Neuronal sensitivity to tetanus toxin requires gangliosides.

Tetanus toxin produces spastic paralysis in situ by blocking inhibitory neurotransmitter release in the spinal cord. Although di- and trisialogangliosides bind tetanus toxin, their role as productive toxin receptors remains unclear. We examined toxin binding and action in spinal cord cell cultures grown in the presence of fumonisin B(1), an inhibitor of ganglioside synthesis. Mouse spinal cord neurons grown for 3 weeks in culture in 20 microM fumonisin B(1) develop dendrites, axons, and synaptic terminals similar to untreated neurons, even though thin layer chromatography shows a greater than 90% inhibition of ganglioside synthesis. Absence of tetanus and cholera toxin binding by toxin-horseradish peroxidase conjugates or immunofluorescence further indicates loss of mono- and polysialogangliosides. In contrast to control cultures, tetanus toxin added to fumonisin B(1)-treated cultures does not block potassium-stimulated glycine release, inhibit activity-dependent uptake of FM1-43, or abolish immunoreactivity for vesicle-associated membrane protein, the toxin substrate. Supplementing fumonisin B(1)-treated cultures with mixed brain gangliosides completely restores the ability of tetanus toxin to bind to the neuronal surface and to block neurotransmitter release. These data demonstrate that fumonisin B(1) protects against toxin-induced synaptic blockade and that gangliosides are a necessary component of the receptor mechanism for tetanus toxin.     

Analysis of mutants of tetanus toxin Hc fragment: ganglioside binding, cell binding and retrograde axonal transport properties

Tetanus toxin binds neuronal tissue prior to internalization and trafficking to the central nervous system. Binding of the carboxy-terminal 50 kDa HC fragment of tetanus toxin to polysialogangliosides is important for this initial cell binding step. Using the three-dimensional structure of HC, mutants were designed to investigate the role of individual residues in ganglioside binding. Mutant proteins were tested for binding to GT1b gangliosides, to primary motoneurons and for their ability to undergo retrograde transport in mice. Two classes of mutant were obtained: (i) those containing deletions in loop regions within the C-terminal beta-trefoil domain which showed greatly reduced ganglioside and cell binding and did not undergo retrograde transport and (ii) those that showed reduced ganglioside binding, but retained primary neuronal cell binding and retrograde transport. The second class included point mutants of Histidine-1293, previously implicated in GT1b binding. Our deletion analysis is entirely consistent with recent structural studies which have identified sugar-binding sites in the immediate vicinity of the residues identified by mutagenesis. These results demonstrate that ganglioside binding can be severely impaired without abolishing cell binding and intracellular trafficking of tetanus toxin.     

Neuronal binding of tetanus toxin compared to its ganglioside binding fragment (H(c))

The non-toxin 50 kD C-terminus peptide of the heavy chain of tetanus H(c) contains the ganglioside binding domain of tetanus toxin (TTX). H(c) retains much of the capacity of tetanus toxin for binding internalization and transport by neurons. For this reason tetanus H(c) has been studied as a vector for delivery of therapeutic proteins to neurons. We directly compared H(c) and TTX in the capacity to bind and be internalized by neurons by ELISA. Primary cultures of dissociated fetal cortical neurons were incubated with equimolar amounts of TTX or H(c). Neuronal associated tetanus protein was 4-8 fold greater on a molar basis with tetanus toxin compared to H(c) (1 h incubation). This increase in neuronal tetanus protein was evident with incubation in concentrations from 0.1 microM to 2 microM. There were greater amounts of TTX delivered to the cultured cells at both 0 degrees C (representing membrane bound tetanus protein) and 37 degrees C (bound and internalized tetanus protein). Unlike H(c), TTX showed significant continued accumulation of protein with increasing incubation durations. Neuronal associated TTX increased 2-3 fold over incubation times ranging from 1 to 8 h. Tetanus toxin appears to be clearly superior to the ganglioside binding fragment (H(c)) in the capacity for neuronal binding and internalization. Atoxic tetanus proteins containing additional molecular domains as well as H(c) may be more suitable vectors for linkage with therapeutic proteins and delivery to neurons.     

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

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 "physiological" 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.     

