The timing of initial neuropeptide expression by an identified insect neuron does not depend on interactions with its normal peripheral target.

To study the developmental regulation of a neuropeptide phenotype, we have analyzed the biochemical and morphological differentiation of two identifiable neurons in embryos of the moth, Manduca sexta. The central cell, CF, and the peripheral cell, L1, are both neuroendocrine neurons that express neuropeptides related to the molluscan tetrapeptide FMRFamide. Both neurons project axons to the transverse nerve in each thoracic segment. Within the CF and L1 cells, neuropeptide-like immunoreactivity was localized to secretory granules that had cell-specific morphologies and sizes. The onset of neuropeptide expression in the two cell types displayed a similar pattern: immunoreactivity was first detected in distal processes and soon after within cell bodies. However, the onsets occurred at different times: for the CF cell, neuropeptides were first seen at 60%-63% of embryonic development, after the neuron had extended a long axon into the periphery, while L1 neuropeptide expression began at approximately 42%, as it first extended its growth cone. These times were related in that they corresponded to the arrival times of the respective growth cones at a similar position in the developing peripheral nerve. Within this region of the nerve, the growth cones of both cell types-exhibited a transient and cell-specific interaction with an identified mesodermal cell, called the Syncytium. Like the L1 and B neurons (Carr and Taghert, 1988b), the CF growth cones typically grew past this cell, yet remained attached to it by lamellipodial and filopodial processes of the axon. Ultrastructurally, the interaction involved filopodial adhesion to and insertion within the Syncytial cell. Two other nonneuroendocrine cell types grew axons past this same region, but showed no such tendencies. To test the hypothesis that the morphological and biochemical differentiation of these cells was somehow linked, central ganglia were isolated (as individuals or connected as ganglionic chains) in tissue culture, prior to the time when CF growth cones entered the periphery and prior to the development of CF neuropeptide expression. In the majority of cases, CF neurons nevertheless displayed their neuropeptide phenotype at a normal and cell-specific stage. We conclude that the initiation of neuropeptide expression is highly correlated with schedules of morphological differentiation in these neurons, but that, in the case of the CF neuron, it is not regulated by interactions of the growth cone with peripheral structures.     

Differential expression of neuropeptide gene mRNA within the LUQ cells of Aplysia californica

Two neuropeptide precursor cDNAs (LUQ-1 and L5-67) have been recently isolated from the Left Upper Quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica (Shyamala, Fisher, and Scheller, 1986; Wickham and DesGroseillers, 1991). Using in situ hybridization techniques as well as dot blot and polymerase chain reaction (PCR) assays, we have studied the expression of these genes in the central nervous system (CNS) of Aplysia californica. The LUQ-1 gene was found to be expressed in neuron L5 in the abdominal ganglion, whereas the expression of the L5-67 gene was observed in the other four LUQ cells (L2-4 and L6). When in situ hybridization was performed on paraffin sections of the abdominal ganglion, clusters of smaller cells located in the left hemiganglion, were also found to express either the LUQ-1 on the L5-67 gene, never both. In many sections, the mRNAs coding for the two neuropeptides were found not only in cell bodies but also in the axon of individual LUQ neurons and even as far as the pericardial nerve. The presence of neuropeptide mRNA in axons, pericardial nerve, and kidney has been confirmed by polymerase chain reaction. A specific, although diffuse hybridization in the left upper quadrant also suggests that mRNA is present in the neuritic field. Taken together these results indicate that neuron L5 is the only giant neuron expressing the LUQ-1 gene and might therefore have a physiological function different from the other four LUQ cells. Neuropeptide mRNAs were also found in the axon and/or the neuritic field of giant neurons and could play important roles related to cell signalling in axons and nerve termini.     

Modulation of growth cone morphology by substrate-bound adhesion molecules.

