Pattern Of Neurite Outgrowth Research Articles

Hypoxia-Induced Neurite Outgrowth Involves Regulation Through TRPM7.

Transient receptor potential melastatin 7 (TRPM7) is a ubiquitously expressed divalent cation channel that plays a key role in cell functions such as ion homeostasis, cell proliferation, survival, and cytoskeletal dynamics and mediates cells death in hypoxic and ischemic conditions. Previously, TRPM7 was found to play a role in the neurite outgrowth and maturation of primary hippocampal neurons. Either knockdown of TRPM7 with target-specific shRNA or blocking channel conductance by a specific blocker waixenicin A enhanced axonal outgrowth in the primary neuronal culture. In this study, we investigated whether and how TPRM7 is involved in hypoxia-altered neurite outgrowth patterns in E16 hippocampal neuron cultures. We demonstrate that short-term hypoxia activated the MEK/ERK and PI3K/Akt pathways, reduced TRPM7 activity, and enhanced axonal outgrowth of neuronal cultures. On the other hand, long-term hypoxia caused a progressive retraction of axons and dendrites that could be attenuated by the TRPM7-specific inhibitor waixenicin A. Further, we demonstrate that in the presence of astrocytes, axonal retraction in long-term hypoxic conditions was enhanced, and TRPM7 block by waixenicin A prevented this retraction. Our data demonstrate the effect of hypoxia on TRPM7 activity and axonal outgrowth/retraction in cultures with or without astrocytes present.

Alterations in Dendritic Spine Maturation and Neurite Development Mediated by FAM19A1.

Neurogenesis and functional brain activity require complex associations of inherently programmed secretory elements that are regulated precisely and temporally. Family with sequence similarity 19 A1 (FAM19A1) is a secreted protein primarily expressed in subsets of terminally differentiated neuronal precursor cells and fully mature neurons in specific brain substructures. Several recent studies have demonstrated the importance of FAM19A1 in brain physiology; however, additional information is needed to support its role in neuronal maturation and function. In this study, dendritic spine morphology in Fam19a1-ablated mice and neurite development during in vitro neurogenesis were examined to understand the putative role of FAM19A1 in neural integrity. Adult Fam19a1-deficient mice showed low dendritic spine density and maturity with reduced dendrite complexity compared to wild-type (WT) littermates. To further explore the effect of FAM19A1 on neuronal maturation, the neurite outgrowth pattern in primary neurons was analyzed in vitro with and without FAM19A1. In response to FAM19A1, WT primary neurons showed reduced neurite complexity, whereas Fam19a1-decifient primary neurons exhibited increased neurite arborization, which was reversed by supplementation with recombinant FAM19A1. Together, these findings suggest that FAM19A1 participates in dendritic spine development and neurite arborization.

Co-culture methods to study neuronal function and disease.

Co-culture methods to study neuronal function and disease.

Human placental laminin: Role in neuronal differentiation, cell adhesion and proliferation

Human placental extract has wound healing potential. Immuno-blots revealed presence of laminin in placental extract (70 +/- 0.257 μg/ml; n=3). It was purified using immuno-affinity chromatography. SDS-PAGE and SEHPLC indicated a188 kDa protein with some small peptides. Since placental laminin existed in its truncated form, its roles in cellular migration, differentiation and wound healing were verified. Induction of cellular migration and motility in rat fibroblasts were enhanced by placental laminin as observed from scratch wound assay. Promotion of neuronal differentiation of PC12 cells by placental laminin was observed by phase contrast microscopy. Confocal images showed presence of laminin on the cell surface and along the axonal processes. Significant interaction between integrin receptors and laminin responsible for cellular differentiation was demonstrated from co-localization experiments. Union between integrin receptor and its synthetic antagonist revealed retarded pattern of neurite outgrowth in laminin treated cells. Animal model studies revealed faster wound healing in the presence of placental laminin. Induction of re-epithelialization and angiogenesis in wound area by cellular proliferation and adhesion were observed. The cytokine levels showed an initial rise and gradual fall over the duration of wound healing on application of the fragmented laminin. Thus, roles of placental laminin in neuronal differentiation and wound healing were indicated.

