GABAergic Growth Cones: Release of Endogenous γ-Aminobutyric Acid Precedes the Expression of Synaptic Vesicle Antigens

Growth cone fractions isolated from neonatal [postnatal day 3 (P3)] rat forebrain contain GABAergic growth cones as demonstrated by immunofluorescence staining with monospecific antibodies to gamma-aminobutyric acid (GABA). HPLC analysis shows that GABAergic growth cones release this endogenous GABA when stimulated with high K+. Endogenous GABA release is Ca2(+)-independent and, in this respect, similar to that seen previously with [3H]GABA. Isolated growth cone fractions also exhibit a K(+)-stimulated, Ca2(+)-independent release of endogenous taurine. None of the other amino acids shown to be present in isolated growth cone fractions were released, including glutamate, aspartate, and glycine. A population of dissociated cerebral cortical neurones prepared from P1 rat forebrain were GABA-immunoreactive after 1 day in culture. The cell body, neurites, and growth cones of these neurones were all stained with GABA antibodies. At this time in culture, neurones did not stain with either of two antibodies to synaptic vesicle antigens, i.e., p65 and synaptophysin. Growth cones isolated from P3 rat forebrain were also not immunoreactive with these antibodies. After about 8 days in culture, when neurones had established extensive networks of long, varicose axons and elaborately branched dendrites, many neurones and their neurites were immunoreactive for GABA antibodies. At this time in culture, p65 and synaptophysin antibodies did stain neuronal cell bodies and particularly their varicose axons. Dendrites were not stained with synaptic vesicle antibodies. These results suggest that GABAergic neurones synthesize GABA during neurite outgrowth and that GABA is present in, and can be released from, the growth cones of these neurones. The presence of GABA in GABAergic growth cones is not associated with synaptic vesicles, which explains the Ca2+ independency of both endogenous and [3H]GABA release from these growth cones.     

Developmental Changes in the Calcium Dependency of γ-Aminobutyric Acid Release from Isolated Growth Cones: Correlation with Growth Cone Morphology

We have investigated the development of Ca2+-dependent gamma-[3H]aminobutyric acid [( 3H]GABA) release in superfused growth cone fractions isolated from rats between the postnatal ages of 1 and 11 days. We have compared this release with the overall morphology of the subcellular fractions, and identified those structures taking up [3H]GABA by electron microscopical autoradiography. In fractions isolated from rats between 1 and 5 days, K+-evoked [3H]GABA release was completely independent of extracellular Ca2+. After 5 days a Ca2+ dependency appeared, which increased with age, such that by 10 days approximately 50% of the K+-evoked release was Ca2+ dependent. Electron microscopical analysis showed that, at all ages, large numbers of GABAergic growth cones were present in the subcellular fractions. Up to postnatal day 5, the growth cones were synaptic vesicle sparse but, after this age, increasing numbers of synaptic vesicle-containing growth cones were seen. These results suggest that during maturation of GABAergic growth cones into synapses there is, initially, a mechanism for release that is independent of extracellular Ca2+ and that the appearance of a Ca2+-dependent [3H]GABA release from growth cones correlates with the appearance of synaptic vesicles.     

Neurotrophin regulation of beta-actin mRNA and protein localization within growth cones.

Neurotrophins play an essential role in the regulation of actin-dependent changes in growth cone shape and motility. We have studied whether neurotrophin signaling can promote the localization of beta-actin mRNA and protein within growth cones. The regulated localization of specific mRNAs within neuronal processes and growth cones could provide a mechanism to modulate cytoskeletal composition and growth cone dynamics during neuronal development. We have previously shown that beta-actin mRNA is localized in granules that were distributed throughout processes and growth cones of cultured neurons. In this study, we demonstrate that the localization of beta-actin mRNA and protein to growth cones of forebrain neurons is stimulated by neurotrophin-3 (NT-3). A similar response was observed when neurons were exposed to forskolin or db-cAMP, suggesting an involvement of a cAMP signaling pathway. NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA. Localization of beta-actin mRNA was blocked by prior treatment of cells with Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A. Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA. These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.     

