Embryonic Chick Dorsal Root Ganglia Research Articles

2'-Deoxy-GTP in the microtubule cytoskeleton of neuronal cells cultured with nerve growth factor.

Tubulin, widely recognized as a GTP/GDP-binding protein, has been isolated in its polymerized state from rat PC12 cells and embryonic chick dorsal root ganglion neurons by Triton X-100 detergent extraction of the cytoskeletal fraction. Perchloric acid extraction and deproteinization of this fraction permitted subsequent analysis of nucleotide identity and content by high performance liquid chromatography. PC12 cells grown in the absence of nerve growth factor (NGF) contained ADP, ATP, GDP, and GTP at levels consistent with the actin and tubulin content of the cytoskeletal fraction. Microtubules from PC12 cells cultured in the presence of NGF contain an additional nucleotide that we have identified as dGTP. Analysis of whole cell nucleotide extracts from PC12 cells grown in the absence or presence of NGF revealed no evidence for the presence of dGTP at 4 and 14 days, respectively. We have determined that embryonic chick dorsal root ganglion neurons also contain this deoxyribonucleotide, and we found virtually no ADP or ATP in the extracted dorsal root ganglion cytoskeletal fraction. On the basis of metabolic labeling studies with [14C] guanine, we have inferred that the presence of dGTP in NGF-treated PC12 cells probably arises either from binding to the nonexchangeable nucleotide site of tubulin undergoing dynamic assembly/disassembly or from binding to the exchangeable site of tubulin subsequently incorporated into highly stabilized microtubules.

P21ras Oncogene Protein Selectively Increases Low-voltage-activated Ca2+ Current Density in Embryonic Chick Dorsal Root Ganglion Neurons.

p21ras protein resembles the alpha subunit of trimeric G-proteins, which regulate ion channel function. We now report a modulation of Ca2+ channels in vertebrate sensory neurons by p21ras in addition to its role in cell growth and differentiation. Quantitative microinjection of oncogenic p21-H-ras into embryonic chick dorsal root ganglion neurons was performed. After 4 h the current density of the low-voltage-activated (LVA; T-type) Ca2+ channels was increased. However, in contrast to trimeric G-proteins, which inhibit high-voltage-activated (HVA) Ca2+ channels in chick dorsal root ganglion neurons, p21ras did not significantly affect HVA Ca2+ currents. To study the time course of p21ras action, guanosine triphosphate-preloaded p21ras was added to the patch pipette. Full-length ras was effective only after a delay of 20 - 30 min. C-terminal modification by cellular enzymes is required to activate full-length ras, and can account for the observed delay. Unexpectedly, C-terminal-truncated p21ras, which was found to be inactive in biological assays, enhanced LVA Ca2+ currents within minutes. This suggests a G-protein-like modulation of the LVA Ca2+ channel by p21ras. In an early phase of neuronal differentiation, dorsal root ganglion neurons express only LVA Ca2+ currents. The regulatory role of p21ras on LVA channels may therefore be particularly important during differentiation.

NGF/BDNF chimeric proteins: analysis of neurotrophin specificity by homolog-scanning mutagenesis

Despite their extensive sequence identities at the amino acid level (approximately 55%), NGF and brain-derived neurotrophic factor (BDNF) display distinct neuronal specificity toward neurons of both the PNS and CNS. To explore which region(s) within these neurotrophic factors might determine their differential actions on various subpopulations of peripheral neurons, a systematic series (homolog-scanning mutagenesis) of chimeric NGF/BDNF molecules was prepared using PCR overlap-extension techniques. After expression in COS-7 cells, the chimeric proteins were tested for their biological activities in neurite outgrowth and neuronal survival assays. This approach led to the functional expression of 12 NGF/BDNF chimeras. Surprisingly, despite replacing successive amino acid segments throughout the entire length of NGF with the corresponding parts of BDNF, all chimeras displayed full NGF-like activity in bioassays carried out with PC12 cells, embryonic chick dorsal root ganglion explants, sympathetic ganglion explants, and dissociated cultures of dorsal root ganglion neurons. Most of the chimeras additionally showed BDNF-like activity as defined by neurite outgrowth on chick nodose ganglion explants. However, none of the chimeras supported the survival of dissociated nodose ganglion neurons. Our results suggest that NGF and BDNF must share very similar higher-order protein structures, and we propose that the overall structure or conformation of NGF, in contrast to short amino acid "active-site" segments, may determine its exact neuronal specificity.

