Depolarization-induced changes in neurite elongation and intracellular Ca2+ in isolated Helisoma neurons.

This study focuses on the effects of K+ depolarization on neurite elongation of identified Helisoma neurons isolated into culture. Application of K+ to the external medium caused a dose-dependent suppression of neurite elongation. Lower concentrations of K+ were associated with a slowing in the rate of neurite elongation, whereas higher concentrations produced neurite retraction. Surprisingly, the effects of K+ depolarization were transient, and neurite elongation rates recovered towards control levels within 90 min even though the neurons remained in high-K+ solution. Identified neurons differed in the magnitude of their response to K+ depolarization; neurite elongation of buccal neuron B4 was inhibited at 5 mM K+, but elongation in B5 and B19 was not affected until concentrations of 25 mM. Electrophysiologically, K+ application evoked a brief period (5-10 s) of action potential activity that was followed by a steady-state membrane depolarization lasting 2 h or more. The changes in neurite elongation induced by K+ depolarization occurred in isolated growth cones severed from their neurites and were blocked by application of calcium antagonists. Intracellular free Ca2+ levels in growth cones of B4 and B19 increased and then decreased during the 90-min depolarization, corresponding to the changes in elongation. B4 and B19 showed differences in the magnitude, time course, and spatial distribution of the Ca2+ change during depolarization, reflecting their different sensitivities to depolarization.     

Acetylcholine elongates neuronal growth cone filopodia via activation of nicotinic acetylcholine receptors

In addition to acting as a classical neurotransmitter in synaptic transmission, acetylcholine (ACh) has been shown to play a role in axonal growth and growth cone guidance. What is not well understood is how ACh acts on growth cones to affect growth cone filopodia, structures known to be important for neuronal pathfinding. We addressed this question using an identified neuron (B5) from the buccal ganglion of the pond snail Helisoma trivolvis in cell culture. ACh treatment caused pronounced filopodial elongation within minutes, an effect that required calcium influx and resulted in the elevation of the intracellular calcium concentration ([Ca]_i). Whole‐cell patch clamp recordings showed that ACh caused a reduction in input resistance, a depolarization of the membrane potential, and an increase in firing frequency in B5 neurons. These effects were mediated via the activation of nicotinic acetylcholine receptors (nAChRs), as the nAChR agonist dimethylphenylpiperazinium (DMPP) mimicked the effects of ACh on filopodial elongation, [Ca]_i elevation, and changes in electrical activity. Moreover, the nAChR antagonist tubucurarine blocked all DMPP‐induced effects. Lastly, ACh acted locally at the growth cone, because growth cones that were physically isolated from their parent neuron responded to ACh by filopodial elongation with a similar time course as growth cones that remained connected to their parent neuron. Our data revealed a critical role for ACh as a modulator of growth cone filopodial dynamics. ACh signaling was mediated via nAChRs and resulted in Ca influx, which, in turn, caused filopodial elongation. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 487–501, 2013     

The regulation of neurite outgrowth, growth cone motility, and electrical synaptogenesis by serotonin

Identified neurons of the buccal ganglion of the snail Helisoma when isolated from their ganglionic environment and plated in cell culture grow new neurites that are tipped with motile growth cones. Addition of the neurotransmitter serotonin to the culture medium surrounding actively growing neurons causes an immediate, premature cessation of neurite elongation in specific identified neurons. Serotonin selectively inhibits neurite extension of neurons B19 and P5 while having no effect on the extension of neuron B5. Coincident with the serotonin evoked inhibition of neurite elongation is an inhibition of growth cone motile activities and a retraction of growth cone filopodia and lamellipodia. One site of serotonin's growth inhibitory actions is directly at the growth cone rather than at the neurites or cell body. A second area of this study concerns connectivity. In Helisoma neurons the formation of electrical synaptic connections critically relies on both potential partner neurons having a mutual interaction of actively growing neurites. Neurons in a nongrowing state do not form electrical synapses (Hadley et al., 1983). As a result of inhibiting neurite extension, serotonin is able to affect synaptogenesis by preventing certain neurons (neurons B19) from forming electrical synaptic connections with other neurons (neurons B5) that are themselves competent to interconnect. Thus, by inhibiting neurite extension, serotonin is capable of regulating both the development of arborizations and the formation of connectivity.     