Differential Activities of Protein Phosphatase Types 1 and 2A in Cytosolic and Participate Fractions from Rat Forebrain

Abstract: The activities and concentrations of protein phosphates type 1 (PP1) and type 2A (PP2A) were compared in cytosol and particulate fractions of rat forebrain. Although the activity of PP2A was highest in the cytosol, immunoblot analysis with a PP2A-specific antibody showed that there were significant levels of the enzyme in the particulate fraction. There was no significant difference between the concentration of PP2A in the cytosol and particulate fractions such that the low activity of PP2A in the particulate fraction represents an inactivation of this form of the enzyme. Similar analysis in skeletal muscle, heart, and liver showed this finding was unique to the brain. Similarly, the majority of PP1 activity was recovered in the cytosol, but most PP1 enzyme was associated with the particulate fraction. Comparison with other tissues showed that the activities of PP1 in the particulate fractions were similar but that the forebrain contained significantly more enzyme than the other tissues. Thus, like PP2A it appears that the specific activity of PP1 in the particulate fraction of rat forebrain is much lower than that of the cytosol and of the particulate fractions of other tissues. Elution of PP1 and PP2A from membranes with 0.5 M NaCl plus 0.3% Triton X-100 resulted in severalfold activation of both enzymes. That the majority of PP1 and PP2A in rat forebrain are associated with membrane structures but in a low activity state suggests that novel regulatory mechanisms exist that have considerable and unique potential for activation of protein dephosphorylation.     

Calcium-Independent γ-Aminobutyric Acid Release from Growth Cones: Role of γ-Aminobutyric Acid Transport

Neuronal growth cones isolated in bulk from neonatal rat forebrain have uptake and K(+)-stimulated release mechanisms for gamma-aminobutyric acid (GABA). Up to and including postnatal day 5, the K(+)-stimulated release of [3H]GABA and endogenous GABA is Ca2+ independent. At these ages, isolated growth cones neither contain synaptic vesicles nor stain for synaptic vesicle antigens. Here we examined the possibility that the release mechanism underlying Ca2(+)-independent GABA release from isolated growth cones is by reversal of the plasma membrane GABA transporter. The effects of two GABA transporter inhibitors, nipecotic acid and an analogue of nipecotic acid, SKF 89976-A, on K(+)-stimulated release of [3H]GABA from superfused growth cones were examined. Nipecotic acid both stimulated basal [3H]GABA release and enhanced K(+)-stimulated release of [3H]GABA, which indicates that this agent can stimulate GABA release and is, therefore, not a useful inhibitor with which to test the role of the GABA transporter in K(+)-stimulated GABA release from growth cones. In contrast, SKF 89976-A profoundly depressed both basal and K(+)-stimulated [3H]GABA release. This occurred at similar concentrations at which uptake was blocked. These observations provide evidence for a major role of the GABA transporter in GABA release from neuronal growth cones.     

Measurements of growth cone adhesion to culture surfaces by micromanipulation

Neurons were grown on plastic surfaces that were untreated, or treated with polylysine, laminin, or L1 and their growth cones were detached from their culture surface by applying known forces with calibrated glass needles. This detachment force was taken as a measure of the force of adhesion of the growth cone. We find that on all surfaces, lamellipodial growth cones require significantly greater detachment force than filopodial growth cones, but this differences is, in general, due to the greater area of lamellipodial growth cones compared to filopodial growth cones. That is, the stress (force/unit area) required for detachment was similar for growth cones of lamellipodial and filopodial morphology on all surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces, which had a significantly lower stress of detachment than on other surfaces. Surprisingly, the forces required for detachment (760-3,340 mudynes) were three to 15 times greater than the typical resting axonal tension, the force exerted by advancing growth cones, or the forces of retraction previously measured by essentially the same method. Nor did we observe significant differences in detachment force among growth cones of similar morphology on different culture surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces. These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture. Our micromanipulations revealed that the most mechanically resistant regions of growth cone attachment were confined to quite small regions typically located at the ends of filopodia and lamellipodia. Detached growth cones remained connected to the substratum at these regions by highly elastic retraction fibers. The closeness of contact of growth cones to the substratum as revealed by interference reflection microscopy (IRM) did not correlate with our mechanical measurements of adhesion, suggesting that IRM cannot be used as a reliable estimator of growth cone adhesion.     

