM, two known binding proteins for agrin, mediate this effect. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 164–179, 2002; DOI 10.1002/neu.10025     

Varicones and Growth Cones: Two Neurite Terminals in PC12 Cells

The rat adrenal pheochromocytoma PC12 cell line is one of the traditional models for the study of neurite outgrowth and growth cone behavior. To clarify to what extent PC12 neurite terminals can be compared to neuronal growth cones, we have analyzed their morphology and protein distribution in fixed PC12 cells by immunocytochemistry. Our results show that that PC12 cells display a special kind of neurite terminal that includes a varicosity in close association with a growth cone. This hybrid terminal, or “varicone”, is characterized by the expression of specific markers not typically present in neuronal growth cones. For example, we show that calpain-2 is a specific marker of varicones and can be detected even before the neurite develops. Our data also shows that a fraction of PC12 neurites end in regular growth cones, which we have compared to hippocampal neurites as a control. We also report the extraordinary incidence of varicones in the literature referred to as “growth cones”. In summary, we provide evidence of two different kinds of neurite terminals in PC12 cells, including a PC12-specific terminal, which implies that care must be taken when using them as a model for neuronal growth cones or neurite outgrowth.     

Strength in the periphery: growth cone biomechanics and substrate rigidity response in peripheral and central nervous system neurons

There is now considerable evidence of the importance of mechanical cues in neuronal development and regeneration. Motivated by the difference in the mechanical properties of the tissue environment between the peripheral (PNS) and central (CNS) nervous systems, we compare substrate-stiffness-dependent outgrowth and traction forces from PNS (dorsal root ganglion (DRG)) and CNS (hippocampal) neurons. We show that neurites from DRG neurons display maximal outgrowth on substrates with a Young's modulus of ～1000 Pa, whereas hippocampal neurite outgrowth is independent of substrate stiffness. Using traction force microscopy, we also find a substantial difference in growth cone traction force generation, with DRG growth cones exerting severalfold larger forces compared with hippocampal growth cones. The traction forces generated by DRG and hippocampal growth cones both increase with increasing stiffness, and DRG growth cones growing on substrates with a Young's modulus of 1000 Pa strengthen considerably after 18–30 h. Finally, we find that retrograde actin flow is almost three times faster in hippocampal growth cones than in DRG. Moreover, the density of paxillin puncta is significantly lower in hippocampal growth cones, suggesting that stronger substrate coupling of the DRG cytoskeleton is responsible for the remarkable difference in traction force generation. These findings reveal a differential adaptation of cytoskeletal dynamics to substrate stiffness in growth cones of different neuronal types, and highlight the potential importance of the mechanical properties of the cellular environment for neuronal navigation during embryonic development and nerve regeneration.     

Poster Sessions CP06: Cell Surface,Cell Membrane. Glucuronyl glycoconjugates replace sulfoglucuronyl glycoconjugates in HNK‐1 knockout mice

Sulfoglucuronyl carbohydrate (SGC), reactive with HNK‐1 antibody, is expressed in several glycolipids, glycoproteins and proteoglycans of the nervous system. The interaction of SGC with SGC‐binding protein, SBP‐1 has been implicated in cell‐cell recognition, neurite outgrowth and neuronal migration during development. In sulfotransferase (ST) null mutant mice, which lack SGC, synaptic transmission in pyramidal cells of the hippocampus was increased and long‐term potentiation was reduced. However, ST null mice are viable, fertile and have wild type anatomy of all major brain areas and many non‐neural organs. Failure to observe severe phenotype in the ST null mice prompted us to determine the compensatory molecular replacement of SGC by analyzing the carbohydrate of glycolipids and glycoprotefins of the mutant nervous system. In the ST null mice, SGC containing molecules were absent and they were replaced by the precursor glucuronyl carbohydrate (GC) containing molecules. Other relevant glycolipids and proteins were not affected. The GC molecules in the mutant were localized at the same anatomical sites as the SGC molecules in the wild type. In vitro binding studies showed that similar to sulfoglucuronyl glycolipids, glucuronyl glycolipids interacted with SBP‐1, but with a lower binding capacity. In vitro studies with explant cultures of cerebellum indicated that neurite outgrowth and cell migration were not significantly affected, possibly due to interaction of SBP‐1 with the GC molecules. The results indicated that in vivo SBP‐1–GC interaction was sufficient enough for normal neurite outgrowth and cell migration in the mutant and thus having a minimal abnormal phenotype.     

Modulators of neuronal migration and neurite growth.

