Growth Cone Calcium Research Articles

Computational modeling of neurons: intensity-duration relationship of extracellular electrical stimulation for changes in intracellular calcium.

In many instances of extensive nerve damage, the injured nerve never adequately heals, leaving lack of nerve function. Electrical stimulation (ES) has been shown to increase the rate and orient the direction of neurite growth, and is a promising therapy. However, the mechanism in which ES affects neuronal growth is not understood, making it difficult to compare existing ES protocols or to design and optimize new protocols. We hypothesize that ES acts by elevating intracellular calcium concentration ([Ca(2+)]i) via opening voltage-dependent Ca(2+) channels (VDCCs). In this work, we have created a computer model to estimate the ES Ca(2+) relationship. Using COMSOL Multiphysics, we modeled a small dorsal root ganglion (DRG) neuron that includes one Na(+) channel, two K(+) channels, and three VDCCs to estimate [Ca(2+)]i in the soma and growth cone. As expected, the results show that an ES that generates action potentials (APs) can efficiently raise the [Ca(2+)]i of neurons. More interestingly, our simulation results show that sub-AP ES can efficiently raise neuronal [Ca(2+)]i and that specific high-voltage ES can preferentially raise [Ca(2+)]i in the growth cone. The intensities and durations of ES on modeled growth cone calcium rise are consistent with directionality and orientation of growth cones experimentally shown by others. Finally, this model provides a basis to design experimental ES pulse parameters, including duration, intensity, pulse-train frequency, and pulse-train duration to efficiently raise [Ca(2+)]i in neuronal somas or growth cones.

Cell-autonomous axon growth of young motoneurons is triggered by a voltage-gated sodium channel

Spontaneous electrical activity preceding synapse formation contributes to the precise regulation of neuronal development. Examining the origins of spontaneous activity revealed roles for neurotransmitters that depolarize neurons and activate ion channels. Recently, we identified a new molecular mechanism underlying fluctuations in spontaneous neuronal excitability. We found that embryonic motoneurons with a genetic loss of the low-threshold sodium channel NaV1.9 show fewer fluctuations in intracellular calcium in axonal compartments and growth cones than wild-type littermates. As a consequence, axon growth of NaV1.9-deficient motoneurons in cell culture is drastically reduced while dendritic growth and cell survival are not affected. Interestingly, NaV1.9 function is observed under conditions that would hardly allow a ligand- or neurotransmitter-dependent depolarization. Thus, NaV1.9 may serve as a cell-autonomous trigger for neuronal excitation. In this addendum, we discuss a model for the interplay between cell-autonomous local neuronal activity and local cytoskeleton dynamics in growth cone function.

NCS‐1 differentially regulates growth cone and somata calcium channels in Lymnaea neurons

Local voltage-gated calcium channels, which regulate intracellular Ca2+ levels by allowing Ca2+ influx, play an important role in guiding and shaping growth cones, and in regulating the outgrowth and branching of neurites. Therefore, elucidating the mechanisms that regulate the biophysical properties of whole-cell calcium currents in the growth cones and somata of growing neurons is important to improving our understanding of neuronal development and regeneration. In this study, taking advantage of the large size of the pedal A (PeA) neurons in Lymnaea stagnalis, we compared the biophysical properties of somata and growth cone whole-cell calcium channel currents using Ba2+ and Ca2+ as current carriers. We found that somata and growth cone currents exhibit similar high-voltage activation properties. However, Ba2+ and Ca2+ currents in growth cones and somata are differentially affected by a dominant-negative peptide containing the C-terminal amino acid sequence of neuronal calcium sensor-1 (NCS-1). The peptide selectively reduces the peak and sustained components of current densities and the slope conductance in growth cones, and shifts the reversal potential of the growth cone currents to more hyperpolarized voltages. In contrast, the peptide had no significant effect on the somata calcium channels. Thus, we conclude that NCS-1 differentially modulates Ca2+ currents in the somata and growth cones of regenerating neurons, and may serve as a key regulator to facilitate the growth cone calcium channel activity.