Gangliosides and Other Lipids of the Growth Cone Membrane

Growth cone membranes, derived from growth cone particles isolated from 16- to 18-day-old fetal rat brain, were found to be rich in overall lipid content with a lipid-to-protein ratio of 3.5. The phospholipid-to-cholesterol ratio indicated considerably less cholesterol than plasma membranes from mature neurons. All major classes of phospholipid were present in the usual proportions except sphingomyelin, which could not be detected. Gangliosides expressed in relation to protein were present at somewhat higher levels compared to previously reported values for synaptic plasma membranes (73 versus 44 micrograms/mg protein), but when related to phospholipid their level was well below that of the latter (26 versus 62 micrograms/mg phospholipid). The ganglioside pattern was generally similar to that of mature synaptic membranes except for the presence of relatively more GD3 and less GD1a, a phenomenon also observed in whole fetal brain of the same age. Several neutral glycosphingolipids were detected, glucosylceramide being the major one of this group. Their total level in growth cone membranes was roughly comparable to that of gangliosides, but unlike the latter their concentration in whole brain decreased with development. For comparison we analyzed the ganglioside composition of mixed membrane fractions from the same fetal brains and found no significant differences between these and growth cone membranes, suggesting that these glycoconjugates are not localized specifically in the growth cones. Neutral glycosphingolipids, on the other hand, appeared somewhat more concentrated in growth cones than in the mixed membranes.     

Rapid arrest of axon elongation by brefeldin A: a role for the small GTP-binding protein ARF in neuronal growth cones

Members of the ADP-ribosylation factor (ARF) family of small guanosine triphosphate-binding proteins play an essential role in membrane trafficking which subserves constitutive protein transport along exocytic and endocytic pathways within eukaryotic cell bodies. In growing neurons, membrane trafficking within motile growth cones distant from the cell body underlies the rapid plasmalemmal expansion which subserves axon elongation. We report here that ARF is a constituent of axonal growth cones, and that application of brefeldin A to neurons in culture produces a rapid arrest of axon extension that can be ascribed to inhibition of ARF function in growth cones. Our findings demonstrate a role for ARF in growth cones that is coupled tightly to the rapid growth of neuronal processes characteristic of developmental and regenerative axon elongation, and indicate that ARF participates not only in constitutive membrane traffic within the cell body, but also in membrane dynamics within growing axon endings.     

Growth cone-growth cone interactions in cultures of rat sympathetic neurons

Growth cones of sympathetic neurons from the superior cervical ganglia of neonatal rats were studied using video-microscopy to determine events following contact between growth cones and other cell surfaces, including other growth cones and neurites. A variety of behaviors were observed to occur upon contact between growth cones. Most commonly, one growth cone would collapse and subsequently retract upon establishing filopodial contact with the growth cone of another sympathetic neuron. Contacts resulting in collapse and retraction were often accompanied by a rapid and transient burst of lamellipodial activity along the neurite 30-50 microns proximal to the retracting growth cone. In no instances did interactions between growth cones and either fibroblasts or red blood cells result in the growth cone collapsing, suggesting that a specific recognition event was involved. On several occasions, growth cones were seen to track other growth cones, although fasciculation was rare. In some cases, there was no obvious response between contacting growth cones. Growth cone-growth cone contact was almost four times more likely to result in collapse and retraction than was growth cone-neurite contact (45% vs 12%, respectively). These observations suggest that the superior cervical ganglion may be composed of neurons with different cell surface determinants and that these determinants are more concentrated on the surface of growth cones than on neurites. These results further suggest that contact-mediated inhibition of growth cone locomotion may play an important role in growth cone guidance.     