The growth cone, a terminal structure on developing and regenerating axons, is specialized for motility and guidance functions. In vivo the growth cone responds to environmental cues to guide the axon to its appropriate target. These cues are thought to be responsible for position-specific morphological changes in the growth cone, but the molecules that control growth cone behavior are poorly characterized. We used scanning electron microscopy to analyze the morphology of retinal ganglion cell growth cones in vitro on different adhesion molecules that axons normally encounter in vivo. L1/8D9, N-cadherin, and laminin each induced distinctive morphological characteristics in growth cones. Growth cones elaborated lamellipodial structures in response to the cell adhesion molecules L1/8D9 and N-cadherin, whereas laminin supported filopodial growth cones with small veils. On L1/8D9, the growth cones were larger and produced more filopodia. Filopodial associations between adjacent growth cones and neurites were frequent on L1/8D9 but were uncommon on laminin or N-cadherin. These results demonstrate that different adhesion molecules have profoundly different effects on growth cone morphology. This is consistent with previous reports suggesting that changes in growth cone morphology in vivo occur in response to changes in substrate composition.     

Non-Muscle Myosin II Regulates Neuronal Actin Dynamics by Interacting with Guanine Nucleotide Exchange Factors

Background

Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family.

Principal Findings

NM II colocalized with GEFs, such as βPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II–GEF interactions by overexpression of the DH domains of βPIX or Tiam1, or by βPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II–GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation.

Conclusion

Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF–Rho GTPase signaling pathway.     

Differential expression of neuropeptide gene mRNA within the LUQ cells of Aplysia californica.

Two neuropeptide precursor cDNAs (LUQ-1 and L5-67) have been recently isolated from the Left Upper Quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica (Shyamala, Fisher, and Scheller, 1986; Wickham and DesGroseillers, 1991). Using in situ hybridization techniques as well as dot blot and polymerase chain reaction (PCR) assays, we have studied the expression of these genes in the central nervous system (CNS) of Aplysia californica. The LUQ-1 gene was found to be expressed in neuron L5 in the abdominal ganglion, whereas the expression of the L5-67 gene was observed in the other four LUQ cells (L2-4 and L6). When in situ hybridization was performed on paraffin sections of the abdominal ganglion, clusters of smaller cells located in the left hemiganglion, were also found to express either the LUQ-1 or the L5-67 gene, never both. In many sections, the mRNAs coding for the two neuropeptides were found not only in cell bodies but also in the axon of individual LUQ neurons and even as far as the pericardial nerve. The presence of neuropeptide mRNA in axons, pericardial nerve, and kidney has been confirmed by polymerase chain reaction. A specific, although diffuse hybridization in the left upper quadrant also suggests that mRNA is present in the neuritic field. Taken together these results indicate that neuron L5 is the only giant neuron expressing the LUQ-1 gene and might therefore have a physiological function different from the other four LUQ cells. Neuropeptide mRNAs were also found in the axon and/or the neuritic field of giant neurons and could play important roles related to cell signalling in axons and nerve termini.     

Neuron differentiation and axon growth in the developing wing of Drosophila melanogaster

Sensory neurons in the wing of Drosophila originate locally from epithelial cells and send their axons toward the base of the wing in two major bundles, the L1 and L3 nerves. We have estimated the birth times of a number of identified wing sensory neurons using an X-irradiation technique and have followed the appearance of their somata and axons by means of an immunohistochemical stain. These cells become immunoreactive and begin axon growth in a sequence which mirrors the sequence of their birth times. The earliest ones are born before pupariation and begin axonogenesis within 1 to 2 hr after the onset of metamorphosis; the last are born and differentiate some 12 to 14 hr later. The L1 and L3 nerves are formed in sections, with specific neurons pioneering defined stretches of the pathways during the period between 0 and 4 hr after pupariation (AP), and finally joining together around 12 hr AP. By 16 hr AP the adult complement of neurons is present and the adult peripheral nerve pattern has been established. Pathway establishment appears to be specified by multiple cues. In places where neurons differentiate in close proximity to one another, random filopodial exploration followed by axon growth to a neighboring neuron soma might be the major factor leading to pathway construction. In other locations, filopodial contact between neighboring somata does not appear to occur, and axon pathways joining neural neighbors by the most direct route are not established. We propose that in these cases additional factors, including veins which are already present at the time of axonogenesis, influence the growth of axons through non-neural tissues.     