Depolarization-induced changes in neurite elongation and intracellular Ca2+ in isolated Helisoma neurons

This study focuses on the effects of K⁺ depolarization on neurite elongation of identified Helisoma neurons isolated into culture. Application of K⁺ to the external medium caused a dose-dependent suppression of neurite elongation. Lower concentrations of K⁺ were associated with a slowing in the rate of neurite elongation, whereas higher concentrations produced neurite retraction. Surprisingly, the effects of K⁺ depolarization were transient, and neurite elongation rates recovered towards control levels within 90 min even though the neurons remained in high-K⁺ solution. Identified neurons differed in the magnitude of their response to K⁺ depolarization; neurite elongation of buccal neuron B4 was inhibited at 5 mM K⁺, but elongation in B5 and B19 was not affected until concentrations of 25 mM. Electrophysiologically, K⁺ application evoked a brief period (5–10 s) of action potential activity that was followed by a steady-state membrane depolarization lasting 2 h or more. The changes in neurite elongation induced by K⁺ depolarization occurred in isolated growth cones severed from their neurites and were blocked by application of calcium antagonists. Intracellular free Ca²⁺ levels in growth cones of B4 and B19 increased and then decreased during the 90-min depolarization, corresponding to the changes in elongation. B4 and B19 showed differences in the magnitude, time course, and spatial distribution of the Ca²⁺ change during depolarization, reflecting their different sensitivities to depolarization.     

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.     

The differential regulation of formation of chemical and electrical connections in Helisoma

Novel chemical and electrical connections form between neurons not normally connected in the buccal ganglia of the snail Helisoma. We examined the cellular and environmental conditions required for the formation of each type of connection. Previous work in situ showed that novel electrical connections could form in response to axotomy. We have now found that axotomy can evoke the formation of novel unidirectional chemical connections between neurons B5 and B4 in addition to a novel electrical connection. The novel chemical connections display all of the normal properties of chemical synapses in Helisoma ganglia. These connections, however, are transient in nature and break 4 days following axotomy. Previous work has shown that conjoint outgrowth is required for the formation of electrical connections. In cell culture we have investigated whether conjoint outgrowth is also required for chemical synaptogenesis. Using neurons B5 and B19 we have found that when neuron pairs make contact in cell culture, under conditions of synchronous neurite extension, both electrical and chemical synapses form. However, if one neuron has ceased extension prior to contact by a growing neuron, electrical synapses never form (Hadley et al., 1983, 1985) but chemical synapses do form. Furthermore, the addition of serotonin (10(-6) M) to culture medium to inhibit neurite extension of B19, but not that of B5, selectively prevents the formation of electrical connections while permitting the formation of chemical synapses. Thus, the timing of contact in relation to the state of neurite extension can specify the type of connection a given neuron can form.     

Role of synaptotagmin, a Ca2+ and inositol polyphosphate binding protein, in neurotransmitter release and neurite outgrowth.

Synaptotagmin I (or II), a possible Ca(2+)-sensor of synaptic vesicles, has two functionally distinct C2 domains: the C2A domain binds Ca2+ and the C2B domain binds inositol high polyphosphates (IP4, IP5, and IP6). Ca(2+)-regulated exocytosis of secretory vesicles is proposed to be activated by Ca2+ binding to the C2A domain and inhibited by inositol polyphosphate binding to the C2B domain. Synaptotagmins now constitute a large family and are thought to be involved in both regulated and constitutive vesicular trafficking. They are classified from their distribution as neuronal (synaptotagmin I-V, X, and XI) and the ubiquitous type (synaptotagmin VI-IX). Among them, synaptotagmins III, V, VI and X are deficient in IP4 binding activity due to the amino acid substitutions in the C-terminal region of the C2B domain, suggesting that these isoforms can work for vesicular trafficking even in the presence of inositol high polyphosphates. Synaptotagmin I is also known to be present in neuronal growth cone vesicles. Antibody against the C2A domain (anti-C2A) that inhibits Ca(2+)-regulated exocytosis also blocked neurite outgrowth of the chick dorsal root ganglion (DRG) neuron, suggesting that Ca(2+)-dependent synaptotagmin activation is also crucial for neurite outgrowth.     