Subtypes of protein kinase C in isolated nerve growth cones: only type II is associated with the membrane skeleton from growth cones.

The growth cone particle (GCP) fraction was isolated from fetal and neonatal rat brains and the distribution of protein kinase C subtypes in the fraction was examined by using subtype-specific antibodies. The main subtype in the GCP fraction from fetal forebrain was type II, and type III was also present, but not type I. The pattern was not altered from embryonic day 17 to postnatal day 5. The membrane skeleton subfraction from the GCP fraction contained type II, but far less amount of type III. Our results suggest that type II and type III may be closely related to the functions of growth cones but that they appear to be associated with distinct signal transduction processes.     

Protein synthesis and breakdown in the mother-cell and forespore compartments during spore morphogenesis in Bacillus megaterium

Recently developed techniques for isolating forespores from bacilli at all stages of spore morphogenesis have been exploited to investigate the contribution of each of the two compartments of the sporulating cell to the overall pattern of protein synthesis and degradation during sporulation in Bacillus megaterium. These studies have shown: (1) that protein synthesis continues in both compartments throughout spore morphogenesis; (2) that the degradation of proteins made at all times during vegetative growth and sporulation is confined to the mother-cell compartment; (3) that proteins synthesized in the mother-cell compartment during sporulation are subsequently degraded more rapidly than proteins synthesized during vegetative growth. This rate of degradation increases the later the proteins are synthesized in the sporulation sequence. Mature spores were disrupted, and the percentage of the total protein in soluble and particulate fractions was determined. Pulse-labelling experiments were performed to investigate the extent to which the proteins of these two fractions are newly synthesized during sporulation. These data were used to calculate the extent of capture of vegetative cell protein at the time of formation of the forespore septum. The value obtained is consistent with evidence from electron micrographs and supports a model for the origin of spore protein in which there is no protein turnover in the developing forespore.     

Cyclic AMP-Dependent Protein Phosphorylation in Isolated Neuronal Growth Cones from Developing Rat Forebrain

We have shown recently that neuronal growth cones isolated from developing rat forebrain possess an appreciable activity of adenylate cyclase, which produces cyclic AMP and can be stimulated by various neurotransmitter receptor agonists and by forskolin. To investigate cyclic AMP-mediated biochemical mechanisms in isolated growth cones, we have centered the present study on cyclic AMP-dependent protein phosphorylation. One-dimensional gel electrophoretic analysis showed that cyclic AMP analogs increased incorporation of 32P into several phosphoproteins in molecular mass ranges of 50-58 and 76-82 kilodaltons, including those of 82, 76, and 51 kilodaltons. Two-dimensional electrophoresis, using isoelectric focusing in the first dimension, resolved phosphorylated alpha- and beta-tubulin species, actin, a very acidic protein (isoelectric point 4.0) with a molecular mass of 93 kilodaltons, and two proteins (x and x') closely neighboring beta-tubulin. Two other phosphoproteins seen in the gels had molecular masses of 56 and 51 kilodaltons (respective isoelectric points, 4.5 and 4.4) and, along with the 93-kilodalton phosphoprotein, were highly enriched in the isolated growth cones. Only the tubulin and actin species were major proteins in the isolated growth cones. Cyclic AMP analogs enhanced incorporation of 32P into phosphoproteins x and x', and, as assessed by immunoprecipitation, into beta-tubulin. Peptide digest experiments suggested that phosphoproteins x and x' are unrelated to beta-tubulin. Nonequilibrium two-dimensional electrophoresis resolved many phosphoproteins, of which a 79- and 75-kilodalton doublet, a 74-kilodalton species, and a 58-kilodalton doublet showed enhanced incorporation of 32P in the presence of cyclic AMP.     

