Tetanus Toxin Binding Sites Research Articles

Characterization of the cholinergic neuronal differentiation of the human neuroblastoma cell line LA-N-5 after treatment with retinoic acid

Analysis of the molecular factors that control cellular differentiation in mammalian embryos is difficult due to the small amount of material available from embryos and their inaccessibility during gestation. One way to circumvent these limitations is to use model systems that allow the study of differentiation in vitro. In this study we have characterized the response of a human neuroblastoma cell line, LA-N-5, to the differentiation-inducing agent, all-trans retinoic acid (RA) using 23 markers that are characteristic of neural crest cells and some of their derivatives. Following induction with RA, the neural crest-like LA-N-5 cells undergo differentiation into cholinergic neurons with increased expression of a variety of neural-specific markers including neurofilaments, growth associated protein-43, tetanus toxin binding sites, receptors for neurotrophic factors, neuropeptides, choline acetyl transferase, vesicular acetylcholine transporter, and acetylcholinesterase with a concomitant decrease in the expression of non-neuronal markers. These results provide the basis for the use of retinoic acid-induced differentiation of LA-N-5 cells as a model system to study molecular events associated with the differentiation of cholinergic neurons.

GABAA receptors modulate early spontaneous excitatory activity in differentiating P19 neurons.

P19 embryonic carcinoma (EC) stem cells are pluripotent and are efficiently induced to differentiate into neurons and glia with retinoic acid (RA) treatment. Within 5 days, a substantial number of differentiating P19 cells express gene products that are characteristic of a neuronal phenotype. P19 neurons were used as a model to explore the relationship between neuronal "differentiation" in vitro and the acquisition of gamma-aminobutyric acid (GABAA) receptors and functional GABA responses. Pulse-labeling experiments using bromodeoxyuridine indicated that all neurons had become postmitotic within 3-4 days after treatment with RA. This was confirmed by a reduction in the immunocytochemical detection of the undifferentiated stem cell antigen SSEA-1. Subsequently, a transient expression of nestin was observed during the first 5 days in vitro (DIV) after exposure to RA. By 5-10 DIV after RA, a significant number of neurons (approximately 80-90%) expressed immunocytochemically detectable glutamate decarboxylase and GABA coincident with the acquisition of membrane binding sites for tetanus toxin. These phenotypic markers were maintained for > 30 DIV after RA. Under current-clamp conditions, random, low-amplitude, spontaneous electrical activity appeared in neurons within the first few days after RA treatment and this was blocked by the specific GABAA receptor antagonist bicuculline. Thereafter, the appearance and progressive increases in the frequency of spontaneous action potentials in P19 neurons were observed that were similarly attenuated by bicuculline. In neurons > 5 DIV after RA, exogenous application of GABA elicited similar action potentials. The onset of excitatory responses to GABA or muscimol in voltage-clamped neurons appeared immediately after the cessation of neuronal mitosis and before the previously reported acquisition of responses to glutamate. In fura-2 imaging studies, the exogenous application of GABA resulted in neuron-specific increases in intracellular Ca2+. Thus, P19 neurons provide an in vitro model for the study of the early acquisition and properties of electrical excitability to GABA and the expression of functional GABAA receptors.

Gamma Interferon‐Induced Specific Binding of Tetanus Toxin on the GG2EE Macrophage Cell Line

We have recently demonstrated that tetanus toxin (TT) selectively inhibited gamma interferon-(IFN-gamma)-induced, but not basal, lysozyme activity in the GG2EE macrophage cell line. By an indirect immunofluorescence technique, we show that tetanus toxin, as well as the BIIb tetanus toxin-derived fragment, selectively binds to IFN-gamma-treated cells but fails to bind to control cells. Moreover, BIIb fragment and TT share the same binding sites, as demonstrated by competition/displacement experiments. These data indicate that IFN-gamma pretreatment results in the acquisition of tetanus toxin binding sites on GG2EE cells by a mechanism(s) yet to be identified.

Preparation of affinity-purified, biotinylated tetanus toxin, and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 1:1 to 20:1 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60-80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using 125I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2-3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these "primed" cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.

A monoclonal antibody directed against quail tyrosine hydroxylase: description and use in immunocytochemical studies on differentiating neural crest cells.

Catecholamine (CA) synthesis is one of the phenotypic traits expressed by some neural crest-derived cells in vivo and in vitro. In the present study, we have evidenced, in quail embryos, the expression of the first enzyme of CA metabolism, tyrosine hydroxylase (TOH), using a monoclonal antibody raised against the quail enzyme. This antibody also recognizes TOH from chick and pleurodele, but not from several mammalian species (rat, human). We have also investigated the extent to which TOH-positive cells, differentiated in neural crest cultures, express structural neuronal markers and display vasoactive intestinal polypeptide (VIP) and substance P (SP) immunoreactivity. Double-immunolabeling experiments show that, in vitro, half of the population of TOH-positive cells exhibits tetanus toxin binding sites but none of them are recognized by a neurofilament antibody. On the other hand, some TOH-positive cells contain VIP or SP. These observations suggest that under our culture conditions autonomic neural crest precursors differentiate only into immature sympathoblasts, but are able to synthesize peptides in addition to CA.