Neurotrophic activity of S-100β in cultures of dorsal root ganglia from embryonic chick and fetal rat

We report here that S-100β, a protein with neurotrophic activity on central nervous system neurons, stimulates neuritic outgrowth from cultures of dorsal root ganglia (DRG). S-100β elicited neurites from explant and dissociated cell cultures of embryonic chick DRG, and the extent of the response varied with the age of the embryo. Specificity was demonstrated by the observation that incubation of S-100β with antibodies directed against S-100β reduced the neurite outgrowth, whereas incubation of S-100β with normal rabbit serum had little effect. S-100β also stimulated the area of neuritic outgrowth from organotypic cultures of fetal rat DRG, showing that the activty of the protein is not restricted to a particular species or culture condition. A mutant S-100β lacking neurotrophic activity on cerebral cortex neurons was unable to effectively stimulate neurite outgrowth from DRG cultures. These studies suggest that S-100β may play a role in neuronal growth and/or maintenance in the peripheral nervous system.

Elevated potassium shortens action potential duration by altering outward currents in chick dorsal root ganglia neurons

Dissociated embryonic chick dorsal root ganglion (DRG) neurons maintained in culture exhibit a mixed Na+/Ca2+ action potential. The characteristic "shoulder" on the repolarizing phase is due to the relatively prolonged inward Ca2+ current. DRG neurons grown in an elevated K+ medium (25 versus. 5 mM) lack the plateau phase of the action potential. Voltage-clamp analysis showed that this plastic change in action potential duration is not due to the loss of the inward Ca2+ current but is partly due to the appearance of a Ca2(+)-dependent, 4-aminopyridine-(4-AP)-sensitive transient outward current. Faster activation of the purely voltage-dependent delayed rectifier outward current also contributes to the rapid repolarization observed in neurons cultured in elevated K+ medium.

G proteins couple alpha-adrenergic and GABAb receptors to inhibition of peptide secretion from peripheral sensory neurons

Regulation of neuronal calcium channels by GTP-binding proteins (G proteins) is likely to be an important mechanism by which inhibitory transmitters influence excitation-secretion coupling in presynaptic nerve endings. Here, we report that in peripheral sensory neurons from embryonic chick dorsal root ganglia (DRG), the G protein-mediated inhibition of voltage-dependent calcium channels may best explain how norepinephrine (NE) and GABA inhibit the electrically evoked, calcium-dependent release of substance P (SP). As is the case for the previously reported inhibitory actions of these transmitters on DRG cell calcium channels, we demonstrate that NE and GABA inhibit peptide secretion through activation of alpha-adrenergic and GABAb receptors that are functionally coupled to pertussis toxin (PTX)-sensitive G proteins. Pretreatment of DRG cell cultures with PTX blocked the ability of NE and GABA to inhibit the release of SP, an action correlated with PTX-catalyzed ADP-ribosylation of membrane proteins with apparent molecular weight (Mr) of 40-41 kDa. Western immunoblot analysis of chick DRG cell membrane proteins using antisera directed against synthetic peptides corresponding to amino acid sequences predicted from cDNAs for PTX-sensitive G protein alpha subunits revealed a minimum of 2 Gi-like proteins (Mr 40 and 41 kDa) and a third Go-like protein (Mr 40 kD). Significantly, these findings implicate Gi- and/or Go-like GTP-binding proteins as mediators of presynaptic inhibition in peripheral sensory neurons.

Immunoelectron microscopic localization of actin in neurites of cultured embryonic chick dorsal root ganglia: actin is a component of granular, microtubule-associated crossbridges

Axons have been shown to contain substantial quantities of actin distributed along their length. However, the general lack of information on the structure and organizational state of this protein in axons has made it difficult to assign it a functional role. In the present study, we used electron microscopic immunocytochemistry (immunogold labeling) on neurites from cultured embryonic chick dorsal root ganglia to: (1) determine the distribution of actin in neurites: (2) identify actin-containing structures; and (3) reveal whether or not actin is associated preferentially with microtubules. Results show that actin is present throughout neurites but is organized primarily into short filaments that are localized almost exclusively to granular, microtubule-associated crossbridges. We propose that these short actin filaments are part of the framework of a supramolecular ‘carrier complex’ for the slow component b polypeptides. In addition, actin-containing crossbridges are often closely associated with the surfaces of membrane-bound organelles. This suggests that actin and microtubule-associated crossbridges are involved somehow in fast axonal transport, although the nature of their participation in this process still needs to be resolved.