SV2 mediates entry of tetanus neurotoxin into central neurons

Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The toxin acts by inhibiting the release of neurotransmitters from inhibitory neurons in the spinal cord that innervate motor neurons and is unique among the clostridial neurotoxins due to its ability to shuttle from the periphery to the central nervous system. Tetanus neurotoxin is thought to interact with a high affinity receptor complex that is composed of lipid and protein components; however, the identity of the protein receptor remains elusive. In the current study, we demonstrate that toxin binding, to dissociated hippocampal and spinal cord neurons, is greatly enhanced by driving synaptic vesicle exocytosis. Moreover, tetanus neurotoxin entry and subsequent cleavage of synaptobrevin II, the substrate for this toxin, was also dependent on synaptic vesicle recycling. Next, we identified the potential synaptic vesicle binding protein for the toxin and found that it corresponded to SV2; tetanus neurotoxin was unable to cleave synaptobrevin II in SV2 knockout neurons. Toxin entry into knockout neurons was rescued by infecting with viruses that express SV2A or SV2B. Tetanus toxin elicited the hyper excitability in dissociated spinal cord neurons - due to preferential loss of inhibitory transmission - that is characteristic of the disease. Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons. Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals. Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms. In summary, the findings reported here indicate that SV2A and SV2B mediate binding and entry of tetanus neurotoxin into central neurons.     

构象稳定的破伤风毒素Hc 片段突变体 (HcM) 的制备及其免疫原性分析

破伤风是由破伤风杆菌侵入人体伤口、生长繁殖、产生毒素而引起的一种急性特异性感染,其死亡率高,严重危害人民生命健康。研究证实破伤风毒素重链C端 (Hc) 具有与毒素受体结合的活性,完全保留了全分子的免疫原性,有望开发成为新的基因工程破伤风亚单位疫苗以替换传统的甲醛灭活类毒素疫苗。由于野生型Hc蛋白 (HcW) 易形成分子间及分子内二硫键,且各构象分子之间易发生不稳定的转换,为疫苗的生产工艺带来困难,因此,通过将破伤风HcW蛋白的869位半胱氨酸突变为丙氨酸,构建构象稳定的破伤风亚单位疫苗突变体HcM,对Hc     

Lipid rafts act as specialized domains for tetanus toxin binding and internalization into neurons

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Although its intracellular catalytic activity is well established, the mechanism by which this neurotoxin interacts with the neuronal surface is not known. In this study, we characterize p15s, the first plasma membrane TeNT binding proteins and we show that they are glycosylphosphatidylinositol-anchored glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, spinal cord cells, and purified motor neurons. We identify p15 as neuronal Thy-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microscopy measurements confirm the close association of the binding domain of TeNT and Thy-1 at the plasma membrane. We find that TeNT is recruited to detergent-insoluble lipid microdomains on the surface of neuronal cells. Finally, we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indicate that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neurons.     

Affinity purified tetanus toxin binds to isolated chromaffin granules and inhibits catecholamine release in digitonin-permeabilized chromaffin cells

Tetanus toxin, a potent neurotoxin which blocks neurotransmitter release in the CNS, also inhibits Ca2+-induced catecholamine release from digitonin-permeabilized, but not from intact bovine chromaffin cells. In searching for intracellular targets for the toxin we studied the binding of affinity-purified tetanus toxin to bovine adrenal chromaffin granules. Tetanus toxin bound in a neuraminidase-sensitive fashion to intact granules and to isolated granule membranes, as assayed biochemically and visualized by electron microscopic techniques. The binding characteristics of the toxin to chromaffin granule membranes are very similar to the binding of tetanus toxin to brain synaptosomal membranes. We suggest that the toxin-binding site is a glycoconjugate of the G1b type (a polysialoganglioside or a glycoprotein-proteoglycan) which is localized on the cytoplasmic face of the granule membrane and might directly be involved in exocytotic membrane fusion.     

Oxygen or glucose deprivation-induced neuronal injury in cortical cell cultures is reduced by tetanus toxin.

We examined glutamate-mediated neurotoxicity in cortical cell cultures pretreated with 1-5 micrograms/ml tetanus toxin to attenuate the Ca(2+)-dependent release of neurotransmitters. Efficacy of the tetanus toxin pretreatment was suggested by blockade of electrical burst activity induced by Mg2+ removal and by reduction of glutamate efflux induced by high K+. Tetanus toxin reduced neuronal injury produced by brief exposure to elevated extracellular K+ or to glutamate, situations in which release of endogenous excitatory neurotransmitter is likely to play a role. Furthermore, although glutamate efflux evoked by anoxic conditions may occur largely via Ca(2+)-independent transport, tetanus toxin attenuated both glutamate efflux and neuronal injury following combined oxygen and glucose deprivation. With prolonged exposure periods, the neuroprotective efficacy of tetanus toxin was comparable to that of NMDA receptor antagonists. Presynaptic inhibition of Ca(2+)-dependent glutamate release may be a valuable approach to attenuating hypoxic-ischemic brain injury.     