Tetanus toxin binds with high affinity to neuroblastoma × glioma hybrid cells NG 108-15 and impairs their stimulated acetylcholine release.

Abstract Differentiated neuroblastoma x glioma hybrid cells NG 108-15 express on their surface specific binding sites for tetanus toxin. 450 sites/cell with a KD of 2 x 10(-11) M were found under conditions of pH and salt concentrations. A Hill coefficient of 1.1 indicated noncooperative binding. Specific binding of 125I-toxin to its sites could be prevented either by preincubation of the toxin with a neutralizing monoclonal antibody or by pretreatment of the cells with neuraminidase (Vibrio cholerae). To quantify the action of tetanus toxin on the stimulated release of 14C activity from differentiated cells preincubated with [14C]choline, a new type of perfusion device was designed which could be filled with cells growing in monolayers on Cytodex-3 microbeads. Tetanus toxin inhibited the stimulated 14C release in a time- and dose-dependent manner. A greater than 50% inhibition was found after 2 h of incubation with 10(-12) M toxin. The inhibitory action of tetanus toxin could be prevented with a monoclonal antibody to the toxin or with neuraminidase treatment of the cells. These results suggest that the neuraminidase-sensitive 2 x 10(-11) KD receptors are the productive receptors for tetanus intoxication in differentiated NG 108-15 cells. The possible chemical composition of these receptors is discussed. Differentiated NG 108-15 cells provide a useful model in which picomolar tetanus concentrations produce both measurable saturable binding and inhibition of potassium-evoked, acetylcholine release under physiological conditions of pH and salt concentrations.

Cell lineages in peripheral nervous system ontogeny: Medium-induced modulation of neuronal phenotypic expression in neural crest cell cultures

Neural crest, taken from cephalic and trunk levels of quail embryos, was grown in vitro in conventional tissue culture medium (Dulbecco's modified Eagle's medium containing 15% fetal calf serum and either 2 or 15% chick embryo extract (CEE)) or in a chemically defined serum- and CEE-free medium. Depending on the conditions employed, different types of neuronal or neuronlike cells developed in the cultures. Thus, in medium containing 15% CEE, adrenergic cells (identified by tyrosine hydroxylase immunoreactivity and catecholamine histofluorescence) emerged after 5–6 days. These cells lacked tetanus toxin binding sites and did not react with an antibody directed against 70-kDa neurofilament protein. In the fully defined medium, a neuronal cell type exhibiting neurofilament and substance P (SP) immunoreactivity differentiated from noncycling precursors within 1 or 2 days of culture. If serum was added to the medium, the neurites disintegrated and the neuronal cells ultimately died. By sequentially culturing neural crest, first in the wholly synthetic medium for 1–3 days and then in the conventional medium supplemented with serum and 15% CEE, the disappearance of the SP-positive neurons was followed, several days later, by the emergence of adrenergic cells. The majority of these cells and/or their precursors were found to undergo cell division in culture. We conclude that the cells expressing the adrenergic phenotype (characteristic of the sympathetic nervous system) and those displaying SP immunoreactivity, comparable to a category of neurons in dorsal root and cranial sensory ganglia, derive from distinct sets of precursors. Our results reinforce the contention, deduced from in ovo transplantation experiments (see N. M. Le Douarin, (1984) In Cellular and Molecular Biology of Neuronal Development (I. Black, Ed.), pp. 3–28. Plenum, New York), that at least two lineages, from which sensory and autonomic cell types are derived respectively, are segregated early during neural crest ontogeny and have extremely different survival and trophic requirements.

Neural induction: embryonic determination elicits full expression of specific neuronal traits

ABSTRACT In Pleurodeles waltl, the early neuronal differentiation of precursor cells from late gastrula stage has been studied by culture in vitro from either isolated neural plate (NP) or isolated neural fold (NF). The aim of this study was to delineate the information acquired by ectodermal target cells during neural induction. By culturing these cells in vitro either with or without the underlying chordamesoderm, we showed that in the absence of chordamesodermal influence such NP or NF cells exhibited a high degree of biochemical and morphological differentiation as revealed by the synthesis and the storage of neurotransmitters, the activity of specific enzymes, as well as by the expression of neuronal markers: specific changes in cell surface carbohydrates, tetanus toxin binding sites and neuro filament polypeptides. Remarkable changes in the cell adhesive properties were the first events observed in the different central (NP) and peripheral (NF) types. In cocultures the chordamesodermal cells exert a beneficial influence on this differentiation, specially increasing acetylcholine synthesis. There are some differences between central (NP) or peripheral (NF) neuroblast response to this further notochord or mesodermal influence.