Growth cones isolated from identified Aplysia neurons in vitro: biochemical and morphological characterization

The right upper quadrant (RUQ) cells (R3-R13) of Aplysia regenerating in dissociated cell culture form unusually large growth cones. The movement of these growth cones was observed by time-lapse phase microscopy and their ultrastructure was examined by transmission electron microscopy. Their behavior and ultrastructure have features that are typical of growth cones in vitro. Additionally, they contain neurosecretory granules similar to those found in these cells in vivo. Because RUQ growth cones are large, they can be isolated by manual dissection. RUQ cells were grown in the presence of [35S]methionine and the labeled proteins transported to the growth cones were analyzed by SDS-PAGE. These proteins were compared to those in RUQ cell bodies, RUQ neurites, and to those in the neurites and cell bodies of other identified neurons grown in vitro. Most proteins synthesized by RUQ cells in vitro are transported to their growth cones, including several glycoproteins and the precursor to the R3-R14 neuropeptide. Neuropeptides are also synthesized by a number of other Aplysia neurons growing in vitro. We examined R2, LPL1, R15, and left upper quadrant neurons and found that their precursor peptides, like those of R3-R14, are readily recognized as major cell-specific radiolabeled bands on SDS gels. The presence in regenerating growth cones of neuropeptides, neurosecretory granules, and glycoproteins known to be rapidly transported toward synapses in vivo supports the emerging view that the growth cone in vitro contains not only a motility apparatus but also a macromolecular assembly capable of forming an active synapse immediately upon or shortly after contacting targets.     

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 filopodiaare 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. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 179–192, 1998     

Growth cone localization of neural cell adhesion molecule on central nervous system neurons in vitro

Ultrastructural analysis of colloidal gold immunocytochemical staining and immunofluorescence microscopy has been used to study the presence of neural cell adhesion molecule (NCAM) on the surface of neuronal growth cones. The studies were carried out with cultures of rat hypothalamic and ventral mesencephalic cells, using morphology and expression of tyrosine hydroxylase, neurofilaments, and glial fibrillary acidic protein as differential markers for neurons and glia. NCAM was found on all plasmalemmal surfaces of neurons including perikarya and neurites. The density of NCAM varied for different neurons growing in the same culture dish, and neurons had at least 25 times more colloidal gold particles on their plasmalemmal membranes than astroglia. Of particular interest in the present study was a strong labeling for NCAM on all parts of neuritic growth cones, including the lamellar and filopodial processes that extend from the tip of the axon. The density of NCAM was similar on different filopodia of the same growth cone. Therefore, in situations where homophilic (NCAM-NCAM) binding might contribute to axon pathfinding, a choice in direction is more likely to reflect differences in the NCAM content of the environment, rather than the distribution of NCAM within a growth cone. On the other hand, the variation in NCAM levels between single neurons in culture was significant and could provide a basis for selective responses of growing neurites.     