Ganglioside potentiation of NGF-independent trophic agents on sensory ganglia

The ability of gangliosides to potentiate nerve growth factor (NGF)-independent trophic agents was determined by examining the capacity of an exogenous mixture of bovine brain gangliosides (BBG) and the monosialoganglioside GM1 to enhance the neuritogenic action of conditioned media (CM). CM were prepared with cultures of C 6 glioma cells, neonatal rat astroglial cells, rat L 6 myoblasts and chick embryonic skeletal muscle. Chick embryonic (9 day) dorsal root ganglia (DRG) were cultured on collagen-coated surfaces. The nutrient media with serum added or serum-free N 1 medium were supplemented with 50% of one of the CM with or without BBG (150 μg/ml) or GM1 (150 μg/ml). The neuritogenic responses of DRG 48 h in vitro were evaluated microscopically on the basis of neurite length and number. The neurite promoting action of the factor(s) present in the various CM was potentiated by BBG or GM1 and resulted in increased neurite length and number.

Inhibition of conditioned media-mediated neuritogenesis of sensory ganglia by monoclonal antibodies to GM1 ganglioside

The monosialoganglioside GM1 can potentiate the neuritogenic activity of media conditioned by several cell types: neonatal glia, C 6 glioma, embryonic chick heart or skeletal muscle and the rat myogenic line L 6. To probe further the neuritogenic activity of conditioned media (CM), 5 mouse monoclonal antibodies (mAbs) against GM1, designated B6, C3, C4h2, D1 and D3 were incorporated individually into nutrient medium (NM) supplemented with CM prior to incubation with sensory ganglia. Nine-day embryonic chick dorsal root ganglia were explanted onto collagen-coated coverslips and incubated at 35 °C for 5 h in NM supplemented with 150 μ/ml GM1. After washing with NM, the explants were re-fed with NM + CM containing 20% mAb and cultured for an additional 43 h. The resultant neuritogenesis was evaluated microscopically by determining mean neurite number and length of randomly mixed cultures. The 5 antibodies differed in their capacities to inhibit CM-mediated neuritogenesis of these primed target cells. D1 and D3 were most effective in reducing neurite length and number produced by all sources of the CM, while C3 and C4h2 were intermediate in their inhibition of neurite initiation (number). The effect of B6 on neurite initiation and elongation was the least. The ability of these mAbs to inhibit neuritogenic activity of CM derived from both glial and myogenic tissue suggests that gangliosides play a basic role in neuronal development. The differing responses elicited by the individual mAbs may reflect a relationship between the structural complexity of the GM1 molecule and the neuritogenic mechanisms. Since the various CMs may contain several specific growth factors, the potentiation of such factors by regions of the GM1 molecule may further regulate the character of the neuritogenic response.

Characterization of the electrically evoked release of substance P from dorsal root ganglion neurons: methods and dihydropyridine sensitivity

The mechanism by which dihydropyridines (DHPs) modulate the electrically evoked or KCI-induced release of substance P (SP) from embryonic chick dorsal root ganglion (DRG) neurons was investigated in the present study. The release of SP, as measured by radioimmunoassay (RIA), was characterized in terms of its dependence on extracellular calcium ion, its stimulus-response relationship, its sensitivity to the calcium-channel blocker omega conus toxin (omega-CgTx), and its modulation by the DHPs Bay K 8644 and nifedipine. Here it is reported that omega-CgTx (1 microM) blocked the electrically evoked release of SP. In contrast, the calcium-channel agonist Bay K 8644 (5 microM) facilitated the release of SP (by 45%), whereas the calcium-channel antagonist nifedipine (5 microM) was without effect. When the release of SP was triggered by depolarization of cultures with 60 mM KCI, the actions of the DHPs became much more pronounced. Under these conditions, Bay K 8644 facilitated (by 115%), whereas nifedipine inhibited (by 58%), peptide secretion. Voltage-clamp analysis of DRG cell calcium currents demonstrated that these actions of omega-CgTx, Bay K 8644, and nifedipine are explicable in terms of their effects on the slowly inactivating (L-type) calcium current. On the basis of these findings, it is suggested that the SP release mechanism exhibits DHP sensitivity due to the involvement of L-type calcium channels in the neurosecretory process. This model predicts that the voltage and time-dependent antagonist actions of nifedipine are sufficient to explain its failure to inhibit the electrically evoked release of SP.