Rapid arrest of axon elongation by brefeldin A: A role for the small GTP‐binding protein ARF in neuronal growth cones

Members of the ADP‐ribosylation factor (ARF) family of small guanosine triphosphate–binding proteins play an essential role in membrane trafficking which subserves constitutive protein transport along exocytic and endocytic pathways within eukaryotic cell bodies. In growing neurons, membrane trafficking within motile growth cones distant from the cell body underlies the rapid plasmalemmal expansion which subserves axon elongation. We report here that ARF is a constituent of axonal growth cones, and that application of brefeldin A to neurons in culture produces a rapid arrest of axon extension that can be ascribed to inhibition of ARF function in growth cones. Our findings demonstrate a role for ARF in growth cones that is coupled tightly to the rapid growth of neuronal processes characteristic of developmental and regenerative axon elongation, and indicate that ARF participates not only in constitutive membrane traffic within the cell body, but also in membrane dynamics within growing axon endings. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 105–115, 1999     

Morphological and biochemical evidence showing neuronal properties in AtT-20 cells and their growth cones

AtT-20 cells, which were derived from a murine pituitary tumor and produce ACTH, have until now been considered to originate from pituitary corticotrophs. Here we show that AtT-20 cells constitutively express several neuronal features. First, AtT-20 cells develop cytoplasmic processes whose fine structure is essentially identical to that of neurites and neuronal growth cones. These growth cones (i) are characterized by an extensive membranous reticulum which is derived from the endoplasmic reticulum (ER) since it contains immunoglobulin heavy chain binding protein, protein disulfide isomerase and glucose-6-phosphatase; (ii) are a major site of endocytosis; (iii) form cell-to-cell contacts resembling immature synapses. Second, AtT-20 cells, in contrast to pituitary corticotrophs, contain neurofilaments and express all three neurofilament polypeptides. They also contain the high molecular weight form of microtubule-associated protein 2 and tau protein. Third, AtT-20 cells express the neuron-specific phosphoprotein synapsin I which accumulates in the growth cones prior to contacts forming between growth cones and cells. Our results show that AtT-20 cells exhibit several properties of peptidergic neuronal cells and that the constitutive expression of a variety of these properties is compatible with continuous cell division.     

Growth cone collapse through coincident loss of actin bundles and leading edge actin without actin depolymerization

Repulsive guidance cues can either collapse the whole growth cone to arrest neurite outgrowth or cause asymmetric collapse leading to growth cone turning. How signals from repulsive cues are translated by growth cones into this morphological change through rearranging the cytoskeleton is unclear. We examined three factors that are able to induce the collapse of extending Helisoma growth cones in conditioned medium, including serotonin, myosin light chain kinase inhibitor, and phorbol ester. To study the cytoskeletal events contributing to collapse, we cultured Helisoma growth cones on polylysine in which lamellipodial collapse was prevented by substrate adhesion. We found that all three factors that induced collapse of extending growth cones also caused actin bundle loss in polylysine-attached growth cones without loss of actin meshwork. In addition, actin bundle loss correlated with specific filamentous actin redistribution away from the leading edge that is characteristic of repulsive factors. Finally, we provide direct evidence using time-lapse studies of extending growth cones that actin bundle loss paralleled collapse. Taken together, these results suggest that actin bundles could be a common cytoskeletal target of various collapsing factors, which may use different signaling pathways that converge to induce growth cone collapse.     