Investigation of neuronal pathfinding and construction of artificial neuronal networks on 3D-arranged porous fibrillar scaffolds with controlled geometry

Herein, we investigated the neurite pathfinding on electrospun microfibers with various fiber densities, diameters, and microbead islands, and demonstrated the development of 3D connected artificial neuronal network within a nanofiber-microbead-based porous scaffold. The primary culture of rat hippocampal embryonic neurons was deposited on geometry-controlled polystyrene (PS) fiber scaffolds while growth cone morphology, neurite outgrowth patterns, and focal adhesion protein expression were cautiously examined by microscopic imaging of immunostained and live neuronal cells derived from actin-GFP transgenic mice. It was demonstrated that the neurite outgrowth was guided by the overall microfiber orientation, but the increase in fiber density induced the neurite path alteration, thus, the reduction in neurite linearity. Indeed, we experimentally confirmed that growth cone could migrate to a neighboring, but, spatially disconnected microfiber by spontaneous filopodium extrusion, which is possibly responsible for the observed neurite steering. Furthermore, thinner microfiber scaffolds showed more pronounced expression of focal adhesion proteins than thicker ones, suggesting that the neuron-microfiber interaction can be delicately modulated by the underlying microfiber geometry. Finally, 3D connected functional neuronal networks were successfully constructed using PS nanofiber-microbead scaffolds where enhanced porosity and vertical fiber orientation permitted cell body inclusion within the scaffold and substantial neurite outgrowth in a vertical direction, respectively.

Nerve guidance conduits based on double-layered scaffolds of electrospun nanofibers for repairing the peripheral nervous system.

Compared to the nerve guidance conduits (NGCs) constructed from a single layer of aligned nanofibers, bilayer NGCs with random and aligned nanofibers in the outer and inner layers are more robust and tear-resistant during surgical procedures thanks to an isotropic mechanical property provided by the random nanofibers. However, it remains unclear whether the random nanofibers will interfere with the aligned nanofibers to alter the extension pattern of the neurites and impede regeneration. To answer this question, we seeded dorsal root ganglia (DRG) on a double-layered scaffold, with aligned and random nanofibers on the top and bottom layers, respectively, and evaluated the outgrowth of neurites. The random nanofibers in the bottom layer exerted a negative impact on the extension of neurites projecting from the DRG, giving neurites a less ordered structure compared to those cultured on a single layer of aligned nanofibers. The negative impact of the random nanofibers could be effectively mitigated by preseeding the double-layered scaffold with Schwann cells. DRG cultured on top of such a scaffold exhibited a neurite outgrowth pattern similar to that for DRG cultured on a single layer of aligned nanofibers. We further fabricated bilayer NGCs from the double-layered scaffolds and tested their ability to facilitate nerve regeneration in a rat sciatic nerve injury model. Both histomorphometric analysis and functional characterization demonstrated that bilayer NGCs with an inner surface that was preseeded with Schwann cells could reach 54%, 64.2%, and 74.9% of the performance of isografts in terms of nerve fiber number, maximum isometric tetanic force, and mass of the extensor digitorum longus muscle, respectively. It can be concluded that the bilayer NGCs hold great potential in facilitating motor axon regeneration and functional motor recovery.

Analyzing neurite outgrowth from explants by fitting ellipses

The establishment of appropriate connectivity in the developing nervous system depends on many molecular guidance cues. A key method for studying the response of nerve fibers to such guidance cues is to culture explants of neural tissue in three-dimensional collagen gels. However, most previous analyses of the neurite outgrowth patterns from these explants have been very simple, often measuring only one or two parameters. Here we introduce a more sophisticated method for characterizing neurite outgrowth from explants, based on fitting an ellipse to the pattern of outgrowth. This provides 5 parameters describing the outgrowth: x and y position of the center of the ellipse, the elongation, the area and the tilt. We then apply this method to a large dataset of dorsal root ganglion explants grown in the presence of precisely controlled gradients of nerve growth factor. This analysis reveals a number of new features of these data. For instance, we find that it is the position of the center of the ellipse rather than the shape of the ellipse that is correlated with the strength of the gradient. Together these results show that ellipse-fitting of explant data can give new insights into the biological processes underlying neurite guidance by molecular cues.