A multitude of molecules have been identified over the past few years that promote neurite outgrowth in vitro. The concept that these molecules work mainly by providing an adhesive surface for neuronal growth cones has been challenged by evidence from recent experiments. Some of the substrate molecules have diverse actions on cell migration and neurite growth. In addition, there is now evidence that there are molecules that specifically inhibit growth cone locomotion. This has given rise to the hypothesis that growth cones integrate a variety of growth-promoting and inhibitory signals and translate them into directed locomotion.     

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

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

Activation of PI3K and R‐ras signaling promotes the extension of sensory axons on inhibitory chondroitin sulfate proteoglycans

Chondroitin sulfate proteoglycans (CSPGs) are extracellular inhibitors of axon extension and plasticity, and cause growth cones to exhibit dystrophic behaviors. Phosphoinositide 3‐kinase (PI3K) is a lipid kinase activated by axon growth promoting signals. In this study, we used embryonic chicken dorsal root ganglion neurons to determine if CSPGs impair signaling through PI3K. We report that CSPGs inhibit PI3K signaling in axons and growth cones, as evidenced by decreased levels of phosphorylated downstream kinases (Akt and S6). Direct activation of PI3K signaling, using a cell permeable phosphopeptide (PI3Kpep), countered the effects of CSPGs on growth cones and axon extension. Both overnight and acute treatment with PI3Kpep promoted axon extension on CSPG‐coated substrates. The R‐Ras GTPase is an upstream positive regulator of PI3K signaling. Expression of constitutively active R‐Ras promoted axon extension and growth cone elaboration on CSPGs and permissive substrata. In contrast, an N‐terminus‐deleted constitutively active R‐Ras, deficient in PI3K activation, promoted axon extension but not growth cone elaboration on CSPGs and permissive substrata. These data indicate that activation of R‐Ras‐PI3K signaling may be a viable approach for manipulating axon extension on CSPGs. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 918–933, 2014     

Grit,a GTPase-activating protein for the Rho family,regulates neurite extension through association with the TrkA receptor and N-Shc and CrkL/Crk adapter molecules

Neurotrophins are key regulators of the fate and shape of neuronal cells and act as guidance cues for growth cones by remodeling the actin cytoskeleton. Actin dynamics is controlled by Rho GTPases. We identified a novel Rho GTPase-activating protein (Grit) for Rho/Rac/Cdc42 small GTPases. Grit was abundant in neuronal cells and directly interacted with TrkA, a high-affinity receptor for nerve growth factor (NGF). Another pool of Grit was recruited to the activated receptor tyrosine kinase through its binding to N-Shc and CrkL/Crk, adapter molecules downstream of activated receptor tyrosine kinases. Overexpression of the TrkA-binding region of Grit inhibited NGF-induced neurite elongation. Further, we found some tendency for neurite promotion in full-length Grit-overexpressing PC12 cells upon NGF stimulation. These results suggest that Grit, a novel TrkA-interacting protein, regulates neurite outgrowth by modulating the Rho family of small GTPases.     

Two distinct filopodia populations at the growth cone allow to sense nanotopographical extracellular matrix cues to guide neurite outgrowth

Background

The process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth.

Methodology/Principal Findings

We show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth.

Conclusions/Significance

We propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.     

Munc18 and Munc13 regulate early neurite outgrowth

Background information. During development, growth cones of outgrowing neurons express proteins involved in vesicular secretion, such as SNARE (soluble N‐ethylmaleimide‐sensitive fusion protein‐attachment protein receptor) proteins, Munc13 and Munc18. Vesicles are known to fuse in growth cones prior to synapse formation, which may contribute to outgrowth. Results. We tested this possibility in dissociated cell cultures and organotypic slice cultures of two release‐deficient mice (Munc18‐1 null and Munc13‐1/2 double null). Both types of release‐deficient neurons have a decreased outgrowth speed and therefore have a smaller total neurite length during early development [DIV1–4 (day in vitro 1–4)]. In addition, more filopodia per growth cone were observed in Munc18‐1 null, but not WT (wild‐type) or Munc13‐1/2 double null neurons. The smaller total neurite length during early development was no longer observed after synaptogenesis (DIV14–23). Conclusion. These data suggest that the inability of vesicle fusion in the growth cone affects outgrowth during the initial phases when outgrowth speed is high, but not during/after synaptogenesis. Overall, the outgrowth speed is probably not rate‐limiting during neuronal network formation, at least in vitro. In addition, Munc18, but not Munc13, regulates growth cone filopodia, potentially via its previously observed effect on filamentous actin.     

