Localized gene expression of axon guidance molecules in neuronal co-cultures.

Axonal growth cones are guided to their targets by contact-dependent mechanisms or by diffusible chemotropic factors. Axon guidance by these factors typically involves culturing neurons on an acellular substrate which may not represent the in vivo biological environment. We developed two novel in vitro methods to create patterned gene expression of guidance molecules in a physiologically-relevant cellular environment. In the Matrigel assay, a droplet of adenovirus-Matrigel suspension was placed on astrocytes grown in Matrigel. The adenovirus diffused through the gel and transduced underlying astrocytes, creating a radial infection gradient within a localized area. In the second model, recombinant adenovirus was bound to an anti-hexon antibody adsorbed onto stripe patterns of nitrocellulose. Once the cells were added, only those contacting the adenovirus were infected. The outgrowth pattern of chick DRG neurons on NGF, semaphorin 3A and brevican were studied. As expected, results showed robust axonal growth toward NGF as opposed to either secreted Sema 3A or membrane bound brevican, however subtle differences in axonal growth responses were observed in comparison to those obtained with less physiologically-relevant methods. Novel to this technology, the location and area of molecule expression can be controlled and manipulated in an intricate cellular environment.

Cocaine exposure in vitro induces apoptosis in fetal locus coeruleus neurons by altering the Bax/Bcl-2 ratio and through caspase-3 apoptotic signaling.

Cocaine inhibits survival and growth of rat locus coeruleus (LC) neurons, which may mediate alterations in attention, following in utero exposure to cocaine. These effects are most severe in early gestation during peak neuritogenesis. Prenatal cocaine exposure may specifically decrease LC survival through an apoptotic pathway involving caspases. Dissociated fetal LC neurons or substantia nigra (SN) neurons (control) were exposed in vitro to a pharmacologically active dose of cocaine hydrochloride (500 ng/ml) and assayed for apoptosis using terminal deoxynucleotidyl transferase mediated DNA nick end labeling and Hoechst methodologies. Cocaine exposure decreased survival and induced apoptosis in LC neurons, with no changes in survival of SN neurons. Activation of apoptotic signal transduction proteins was determined using enzyme assays and immunoblotting at 30 min, 1 h, 4 h and 24 h. In LC neurons, Bax levels were induced at 30 min and 1 h, following cocaine treatment, and Bcl-2 levels remained unchanged at all time points, altering the Bax/Bcl-2 ratio. The ratio was reversed for SN neurons (elevated Bcl-2 levels and transient reduction of Bax levels). Further, cocaine exposure significantly increased caspase-9 and caspase-3 activities at all time points, without changes in caspase-8 activity in LC neurons. In addition, cleavage of caspase-3 target proteins, alpha-fodrin and poly (ADP-ribose) polymerase (PARP) were observed following cocaine treatment. In contrast, SN neurons showed either significant reductions, or no significant changes, in caspase-3, -8 or -9 activities or caspase-3 target proteins, alpha-fodrin and PARP. Thus, cocaine exposure in vitro may preferentially induce apoptosis in fetal LC neurons putatively regulated by Bax, via activation of caspases and their downstream target proteins.

Specificity of prenatal cocaine on inhibition of locus coeruleus neurite outgrowth.

Prenatal cocaine exposure induces alterations in attentional function that presumably involve locus coeruleus noradrenergic neurons and their projections. Previous reports indicate that embryonic rat locus coeruleus neurons exposed to cocaine, both in vitro and in vivo, showed in decreased cell survival and inhibition of neurite outgrowth, and that the effects were most deleterious during early gestation. The present study performed in vitro addressed the specificity of the inhibitory effects of cocaine by comparing locus coeruleus neurite formation and extension to that of dopaminergic substantia nigra neurons following exposure to a physiologically-relevant dose of cocaine (500 ng/ml, two times a day, for four days) during peak neuritogenesis. Following cocaine treatment, immunocytochemistry (anti-norepinephrine antibody to locus coeruleus; anti-tyrosine hydroxylase antibody to substantia nigra) and image analysis were performed to measure a variety of neurite outgrowth parameters. For locus coeruleus neurons, cocaine treatment decreased the 1) number of cells initiating neurites [P<0.001], 2) mean number [P<0.05] and length of neurites [P<0.0001], 3) mean number [P<0.0016] and length of branched neurites [P<0.0006], and 4) mean length of the longest neurites [P<0.0001]. In comparison, substantia nigra neurons were not significantly affected by cocaine for any of the parameters examined. More importantly, a significant interaction between cocaine treatment and brain region was observed [P<0.0002] indicating greater vulnerability of locus coeruleus, relative to substantia nigra neurons, to cocaine exposure. These data support our hypothesis that cocaine targets the noradrenergic system by negatively regulating locus coeruleus neuronal outgrowth, which likely affects pathfinding, synaptic connectivity, and ultimately attentional behavior in cocaine-exposed offspring.

