Ultrastructural changes in differentiating neuroblastoma x glioma hybrid cells

The ultrastructure of differentiating neuroblastoma x glioma hybrid cells NG108-15 was observed. Cells cultured in growth medium showed undifferentiated features, while cells treated with dBcAMP became round and large, and extended thick long neurites. After 1 week in culture, cells showed features similar to those of normal neurons. The dense cored vesicles with diameters ranging from 60 to 170 nm were observed in differentiated NG108-15 cells, but clear vesicles were usually rare. However, in the case of co-culture with striated myotubes, clusters of clear vesicles appeared in the neurites and terminals. The timecourse of the differentiation process was correlated with results obtained by the electrophysiology and freeze-fracture.     

Ultrastructural changes in differentiating neuroblastoma × glioma hybrid cells

The ultrastructure of differentiating neuroblastoma × glioma hybrid cells NG108-15 was observed. Cells cultured in growth medium showed undifferentiated features, while cells treated with dBcAMP became round and large, and extended thick long neurites. After 1 week in culture, cells showed features similar to those of normal neurons. The dense cored vesicles with diameters ranging from 60 to 170 nm were observed in differentiated NG108-15 cells, but clear vesicles were usually rare. However, in the case of co-culture with striated myotubes, clusters of clear vesicles appeared in the neurites and terminals. The timecourse of the differentiation process was correlated with results obtained by the electrophysiology and freeze-fracture.     

The time course of synapse formation of mouse neuroblastoma cells in monolayer cultures

Summary Neuroblastoma cells grown on substrates in culture develop long processes and assume the morphology of normal neurons as judged light microscopically. The development of synapses in the cultured tissue is studied by periodic electron microscopic examination of the areas of contact between cells. The initial expiants are free of any apparent synaptic contacts. After 48 h in culture, simple swellings or boutons are detected at the periphery of the cells or at the end of the fine processes. These initial synaptic profiles contain a few vesicles but lack mitochondria. The synaptic vesicles appear to originate from the smooth endoplasmic reticulum. Further expiants remain primitive, only the number of vesicles in the cytoplasmic swellings or boutons increases. These clusters of vesicles are 40–60 nm in diameter and morphologically distinguishable from the synaptic vesicles of normal neurons. There are no postsynaptic folds or membrane thickenings. Specialized cell contacts between cells are also present.     

Neuronal galvanotropism is independent of external Ca(2+) entry or internal Ca(2+) gradients

The mechanism by which growing neurites sense and respond to small applied electrical fields is not known, but there is some evidence that the entry of Ca(2+) from the external medium, with the subsequent formation of intracellular Ca(2+) gradients, is important in this process. We have employed two approaches to test this idea. Xenopus spinal neurites were exposed to electrical fields in a culture medium in which Ca(2+) was chelated to very low levels compared to the normal extracellular concentration of 2 mM. In other experiments, loading the neurites with the calcium buffer, 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), disrupted the putative internal Ca(2+) gradients, and the effects on the electrical response were determined. Fields of 100 mV/mm were applied for 12 h, and no difference was detected in the cathodal turning response between the treated neurites and the untreated controls. Using the Differential Growth Index (DGI), an asymmetry index, to quantitate the turning response, we recorded DGIs of -0.64, -0.65, and -0.62 for control cells, cells in Ca(2+)-free medium, and cells preloaded with BAPTA, respectively. Furthermore, we detected an increase in neurite length for those neurons cultured in Ca(2+)-free medium; they were 1.5-1.7 times as long as neurites from neurons cultured in normal Ca(2+) medium. Likewise, we found that BAPTA-loaded neurites were longer than control neurites. Our data indicate that neuronal galvanotropism is independent of the entry of external Ca(2+) or of internal Ca(2+) gradients. Both cell-permeant agonistic and antagonistic analogs of cyclic 3',5'-adenosine monophosphate (cAMP) increased the response to applied electrical fields.     

Factors influencing the release of proteins by cultured schwann cells

Cultured rat schwann cells grown in association with sensory neurons when labeled with [(3)H]leucinem, [(3)H]glucosamine, or [(35)S]methionine release labeled polypeptides into the culture medium. Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the culture medium reveals a reproducible pattern of more than 20 polypeptides with molecular weights ranging from 15,000 to more than 250,000. Five major polypeptides (apparent molecular weights 225,000, 210,000, 90,000, 66,000, 50,000, and 40,000) account for approximately 40 percent of the leucine or methionine radioactivity in medium polypeptide. Schwann cells grown in a serum-free defined medium, in which schwann cells do not relate normally to axons, release approximately four times less labeled medium polypeptides tha cultures grown in medium supplemented with serum and chick embryo extract. In addition, there is a qualitative difference in the pattern of medium polypeptides resolved by SDS-PAGE, so that a single polypeptide (mol wt 40,000) accounts for nearly all of the label in medium polypeptides. Switching of cultures grown in defined medium to supplemented medium for 2 d results in a fourfold increase in the amount of labeled polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides as resolved by SDS-PAGE. This change in the pattern of polypeptides release by schwann cells is accompanied by changes in the association between schwann cells and axons. An early step in the establishment of normal axon-schwann cell relations appears to be an inward migration of schwann cells into axonal bundles and spreading of schwann cells along neurites. These changes are evident within 48 h after medium shift. Our results thus suggest that the release of proteins by schwann cells may be important for the development of normal axonal ensheathment.     