Gene delivery to overcome astrocyte inhibition of axonal growth: An in vitro Model of the glial scar

After injury to the central nervous system, a glial scar develops that physically and biochemically inhibits axon growth. In the scar, activated astrocytes secrete inhibitory extracellular matrix, of which chondroitin sulfate proteoglycans (CSPGs) are considered the major inhibitory component. An inhibitory interface of CSPGs forms around the lesion and prevents axons from traversing the injury, and decreasing CSPGs can enhance axon growth. In this report, we established an in vitro interface model of activated astrocytes and subsequently investigated gene delivery as a means to reduce CSPG levels and enhance axon growth. In the model, a continuous interface of CSPG producing astrocytes was created with neurons seeded opposite the astrocytes, and neurite crossing, stopping, and turning were evaluated as they approached the interface. We investigated the efficacy of lentiviral delivery to degrade or prevent the synthesis of CSPGs, thereby removing CSPG inhibition of neurite growth. Lentiviral delivery of RNAi targeting two key CSPG synthesis enzymes, chondroitin polymerizing factor and chondroitin synthase‐1, decreased CSPGs, and reduced inhibition by the interface. Degradation of CSPGs by lentiviral delivery of chondroitinase also resulted in less inhibition and more neurites crossing the interface. These results indicate that the interface model provides a tool to investigate interventions that reduce inhibition by CSPGs, and that gene delivery can be effective in promoting neurite growth across an interface of CSPG producing astrocytes. Biotechnol. Bioeng. 2013; 110: 947–957. © 2012 Wiley Periodicals, Inc.     

Semaphorin 5A is a bifunctional axon guidance cue regulated by heparan and chondroitin sulfate proteoglycans

The response of neuronal growth cones to axon guidance cues depends on the developmental context in which these cues are encountered. We show here that the transmembrane protein semaphorin 5A (Sema5A) is a bifunctional guidance cue exerting both attractive and inhibitory effects on developing axons of the fasciculus retroflexus, a diencephalon fiber tract associated with limbic function. The thrombospondin repeats of Sema5A physically interact with the glycosaminoglycan portion of both chondroitin sulfate proteoglycans (CSPGs) and heparan sulfate proteoglycans (HSPGs). CSPGs function as precisely localized extrinsic cues that convert Sema5A from an attractive to an inhibitory guidance cue. Therefore, glycosaminoglycan bound guidance cues provide a molecular mechanism for CSPG-mediated inhibition of axonal extension. Further, axonal HSPGs are required for Sema5A-mediated attraction, suggesting that HSPGs are components of functional Sema5A receptors. Thus, neuronal responses to Sema5A are proteoglycan dependent and interpreted according to the biological context in which this membrane bound guidance cue is presented.     

Localization of ganglioside 9-O-acetyl GD3 in point contacts of neuronal growth cones

Gangliosides are a large group of sialylated glycosphingolipids widely expressed in mammalian tissues. We have shown previously that the expression of 9-O-acetyl GD3 is highly correlated with periods of neurite outgrowth in the developing nervous system, and that the advance of dorsal root ganglia growth cones on laminin was halted in presence of an antibody specific for 9-O-acetyl GD3. In this work, we examined by immunocytochemistry and confocal microscopy whether this ganglioside is localized in point contacts in neuronal growth cones. We identified point contacts by immunoreactions with proteins, such as vinculin and beta1 integrin, known to be associated with these structures in growth cones. Our observations indicate that 9-O-acetyl GD3 is specifically associated with vinculin and beta1 integrin in point contacts of growth cones, suggesting a possible role for this particular ganglioside in the modulation of these contacts during neurite outgrowth.     

Roles of 5-HT on phase transition of neurite outgrowth in the identified serotoninergic neuron C1, <Emphasis Type="Italic">Helisoma trivolvis</Emphasis>

Neurite outgrowth is a morphological marker of neuronal differentiation and neuroregeneration, and the process includes four essential phases, namely initiation, elongation, guidance and cessation. Intrinsic and extrinsic signaling molecules seem to involve morphological changes of neurite outgrowth via various cellular signaling cascades phase transition. Although mechanisms associated with neurite outgrowth have been studied extensively, little is known about how phase transition is regulated during neurite outgrowth. 5-HT has long been studied with regard to its relationship to neurite outgrowth in invertebrate and vertebrate culture systems, and many studies have suggested 5-HT inhibits neurite elongation and growth cone motility, in particular, at the growing parts of neurite such as growth cones and filopodia. However, the underlying mechanisms need to be investigated. In this study, we investigated roles of 5-HT on neurite outgrowth using single serotonergic neurons C1 isolated from Helisoma trivolvis. We observed that 5-HT delayed phase transitions from initiation to elongation of neurite outgrowth. This study for the first time demonstrated that 5-HT has a critical role in phase-controlling mechanisms of neurite outgrowth in neuronal cell cultures.