Lateral plantar nerve injury following steroid injection for plantar fasciitis.

A 41 year old man presented with pain and numbness affecting the lateral aspect of his foot after a steroid injection for plantar fasciitis. Examination confirmed numbness and motor impairment of the lateral plantar nerve. The findings were confirmed by electromyographic studies. The anatomy of the lateral plantar nerve and correct technique for injection to treat plantar fasciitis are discussed.

L- and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) inhibit neurite outgrowth from SH-SY5Y cells.

Gangliosides and extracellular matrix molecules influence neurite outgrowth, but the combinatorial effects of these endogenous agents on outgrowth are unclear. Exogenous gangliosides inhibit neurite outgrowth from SH-SY5Y cells stimulated with platelet-derived growth factor-BB, and different isoforms of the ceramide analog threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) stimulate (L-PDMP) or inhibit (D-PDMP) glycosphingolipid biosynthesis. In this study, we determined whether altering the endogenous ganglioside levels with PDMP in SH-SY5Y cells regulates neurite outgrowth on the outgrowth-supporting extracellular matrix molecule, laminin. In cells stimulated with 20 ng/ml platelet-derived growth factor-BB to promote outgrowth, we used image analysis to evaluate neurite outgrowth from SH-SY5Y cells grown on endogenous matrix or laminin and exposed to L- or D-PDMP. Both L- and D-PDMP decreased neurite initiation (the number of neurites/cell, the percent of neurite-bearing cells), elongation (the length of the longest neurite/cell, the total neurite length/cell), and branching (the number of branch points/neurite) from SH-SY5Y cells on endogenous matrix or laminin in a dose-dependent manner in serum-free or serum-containing medium. The inhibitory effects of each PDMP isoform were reversible. Inhibition of neurite outgrowth by L-PDMP could be mimicked by addition of exogenous gangliosides or C2-ceramide. Our analyses of neurite outgrowth in SH-SY5Y cells, a model of developing or regenerating noradrenergic neurons, demonstrate that increasing or decreasing endogenous ganglioside levels decreases neurite outgrowth. These results may indicate that SH-SY5Y cells undergo tight regulation by gangliosides, possibly through modulation of growth/trophic factor- and/or extracellular matrix-activated signaling cascades.

Fibronectin and laminin elicit differential behaviors from SH-SY5Y growth cones contacting inhibitory chondroitin sulfate proteoglycans.

Neuronal growth cones integrate signals from outgrowth-promoting molecules, e.g., laminin (LN) or fibronectin (FN), and outgrowth-inhibiting molecules, e.g., chondroitin sulfate proteoglycans (CSPGs), to navigate through extracellular matrix (ECM). Sensory neurons on LN typically turn to avoid areas rich in inhibitory CSPGs, whereas neuron-like cells of human origin (SH-SY5Y) preferentially stop/stall. These different behaviors may reflect differences in neuron type, response to outgrowth-promoters, or the mechanisms involved in outgrowth vs. inhibition. We used image analysis to determine the effects of different outgrowth promoters on the response of SH-SY5Y cells to inhibitory CSPGs. LN increased neurite initiation and elongation compared to cells plated either on endogenous matrix or FN. On a patterned substratum consisting of alternating stripes of FN and CSPGs, 59.6 +/- 9.3% of SH-SY5Y growth cones turned upon CSPG contact, whereas only 31.9 +/- 8.2% of growth cones turned at a LN/CSPG border. Growth cones on LN spread more upon contact with CSPG than growth cones on FN, whereas growth cones on LN or FN not contacting CSPGs were morphologically similar. Because it is known that integrins are involved in outgrowth on promoters, we analyzed integrin expression in response to inhibitory CSPGs in a choice assay. CSPGs did not induce increases or redistribution of several integrin subunits in SH-SY5Y cells. Furthermore, an anti-beta1 integrin function-blocking antibody did not alter growth cone behavior at a CSPG border. These results indicate that significant mechanistic differences may exist between outgrowth on homogenous outgrowth promoters and growth cone turning at inhibitory molecules.