Tetanus toxin labeling as a novel rapid and highly specific tool in human neuroblastoma differential diagnosis

Tetanus toxin (TT) was used as a diagnostic marker for human neuroblastoma (NB) cells. TT binding sites visualized by TT and FITC-conjugated anti-TT antibodies were present on NB cells from all 13 cases studied comprising Stages II, III, IV, IVS and histologic grades 1 through 3. NB cells from both bone marrow aspirates and tumor biopsies as well as cultured NB cells were TT-positive. Diagnosis of NB was further ascertained by electron microscopy, cell culture, and quantitative determinations of catecholamines in tumor material. Only electron microscopic diagnoses had an accuracy comparable to that of TT labeling. None of the non-NB tumors (Ewing's sarcoma, acute lymphatic and myeloic leukemia, acute monocyte leukemia, chronic myeloic leukemia, Hodgkin's disease, oat cell carcinoma of the lung, pheochromocytoma), except for the pheochromocytoma, were found to bind TT specifically. These results suggest that TT may be profitably employed as a diagnostic marker of human NB cells. The advantages of the methods are its high discriminative capacity against non-NB cells and rapid applicability.

Tetanus toxin affects the K +-stimulated release of catecholamines from nerve growth factor-treated PC12 cells

Tetanus toxin specifically binds to neuronal surfaces and interferes with the release of transmitters. The effect of tetanus toxin pretreatment of PC12 cell line, taken as a model of neuronal cells in culture, was studied and found that it depresses depolarization-dependent catecholamines secretion. This effect is limited to PC12 cells fully differentiated by the action of Nerve Growth Factor (NGF) and is indicative of the expression of specific binding sites for tetanus toxin during transition from the undifferentiated state. Specific binding of [ 125I] tetanus toxin to NGF-treated PC12 was demonstrable. The toxin has no effect on the 45Ca accumulation coupled with the depolarization dependent release of catecholamines.

Ultrastructural Immunocytochemistry of Tetanus Toxin Binding Sites to Spinal Ganglia of Chick Embryo in Tissue Culture

By the use of immunoperoxidase technique, tetanus toxin binding sites were ultrastructurally demonstrated in monolayer culture after three days in cultures derived from the spinal ganglia of the 10 daychick embryo. The toxin binding sites, seen as electron dense reaction products, were clearly limited to the surface membranes of the neuronal processes, but not of the nerve cell bodies.

Neuronal acquisition of tetanus toxin binding sites: Relationship with the last mitotic cycle

In an earlier study on the developing nervous system, the existence of a temporal correlation between the appearance of tetanus toxin-binding cells and neurogenesis was reported (A. Koulakoff, B. Bizzini, and Y. Berwald-Netter (1982). Dev. Brain Res. 5, 139–147) . Using a combined approach of immunocytochemistry and [ 3H]thymidine autoradiography it is shown that, in the fetal mouse central nervous system, dividing cells do not express membrane binding sites for tetanus toxin. A time-course quantitative autoradiography revealed that the toxin-binding sites become apparent within 7 ± 1 hr, following the last S phase, on cells undergoing the conversion from dividing to postmitotic state. The acquisition of surface binding sites for tetanus toxin may thus be an early property of nascent central neurons, marking the transition from cycling precursor neuroblasts to postmitotic neuronal cells. Parallel studies on in vivo-developing dorsal root ganglia disclosed that at least some peripheral nervous system cells are endowed with tetanus toxin-binding capacity while still capable of DNA synthesis and undergo one or more divisions.

Expression of differentiation markers by chick embryo neuroretinal cells in vivo and in culture

Several markers of chick neuroretinal differentiation were monitored in vivo and in culture. All increase markedly between 7 and 20 days of embryonic development in vivo. In vitro, endogenous GABA levels decrease almost immediately, while other neuronal markers increase as in vivo for 2 to 5 days before declining (choline acetyltransferase, acetyl cholinesterase, glutamic acid decarboxylase). Neuronal cell surface markers (binding sites for tetanus toxin, alpha-bungarotoxin, muscimol), however, reach maximal levels only after 8 days in vitro. Glial markers such as carbonic anhydrase and hydrocortisone-induced glutamine synthetase activities are also expressed only transiently in culture.