Fibronectin-like immunoreactivity in Helisoma buccal ganglia: evidence that an endogenous fibronectin-like molecule promotes neurite outgrowth

We examined the distribution of fibronectin-like (FNL) immunoreactivity associated with intact buccal ganglia, cell-cultured buccal ganglia neurons and nonneuronal cells, and brain-conditioned medium from the snail Helisoma. In addition, the possible roles of fibronectin in the regulation of neurite outgrowth were studied. Immunofluorescent staining for FNL antigens revealed intense staining in patches and fibrous arrays over the connective tissue sheaths of buccal ganglia and nerve trunks. Within the ganglia, heavy staining was seen surrounding neurons and in track-like arrangements. In cell cultures, specific staining was associated with nonneuronal cell surfaces and to a lesser degree with the surface of identified neurons. In addition, a noncellular, substrate-bound component of brain-conditioned medium displayed FNL immunoreactivity. Since cultured Helisoma neurons require a substrate-associated, brain-derived conditioning factor (CF) in order to elaborate neurites with motile growth cones, we tested whether the FNL immunoreactive substance might act as a neuritotropic agent. Fibronectin antiserum suppressed, in a dose-dependent manner, the CF-induced sprouting of identified neurons in isolated cell culture. When added at increasing concentrations to neurons already growing in response to CF, fibronectin antiserum exerted a biphasic effect on neurite elongation; outgrowth was accelerated at low, but inhibited at high, antiserum concentrations. In contrast, growth cone structures associated with motility (filopodia and lamellipodia) were progressively reduced by increasing levels of antiserum. A short peptide derived from fibronectin's cell-binding domain (Arg-Gly-Asp-Ser) also greatly reduced neurite outgrowth. The combined results of this study indicate an abundance of FNL immunoreactive molecules within the CNS of Helisoma, their probable production by nonneuronal cells, and their function as a substrate-associated component of CF which promotes growth cone filopodial and lamellipodial activity.     

Cdc42 stimulates neurite outgrowth and formation of growth cone filopodia and lamellipodia

To assess the role of cdc42 during neurite development, cmyc-tagged constitutively active (CA) and dominant negative (DN) cdc42 were expressed in dissociated primary chick spinal cord neurons using adenoviral-mediated gene transfer. Three days after infection, >85% of the neurons in infected cultures expressed cdc42 proteins, as detected by indirect immunofluorescence against cmyc. Growth cones of infected neurons displayed 1.83- (CAcdc42) and 1.93-fold (DNcdc42) higher cmyc immunofluorescence per square micrometer than uninfected controls. CAcdc42 expression stimulated growth cones, almost doubling growth cone size and number of filopodia, and increased neurite growth rates by 65-89%. In neurons plated onto fibronectin, the percent of growth cones with both filopodia and lamellipodia increased from 71 to 92%. Total Texas Red-phalloidin staining in these growth cones doubled, and the percent of growth cones with F-actin localized to peripheral regions increased from 52% in controls to 78% after CAcdc42 expression. Expression of DNcdc42 did not significantly alter growth cone morphology or neurite growth rates. Addition of soluble laminin to spinal cord neurons resulted in the identical phenotype as CAcdc42 expression, including changes in growth cone morphology, F-actin localization, and neurite growth rates. Significantly, expression of DNcdc42 blocked the effects of laminin on growth cones. These results show that cdc42 promotes neurite outgrowth and filopodial and lamellipodial formation in growth cones and suggests that cdc42 and laminin share a common signaling pathway during neurite development. Addition of laminin to CAcdc42-expressing neurons is inhibitory to growth cones, indicating that laminin also may activate some other pathways.     

Shootin1 interacts with actin retrograde flow and L1-CAM to promote axon outgrowth

Actin polymerizes near the leading edge of nerve growth cones, and actin filaments show retrograde movement in filopodia and lamellipodia. Linkage between actin filament retrograde flow and cell adhesion molecules (CAMs) in growth cones is thought to be one of the mechanisms for axon outgrowth and guidance. However, the molecular basis for this linkage remains elusive. Here, we show that shootin1 interacts with both actin filament retrograde flow and L1-CAM in axonal growth cones of cultured rat hippocampal neurons, thereby mediating the linkage between them. Impairing this linkage, either by shootin1 RNA interference or disturbing the interaction between shootin1 and actin filament flow, inhibited L1-dependent axon outgrowth, whereas enhancing the linkage by shootin1 overexpression promoted neurite outgrowth. These results strengthen the actin flow-CAM linkage model ("clutch" model) for axon outgrowth and suggest that shootin1 is a key molecule involved in this mechanism.     