Regulation of neuronal growth cone filopodia by nitric oxide depends on soluble guanylyl cyclase

Nitric oxide has been proposed to play an important role in neuronal development. We have previously shown that growth cones from an identified neuron, B5, in the snail Helisoma trivolvis, respond to nitric oxide (NO) donors by increasing the length of their filopodia within minutes of application (Van Wagenen and Rehder, 1999). This effect was mediated through a cGMP-induced increase in [Ca2+]i and resulted in an enlargement of the growth cone's action radius, suggesting that NO could function as a signaling molecule during neuronal pathfinding. We show here that NO functions as a specific rather than a general regulator of growth cone filopodia, because another identified neuron from the same ganglion, B19, failed to respond to NO with an increase in filopodial length. We found that, contrary to B5 neurons, B19 growth cones contained little or no soluble guanylyl cyclase (sGC) immunoreactivity, presumably preventing their response to NO. This hypothesis was supported by the finding that the sGC activator YC-1 (10 microM) had no effect on B19 filopodia but induced elongation of B5 filopodia. These results indicate that the effects of NO can be quite specific, and raise the interesting possibility that neurons could selectively tune in to NO by differentially expressing the target enzyme sGC in the appropriate cellular location during critical developmental stages. In addition, our NADPH-diaphorase staining and anti-NOS immunohistochemisty suggest that growth cones of B5 neurons, but not of B19 neurons, could be a source of NO, making NO a potential intra- and transcellular messenger.     

A Highlights from MBoC Selection: Calcineurin-dependent cofilin activation and increased retrograde actin flow drive 5-HT–dependent neurite outgrowth in Aplysia bag cell neurons

Neurite outgrowth in response to soluble growth factors often involves changes in intracellular Ca²⁺; however, mechanistic roles for Ca²⁺ in controlling the underlying dynamic cytoskeletal processes have remained enigmatic. Bag cell neurons exposed to serotonin (5-hydroxytryptamine [5-HT]) respond with a threefold increase in neurite outgrowth rates. Outgrowth depends on phospholipase C (PLC) → inositol trisphosphate → Ca²⁺ → calcineurin signaling and is accompanied by increased rates of retrograde actin network flow in the growth cone P domain. Calcineurin inhibitors had no effect on Ca²⁺ release or basal levels of retrograde actin flow; however, they completely suppressed 5-HT–dependent outgrowth and F-actin flow acceleration. 5-HT treatments were accompanied by calcineurin-dependent increases in cofilin activity in the growth cone P domain. 5-HT effects were mimicked by direct activation of PLC, suggesting that increased actin network treadmilling may be a widespread mechanism for promoting neurite outgrowth in response to neurotrophic factors.     

The role of action potentials in determining neuron‐type‐specific responses to nitric oxide

The electrical activity in developing and mature neurons determines the intracellular calcium concentration ([Ca²⁺]_i), which in turn is translated into biochemical activities through various signaling cascades. Electrical activity is under control of neuromodulators, which can alter neuronal responses to incoming signals and increase the fidelity of neuronal communication. Conversely, the effects of neuromodulators can depend on the ongoing electrical activity within target neurons; however, these activity‐dependent effects of neuromodulators are less well understood. Here, we present evidence that the neuronal firing frequency and intrinsic properties of the action potential (AP) waveform set the [Ca²⁺]_i in growth cones and determine how neurons respond to the neuromodulator nitric oxide (NO). We used two well‐characterized neurons from the freshwater snail Helisoma trivolvis that show different growth cone morphological responses to NO: B5 neurons elongate filopodia, while those of B19 neurons do not. Combining whole‐cell patch clamp recordings with simultaneous calcium imaging, we show that the duration of an AP contributes to neuron‐specific differences in [Ca²⁺]_i, with shorter APs in B19 neurons yielding lower growth cone [Ca²⁺]_i. Through the partial inhibition of voltage‐gated K⁺ channels, we increased the B19 AP duration resulting in a significant increase in [Ca²⁺]_i that was then sufficient to cause filopodial elongation following NO treatment. Our results demonstrate a neuron‐type specific correlation between AP shape, [Ca²⁺]_i, and growth cone motility, providing an explanation to how growth cone responses to guidance cues depend on intrinsic electrical properties and helping explain the diverse effects of NO across neuronal populations. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 435–451, 2015     