Converting Attraction to Repulsion

During nervous system development, axonal growth cones respond to attractive and repulsive cues that help them navigate to their targets. Wen et al . discovered that, whereas embryonic Xenopus spinal axons in an in vitro culture system were attracted to a gradient of bone morphogenetic protein 7 (BMP7) presented 4 to 8 hours after being placed in culture, BMP7 repelled the same axons after they had been cultured overnight. The type II BMP receptor (BMPRII) mediated both attractive and repulsive responses, and overexpression of a shortened form of BMPRII [which lacks a region that mediates interactions with LIM kinase I (LIMKI) but is dispensable for activation of Smad signaling] abolished BMP7-induced turning. A 32-amino-acid peptide that competitively inhibits LIMKI abolished attractive turning to BMP7, as did a dominant-negative LIMKI mutant, whereas neither affected repulsive turning. In neurons cultured overnight that overexpressed a dominant-negative mutant of the phosphatase Slingshot (SSH), however, the response to BMP7 was converted from repulsion to attraction. A combination of live imaging of the actin cytoskeleton, immunofluorescence analysis of the phosphorylation of XAC [ Xenopus actin-depolymerizing factor (ADF)/cofilin], and expression of wild-type and mutant forms of XAC revealed that LIMKI and SSH regulated growth cone turning through phosphorylation of XAC. BMP7 elicited an increase in growth cone calcium concentration in neurons cultured overnight but not in those cultured for 6 hours. Manipulations designed to block calcium signaling failed to affect attractive turning in 4- to 8-hour cultures, whereas in overnight cultures these treatments abolished the BMP-dependent decrease in XAC phosphorylation and converted repulsive to attractive turning, as did pharmacological inhibition of calcineurin. The transient receptor potential channel TRPC1 was more abundant in overnight cultures than in 4- to 8-hour cultures, and expression of a dominant-negative TRPC1 mutant abolished BMP7-dependent repulsion in overnight cultures, whereas overexpression of wild-type TRPC1 elicited BMP7-dependent repulsion in 4- to 8-hour cultures. Thus, opposing actions of LIMKI and SSH on XAC phosphorylation appear to regulate the actin cytoskeleton and thereby growth cone turning, with calcium influx through TRPC1 switching attraction to repulsion by shifting the balance toward SSH. Z. Wen, L. Han, J. R. Bamburg, S. Shim, G.-l. Ming, J. Q. Zheng, BMP gradients steer nerve growth cones by a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofilin. J. Cell Biol. 178 , 107-119 (2007). [Abstract] [Full Text]

Sensing the Right Path

During nervous system development, axonal growth cones find their way to their targets with the help of various chemoattractant and chemorepellant cues. Several neurite guidance signals modulate calcium influx into the growth cone, but the underlying mechanisms and the specific channels involved have been unclear (see Gomez). Now two groups, Li et al. and Wang and Poo, provide evidence implicating transient receptor potential canonical (TRPC) channels--a family of channels known for mediating sensory responses--in the response to the chemoattractants brain-derived neurotrophic factor (BDNF) and netrin-1. Wang and Poo performed perforated whole-cell patch clamp analysis of cultured Xenopus spinal neurons and found that netrin-1 elicited membrane depolarization that was inhibited by an antibody to its receptor, DDC. Netrin-induced current present during pharmacological blockade of most voltage-dependent ion channels was inhibited by SKF-96365, which blocks TRP channels, as was a similar current produced by BDNF. Further, growth cone turning in response to netrin-1 or BDNF was abolished by SKF-96365. Injection of morpholino oligonucleotides (MOs) that knocked down xTRPC1 expression inhibited the putative TRP currents and abolished growth cone turning in response to a netrin-1 gradient. Moreover, MO or SKF-96365 abolished the netrin-induced increase in growth cone calcium concentration. Li et al. investigated TRPC channels in BDNF-mediated guidance of cultured cerebellar granule cells, which was blocked by antibodies to the TrkB receptor, incubation with a membrane-permeable calcium chelator, or pharmacological depletion of intracellular calcium stores. Although pharmacological blockade of several voltage-sensitive sodium and calcium channels had no effect on growth cone turning elicited by BDNF, turning was abolished by SKF-96365, as well as by pharmacological inhibition of phospholipase C (PLC), phosphatidylinositol 3-kinase (PI3K), or blockade of the inositol 1,4,5-trisphosphate receptor (IP 3 R). In contrast, a PLC activator elicited turning, an effect that depended on extracellular calcium and was abolished by SKF-96365. BDNF increased growth cone calcium concentration, an effect that depended on both calcium influx and release from internal stores and that was reduced by SKF-96365, pharmacological inhibition of PLC, or IP 3 R blockade. BDNF-induced turning was abolished by siRNA directed against TRPC3 or by overexpression of dominant-negative mutant forms of TRPC3 or TRPC6. Thus, activation of TRPC channels appears to play a key role in calcium-dependent neurite pathfinding in response to both netrin-1 and BDNF. Y. Li, Y.-C. Jia, K. Cui, N. Li, Z.-Y. Zheng, Y.-Z. Wang, X.-B. Yuan, Essential role of TRPC channels in the guidance of nerve growth cones by brain-derived neurotrophic factor. Nature 434 , 894-898 (2005). [PubMed] G. X. Wang, M.-M. Poo, Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones. Nature 434 , 898-904 (2005). [PubMed] T. Gomez, Channels for pathfinding. Nature 434 , 835-838 (2005). [PubMed]