Neurite Outgrowth on Nanofiber Scaffolds with Different Orders, Structures, and Surface Properties

Electrospun nanofibers can be readily assembled into various types of scaffolds for applications in neural tissue engineering. The objective of this study is to examine and understand the unique patterns of neurite outgrowth from primary dorsal root ganglia (DRG) cultured on scaffolds of electrospun nanofibers having different orders, structures, and surface properties. We found that the neurites extended radially outward from the DRG main body without specific directionality when cultured on a nonwoven mat of randomly oriented nanofibers. In contrast, the neurites preferentially extended along the long axis of fiber when cultured on a parallel array of aligned nanofibers. When seeded at the border between regions of aligned and random nanofibers, the same DRG simultaneously expressed aligned and random neurite fields in response to the underlying nanofibers. When cultured on a double-layered scaffold where the nanofibers in each layer were aligned along a different direction, the neurites were found to be dependent on the fiber density in both layers. This biaxial pattern clearly demonstrates that neurite outgrowth can be influenced by nanofibers in different layers of a scaffold, rather than the topmost layer only. Taken together, these results will provide valuable information pertaining to the design of nanofiber scaffolds for neuroregenerative applications, as well as the effects of topology on neurite outgrowth, growth cone guidance, and axonal regeneration.

Analysis of gene expression during neurite outgrowth and regeneration

BackgroundThe ability of a neuron to regenerate functional connections after injury is influenced by both its intrinsic state and also by extrinsic cues in its surroundings. Investigations of the transcriptional changes undergone by neurons during in vivo models of injury and regeneration have revealed many transcripts associated with these processes. Because of the complex milieu of interactions in vivo, these results include not only expression changes directly related to regenerative outgrowth and but also unrelated responses to surrounding cells and signals. In vitro models of neurite outgrowth provide a means to study the intrinsic transcriptional patterns of neurite outgrowth in the absence of extensive extrinsic cues from nearby cells and tissues.ResultsWe have undertaken a genome-wide study of transcriptional activity in embryonic superior cervical ganglia (SCG) and dorsal root ganglia (DRG) during a time course of neurite outgrowth in vitro. Gene expression observed in these models likely includes both developmental gene expression patterns and regenerative responses to axotomy, which occurs as the result of tissue dissection. Comparison across both models revealed many genes with similar gene expression patterns during neurite outgrowth. These patterns were minimally affected by exposure to the potent inhibitory cue Semaphorin3A, indicating that this extrinsic cue does not exert major effects at the level of nuclear transcription. We also compared our data to several published studies of DRG and SCG gene expression in animal models of regeneration, and found the expression of a large number of genes in common between neurite outgrowth in vitro and regeneration in vivo.ConclusionMany gene expression changes undergone by SCG and DRG during in vitro outgrowth are shared between these two tissue types and in common with in vivo regeneration models. This suggests that the genes identified in this in vitro study may represent new candidates worthy of further study for potential roles in the therapeutic regrowth of neuronal connections.

Transmembrane agrin regulates filopodia in rat hippocampal neurons in culture

Filopodia mediate axon guidance, neurite branching and synapse formation, but the membrane molecules that regulate neuronal filopodia in response to extracellular cues are largely unknown. The transmembrane isoform of the proteoglycan agrin, expressed predominantly in the CNS, may regulate neurite outgrowth, synapse formation and excitatory signaling. Here we demonstrate that agrin positively regulates neuronal filopodia. Over-expression of TM-agrin caused the formation of excess filopodia on neurites of hippocampal neurons cultured 1–6 days. Conversely, suppression of agrin expression by siRNA reduced the number of filopodia. Time lapse analysis indicated that endogenous TM-agrin regulates filopodia by increasing their stability and initiation. The N-terminal half of agrin was necessary for induction of filopodia, and over-expression of TM-agrin in a neuronal cell line increased Cdc42 activation, suggesting a role for Cdc42 downstream of agrin. By positively regulating filopodia in developing neurons, TM-agrin may influence the pattern of neurite outgrowth and synapse formation.