Neurons from chick embryo cerebrum: Ultrastructural and biochemical development in vitro

Primary cultures of neurons prepared from dissociated telencephalon of 8-day-old chick embryos exhibited characteristic differentiated morphology with an interconnecting neurofibrillary network that became increasingly ramified during the 15-day period of culture. All the cells in the culture can be stained positively using the Sevier-Munger silver impregnation technique. The cells and fibers also contain tetanus toxin binding sites and are heavily stained by antitubulin, as visualized by indirect immunofluorescence. Cell processes containing abundant microtubules organized in parallel arrays were observed by immunofluorescence and electron microscopy. Transmission electron microscopy of processes also revealed vesicle-containing boutons with synaptic attachments to other cells. These cultures are free of glial contamination since significant levels of glial markers, glial fibrillary acidic protein or glutamine synthetase activity, were not detected. In addition, cells with the microtubular pattern characteristic of glia or fibroblasts were not observed in typical microscopic fields. Neuronal glutamic acid decarboxylase activity was comparable to that of adult brain while tyrosine hydroxylase and choline acetyltransferase activities were not detectable. Crude membrane preparations from the cultured neurons contain receptors for γ-aminobutyric acid and benzodiazepine at levels which are 30% and 14% those of adult brain. The chick cerebral cultures thus appear enriched in GABAergic neurons. It can be concluded that development of neuron-specific structural and biochemical markers can occur in the absence of glial cells. The culture system therefore provides a useful model for studies of central neurons in vitro.

Cell surface and cytoskeletal antigens in cerebral cell cultures after chloroform-methanol delipidation.

Fixation and in situ total delipidation of nerve cells in monolayer cultures have been used for immunofluorescence visualization of cellular antigens. Cells grown on poly-L-lysine-coated coverslips are exposed to a chloroform/methanol (2/1 by vol) solvent mixture for 15 min at -65 degrees C. This step results in the fixation of cells and is accompanied by the complete extraction of cellular lipids. About 3.2% of the total 35S-methionine-labeled trichloroacetic acid-insoluble cellular material is extracted into the organic solvent mixture, demonstrating that aqueous soluble proteins are not significantly extracted under these conditions. A total loss of tetanus toxin binding sites, presumably reflecting the removal of polysialogangliosides from the cell surface, is observed. The accessibility of antibodies against tubulin and against intermediate filaments after this treatment has been investigated. Visualization of these cytoskeletal elements by indirect immunofluorescence and by phase microscopy suggests that the removal of cellular lipids does not affect the apparent cytoarchitecture and that extraction with these organic solvents does not interfere with the staining of tubulin and intermediate filaments, the latter being exclusively present in nonneuronal cells. The advantage of this method of cell fixation and lipid extraction for immunofluorescence of cells in monolayer culture is discussed.

Expression of neuronal markers in chick and quail embryo neuroretina cultures infected with rous sarcoma virus

Cultures of neuroretina (NR) cells from 7-day chick and quail embryos were infected with ts NY-68, a thermosensitive mutant of Rous sarcoma virus (RSV) which transformed NR cells at 36°C. The following differentiation markers for neurones were studied: tetanus toxin-binding sites at the cell surfaces, presence of synapses, and the specific activity of the enzymes choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD). Appearance of synapses and expression of CAT were similar in control and transformed cultures. Tetanus toxin-binding cells were observed in transformed primary cultures and also in quail NR subcultures. GAD-specific activity was markedly stimulated in chick and quail primary cultures transformed by ts NY-68 and further increased in sub-cultures of ts NY-68-transformed quail NR cells. Stimulation of GAD activity is controlled by the transforming ( src) gene of RSV since it was not observed in cultures infected with RAV-1, a leukosis virus which lacks the src gene. These data show that infection of chick quail NR cultures with RSV results in the transformation of cells with neuronal markers.

Neurotoxins as probes in the study of neuronal development

We have investigated the expression of surface membrane binding sites for tetanus toxin and α-scorpion toxin (AaHII) on cells of the in vivo developing mouse nervous system. There is a close temporal correlation in the pattern of emergence and accumulation of tetanus toxin bindin cells (TBC) and that of post-mitotic neurons. In different nervous system areas, the fluctuations in relative TBC abundance reflect regional changes in the dynamics of neuronal subpopulations. The results indicate that the acquisition of membrane tetanus toxin binding sites may represent one of the earliest detectable characteristics of nascent neurons. The Na + channel-associated scorpion toxin receptors become detectable in fetal mouse brain two days after the appearance of TBC. Their density increases with fetal age without change in receptor properties. At all stages, scorpion toxin binds to a single class of noninteracting sites with a K D = 0. 1 − 0.5 nM. The affinity of binding is voltage-dependent. Studies on brain cells and various cell lines grown in vitro suggest a selective association of the high affinity scorpion toxin receptors with neuronal phenotype. In culture, as in vivo, there is a time dependent increase in receptor density. These results indicate that both tetanus toxin and scorpion toxin can be used as qualitative markers of neuronal differentiation; moreover, estimates of the density of scorpion toxin binding sites provide a quantitative index of neuronal maturation.