Axonal synthesis of phosphatidylcholine is required for normal axonal growth in rat sympathetic neurons

The goal of this study was to assess the relative importance of the axonal synthesis of phosphatidylcholine for neurite growth using rat sympathetic neurons maintained in compartmented culture dishes. In a double-labeling experiment [14C]choline was added to compartments that contained only distal axons and [3H]choline was added to compartments that contained cell bodies and proximal axons. The specific radioactivity of labeled choline was equalized in all compartments. The results show that approximately 50% of phosphatidylcholine in distal axons is locally synthesized by axons. The requirement of axonal phosphatidylcholine synthesis for neurite growth was investigated. The neurons were supplied with medium lacking choline, an essential substrate for phosphatidylcholine synthesis. In the cells grown in choline-deficient medium for 5 d, the incorporation of [3H]palmitate into phosphatidylcholine was reduced by 54% compared to that in cells cultured in choline-containing medium. When phosphatidylcholine synthesis was reduced in this manner in distal axons alone, growth of distal neurites was inhibited by approximately 50%. In contrast, when phosphatidylcholine synthesis was inhibited only in the compartment containing cell bodies with proximal axons, growth of distal neurites continued normally. These experiments imply that the synthesis of phosphatidylcholine in cell bodies is neither necessary nor sufficient for growth of distal neurites. Rather, the local synthesis of phosphatidylcholine in distal axons is required for normal growth.     

Neuronal galvanotropism is independent of external Ca2+ entry or internal Ca2+ gradients

The mechanism by which growing neurites sense and respond to small applied electrical fields is not known, but there is some evidence that the entry of Ca²⁺ from the external medium, with the subsequent formation of intracellular Ca²⁺ gradients, is important in this process. We have employed two approaches to test this idea. Xenopus spinal neurites were exposed to electrical fields in a culture medium in which Ca²⁺ was chelated to very low levels compared to the normal extracellular concentration of 2 mM. In other experiments, loading the neurites with the calcium buffer, 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA), disrupted the putative internal Ca²⁺ gradients, and the effects on the electrical response were determined. Fields of 100 mV/mm were applied for 12 h, and no difference was detected in the cathodal turning response between the treated neurites and the untreated controls. Using the Differential Growth Index (DGI), an asymmetry index, to quantitate the turning response, we recorded DGIs of −0.64, −0.65, and −0.62 for control cells, cells in Ca²⁺‐free medium, and cells preloaded with BAPTA, respectively. Furthermore, we detected an increase in neurite length for those neurons cultured in Ca²⁺‐free medium; they were 1.5–1.7 times as long as neurites from neurons cultured in normal Ca²⁺ medium. Likewise, we found that BAPTA‐loaded neurites were longer than control neurites. Our data indicate that neuronal galvanotropism is independent of the entry of external Ca²⁺ or of internal Ca²⁺ gradients. Both cell‐permeant agonistic and antagonistic analogs of cyclic 3′,5′‐adenosine monophosphate (cAMP) increased the response to applied electrical fields. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 30–38, 2000     

Morphological differentiation of neuroblastoma cells induced by dimethylsulfoxide

Morphological features of neuroblastoma cells grown in culture in the presence of dimethylsulfoxode (DMSO) were studied. Morphological differentiation, expressed as the appearance of long axon-like processes (neurites), an increase in size of the cells, and inhibition of cell division, was observed in neuroblastoma cells of line C 1300, subline N-18-TG₂A₁, incubated in medium containing 1% DMSO. In the early stages of culture in normal growth medium the cells possess primary features of morphological differentiation. Quantitative criteria for the development of these features depending on duration of culture in modified medium were worked out. An increase in the total length of the neurites of cells differentiating under the influence of DMSO is a linear function of time. The rate of growth of the neurites is 20.0±3.0 µ/h. The area of cross-section of the soma of the differentiated cells is 6–7 times greater than the corresponding parameter in the control. An increase in the DMSO concentration in the culture medium (1.5 and 2.0%) does not induce rapid growth of the neurites or an increase in size of the cell soma, but it does block mitosis. Characteristics of morphological differentiation of neuroblastoma cells are compared with probable functional changes in these cells.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 519–527, July–August, 1984.     