Concentration-dependent stimulation and inhibition of growth cone behavior and neurite elongation by protein kinase inhibitors KT5926 and K-252a

We examined the concentration- and time-dependent effects of two related protein kinase inhibitors, KT5926 and K-252a, on neurite formation and nerve growth cone migration of chick embryo sensory neurons. The effects of these drugs on neurite formation over an 18-h period were dissimilar. KT5926 stimulated neurite formation at concentrations between 100 and 500 nM and inhibited neurite formation at 5 μM. K-252a had no stimulatory effects on neurite formation, and it inhibited neurite formation at concentrations above 50 nM. This difference may occur because K-252a inhibits activation of the nerve growth factor receptor trk A, while KT5926 does not inhibit trk A. Both drugs, however, had similar immediate effects on growth cone migration. Growth cone migration and lamellipodial spreading were rapidly stimulated by 500 nM concentrations of KT5926 and K-252a. At 2 μM levels of either drug, growth cone spreading was still stimulated, but growth cone migration was inhibited by both drugs. These results show that changes in protein phosphorylation/dephosphorylation can rapidly regulate the cellular machinery that is responsible for driving growth cone migration and neurite elongation. The different effects of 2 μM concentrations of either KT5926 or K-252a on growth cone spreading versus migration suggests that the actin-dependent protrusive motility of the growth cone leading margin is regulated differently by changes in protein phosphorylation and dephosphorylation than the cytoskeletal mechanism that drives neurite elongation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 161–171, 1997     

Phosphorylation by Rho kinase regulates CRMP-2 activity in growth cones

Collapsin response mediator protein 2 (CRMP-2) enhances the advance of growth cones by regulating microtubule assembly and Numb-mediated endocytosis. We previously showed that Rho kinase phosphorylates CRMP-2 during growth cone collapse; however, the roles of phosphorylated CRMP-2 in growth cone collapse remain to be clarified. Here, we report that CRMP-2 phosphorylation by Rho kinase cancels the binding activity to the tubulin dimer, microtubules, or Numb. CRMP-2 binds to actin, but its binding is not affected by phosphorylation. Electron microscopy revealed that CRMP-2 localizes on microtubules, clathrin-coated pits, and actin filaments in dorsal root ganglion neuron growth cones, while phosphorylated CRMP-2 localizes only on actin filaments. The phosphomimic mutant of CRMP-2 has a weakened ability to enhance neurite elongation. Furthermore, ephrin-A5 induces phosphorylation of CRMP-2 via Rho kinase during growth cone collapse. Taken together, these results suggest that Rho kinase phosphorylates CRMP-2, and inactivates the ability of CRMP-2 to promote microtubule assembly and Numb-mediated endocytosis, during growth cone collapse.     