A multiplexed proteomics approach to differentiate neurite outgrowth patterns

We report here a method for proteomics pattern discovery by utilizing a self-organizing map approach to analyze data obtained from a novel multiplex iTRAQ™ proteomics method. Through the application of this technique, we were able to delineate the early molecular events preceding dorsal root ganglia neurite outgrowth induced by either nerve growth factor (NGF) or an immunophilin ligand, JNJ460. Following pattern analysis we discovered that each neurotrophic agent promoted mostly distinct increases in protein expression with few overlapping patterns. In the NGF-treated group, proteins possessing “biosynthesis function” ( p < 0.002) and “ribosome localization” ( p < 0.0003) were increased, while proteins promoting “organogenesis” ( p < 0.004) and related “signal transduction” ( p < 0.008) functions were notably increased in the JNJ460-treated group. This study suggests that the properties of neurite outgrowth triggered by NGF and JNJ460 can be distinguished at the proteome level. Multiplexed proteomics analysis, along with pattern discovery bioinformatics tools, has the capability to differentiate subtle neuroproteomics patterns.

Regulation of filopodia by transmembrane agrin

Filopodial activity is implicated in control of growth and movement in many cell types. Neuronal filopodia are involved in control of axon guidance, neurite branching and in synapse formation. GTPase molecular switches and their effectors of actin assembly play a recognized role in filopodia formation but the upstream molecules that control filopodia formation and activity in response to extracellular cues are much less well characterized. Here we describe a novel function of the transmembrane isoform of the proteoglycan agrin (TM-agrin), which is expressed predominantly in the CNS. Roles for agrin have been suggested in the regulation of neurite outgrowth, excitatory signaling and synapse formation. We found that over-expression of TM-agrin caused the formation of excess filopodia on neurites of hippocampal neurons cultured for up to a week. Conversely, suppression of agrin expression by transfection with a siRNA plasmid reduced the number of filopodia. Time-lapse analysis suggested that endogenous TM-agrin regulates filopodia number by increasing the stability of filopodia and increasing initiation. Over-expression of TM-agrin induced the formation of filopodia in several cell lines and branched retraction fibers in most cell lines tested. Analysis of filopodia induction by deletion mutants of TM-agrin in COS-7 cells and hippocampal neurons indicated that motifs in the extracellular portion of the N-terminal half of TM-agrin are essential. Cdc42 and Rac activation were strongly increased in COS-7 cells transfected with TM-agrin, suggesting a role for these GTPases downstream of agrin. By positively regulating the number and stability of filopodia in developing neurons, TM-agrin may influence the pattern of neurite outgrowth and synapse formation. This work was funded by the NHLBI Intramural Research Program.

Alpha-synuclein up-regulates expression of caveolin-1 and down-regulates extracellular signal-regulated kinase activity in B103 neuroblastoma cells: role in the pathogenesis of Parkinson's disease.

alpha-Synuclein accumulation plays an important role in the pathogenesis of Lewy body disease (LBD) and Parkinson's disease (PD). Although the mechanisms are not yet clear, it is possible that dysregulation of the extracellular signal-regulated kinase (ERK) might play a role. As caveolins form scaffolds onto which signaling molecules such as ERK can assemble, we propose that signaling alterations associated with alpha-synuclein accumulation and neurodegeneration, might be mediated via caveolae. Therefore, the objective of the present study was to investigate the potential contribution of alterations in the caveolar system in mediating alpha-synuclein effects on the ERK signaling pathway. For this, synuclein-transfected B103 neuroblastoma cells were used as a model system. In this cell line, caveolin-1 expression was up-regulated, whereas, ERK was down-regulated. ERK was weakly but consistently co-immunoprecipitated with alpha-synuclein but caveolin-1 did not co-immunoprecipitate with alpha-synuclein. Moreover, treatment of alpha-synuclein- overexpressing cells with caveolin-1 antisense oligonucleotides resulted in stimulation of ERK activity, with amelioration of the neuritic alterations. Transduction of alpha-synuclein-overexpressing cells, with an adenoviral vector directing the expression of ERK, resulted in suppression of caveolin-1 expression and re-establishment of the normal patterns of neurite outgrowth. These results suggest that alpha-synuclein may also interfere with ERK signaling by dysregulating caveolin-1 expression. Thus, the caveolin-1/ERK pathway could be a therapeutic target for the alpha-synuclein-related neurodegenerative disorders.