Peanut agglutinin and chondroitin-6-sulfate are molecular markers for tissues that act as barriers to axon advance in the avian embryo

Axon outgrowth between the spinal cord and the hindlimb of the chick embryo is constrained by three tissues that border axon pathways. Growth cones turn to avoid the posterior sclerotome, perinotochordal mesenchyme, and pelvic girdle precursor during normal development and after experimental manipulation. We wanted to know if these functionally similar barriers to axon advance also share a common molecular composition. Since the posterior sclerotome differentially binds peanut agglutinin (PNA) and since PNA binding is also typical of prechondrogenic differentiation, we examined the pattern of expression of PNA binding sites and cartilage proteoglycan epitopes in relation to axon outgrowth. We found that all three barrier tissues preferentially express both PNA binding sites and chondroitin-6-sulfate (C-6-S) immunoreactivity at the time when growth cones avoid these tissues. Moreover, both epitopes are expressed in the roof plate of the spinal cord and in the early limb bud, two additional putative barriers to axon advance. In contrast, neither epitope is detected in peripheral axon pathways. In the somites, this dichotomous pattern of expression clearly preceded the invasion of the anterior sclerotome by either motor growth cones or neural crest cells. However, in the limb, barrier markers disappeared from presumptive axon pathways in concert with the invasion of axons. Since this coordinate pattern suggested that the absence of barrier markers in these axon pathways requires an interaction with growth cones, we analyzed the pattern of barrier marker expression following unilateral neural tube deletions. We found that PNA-negative axon pathways developed normally even in the virtual absence of axon outgrowth. We conclude that the absence of staining with carbohydrate-specific barrier markers is an independent characteristic of the cells that comprise axon pathways. These results identify two molecular markers that characterize known functional barriers to axon advance and suggest that barrier tissues may impose patterns on peripheral nerve outgrowth by virtue of their distinct molecular composition.     

Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons

The identification of surface proteins restricted to subsets of embryonic axons and growth cones may provide information on the mechanisms underlying axon fasciculation and pathway selection in the vertebrate nervous system. We describe here the characterization of a 135 kd cell surface glycoprotein, TAG-1, that is expressed transiently on subsets of embryonic spinal cord axons and growth cones. TAG-1 is immunochemically distinct from the cell adhesion molecules N-CAM and L1 (NILE) and is expressed on commissural and motor neurons over the period of initial axon extension. Moreover, TAG-1 and L1 appear to be segregated on different segments of the same embryonic spinal axons. These observations provide evidence that axonal guidance and pathway selection in vertebrates may be regulated in part by the transient and selective expression of distinct surface glycoproteins on subsets of developing neurons.     

Embryogenesis of peripheral nerve pathways in grasshopper legs. I. The initial nerve pathway to the CNS

The founding of the first nerve path of the grasshopper metathoracic leg was examined at the level of identified neurons, using intracellular dye fills, immunohistochemistry, Nomarski optics, and scanning and transmission electron microscopy. The embryonic nerve is established by the axonal trajectory of a pair of afferent pioneer neurons, the tibial 1 (Ti1) cells. Following a period of profuse filopodial sprouting, the Ti1 axonal growth cones, possessing 75- to 100-microns-long filopodia, navigate a stereotyped path across the limb bud epithelium to the base of the appendage and into the CNS. The Ti1 axons grow from cell to cell along a chain of preaxonogenesis neurons spaced at intervals along the pathway, forming dye-passing junctions with them. The contacted neurons subsequently undergo axonogenesis and follow the pioneer axons into the CNS. Later arising neurons project their axons onto the cell bodies of the chain, thereby establishing the principal branch points of the nerve. Among the later arising afferents are the sensory neurons of the femoral chordotonal and subgenual organs. The morphology of the adult nerve appears to be determined by the stereotyped positioning of neurons in the differentiating limb bud and by the resultant axonal trajectories established during the first 10% of peripheral neurogenesis.     