VAMP2, but not VAMP3/cellubrevin, mediates insulin-dependent incorporation of GLUT4 into the plasma membrane of L6 myoblasts

Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.     

Isolated Light Chain of Tetanus Toxin Inhibits Exocytosis: Studies in Digitonin-Permeabilized Cells

Previous work indicates that the heavy chain of tetanus toxin is responsible for the binding of the toxin to the neuronal membrane and its subsequent internalization. In the present study, the light chain of tetanus toxin mimicked the holotoxin in inhibiting Ca2+-dependent secretion of [3H]norepinephrine from digitonin-permeabilized adrenal chromaffin cells. Preincubation of tetanus toxin with monoclonal antibodies to the light chain prevented the inhibition by tetanus toxin. Preincubation of tetanus toxin with nonimmune ascites fluid or with monoclonal antibodies directed against the C fragment (the C-terminal of the heavy chain) or the heavy-chain portion of the B fragment did not prevent inhibition by tetanus toxin. The data indicate that the light chain is responsible for the intracellular blockade of exocytosis.     

Studies on the intoxication pathway of tetanus toxin in the rat pheochromocytoma (PC12) cell line. Binding, internalization, and inhibition of acetylcholine release

Tetanus toxin was found to be a potent inhibitor of neurosecretion in the rat pheochromocytoma cell line PC12, a system in which biochemical and functional studies could be performed in parallel. Incubation of the cells with 10 nM tetanus toxin (3 h) led to an inhibition of acetylcholine release by 75-80% when evoked by 200 microM veratridine, 1 mM carbachol, or 2 mM Ba2+. The main characteristics of the inhibition process are: 1) the toxin is very potent, with threshold doses of 10 pM; 2) the action of toxin is blocked at low temperature (0 degrees C) and by antitoxin; 3) the effects are dose- and time-dependent; 4) a concentration-dependent lag phase precedes the onset of the inhibitory effects. Thus the PC12 cultures are a valid system for studies on the underlying molecular process in tetanus action. This system was exploited by the use of long term incubation studies to examine the processes responsible for the lag phase. When cells were incubated with 0.1 nM 125I-tetanus toxin, cell-associated toxin reached a plateau of 16 fmol of toxin/mg of protein, yet the toxic effects did not appear until 12 h. Further, PC12 cells were found to rapidly internalize tetanus toxin, with a half-life of 1-2 min, once it was bound to the surface of the cells. Thus, the lag phase results from steps that occur in the intracellular compartment after internalization. An important discovery was that the differentiation state of the PC12 cells was a critical factor in determining sensitivity to tetanus toxin. Cells that were cultured with nerve growth factor for 8-12 days were very sensitive to toxin. In contrast, acetylcholine release from nondifferentiated, autodifferentiated, or dexamethasone-treated cultures was insensitive to tetanus toxin. Since differential expression of high affinity tetanus toxin receptors cannot explain these results, it is concluded that PC12 cells are capable of expressing different forms of excitation-secretion coupling mechanisms. Tetanus toxin should prove a valuable probe to further distinguish these processes.     

Two carbohydrate binding sites in the H(CC)-domain of tetanus neurotoxin are required for toxicity

Tetanus neurotoxin binds via its carboxyl-terminal H(C)-fragment selectively to neurons mediated by complex gangliosides. We investigated the lactose and sialic acid binding pockets of four recently discovered potential binding sites employing site-directed mutagenesis. Substitution of residues in the lactose binding pocket drastically decreased the binding of the H(C)-fragment to immobilized gangliosides and to rat brain synaptosomes as well as the inhibitory action of recombinant full length tetanus neurotoxin on exocytosis at peripheral nerves. The conserved motif of S(1287)XWY(1290) em leader G(1300) assisted by N1219, D1222, and H1271 within the lactose binding site comprises a typical sugar binding pocket, as also present, for example, in cholera toxin. Replacement of the main residue of the sialic acid binding site, R1226, again caused a dramatic decline in binding affinity and neurotoxicity. Since the structural integrity of the H(C)-fragment mutants was verified by circular dichroism and fluorescence spectroscopy, these data provide the first biochemical evidence that two carbohydrate interaction sites participate in the binding and uptake process of tetanus neurotoxin. The simultaneous binding of one ganglioside molecule to each of the two binding sites was demonstrated by mass spectroscopy studies, whereas ganglioside-mediated linkage of native tetanus neurotoxin molecules was ruled out by size exclusion chromatography. Hence, a subsequent displacement of one ganglioside by a glycoprotein receptor is discussed.     