Frequency-dependent and cell-specific effects of electrical activity on growth cone movements of cultured Helisoma neurons

The present experiments address the question of how stimulation parameters, which evoke action potentials in neuronal cell bodies, influence growth cone movements of different identified neurons. The motility of growth cones of Helisoma buccal neurons B19 and B4 was monitored while somata were stimulated simultaneously via an intracellular microelectrode. The findings show that the responses of growth cones of B19 and B4 contain components that are common as well as unique to each neuron. Whereas rates of growth cone advance were suppressed in a graded fashion by stimulus frequencies beyond a threshold of 2 s-1 for both neurons, B4 was more sensitive to electrical stimulation and exhibited a new response, namely, growth rates were enhanced during the poststimulation recovery period after stimulation at specific frequencies. Thus, electrical activity can result in enhancement as well as in inhibition of growth cone movement. Changes in number of filopodia on B19 and B4 were graded also, with B4 again displaying greater sensitivity. The frequency dependence of filopodia compared to growth rate changes was different and suggests a possible dissociation between filopodial activity and growth cone motility. Patterned electrical activity produced effects similar to constant stimulation for B19 growth cones, whereas it decreased the threshold frequency and eliminated the growth enhancement effect for B4. Taken together, these data demonstrate that the quantitative features of electrical activity as well as intrinsic properties of neurons both determine the growth cone response to changes in neuronal activity.     

Identification of a proline-rich inositol polyphosphate 5-phosphatase (PIPP)•collapsin response mediator protein 2 (CRMP2) complex that regulates neurite elongation

Neuron polarization is essential for the formation of one axon and multiple dendrites, establishing the neuronal circuitry. Phosphoinositide 3-kinase (PI3K) signaling promotes axon selection and elongation. Here we report in hippocampal neurons siRNA knockdown of the proline-rich inositol polyphosphate 5-phosphatase (PIPP), which degrades PI3K-generated PtdIns(3,4,5)P(3), results in multiple hyperelongated axons consistent with a polarization defect. We identify collapsin response mediator protein 2 (CRMP2), which regulates axon selection by promoting WAVE1 delivery via Kinesin-1 motors to the axon growth cone, as a PIPP-interacting protein by Y2H screening, direct binding studies, and coimmunoprecipitation of an endogenous PIPP, CRMP2, and Kinesin-1 complex from brain lysates. The C-terminal growth cone-targeting domain of PIPP facilitates its interaction with CRMP2. PIPP growth cone localization is CRMP2-dependent. PIPP knockdown in PC12 cells promotes neurite elongation, WAVE1 and Kinesin-1 growth cone localization, whereas knockdown of CRMP2 exhibits the opposite phenotype, with shorter neurites and decreased WAVE1/Kinesin-1 at the growth cone. In contrast, CRMP2 overexpression promotes neurite elongation, a phenotype rescued by full-length PIPP, or expression of the CRMP2-binding PIPP domain. Therefore this study identifies PIPP and CRMP2 exert opposing roles in promoting axon selection and neurite elongation and the complex between these proteins serves to regulate the localization of effectors that promote neurite extension.     

Cyclic GMP-gated CNG channels function in Sema3A-induced growth cone repulsion

Cyclic nucleotide-gated channels (CNGCs) transduce external signals required for sensory processes, e.g., photoreception, olfaction, and taste. Nerve growth cone guidance by diffusible attractive and repulsive molecules is regulated by differential growth cone Ca2+ signaling. However, the Ca2+-conducting ion channels that transduce guidance molecule signals are largely unknown. We show that rod-type CNGC-like channels function in the repulsion of cultured Xenopus spinal neuron growth cones by Sema3A, which triggers the production of the cGMP that activates the Xenopus CNGA1 (xCNGA1) subunit-containing channels in interneurons. Downregulation of xCNGA1 or overexpression of a mutant xCNGA1 incapable of binding cGMP abolished CNG currents and converted growth cone repulsion to attraction in response to Sema3A. We also show that Ca2+ entry through xCNGCs is required to mediate the repulsive Sema3A signal. These studies extend our knowledge of the function of CNGCs by demonstrating their requirement for signal transduction in growth cone guidance.     