Growth promotion of rice seedlings by allelopathic polysaccharide from Welsh onion

The underground portion of the Welsh onion (Allium fistulosum) was extracted and separated into seven fractions to purify allelopathic compounds that were found to promote the growth of rice seedlings (Oryza sativa). The 80% (v/v) ethanol-insoluble fraction of the hot water extract showed the highest growth-promoting effect. Purification by DEAE-cellulose chromatography gave four fractions, AD-1, 2, 3 and 4. AD-3 at 1000 ppm stimulated root and shoot growth by about 2.4 and 1.5 times over the respective water and sucrose-treated controls. The molecular weight of AD-3 was roughly estimated as 630 kDa by gel permeation chromatography. Seventy two percent (by wt.) of AD-3 was a carbohydrate and no proteins were detected. A GC analysis of the neutral sugar composition revealed the presence of Gal (50.1%), Man (17.9%), Ara (10.4%), Rha (8.8%), Glc (7.2%) and Xyl (5.6%). About 3% (by wt.) of the total carbohydrate was uronic acid, which was identified as GalU by a GC analysis. The remainder of AD-3 was extracted in ethyl acetate after its hydrolysis with 2 M HCl. The major component of the ethyl acetate-soluble fraction was identified as unsaturated linear primary alcohols, 1-tetradecanol and 1-octadecanol (16% and 84% by peak areas from GC, respectively) by means of NMR and GC-MS. Sole or combined treatment of these alcohols did not affect the growth of rice seedlings. After partial hydrolysis with 0.2 M trifluoroacetic acid or by a sodium periodate treatment, the activity of AD-3 had completely disappeared, suggesting that the sugar moiety and/or molecular size were important for the activity.     

Slit2-Mediated chemorepulsion and collapse of developing forebrain axons

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptor and its secreted ligand Slit. In rodents, Robo and Slit are expressed in the spinal cord and Slit can repel spinal motor axons in vitro. Here, we extend these findings into higher brain centers by showing that Robo1 and Robo2, as well as Slit1 and Slit2, are often expressed in complementary patterns in the developing forebrain. Furthermore, we show that human Slit2 can repel olfactory and hippocampal axons and collapse their growth cones.     

Smn, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of beta-actin mRNA in growth cones of motoneurons

Spinal muscular atrophy (SMA), a common autosomal recessive form of motoneuron disease in infants and young adults, is caused by mutations in the survival motoneuron 1 (SMN1) gene. The corresponding gene product is part of a multiprotein complex involved in the assembly of spliceosomal small nuclear ribonucleoprotein complexes. It is still not understood why reduced levels of the ubiquitously expressed SMN protein specifically cause motoneuron degeneration. Here, we show that motoneurons isolated from an SMA mouse model exhibit normal survival, but reduced axon growth. Overexpression of Smn or its binding partner, heterogeneous nuclear ribonucleoprotein (hnRNP) R, promotes neurite growth in differentiating PC12 cells. Reduced axon growth in Smn-deficient motoneurons correlates with reduced beta-actin protein and mRNA staining in distal axons and growth cones. We also show that hnRNP R associates with the 3' UTR of beta-actin mRNA. Together, these data suggest that a complex of Smn with its binding partner hnRNP R interacts with beta-actin mRNA and translocates to axons and growth cones of motoneurons.     

BDNF Reduces eEF2 Phosphorylation and Enhances Novel Protein Synthesis in the Growth Cones of Dorsal Root Ganglia Neurons

The local translation, which is regulated by extracellular stimuli such as guidance molecules, in growth cones of neurons provides a molecular mechanism for axonal development. In this study, we performed immunocytochemistry together with atomic force microscopy to investigate the localization of ribosomal proteins in the growth cones of rat dorsal root ganglion (DRG) neurons. The immunoreactivity of ribosomal protein P0/1/2 and S6, and novel protein synthesis were observed in the central, sterically bulky region of growth cones. Brain derived neurotrophic factor (BDNF) reduced the eEF2 phosphorylation, indicating its activation, and enhanced protein synthesis within 30 min. The effects of BDNF were completely inhibited by rapamycin, an inhibitor of mammalian target of rapamycin (mTOR). These results indicated that BDNF rapidly activates translation and enhances novel protein synthesis in growth cones of DRG though the mTOR signaling.