Peptidergic nerves in human dental pulp

Summary The peptidergic innervation of human dental pulp was studied with indirect immunofluorescence and immunoperoxidase techniques. Pulpal nerve fibres displaying immunoreactivity for cholecystokinin, calcitonin gene-related peptide, C-terminal flanking peptide of neuropeptide tyrosine, leucine-enkephalin, methionine-enkephalin, neuropeptide K, neuropeptide tyrosine, peptide with N-terminal histidine and C-terminal isoleucine, somatostatin-28, substance P and vasoactive intestinal polypeptide were observed. Immunoreactive axon varicosities were detectable within radicular and coronal nerve trunks and within the nerve plexus of Raschkow in the para-odontoblastic region. Many peptidergic nerve fibres were observed in association with blood vessels of various sizes. Substance P- and calcitonin-gene-related peptide-immunoreactive axons were visible in the odontoblastic layer. The occurrence of VIP- and PHI-immunoreactive fibres lends support to the hypothesis that human tooth may be supplied by parasympathetic nerves. The immunocytochemical results here shown provide a morphological basis to previous experimental studies concerning the possible roles of neuropeptides in nociception mechanisms, control of the blood flow and modulation of the inflammatory response in dental tissues.     

Regulation of neuropeptide expression in the brain by neurotrophins

Neurotrophins, which are structurally related to nerve growth factor, have been shown to promote survival of various neurons. Recently, we found a novel activity of a neurotrophin in the brain: Brain-derived neurotrophic factor (BDNF) enhances expression of various neuropeptides. The neuropeptide differentiation activity was then compared among neurotrophins both in vivo and in vitro. In cultured neocortical neurons, BDNF and neurotrophin-5 (NT-5) remarkably increased levels of neuropeptide Y and somatostatin, and neurotrophin-3 (NT-3) also increased these peptides but required higher concentrations. At elevating substance P, however, NT-3 was as potent as BDNF. In contrast, NGF had negligible or no effect. Neurotrophins administered into neonatal brain exhibited slightly different potencies for increasing these neuropeptides: The most marked increase in neuropeptide Y levels was obtained in the neocortex by NT-5, whereas in the striatum and hippocampus by BDNF, although all three neurotrophins increased somatostatin similarly in all the brain regions examined. Overall spatial patterns of the neuropeptide induction were similar among the neurotrophins. Neurons in adult rat brain can also react with the neurotrophins and alter neuropeptide expression in a slightly different fashion. Excitatory neuronal activity and hormones are known to change expression of neurotrophins. Therefore, neurotrophins, neuronal activity, and hormones influence each other and all regulate neurotransmitter/peptide expression in developing and mature brain. Physiological implication of the neurotransmitter/peptide differentiation activities is also discussed.     

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     

Axon initiation by ciliary neurons in culture

A nerve culture system for the study of axon initiation is described. A population of individual chick embryo ciliary neurons, free from contact with other cells and attached to a polyornithinecoated culture dish, is exposed to heart cell-conditioned medium (HCM). Within 30 min after the addition of HCM the majority of neurons have formed growth cones, and by 90 min more than 80% of the neurons bear at least one axon longer than 15 μm. Before the addition of HCM, ciliary neurons generate membrane ruffles and extend filopodia around the entire periphery of the rounded cell body. Axon initiation, following addition of HCM, consists of two distinctive changes in the cell surface: (1) organization of the randomly distributed surface movements into localized highly active growth cones, which then form axons; and (2) the cessation of surface movements elsewhere on the cell periphery. Heart cell-conditioned medium may induce these changes by increasing the adhesion between parts of the nerve cell surface and the substratum.