Dorsal Root Ganglion Neurons React to Semaphorin 3A Application through a Biphasic Response that Requires Multiple Myosin II Isoforms

Growth cone responses to guidance cues provide the basis for neuronal pathfinding. Although many cues have been identified, less is known about how signals are translated into the cytoskeletal rearrangements that steer directional changes during pathfinding. Here we show that the response of dorsal root ganglion (DRG) neurons to Semaphorin 3A gradients can be divided into two steps: growth cone collapse and retraction. Collapse is inhibited by overexpression of myosin IIA or growth on high substrate-bound laminin-1. Inhibition of collapse also prevents retractions; however collapse can occur without retraction. Inhibition of myosin II activity with blebbistatin or by using neurons from myosin IIB knockouts inhibits retraction. Collapse is associated with movement of myosin IIA from the growth cone to the neurite. Myosin IIB redistributes from a broad distribution to the rear of the growth cone and neck of the connecting neurite. High substrate-bound laminin-1 prevents or reverses these changes. This suggests a model for the Sema 3A response that involves loss of growth cone myosin IIA to facilitate actin meshwork instability and collapse, followed by myosin IIB concentration at the rear of the cone and neck region where it associates with actin bundles to drive retraction.     

Formation and modulation of chemical connections: evoked acetylcholine release from growth cones and neurites of specific identified neurons

The ability to release acetylcholine (ACh) from cultured neurons of Helisoma was assessed by micromanipulating ACh-sensitive somata into contact with presynaptic neurons. ACh release was reliably detected from neurites and growth cones of cholinergic neuron B5, but not neuron B19, as early as 3 s after contact with novel target neurons. The rapid onset of transmission correlates with the ability of neuron B5, but not neuron B19, to indiscriminately form chemical connections and may be related to the specificity of synaptogenesis. The neuropeptide FMRFamide reduces ACh release at early chemical connections. The rapid onset of functional transmission and the ability of FMRFamide to modulate chemical transmission at this early chemical connection suggest that neuron B5 acquires its presynaptic apparatus through an intrinsic program independently of target contact.     

Antagonism of relaxation to isoproterenol caused by agonist interactions

The interaction of contractile agonists on the relaxation elicited with isoproterenol (ISO) was studied in 112 tracheal smooth muscle (TSM) strips from 20 dogs in vitro. Strips were contracted to the same active target tension (TT) with acetylcholine (ACh), histamine (HIS), serotonin (5-hydroxytryptamine, 5-HT), potassium chloride (KCl), or the combinations of ACh + HIS, ACh + 5-HT, HIS + KCl, HIS + 5-HT (50% TT from each agonist). Although a less potent agonist, adding HIS to cause 50% of the TT reduced the concentration of ACh to elicit the remaining 50% TT and substantially altered relaxation by ISO compared with HIS alone [concentration required to achieve 50% relaxation (RC50) = 9.2 +/- 2.4 X 10(-8) vs. 9.0 +/- 4.4 X 10(-9) M to HIS alone; P less than 0.003]. Relaxation for TSM strips contracted with ACh + HIS was comparable to that elicited from the same TT with ACh alone, although concentrations required in combination were lower than for either agonist alone. Trachealis strips contracted equivalently with KCl + HIS also had augmented contraction and attenuated relaxation (RC50 = 3.7 +/- 0.8 X 10(-8) M; P less than 0.015 vs. HIS alone). However, combinations of 5-HT + ACh and 5-HT + HIS did not alter relaxation to ISO from that elicited by the weaker agonist alone. We demonstrate that TSM relaxation depends on the combination of agonists eliciting contraction and may be inhibited substantially by interactions among contractile agonists.     

Presynaptic regulation of acetylcholine release in the CNS

The release of ACh appears to be under the control of autoreceptors localized on cholinergic nerve terminals. Moreover, the process can be regulated by transmitters other than ACh or by modulators either through receptor-mediated or carrier-mediated mechanisms. In this chapter we report on our recent results concerning the regulation of the release of ACh by ACh itself, 5-HT and GABA in the rat hippocampus. In particular it will be shown: 1) that the release of the cholinergic transmitter can be inhibited through muscarinic receptors of the M3 subtype; 2) that 5-HT can interact with ACh by depressing ACh release through the activation of receptors of the 5-HT1B subtype; 3) that the release of ACh can be enhanced by GABA by a novel mechanism involving a selective penetration of the amino acid into the cholinergic terminals.