Substrate effects on the dynamics of neurite growth in vitro: A quantitative multi-parametric analysis

Embryonic chick dorsal root ganglia were cultured in serum-free medium on natural (collagen, fibronectin and hyaluronic acid) and artificial (polylysine and polyornithine) substrata. The movement of individual growth cones was quantified by measuring five parameters using time-lapse cinematography combined with a digitizing-computer system, and the neurite behaviour was compared between the different substrata with multivariate statistical methods. For each substratum, the morphometry of the growth cone was quantified by measuring six morphological parameters. The most discriminative parameters proved to be mean velocity and straightness index for neurite extension, and projected area and cumulated length of filopodia for growth cone morphometry. A good correlation was obtained between behavioural and morphological parameters and the larger the cone area and the filopodia length, the faster and the straighter the neuritic growth. Both quantitative analyses showed highest values for polyornithine and the lowest for hyaluronic acid, and divided the substrata into two opposite groups, artificial and natural. It is concluded that growth cone behaviour and conformation is modulated by substratum properties.

Dihydropyridine inhibition of neuronal calcium current and substance P release

Dihydropyridine (DHP) calcium channel antagonists, which inhibit the slowly inactivating or L-type cardiac calcium (Ca) current, have been shown to be ineffective in blocking 45Ca influx and Ca-dependent secretion in a number of neuronal preparations. In the studies reported here, however, the antagonist DHP nifedipine inhibited both the L-type Ca current and potassium-evoked substance P (SP) release from embryonic chick dorsal root ganglion (DRG) neurons. These results suggest that, in DRG neurons, Ca entry through L-type channels is critical to the control of secretion. The inhibition of Ca current by nifedipine was both voltage and time-dependent, significant effects being observed only on currents evoked from relatively positive holding potentials maintained for several seconds. As expected from these results, nifedipine failed to inhibit L-type Ca current underlying the brief plateau phase of the action potential generated from the cell's normal resting potential; likewise, no significant effect of the drug was observed on action potential-stimulated SP release evoked by electrical field stimulation. The results of this work are discussed in terms of an assessment of the role of L-type Ca channels in neurosecretion.

Human amnion membrane as a substratum for cultured peripheral and central nervous system neurons

We report here on the use of human amnion membrane as a substratum for the culture of neuronal cells. Pieces of amnion membrane were bound to nitrocellulose paper as a supporting material, seeded with neurons, and cultured for 1–4 days. Neurons and neurites were visualized after fixation by immunoperoxidase staining using an anti-neurofilament monoclonal antibody. Neurons from embryonic chick ciliary and dorsal root ganglia and fetal rat hippocampus were cultured on either the basement membrane or stromal surface of the amnion membrane. Neurons adhered to both surfaces but extended neurites only on the basement membrane surface. Neurons survived and continued to grow neurites in serum-free medium for at least 4 days. When cultured for 4 days on the basement membrane surface in the presence of 10% fetal calf serum, the ciliary ganglion neurons survived but neurite growth was markedly inhibited, while dorsal root ganglion neurons survived, hypertrophied and grew an extensive network of neurites. Other experiments addressed the question whether the basement membrane surface had an ability to guide growing neurites. Amnion membranes were folded, frozen, cross-sectioned using a cryostat, and placed on the nitrocellulose to give irregular patterns of basement membrane juxtaposed to the collagenous stroma. Ciliary ganglion neurons after 4 days in culture had initiated and extended neurites in patterns which corresponded and were limited to those areas visualized by indirect immunofluorescence staining using anti-laminin antibodies. Thus, in vivo assembled human amnion basement membranes appear to contain signals that both promote and guide neuritic growth from previously axotomized embryonic peripheral and central nervous system neurons. The amnion membrane represents a novel tool for the culture of neuronal cells in vitro and potentially could be used as a neurite-promoting bridging material in vivo for regeneration studies.