Coordinated Motor Neuron Axon Growth and Neuromuscular Synaptogenesis Are Promoted by CPG15 In Vivo

We have used in vivo time-lapse two-photon imaging of single motor neuron axons labeled with GFP combined with labeling of presynaptic vesicle clusters and postsynaptic acetylcholine receptors in Xenopus laevis tadpoles to determine the dynamic rearrangement of individual axon branches and synaptogenesis during motor axon arbor development. Control GFP-labeled axons are highly dynamic during the period when axon arbors are elaborating. Axon branches emerge from sites of synaptic vesicle clusters. These data indicate that motor neuron axon elaboration and synaptogenesis are concurrent and iterative. We tested the role of Candidate Plasticity Gene 15 (CPG15, also known as Neuritin), an activity-regulated gene that is expressed in the developing motor neurons in this process. CPG15 expression enhances the development of motor neuron axon arbors by promoting neuromuscular synaptogenesis and by increasing the addition of new axon branches.

Activity-dependent neuronal differentiation prior to synapse formation: the functions of calcium transients

Spinal cord neurons become excitable prior to synapse formation, and generate spontaneous calcium transients that regulate aspects of their differentiation before neuronal networks are established. Calcium spikes, generated by calcium-dependent action potentials and calcium-induced calcium release (CICR), regulate transcription. Growth cone calcium transients, produced by calcium influx through unidentified channels that triggers CICR, control the rate of axon outgrowth in response to environmental cues. Filopodial calcium transients, generated by calcium influx through channels activated by β1 integrins, signal information about the molecular identity of the substrate and regulate growth cone turning. All three classes of calcium transients appear to use a frequency code to implement their effects. Oscillations of second messengers in embryonic neurons and perhaps more generally in other differentiating cells may behave like a kinetic quilt, demonstrating patchy fluctuations in concentrations that orchestrate the complex processes of development.

Regulation of Calcineurin by Growth Cone Calcium Waves Controls Neurite Extension

Growth cones generate spontaneous transient elevations of intracellular Ca(2+) that regulate the rate of neurite outgrowth. Here we report that these Ca(2+) waves inhibit neurite extension via the Ca(2+)-dependent phosphatase calcineurin (CN) in Xenopus spinal neurons. Pharmacological blockers of CN (cyclosporin A and deltamethrin) and peptide inhibitors of CN [the Xenopus CN (xCN) autoinhibitory domain and African swine fever virus protein A238L] block the Ca(2+)-dependent reduction of neurite outgrowth in cultured neurons. Time-lapse microscopy of growing neurites demonstrates directly that the reduction in the rate of outgrowth by Ca(2+) transients is blocked by cyclosporin A. In contrast, expression of a constitutively active form of xCN in the absence of waves results in shorter neurite lengths similar to those seen in the presence of waves. The developmental expression pattern of xCN transcripts in vivo coincides temporally with axonal pathfinding by spinal neurons, supporting a role of CN in regulating Ca(2+)-dependent neurite extension in the spinal cord. Ca(2+) wave frequency and Ca(2+)-dependent expression of GABA are not affected by inhibition or activation of CN. However, phosphorylation of the cytoskeletal element GAP-43, which promotes actin polymerization, is reduced by Ca(2+) waves and enhanced by suppression of CN activity. CN ultimately acts on the growth cone actin cytoskeleton, because disrupting actin microfilaments with cytochalasin D or stabilizing them with jasplakinolide negates the effects of suppressing or activating CN. Destabilization or stabilization of microtubules with colcemide or taxol results in Ca(2+)-independent inhibition of neurite outgrowth. The results identify components of the cascade by which Ca(2+) waves act to regulate neurite extension.