Expression and characterisation of the heavy chain of tetanus toxin: reconstitution of the fully-recombinant dichain protein in active form.

Tetanus toxin, composed of a disulphide-linked heavy (HC) and light (LC) chain, preferentially blocks the release of inhibitory neurotransmitters in the spinal cord by Zn2+-dependent proteolytic cleavage of synaptobrevin. This intoxication involves binding via HC to ecto-acceptors on peripheral nerve endings, followed by internalisation and retrograde transportation to its prime site of action in central neurons. To facilitate exploitation of the toxin's unique activities, HC was expressed at a high level in Escherichia coli as a fusion with maltose binding protein; after cleavage by thrombin, free HC was isolated and its identity confirmed by Western blotting and N-terminal microsequencing. The expressed and native HC gave very similar circular dichroism spectra, excluding any gross differences in their folded structures. Recombinant HC antagonised the neuromuscular paralysing activity of the native toxin, by competing for binding to neuronal ecto-acceptors. The HC was reconstituted with bacterially-expressed LC to create disulphide-bridged dichain toxin that blocked neuromuscular transmission. The fully-recombinant toxin produced spastic paralysis in mice characteristic of the blockade of central inhibitory synapses, revealing that it undergoes axonal transport to the spinal cord, like the native toxin but with a reduced efficacy. This first report of the large-scale production of recombinant tetanus toxin in active form should facilitate studies on the use of engineered innocuous forms of the toxin as neuronal transport vehicles.     

Identification and partial characterization of a low affinity metal-binding site in the light chain of tetanus toxin.

Tetanus toxin was shown to contain a metal-binding site for zinc and copper. Equilibrium dialysis binding experiments using 65Zn indicated an association constant of 9-15 microM, with one zinc-binding site/toxin molecule. The zinc-binding site was localized to the toxin light chain as determined by binding of 65Zn to the light chain but not to the heavy chain after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to Immobilon membranes. Copper was an efficient inhibitor of 65Zn binding to tetanus toxin and caused two peptide bond cleavages in the toxin light chain in the presence of ascorbate. These metal-catalyzed oxidative cleavages were inhibited by the presence of zinc. Partial characterization of metal-catalyzed oxidative modifications of a peptide based on a putative metal-binding site (HELIH) in the toxin light chain was used to map the metal-binding site in the protein.     

Affinity purification and partial characterization of the zonulin/zonula occludens toxin (Zot) receptor from human brain

The intercellular tight junctions (TJs) of endothelial cells represent the limiting structure for the permeability of the blood-brain barrier (BBB). Although the BBB has been recognized as being the interface between the bloodstream and the brain, little is known about its regulation. Zonulin and its prokaryotic analogue, zonula occludens toxin (Zot) elaborated by Vibrio cholerae, both modulate intercellular TJs by binding to a specific surface receptor with subsequent activation of an intracellular signaling pathway involving phospholipase C and protein kinase C activation and actin polymerization. Affinity column purification revealed that human brain plasma membrane preparations contain two Zot binding proteins of approximately 55 and approximately 45 kDa. Structural and kinetic studies, including saturation and competitive assays, identified the 55-kDa protein as tubulin, whereas the 45-kDa protein represents the zonulin/Zot receptor. Biochemical characterization provided evidence that this receptor is a glycoprotein containing multiple sialic acid residues. Comparison of the N-terminal sequence of the zonulin/Zot receptor with other protein sequences by BLAST analysis revealed a striking similarity with MRP-8, a 14-kDa member of the S-100 family of calcium binding proteins. The discovery and characterization of this receptor from human brain may significantly contribute to our knowledge on the pathophysiological regulation of the BBB.     