Nervous system-derived chondroitin sulfate proteoglycans regulate growth cone morphology and inhibit neurite outgrowth: a light, epifluorescence, and electron microscopy study.

Proteoglycans influence aging and plasticity in the nervous system. Particularly prominent are the chondroitin sulfate proteoglycans (CSPGs), which are generally inhibitory to neurite outgrowth. During development, CSPGs facilitate normal guidance, but following nervous system injury and in diseases of aging (e.g., Alzheimer's disease), they block successful regeneration, and are associated with axon devoid regions and degenerating nerve cells. Whereas previous studies used non-nervous system sources of CSPGs, this study analyzed the morphology and behavior of sensory (dorsal root ganglia) neurons, and a human nerve cell model (SH-SY5Y neuroblastoma cells) as they contacted nervous system-derived CSPGs, using a variety of microscopy techniques. The results of these qualitative analyses show that growth cones of both nerve cell types contact CSPGs via actin-based filopodia, sample the CSPGs repeatedly without collapse, and alter their trajectory to avoid nervous system-derived CSPGs. Turning and branching are correlated with increased filopodial sampling, and are common to both neurons and Schwann cells. We show that CSPG expression by rat CNS astrocytes in culture is correlated with sensory neuron avoidance. Further, we show for the first time the ultrastructure of sensory growth cones at a CSPG-laminin border and reveal details of growth cone and neurite organization at this choice point. This type of detailed analysis of the response of growth cones to nervous system-derived CSPGs may lead to an understanding of CSPG function following injury and in diseases of aging, where CSPGs are likely to contribute to aberrant neurite outgrowth, failed or reduced synaptic connectivity, and/or ineffective plasticity.

Cocaine decreases cell survival and inhibits neurite extension of rat locus coeruleus neurons.

Cocaine use during pregnancy is affiliated with neurobehavioral abnormalities in offspring that are associated with problems of attention. Given the putative role of the noradrenergic system in attentional processes, impairments in the noradrenergic system may underlie specific attentionally sensitive, neurobehavioral alterations. Recent data using a clinically relevant intravenous (iv) route of administration show that the norepinephrine cell bodies of the locus coeruleus (LC) are a primary target for in utero cocaine exposure. Cell survival and neurite outgrowth of LC neurons were studied using two paradigms: (1) in vitro, using a physiologically relevant concentration of cocaine, and (2) in vivo, using a clinically relevant intravenous rat model. Fetal cocaine exposure significantly decreased neuronal survival (in vitro: P=.0001, n=24; in vivo: P=.0337, n=30), reduced neurite initiation (in vitro: P=.001, n=24; in vivo: P=.0169, n=30), decreased the number of neurites elaborated (in vivo: P=.0031, n=30), and reduced total neurite length (in vivo: P=.0237, n=30). The results of this novel approach toward an understanding of noradrenergic neurons as they respond to cocaine during development suggest that cocaine may affect behavior by negatively regulating neuronal pathfinding and synaptic connectivity.

Chondroitin sulfate proteoglycan expression pattern in hippocampal development: potential regulation of axon tract formation.

A variety of molecular influences in the extracellular matrix (ECM) interact with developing axons to guide the formation of hippocampal axon pathways. One of these influences may be chondroitin sulfate proteoglycans (CSPGs), which are known to inhibit axonal extension during development and following central nervous system injury. In this study, we examined the role of CSPGs and cell adhesion molecules in the regulation of axon tract formation during hippocampal development. We used indirect immunofluorescence to examine the developmental pattern of CSPG expression relative to axon tracts that express the cell adhesion molecule L1. Additionally, we used dissociated and explant cell cultures to examine the effects of CSPGs on hippocampal axon development in vitro. In vivo, we found that the CSPG neurocan is expressed throughout the alveus, neuropil layers, and parts of the dentate gyrus from E16 to P2. The CSPG phosphacan is expressed primarily in the neuropil layers at postnatal stages. After E18, intense labeling of neurocan was observed in regions of the alveus surrounding L1-expressing axon fascicles. In vitro, axons from brain regions that project through the alveus during development would not grow across CSPG substrata, in a concentration-dependent manner. In addition, hippocampal axons from dissociated neuron cultures only traveled across CSPG substrata as fasciculated axon bundles. These findings implicate CSPG in the regulation of axon trajectory and fasciculation during hippocampal axon tract formation.