Sensory Neuron Subtypes Have Unique Substratum Preference and Receptor Expression before Target Innervation

The factors controlling the specification and subsequent differentiation of sensory neurons are poorly understood. Data from embryological manipulations suggest that either sensory neuron fates are specified by the targets they encounter or sensory neurons are considerably more "plastic" with respect to specification than are neurons of the CNS. The prevailing view that sensory neurons are specified late in development is not consistent, however, with the directed outgrowth of sensory neurons to their targets and the characteristic spatial distribution of sensory neuron fates within the peripheral ganglia. To address when in development different classes of sensory neurons can first be distinguished, we investigated the interactions of early dorsal root ganglia neurons with the extracellular matrix before neurite outgrowth to targets. We found that subclasses of sensory neurons in early dorsal root ganglia show different patterns of neurite outgrowth and integrin expression that are predictive of their fates. In the absence of neurotrophins, presumptive proprioceptive neurons extend neurites robustly on both laminin and fibronectin, whereas presumptive cutaneous neurons show a strong preference for laminin. Cutaneous afferents that have innervated targets show a similar strong preference for laminin and show higher levels of integrin alpha7beta1 than do proprioceptive neurons. Finally, presumptive proprioceptive neurons express fibronectin receptors, integrin alpha3beta1, alpha4beta1, and alpha5beta1, at higher levels than do presumptive cutaneous neurons. Our results indicate that subtypes of sensory neurons have unique patterns of neurite outgrowth and receptor expression before target innervation.

Histomorphometric assessment of spiral ganglion neurite outgrowth in vitro

During the last years, various groups of growth factors have been identified to influence spiral ganglion cell survival and neurite extension in the mammalian cochlea. To evaluate and compare the effects of different growth factors, a precise histomorphometrical analysis of neurite outgrowth patterns has to be applied. The here presented technique is compared to already published methods, that only approximately estimate the neurite length. A software has been developed to analyse digitalised scans of spiral ganglion cells and to measure the length of the neurites. Therefore, the neurites are being separated in any given number of straight lines. The totals of these lines can then be added like a polygon. This polygon method was compared to a semi-quantitative procedure in which the neurite length was determined by concentric circles that were crossed by the neurites in a certain distance. The accuracy of both methods was analysed. Both methods were performed in 20 specimen of neonatal rat spiral ganglion cells after in vitro stimulation with neurotrophic factors. The semi-quantitative method has shown to involve a systematic error between +/- 10 to 15 %. The polygon method, on the contrary, has a systematic error of around +/- 1 %, which admits much more accurate measurement of spiral ganglion neurite outgrowth. With the described polygon method, spiral ganglion neurite growth patterns in cell culture studies can be characterised more precisely and, thus, helps to better differentiate the action domain of neurotrophic factors.

Dental Pulp Cells Produce Neurotrophic Factors, Interact with Trigeminal Neurons in Vitro, and Rescue Motoneurons after Spinal Cord Injury

Interactions between ingrowing nerve fibers and their target tissues form the basis for functional connectivity with the central nervous system. Studies of the developing dental pulp innervation by nerve fibers from the trigeminal ganglion is an excellent example of nerve–target tissue interactions and will allow specific questions regarding development of the dental pulp nerve system to be addressed. Dental pulp cells (DPC) produce an array of neurotrophic factors during development, suggesting that these proteins might be involved in supporting trigeminal nerve fibers that innervate the dental pulp. We have established an in vitro culture system to study the interactions between the dental pulp cells and trigeminal neurons. We show that dental pulp cells produce several neurotrophic factors in culture. When DPC are cocultured with trigeminal neurons, they promote survival and a specific and elaborate neurite outgrowth pattern from trigeminal neurons, whereas skin fibroblasts do not provide a similar support. In addition, we show that dental pulp tissue becomes innervated when transplanted ectopically into the anterior chamber of the eye in rats, and upregulates the catecholaminergic nerve fiber density of the irises. Interestingly, grafting the dental pulp tissue into hemisected spinal cord increases the number of surviving motoneurons, indicating a functional bioactivity of the dental pulp-derived neurotrophic factors in vivo by rescuing motoneurons. Based on these findings, we propose that dental pulp-derived neurotrophic factors play an important role in orchestrating the dental pulp innervation.