Temporal and spatial pattern of expression of cyclic nucleotide-gated channels in developing rat visual cortex.

Cyclic nucleotide-gated channels, ligand-gated and highly permeable to calcium, are good candidates for transducing signals received by migrating cells, growth cones and developing synapses. The level of calcium in growth cones is important for axon guidance. Further, cyclic nucleotides, whose levels can be altered by nitric oxide and other transmitters, are known to alter growth cone motility. We use rat visual cortex as a model in our semi-quantitative RT-PCR and in situ hybridization studies to determine the developmental time course and localization of all three CNG family members (rod, olfactory and cone/testis). We demonstrate that in the cortex, the three channel subtypes are each expressed in a distinct temporal and spatial pattern in only sensorimotor and occipital regions of the cortex. Specifically, the rod and olfactory subtypes are present at the time of migration and rapid dendritic outgrowth, and the cone/testis subtype is highly expressed after eye opening. These results suggest CNG channels may play a role in both early and late events in visual cortical development.

Regulation of neuronal growth cone filopodia by nitric oxide.

Nitric oxide (NO) has been proposed to play an important role during neuronal development. Since many of its effects occur during the time of growth cone pathfinding and target interaction, we here test the hypothesis that part of NO's effects might be exerted at the growth cone. We found that low concentrations of the NO-donors DEA/NO, SIN-1, and SNP caused a rapid and transient elongation of filopodia as well as a reduction in filopodial number. These effects resulted from distinct changes in filopodial extension and retraction rates. Our novel findings suggest that NO could play a physiological role by temporarily changing a growth cone's morphology and switching its behavior from a close-range to a long-range exploratory mode. We subsequently dissected the pathway by which NO acted on growth cones. The effect of NO donors on filopodial length could be blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase (sGC), indicating that NO acted via sGC. Supporting this idea, injection of cyclic GMP (cGMP) mimicked the effect of NO donors on growth cone filopodia. Moreover, application of NO-donors as well as injection of cGMP elicited a rapid and transient rise in intracellular calcium in growth cones, indicating that NO acted via cGMP to elevate calcium. This calcium rise, as well as the morphological effects of SIN-1 on filopodia, were blocked by preventing calcium entry. Given the role of filopodia in axonal guidance, our new data suggest that NO could function at the neuronal growth cone as an intracellular and/or intercellular signaling molecule by affecting steering decisions during neuronal pathfinding.

Autonomous regulation of growth cone filopodia.

The fan-shaped array of filopodia is the first site of contact of a neuronal growth cone with molecules encountered during neuronal pathfinding. Filopodia are highly dynamic structures, and the "action radius" of a growth cone is strongly determined by the length and number of its filopodia. Since interactions of filopodia with instructive cues in the vicinity of the growth cone can have effects on growth cone morphology within minutes, it has to be assumed that a large part of the signaling underlying such morphological changes resides locally within the growth cone proper. In this study, we tested the hypothesis that two important growth cone parameters-namely, the length and number of its filopodia-are regulated autonomously in the growth cone. We previously demonstrated in identified neurons from the snail Helisoma trivolvis that filopodial length and number are regulated by intracellular calcium. Here, we investigated filopodial dynamics and their regulation by the second-messenger calcium in growth cones which were physically isolated from their parent neuron by neurite transection. Our results show that isolated growth cones have longer but fewer filopodia than growth cones attached to their parent cell. These isolated growth cones, however, are fully capable of undergoing calcium-induced cytoskeletal changes, suggesting that the machinery necessary to perform changes in filopodial length and number is fully intrinsic to the growth cone proper.