Embryonic neurons adapt to the inhibitory proteoglycan aggrecan by increasing integrin expression.

The primary mediators of cell migration during development, wound healing and metastasis, are receptors of the integrin family. In the developing and regenerating nervous system, chondroitin sulfate proteoglycans (CSPGs) inhibit the integrin-dependent migration of neuronal growth cones. Here we report that embryonic sensory neurons cultured on the growth-promoting molecule laminin in combination with the inhibitory CSPG aggrecan rapidly adapt to inhibition. Adaptation is associated with a two- to threefold increase in the levels of RNA and surface protein for two laminin receptors, integrin alpha6beta1 and alpha3beta1, indicating that integrin expression is regulated by aggrecan. Increased integrin expression is associated both with increases in neuronal cell adhesion/outgrowth and with decreases in the ability of aggrecan to inhibit cell adhesion. Directly increasing integrin expression by adenoviral infection is sufficient to eliminate the inhibitory effects of aggrecan, indicating that upregulation of integrin receptors may promote neuronal regeneration in the presence of inhibitory matrix components.

MSE55, a Cdc42 effector protein, induces long cellular extensions in fibroblasts.

Cdc42 is a member of the Rho GTPase family that regulates multiple cellular activities, including actin polymerization, kinase-signaling activation, and cell polarization. MSE55 is a nonkinase CRIB (Cdc42/Rac interactive-binding) domain-containing molecule of unknown function. Using glutathione S-transferase-capture experiments, we show that MSE55 binds to Cdc42 in a GTP-dependent manner. MSE55 binding to Cdc42 required an intact CRIB domain, because a MSE55 CRIB domain mutant no longer interacted with Cdc42. To study the function of MSE55 we transfected either wild-type MSE55 or a MSE55 CRIB mutant into mammalian cells. In Cos-7 cells, wild-type MSE55 localized at membrane ruffles and increased membrane actin polymerization, whereas expression of the MSE55 CRIB mutant showed fewer membrane ruffles. In contrast to these results, MSE55 induced the formation of long, actin-based protrusions in NIH 3T3 cells as detected by immunofluorescence and live-cell video microscopy. MSE55-induced protrusion formation was blocked by expression of dominant-negative N17Cdc42, but not by expression of dominant-negative N17Rac. These findings indicate that MSE55 is a Cdc42 effector protein that mediates actin cytoskeleton reorganization at the plasma membrane.

Determination of beta1,4-galactosyltransferase enzymatic activity by capillary electrophoresis and laser-induced fluorescence detection.

We have developed a nonradioactive method to assay UDP-Gal:beta-d-GlcNAcbeta1,4-galactosyltransferase (beta4GalT-I) enzymatic activity. Capillary electrophoresis combined with laser-induced fluorescence detection (CE-LIF) was employed to provide a baseline separation of FITC-conjugated O-GlcNAc-containing substrate peptides and galactose-capped product peptides, while at the same time allowing a level of detection in the low attomole range (10(-18)). The addition of 2 mM hexamethylene diamine to the borate-based capillary electrophoretic buffer modulated the electroosmotic flow, resulting in optimum separation of the glycopeptide product from reactant. beta4GalT-I activity was dependent upon the addition of both manganese and UDP-galactose. Using this assay, we show that two beta4GalT-I constructs, predicted to localize to different intracellular compartments, are enzymatically active when expressed in vitro using a rabbit reticulocyte transcription-translation system. The high sensitivity of product detection by CE-LIF in combination with in vitro transcription-translation is applicable to the facile determination of the enzymatic activity of other newly cloned glycosyltransferases.

Neurite outgrowth inhibition by chondroitin sulfate proteoglycan: stalling/stopping exceeds turning in human neuroblastoma growth cones.