The astrocyte/meningeal cell interface--a barrier to successful nerve regeneration?

Following injuries to the adult mammalian CNS meningeal cells migrate into the lesion cavity, forming a fibrotic scar and accessory glia limitans. This infiltration re-establishes the meningeal layer that normally surrounds the CNS, and so reforms the barrier between the CNS and external environment, thus protecting the damaged region from events outside it. However, the newly formed meningeal layer and glia limitans may impede subsequent nerve regeneration through the injured region. This structure can be modelled in vitro using an astrocyte/meningeal co-culture system. We have examined patterns of neurite outgrowth on such cultures, and we find that axons cross readily from meningeal cells to astrocytes, but are unwilling to cross in the other direction. The distribution of cell surface and matrix molecules on these cultures is described, and the effect of various pharmacological interventions which can affect axon growth between the two cell types is summarised in this review.

Steps in the formation of neurites and synapses studied in cultured leech neurons

Leech neurons in culture have provided novel insights into the steps in the formation of neurite outgrowth patterns, target recognition and synapse formation. Identified adult neurons from the central nervous system of the leech can be removed individually and plated in culture under well-controlled conditions, where they retain their characteristic physiological properties, grow neurites and form specific chemical or electrical synapses. Different identified neurons develop distinctive outgrowth patterns that depend on their identities and on the molecular composition of the substrate. On native substrates, the patterns displayed by these neurons reproduce characteristics from the adult or the developing neurons. In addition, the substrate may induce selective directed growth between pairs of neurons that normally make contact in the ganglion. Upon contact, pairs of cultured leech neurons form chemical or electrical synapses, or both types depending on the neuronal identities. Anterograde and retrograde signals during membrane contact and synapse formation modify the distribution of synaptic terminals, calcium currents, and responses to 5-hydroxytryptamine.

Directed and enhanced neurite growth with pulsed magnetic field stimulation

Pulsed magnetic field (PMF) stimulation was applied to mammalian neurons in vitro to influence axonal growth and to determine whether induced current would direct and enhance neurite growth in the direction of the current. Two coils were constructed from individual sheets of copper folded into a square coil. Each coil was placed in a separate water-jacketed incubator. One was energized by a waveform generator driving a power amplifier, the other was not energized. Whole dorsal root ganglia (DRG) explant cultures from 15-day Sprague-Dawley rat embryos were established in supplemented media plus nerve growth factor (NGF) at concentrations of 0-100 ng/mL on a collagen-laminin substrate. Dishes were placed at the center of the top and bottom of both coils, so that the DRG were adjacent to the current flowing in the coil. After an initial 12 h allowing DRG attachment to the substrate floor, one coil was energized for 18 h, followed by a postexposure period of 18 h. Total incubation time was 48 h for all DRG cultures. At termination, DRG were histochemically stained for visualization and quantitative analysis of neurite outgrowth. Direction and length of neurite outgrowth were recorded with respect to direction of the current. PMF exposed DRG exhibited asymmetrical growth parallel to the current direction with concomitant enhancement of neurite length. DRG cultures not PMF exposed had a characteristic radial pattern of neurite outgrowth. These results suggest that PMF may offer a noninvasive mechanism to direct and promote nerve regeneration.

Target cell contact suppresses neurite outgrowth from soma-soma paired Lymnaea neurons.

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma-soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non-target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth-promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12-24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non-target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma-soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre- and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma-soma paired cells was transient, and neuronal sprouting began after a delay of 48-72 h. In contrast, when paired with VD1, both RPeD1 and this non-target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma-soma paired cells was target cell contact/synapse specific and Ca(2+) dependent. Specifically, soma-soma pairing in CM containing either lower external Ca(2+) concentration (50% of its control level) or Cd(2+) resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre- and postsynaptic neurons in a time-dependent and cell-specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca(2+) concentration.