Growth cone calcium elevation by GABA

Cytoplasmic calcium plays a key role in neurite growth. In contrast to previous work suggesting that gamma aminobutyrate's (GABA) role in regulating growth cone calcium is primarily to antagonize the effects of glutamate, we report that GABA can act in an excitatory manner on developing hypothalamic neurites, independently raising calcium in growing neurites and their growth cones. Time-lapse digital video and confocal laser microscopy with the calcium-sensitive dyes fluo-3 and fura-2 were used to study the influence of GABA on neurite calcium levels. GABA (10 μM) evoked a calcium rise in both bicarbonate- and Hepes-based buffers. The calcium rise was greatly reduced after chloride transport was blocked. GABA raised calcium by stimulating the cell body, resulting in an increase in calcium throughout the neuronal cell body and dendritic arbor. GABA also acted locally, stimulating a neuritic calcium rise only in a single dendrite or growth cone. In some neurites and growth cones during early development, GABA generated a greater calcium rise than did glutamate. Bicuculline, a GABAA receptor antagonist, reduced calcium levels in neurites of young synaptically coupled neurons, indicating that ongoing synaptic release of GABA raised neuritic calcium. These data suggest that during early development, GABA may play a significant role in regulating process growth and modulating the formation of early connections in the hypothalamus. Our data support the hypothesis that GABA receptors are functionally active and may play a calcium regulating role similar to that of glutamate in neuronal development. This is particularly true in early development, as later in development GABA's role becomes more inhibitory, and glutamate plays the primary excitatory role. © 1996 Wiley-Liss, Inc.

Growth cone calcium elevation by GABA.

Cytoplasmic calcium plays a key role in neurite growth. In contrast to previous work suggesting that gamma aminobutyrate's (GABA) role in regulating growth cone calcium is primarily to antagonize the effects of glutamate, we report that GABA can act in an excitatory manner on developing hypothalamic neurites, independently raising calcium in growing neurites and their growth cones. Time-lapse digital video and confocal laser microscopy with the calcium-sensitive dyes fluo-3 and fura-2 were used to study the influence of GABA on neurite calcium levels. GABA (10 microM) evoked a calcium rise in both bicarbonate- and Hepes-based buffers. The calcium rise was greatly reduced after chloride transport was blocked. GABA raised calcium by stimulating the cell body, resulting in an increase in calcium throughout the neuronal cell body and dendritic arbor. GABA also acted locally, stimulating a neuritic calcium rise only in a single dendrite or growth cone. In some neurites and growth cones during early development, GABA generated a greater calcium rise than did glutamate. Bicuculline, a GABAA receptor antagonist, reduced calcium levels in neurites of young synaptically coupled neurons, indicating that ongoing synaptic release of GABA raised neuritic calcium. These data suggest that during early development, GABA may play a significant role in regulating process growth and modulating the formation of early connections in the hypothalamus. Our data support the hypothesis that GABA receptors are functionally active and may play a calcium regulating role similar to that of glutamate in neuronal development. This is particularly true in early development, as later in development GABA's role becomes more inhibitory, and glutamate plays the primary excitatory role.

Calcium channels in neuroblastoma cell growth cones.

The concentration of free calcium ions in the cytosol has been shown to influence many components of growth cone behaviour, including the extension of filopodia and veils, the addition of new membrane to the plasmalemma, the retraction and disappearance of filopodia, and gross collapse and retraction of the growth cone. A spatially localized modulation of these processes by very local calcium changes has been proposed to underlie the steering of growth cones by gradients of neurotransmitters, voltage and cell adhesion molecules. Such local control can be studied in mouse neuroblastoma cells, where depolarization causes calcium to rise in a limited number of spatially restricted hotspots, triggering a localized advance. We have studied the simple, club-shaped growth cones that are characteristically found on advancing neurites. Depolarization caused calcium to increase most at the distal, leading tip. Agents that disrupt calcium-induced calcium release do not affect growth cone calcium dynamics, ruling out a local release of calcium at the tip as a cause of the gradient. Using cell-attached patch recording, we find that L-type calcium channels are present at a higher density at the distal tip than in the proximal growth cone. Our results show that the calcium gradients seen in depolarized growth cones are a direct consequence of a gradient of calcium channel density.

Asymmetric retraction of growth cone filopodia following focal inactivation of calcineurin.

The neuronal growth cone is thought to be the site of decision making in nerve growth and guidance. One likely mechanism of how the growth cone translates various extracellular cues into directed motility involves rises in intracellular calcium. A variety of physiological cues, such as adhesion molecules and neurotransmitters, increases intracellular calcium, and artificial manipulations of growth cone calcium levels affect growth cone morphology and neurite outgrowth. The molecular events downstream of calcium fluxes are incompletely understood. Here we show that calcineurin, a protein phosphatase enriched in growth cones that is dependent on calcium ions and calmodulin, functions in neurite outgrowth and directed filopodial motility in cultured chick dorsal root ganglia neurons. Cyclosporin A and FK506, inhibitors of calcineurin, delayed neuritogenesis and inhibited neurite extension. Chromophore-assisted laser inactivation of calcineurin in regions of growth cones causes localized filopodial and lamellipodial retraction and influences the direction of subsequent outgrowth. We suggest that a spatial distribution of calcineurin activity within the growth cone can regulate motility and direct outgrowth.