Chondroitin sulfate proteoglycan (CSPG) inhibits outgrowth from embryonic chick and rodent neurons in vivo and in vitro and is upregulated during development and following injury. The role of CSPG in outgrowth from human neurons has been largely untested, but is critical for our understanding of regeneration in humans following nervous system injury. Here we determined the effects of CSPG on platelet-derived growth factor (PDGF)-stimulated neurite outgrowth from SH-SY5Y human neuroblastoma cells, a well-accepted model of neuronal differentiation. Cells were plated on glass coverslips adsorbed with laminin (LN), CSPG, or a patterned substratum consisting of alternating stripes of the two molecules. Similar to other studies using chick or rodent neurons, SH-SY5Y cells extend neurites on LN, displaying a 15.2% increase in the total neurite length/cell as compared to cells plated on glass. Cells plated on CSPG alone exhibited reduced neurite outgrowth compared to cells plated on glass or LN. Interestingly, SH-SY5Y growth cones extending on LN and then encountering a CSPG border display more stopping/stalling (62.3%) than turning (27.9%) behaviors. Soluble CSPG inhibits neurite initiation from SH-SY5Y cells plated on glass, but not on LN. These data demonstrate that several CSPG-elicited responses of human neuron-like cells are similar to those from nonhuman neurons. However, approximately 70% of SH-SY5Y growth cones stop or stall at a CSPG border while over 80% of chick sensory neurons turn at a CSPG border. The experimental difference between these models may well indicate a functional difference between animal and human neuronal regeneration.

Nuclear and cytoplasmic glycosylation.

O-GlcNAcylation is a form of cytoplasmic and nuclear glycosylation that is found on many diverse proteins of the cell including RNA polymerase II and its associated transcription factors, cytoskeletal proteins, nucleoporins, viral proteins, heat shock proteins, tumor suppressors, and oncogenes. It involves the attachment of a single, unmodified N-acetylglucosaminyl residue O-glycosidically linked to the hydroxyl groups of serine and threonine moieties of proteins. It is a highly abundant and dynamic form of posttranslational modification that appears to modulate function in a manner similar to phosphorylation. All O-GlcNAc-containing proteins are phosphoproteins that are involved in the formation of multimeric complexes, suggesting that O-GlcNAc may play a role in mediating protein-protein interactions. O-GlcNAc sites resemble phosphorylation sites and in many cases the two modifications are mutually exclusive; therefore, O-GlcNAcylation may act as an antagonist of phosphorylation and help to mediate many essential functions of the cell.

Dynamic microtubule ends are required for growth cone turning to avoid an inhibitory guidance cue.

Growth cone turning is an important mechanism for changing the direction of neurite elongation during development of the nervous system. Our previous study indicated that actin filament bundles at the leading margin direct the distal microtubular cytoskeleton as growth cones turn to avoid substratum-bound chondroitin sulfate proteoglycan. Here, we investigated the role of microtubule dynamics in growth cone turning by using low doses of vinblastine and taxol, treatments that reduce dynamic growth and shrinkage of microtubule ends. We used time-lapse phase-contrast videomicroscopy to observe embryonic chick dorsal root ganglion neuronal growth cones as they encountered a border between fibronectin and chondroitin sulfate proteoglycan in the presence and absence of 4 nM vinblastine or 7 nM taxol. Growth cones were fixed and immunocytochemically labeled to identify actin filaments and microtubules containing tyrosinated and detyrosinated alpha-tubulin. Our results show that after contact with substratum-bound chondroitin sulfate proteoglycan, vinblastine- and taxol-treated growth cones did not turn, as did controls; instead, they stopped or sidestepped. Even before drug-treated growth cones contacted a chondroitin sulfate proteoglycan border, they were narrower than controls, and the distal tyrosinated microtubules were less splayed and were closer to the leading edges of the growth cones. We conclude that the splayed dynamic distal ends of microtubules play a key role in the actin filament-mediated steering of growth cone microtubules to produce growth cone turning.

Actin filament bundles are required for microtubule reorientation during growth cone turning to avoid an inhibitory guidance cue.

The extracellular matrix through which growth cones navigate contains molecules, such as chondroitin sulfate proteoglycan, that can inhibit growth cone advance and induce branching and turning. Growth cone turning is accompanied by rearrangement of the cytoskeleton. To identify changes in the organization of actin filaments and microtubules that occur as growth cones turn, we used time-lapse phase contrast videomicroscopy to observe embryonic chick dorsal root ganglion neuronal growth cones at a substratum border between fibronectin and chondroitin sulfate proteoglycan, in the presence and absence of cytochalasin B. Growth cones were fixed and immunocytochemically labeled to identify actin filaments and dynamic and stable microtubules. Our results suggest that microtubules are rearranged within growth cones to accomplish turning to avoid chondroitin sulfate proteoglycan. Compared to growth cones migrating on fibronectin, turning growth cones were more narrow, and they contained dynamic microtubules that were closer to the leading edge and were more bundled. Cytochalasin B-treated growth cones sidestepped laterally along the border instead of turning, and in sidestepping growth cones, microtubules were not bundled and aligned. We conclude that actin filament bundles are required for microtubule reorientation and growth cone turning to avoid chondroitin sulfate proteoglycan.