Ganglioside GM1 antibodies and B-cholera toxin bind specifically to embryonic chick dorsal root ganglion neurons but do not modulate neurite regeneration.

Polyclonal antibodies to ganglioside GM1 have been prepared and characterised by direct and competitive enzyme-linked immunoassay. An immunoglobulin fraction was prepared from a rabbit antisera showing high specificity and antibody titre for GM1 relative to the other major brain gangliosides. The anti-GM1 immunoglobulin fraction and B-cholera toxin specifically labelled neurons in primary cultures of embryonic chick dorsal root ganglia and there was a good correlation between the relative increase in binding of anti-GM1 immunoglobulin and B-cholera toxin following neuraminidase treatment of a variety of cell types. At antibody concentrations that show saturable binding to endogenous ganglioside in the neuronal membrane, the anti-GM1 immunoglobulin fraction did not interfere with the nerve growth factor (NGF)-mediated fibre outgrowth and neuronal survival as indexed by measurement of neurofilament protein levels. Similarly, at levels in excess of those shown to stimulate thymocyte proliferation, B-cholera toxin was also without effect. These data are not consistent with GM1 in the neuronal membrane functioning as a receptor molecule for NGF and/or other differentiation factors present in the tissue culture media.

Rat astrocytes and Schwann cells in culture synthesize nerve growth factor-like neurite-promoting factors

Neurite-promoting activity in feeding medium conditioned by rat astrocytes and Schwann cells in culture was examined. The conditioned medium (CM) from both types of glial cultures stimulated extensive neurite outgrowth from embryonic chick dorsal root ganglia (DRG) as well as pheochromocytoma (PC12) cells. Both the DRG and PC12 cells also produce neurite outgrowth in the presence of nerve growth factor (NGF). With the DRG, the neurite growth rates observed with the glial cell CM were identical to growth rates seen with NGF. Although anti-NGF antibody did not inhibit the neurite outgrowth produced by either of the glial CM, a nerve growth factor radioreceptor assay did detect an NGF-like molecule in both CM. Since the extensive neurite outgrowth stimulated by the glial CM was not mimicked by pure laminin alone, we conclude that the glial neurite promoting factors are distinct from laminin.

Noradrenaline modulates calcium channels in avian dorsal root ganglion cells through tight receptor‐channel coupling.

Averaged ensemble Ba currents were recorded from tissue cultured embryonic chick dorsal root ganglion (d.r.g.) cells using the cell-attached patch-clamp technique. Noradrenaline (NA) applied to extrapatch membrane had no clear consistent effect on drug-free patch currents. This finding supports a previous suggestion that second messengers may not be involved in NA-mediated decreases in Ca currents in sensory neurones (Forscher & Oxford, 1985). Cell-attached patch currents sometimes increased slowly after extrapatch application of NA, but were not reversibly decreased by drug treatment. Large patch currents were used to trigger cellular action potentials. NA reversibly decreased action potential duration as reflected in extracellularly recorded patch action currents. Simultaneously recorded inward patch currents were not affected. D.r.g. cell adenylate cyclase activity was assayed. NA did not affect intracellular cyclic AMP levels at concentrations which cause 30-70% decreases in gCa in dialysed cells (Forscher & Oxford, 1985). Treatment with forskolin (50 microM) or isoprenaline (10 microM) resulted in 60- and 2-fold increases respectively in adenylate cyclase activity over basal levels. These results suggest that NA decreases Ca currents by direct NA interactions with the Ca channel or a molecule tightly coupled to channel function in d.r.g. cells.

Steroids and triiodothyronine reduce nerve growth factor concentrations in medium conditioned by L-929 fibroblasts.