Neurite outgrowth stimulated by L1 requires calcium influx into neurons but is not associated with changes in steady state levels of calcium in growth cones.

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N-type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.

Filopodia as detectors of environmental cues: signal integration through changes in growth cone calcium levels

This chapter examines a selected series of environmental stimuli that are known to interact with growth cones and examines how these stimuli are translated by the second messenger systems intrinsic to the growth cone proper. In particular, this chapter emphasizes the role of intracellular calcium ions as the messenger translating extracellular environmental cues into changes in neuronal growth cone behavior. The effects of two distinctive classes of external cues are examined: neurotransmitters and membrane protein NI-35, derived from central nervous system oligodendrocytes. The discussion also describes how these two sets of stimuli interact with intracellular systems within the cell. The text also provides a description of what is now termed the calcium hypothesis for the control of growth cone behavior. Finally, discussion examines an extended set of roles for intracellular calcium and how individual components of the growth cone interact. In doing so, the chapter presents the antennae amplifier model of filopodia/growth cone interaction and how this model incorporates changes in intracellular calcium from distant filopodia into a graded set of actions within the growth cone proper.

Omega-conotoxin prevents myelin-evoked growth cone collapse in neonatal rat locus coeruleus neurons in vitro

The response of neonatal rat locus coeruleus neurons to contact with myelin extracts prepared from the CNS and PNS was examined. The growth cones of these neurons collapsed following contact with central myelin, but continued to elongate on contact with peripheral myelin. Central myelin elicited an increase in the intracellular free calcium concentration in these growth cones, while peripheral myelin did not. This increase appeared to require transmembrane calcium flux, since it was blocked by extracellular EGTA, and also by extracellular cobalt. These neurons express N- and L-type calcium channels. Exposure to 5 microM omega-conotoxin GVIA, a specific blocker of N-type channels, prevented both the myelin-evoked increase in growth cone calcium concentration and the collapse of growth cones.

Modulation of growth cone calcium current is mediated by a PTX-sensitive G protein

Growth cones of isolated neuron B5 of Helisoma were voltage clamped in the whole-cell configuration. Depolarization of growth cones to −20 mV or greater activated a high-voltage-activated (HVA) calcium current. Addition of the neuropeptide FMRFamide (1 μM), which causes a presynaptic inhibition of synaptic transmission, reversibly reduced the calcium current magnitude. This inhibitory effect is mediated by a pertussis toxin (PTX)-sensitive G protein. Dialysis with the non-hydrolyzable GTP analogs GTPγS and Gpp(NH)p caused FMRFamide's effect to become irreversible. Dialysis with GDPβS or preincubation with PTX prevented FMRFamide from reducing the calcium current. Thus, one role of growth cone G proteins is to modulate ion channels in growth cone membrane which in turn may control growth cone motility.

Electrical activity increases growth cone calcium but fails to inhibit neurite outgrowth from rat sympathetic neurons

Previous studies have shown that the growth of axons from both mouse dorsal root ganglion neurons and Helisoma neurons is arrested when the cells are electrically stimulated (Cohan and Kater, 1986; Fields et al., 1990a). Furthermore, in the case of Helisoma neurons, this arrest has been attributed to a rise in the calcium concentration in the growth cones (Cohan et al., 1987). To test the generality of these results, we examined the response of cultured rat superior cervical ganglion (SCG) neurons to electrical stimulation and changes in cytoplasmic calcium. Suprathreshold electrical stimulation of SCG neurons at 10 Hz by extracellular patch electrodes for periods of up to 1 hr had no measurable effect on their rate of growth. In agreement with previous studies, electrical stimulation was accompanied by a rise in the internal calcium concentration: when measured by the fluorescence of fura-2, growth cone calcium levels rose from about 100 nM to greater than 500 nM and then settled to a plateau value of about 350 nM. Despite this increase, however, growth of SCG neurons' processes continued. Our results show that electrical activity is not a universal signal for neurons to stop growing and that a rise in internal calcium does not always arrest the migration of growth cones.