Effects of Tetanus Toxin on Catecholamine Release from Intact and Digitonin-Permeabilized Chromaffin Cells

Abstract: Tetanus exotoxin inhibited Ca²⁺-dependent cate-cholamine secretion in a dose-dependent manner in digito-nin-permeabilized chromaffin cells. The inhibition was specific for tetanus exotoxin and the B fragment of tetanus toxin; the C fragment had no effect. Inhibition required the introduction of toxin into the cell, and was not seen when intact cells were preincubated with the toxin or toxin fragments. The degree of inhibition was related to the length of preincubation with toxin, as well as the concentration of toxin used. A short preincubation with toxin was sufficient to inhibit secretion, and the continued presence of toxin in the incubation medium was not required during the incubation with Ca²⁺. The inhibition of secretion by tetanus toxin or the B fragment was not overcome with increasing Ca²⁺ concentrations. Tetanus toxin also inhibited catechol-amine secretion enhanced by phorbol ester-induced activation of protein kinase C. Thus, the toxin or a proteolytic fragment of the toxin can enter digitonin-permeabilized cells to interact with a component of the Ca²⁺-dependent exocytotic pathway to inhibit secretion.     

Molecular basis for tetanus toxin coreceptor interactions

Tetanus toxin (TeNT) elicits spastic paralysis through the cleavage of vesicle-associated membrane protein-2 (VAMP-2) in neurons at the interneuronal junction of the central nervous system. While TeNT retrograde traffics from peripheral nerve endings to the interneuronal junction, there is limited understanding of the neuronal receptors utilized by tetanus toxin for the initial entry into nerve cells. Earlier studies implicated a coreceptor for tetanus toxin entry into neurons: a ganglioside binding pocket and a sialic acid binding pocket and that GT1b bound to each pocket. In this study, a solid phase assay characterized the ganglioside binding specificity and functional properties of both carbohydrate binding pockets of TeNT. The ganglioside binding pocket recognized the ganglioside sugar backbone, Gal-GalNAc, independent of sialic acid-(5) and sialic acid-(7) and GM1a was an optimal substrate for this pocket, while the sialic acid binding pocket recognized sialic acid-(5) and sialic acid-(7) with "b"series of gangliosides preferred relative to "a" series gangliosides. The high-affinity binding of gangliosides to TeNT HCR required functional ganglioside and sialic acid binding pockets, supporting synergistic binding to coreceptors. This analysis provides a model for how tetanus toxin utilizes coreceptors for high-affinity binding to neurons.     

Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc.

Tetanus and botulinum neurotoxins are the most potent toxins known. They bind to nerve cells, penetrate the cytosol and block neurotransmitter release. Comparison of their predicted amino acid sequences reveals a highly conserved segment that contains the HexxH zinc binding motif of metalloendopeptidases. The metal content of tetanus toxin was then measured and it was found that one atom of zinc is bound to the light chain of tetanus toxin. Zinc could be reversibly removed by incubation with heavy metal chelators. Zn2+ is coordinated by two histidines with no involvement in cysteines, suggesting that it plays a catalytic rather than a structural role. Bound Zn2+ was found to be essential for the tetanus toxin inhibition of neurotransmitter release in Aplysia neurons injected with the light chain. The intracellular activity of the toxin was blocked by phosphoramidon, a very specific inhibitor of zinc endopeptidases. Purified preparations of light chain showed a highly specific proteolytic activity against synaptobrevin, an integral membrane protein of small synaptic vesicles. The present findings indicate that tetanus toxin, and possibly also the botulinum neurotoxins, are metalloproteases and that they block neurotransmitter release via this protease activity.     

Neural induction and in vitro initial expression of neurofilament and tetanus toxin binding site molecules in amphibians

Tetanus toxin (Tt) binding site and neurofilament (NIF), the intermediate-sized filaments, are neuronal markers essentially described in mammals and birds; are these molecular markers present in urodela neuronal cells and are they expressed immediately after neural induction? Our findings are based on immunofluorescent localization of NIF and Tt proteins using three previously characterized antisera against 200 kDa and 70 kDa neurofilament components and against fragment IIc derived from purified tetanus toxin. Embryonic undifferentiated neuronal cells from Pleurodeles waltlii neural plate and/or neural fold (early neurula stage) are cultured isolated in vitro without further chordamesodermal influence. At the beginning of the culture none of the undifferentiated neuronal precursors bind antibodies against NIF or Tt components. The binding is detected when phenotypical differentiation takes place (2/3-day cultures). Both the cell bodies and the cell processes are stained. After 2-3 weeks, immunostaining of the neurones is very distinctive and bright; the non-neuronal cultured cells do not exhibit any labelling. These observations indicate the early acquisition of NIF and Tt binding site expression by neuronal precursor cells (late gastrula stage).