Growth cone behavior in the presence of soluble chondroitin sulfate proteoglycan (CSPG), compared to behavior on CSPG bound to laminin or fibronectin.

Proteoglycans (PGs) are complex macromolecules of the extracellular matrix (ECM) that have a wide variety of effects on developing and regenerating neurons in vivo and in vitro. One hypothesis regarding the mechanisms of PG regulation of neuronal behavior states that the conformation of PGs may be critical, and thus that ECM- or cell surface-bound PGs may operate differently than secreted (soluble) PGs. Therefore, this study examined differences between the effects of soluble chondroitin sulfate proteoglycan (CSPG) and substratum-bound CSPG on neuronal growth cone behavior. Dissociated chicken dorsal root ganglion (DRG) neurons were cultured on either laminin (LN) or fibronectin (FN), both sensory neurite outgrowth-promotin glycoproteins. CSPG (or chondroitin sulfate alone) was either bound to FN or LN, or was added to the culture media. Subsequently, using time lapse video microscopy and image analysis, this study measured: (1) neuronal attachment, (2) neurite outgrowth, (3) rate of neurite elongation, and (4) filopodial length and lifespan. To determine the site of CSPG action, DRG neurons were grown on either: CS-1, a FN peptide [Humphries M. J. et al. (1987) J. biol. Chem. 262, 6886-6892], or a recombinant FN protein, RFNIIIcs (Maejne, submitted), both of which permit DRG attachment and outgrowth but do not have recognized CSPG binding sites, and the resulting neuronal behavior was compared to that of DRG neurons grown on intact FN. The results of these studies confirm that the effect of CSPG on DRG neurons is concentration-, conformation- and substratum-dependent. On I.N, soluble CSPG had little to no effect on neurite initiation or outgrowth, while substratum-bound CSPG inhibited neurite outgrowth. In contrast, on FN, soluble CSPG inhibited neurite outgrowth and decreased the rate of neurite elongation. Soluble CSPG did not affect the length of sensory growth cone filopodia or filopodial lifespan on either LN or FN. From the FN fragment experiments, we found that: (1) soluble CSPG reduces neurite outgrowth on FN or FN fragments, but not on LN, up to 80%, and reduces elongation rate on FN up to 50%, and (2) soluble CSPG regulates neuronal behavior by binding directly to growth cones elongating on FN. Given that substratum-bound CSPG from a variety of sources is inhibitory to neurite outgrowth and to the rate of neurite elongation, while soluble CSPG often has different effects on growth cone behavior, the regulation of growth cone behavior by CSPGs may be dependent upon CSPG conformation. Further, CSPG may affect growth cone behavior by either binding to the substratum or by binding directly to growth cones.

Characterization of spontaneous calcium transients in nerve growth cones and their effect on growth cone migration.

This study examines the mechanisms of spontaneous and induced [Ca2+]i spiking in nerve growth cones and the effect of spikes on growth cone migration. Over a 10-20 min observation period, 29% of DRG growth cones undergo spontaneous and transient elevations in physiological extracellular Ca2+ ((Ca2+)o; 2 mM), whereas 67% of growth cones exposed to 20 mM (Ca2+)o exhibit similar [Ca2+]i spikes. Spontaneous [Ca2+]i spiking was not observed in neuronal cell bodies or nonneuronal cells. Ca2+ influx through non-voltage-gated Ca2+ channels was required for spontaneous [Ca2+]i spikes in growth cones, since removal of (Ca2+)o, or addition of the general Ca2+ channel blockers La3+ or Ni2+, reversibly blocked [Ca2+]i spiking, while blockers of the voltage-gated Ca2+ channels did not. Experiments using agents that influence intracellular Ca2+ stores suggest that Ca2+ stores may buffer and release Ca2+ during growth cone [Ca2+]i spikes. Growth cone migration was immediately and transiently inhibited by [Ca2+]i spikes, but eventually returned to prespike rates.