Thyroid hormone, testosterone, and glucocorticoids stimulate the production of nerve growth factor (NGF) in mouse submandibular glands, but little is known about the influences of these hormones on other sources of NGF. In this study we tested the effects of thyroid hormone and steroids on NGF levels in medium conditioned by L-929 fibroblasts (L-cells), a mouse cell line that secretes NGF in culture. NGF concentrations in conditioned medium were measured by RIA and were normalized for cell number. In the absence of hormones, NGF levels in medium averaged 5.57 ng/ml. T3, cortisone, and aldosterone decreased NGF levels below the sensitivity of the RIA (less than 0.37 ng/ml), with respective ID50 values of 1 X 10(-5), 1 X 10(-8), and 5 X 10(-8) M. Testosterone decreased NGF levels by 66%, with an ID50 of 9 X 10(-9) M, and 17 beta-estradiol decreased it by up to 50%, although the hormone's effects had not plateaued at 10(-6) M. NGF concentrations were unaffected by progesterone at doses as high as 10(-6) M. Cell viability was unaffected by hormone treatment, and the total amount of secreted protein in medium decreased only in cultures treated with high doses of cortisone and aldosterone. Qualitatively similar results were obtained by bioassay; steroids markedly reduced, but did not eliminate, the nerve growth-promoting effects of L-cells on embryonic chick dorsal root ganglia in cocultures. These data indicate that hormones that stimulate NGF production in salivary glands inhibit NGF production by L-cells in culture.

Molecular specificity of ganglioside effects on neurite regeneration of sensory neurons in vitro

Highly purified preparations of individual gangliosides have been tested for their ability to modulate the survival and morphological differentiation of embryonic chick dorsal root ganglion neurons. When added at 48 h to established cultures of nerve growth factor (NGF)-dependent neurons, all ganglioside species tested increased the expression of neurofilament protein. Poly- and trisialogangliosides were more effective than di- or monosialogangliosides. In contrast, neither NGF nor an antiserum against NGF influenced neurofilament protein expression over this period of culture. In addition, ganglioside-induced expression of neurofilament protein was not inhibited by the anti-NGF serum.

Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor

The response of embryonic chick nodose ganglion (neural placode-derived) and dorsal root ganglion (neural crest-derived) sensory neurons to the survival and neurite-promoting activity of brain-derived neurotrophic factor (BDNF) was studied in culture. In dissociated, neuron-enriched cultures established from chick embryos between Day 6 (E6) and Day 12 (E12) of development, both nodose ganglion (NG) and dorsal root ganglion (DRG) neurons were responsive on laminin-coated culture dishes to BDNF. In the case of NG, BDNF elicited neurite outgrowth from 40 to 50% of the neurons plated at three embryonic ages; E6, E9, and E12. At the same ages, nerve growth factor (NGF) alone or in combination with BDNF, had little or no effect upon neurite outgrowth from NG neurons. The response of NG neurons to BDNF was dose dependent and was sustainable for at least 7 days in culture. Surprisingly, in view of a previous study carried out using polyornithine as a substrate for neuronal cell attachment, on laminin-coated dishes BDNF also sustained survival and neurite outgrowth from a high percentage (60–70%) of DRG neurons taken from E6 embryos. In marked contrast to NG neurons, the combined effect of saturating levels of BDNF and NGF activity on DRG neurons was greater than the effect of either agent alone at all embryonic ages studied. Under similar culture conditions, BDNF did not elicit survival and neurite outgrowth from paravertebral chain sympathetic neurons or parasympathetic ciliary ganglion neurons. We propose that primary sensory neurons, regardless of their embryological origin, are responsive to a “central-target” (CNS) derived neurotrophic factor—BDNF, while they are differentially responsive to “peripheral-target”-derived growth factors, such as NGF, depending on whether the neurons are of neural crest or placodal origin.

Progenitor cells from embryonic chick dorsal root ganglia differentiate in vitro to neurons: biochemical and electrophysiological evidence.

We have analyzed the appearance of neurons and glial cells in chick dorsal root ganglia during development. Neurons were identified by the presence of polysialogangliosides recognized by tetanus toxin (GD1b, GT1) or by the monoclonal antibody Q211 directed against polysialogangliosides containing four, five and six sialic acid residues. Glial cells were identified by the presence of 04 antigen. A population of undifferentiated cells, i.e., cells which express neither neuronal nor glial cell surface antigens, present in dorsal root ganglia until embryonic day 7, was separated from the neuronal and glial population. This cell population contains neuronal progenitor cells which differentiate to neurons within 1 day in culture. This differentiation process is characterized by the appearance of neuronal morphology, of neuron-specific gangliosides and by the appearance of voltage-dependent sodium and calcium channels.