Laminin induces formation of neurite-like processes and potentiates prolactin secretion by GH3 rat pituitary cells

Tumor-derived GH3 rat pituitary cell lines are widely utilized to study mechanisms of prolactin secretion and responsiveness to secretagogues. These cells served here as a model with which to study relationships between shape and function. When GH3 cells were routinely grown in serum-supplemented medium, they exhibited the polygonal phenotype of epithelial cells, with scarce secretory granules. In contrast, when seeded in a serum-free medium, they attached loosely and contained more secretory granules. In both cases, they released prolactin in a nonpolarized manner. We show in the present work that laminin extracted from Englebreth-Holm-Swarm (EHS) tumors was a potent attachment and spreading factor for GH3/B6 cells seeded in serum-free medium. Moreover, it induced the formation of neurite-like processes, which were increased in number and length by chronic treatment with a specific secretagogue, thyroliberin (TRH). These changes in cell shape were correlated with a potentiation of prolactin secretion, both basal and TRH-stimulated. Furthermore, using immunocytochemistry and electron microscopy, we revealed--at the dilated tip of processes--an accumulation not only of prolactin, but also of synaptophysin, a vesicle membrane marker, and of several organelles, such as secretory granules, smooth vesicles, dense bodies and mitochondria. The cytoplasmic processes contained long parallel bundles of microtubules and showed a strong immunoreactivity for beta 2-tubulin. In addition, we found immunocyto-chemical evidence for the presence of 200-k Da neurofilament protein in GH3/B6 cell processes as well as in neurites of cultured hypothalamic neurons. We conclude that, in GH3/B6 cells, laminin induced the differentiation of neurite-like processes, which were the site of polarized organelle transport and exhibited some neuronal markers.     

Ultrastructural localization of RFamide-like peptides in neuronal dense-cored vesicles in the peduncle of Hydra

The presence of Arg-Phe-amide (RFamide)-like peptides in dense-cored vesicles in neurons of the peduncle of Hydra was demonstrated by immunogold electron microscopy. Thin sections of Lowicryl-embedded tissue labeled with antisera to RFamide and 5-nm gold-conjugated, secondary antibody and of Epon-Araldite-embedded tissue labeled with 15-nm gold particles revealed a concentration of RFamide-like immunoreactivity over the granular cores of vesicles in epidermal ganglion cells. Gold-labeled, dense-cored vesicles were present in the perikaryon, long thin neurites, and axon terminals of these neurons. The aggregation of labeled dense-cored vesicles in an axon terminal on the myoneme of an epitheliomuscular cell suggests a possible function of RFamide-like peptides in neuromuscular transmission. Gold staining of dense-cored vesicles completely disappeared when the RFamide antiserum was preabsorbed with 10 micrograms/ml RFamide. These results are the first demonstration that the dense-cored vesicles of Hydra neurons contain a neuropeptide.     

Neurite outgrowth from hippocampal neurons is promoted by choroid plexus ependymal cells <Emphasis Type="Italic">in vitro</Emphasis>

Choroid plexus ependymal cells (CPECs) were known to promote axonal growth when choroid plexus is grafted into the adult rat spinal cord. The present study was carried out to examine whether CPECs promote axonal outgrowth from neurons derived from the CNS in vitro. Hippocampal neurons were cocultured on CPEC monolayers. After 24 h, neurite extension was evaluated using various parameters in comparison with cultures grown on poly-L-lysine (PLL)-coated plates and cocultures grown on astrocyte monolayers. The primary neurite length and total neurite length were longest in the cocultures with CPECs. The number of primary neurites and the number of branches were larger in the cultures with CPECs than in the cultures on PLL-coated plates, but almost the same as in the cocultures with astrocytes. Next, we examined whether the neurite extension-promoting effect occurring within 24 h is due primarily to contact with the CPECs or to factors secreted by CPECs into the culture medium. The CPEC monolayers were killed by ethanol fixation, and neurons cultured on them. The neurons extended long neurites with elaborate branching, as in the case of cocultures grown on living CPECs. On the other hand, CPEC-conditioned medium exhibited less promoting effect on neurite outgrowth from hippocampal neurons. These results indicate that CPECs have a capacity to promote neurite outgrowth from CNS neurons in vitro, and that surface plasma membrane-bound components of CPECs strongly contribute to the enhancement of neurite outgrowth in the present coculture system.     

Experimental and theoretical analysis of neuron-transistor hybrid electrical coupling: the relationships between the electro-anatomy of cultured Aplysia neurons and the recorded field potentials

Understanding the mechanisms that generate field potentials (FPs) by neurons grown on semiconductor chips is essential for implementing neuro-electronic devices. Earlier studies emphasized that FPs are generated by current flow between differentially expressed ion channels on the membranes facing the chip surface, and those facing the culture medium in electrically compact cells. Less is known, however, about the mechanisms that generate FPs by action potentials (APs) that propagate along typical non-isopotential neurons. Using Aplysia neurons cultured on floating gate-transistors, we found that the FPs generated by APs in cultured neurons are produced by current flow along neuronal compartments comprising the axon, cell body, and neurites, rather than by flow between the membrane facing the chip substrate and that facing the culture medium. We demonstrate that the FPs waveform generated by non-isopotential neurons largely depends on the morphology of the neuron.     

Suppression of kinesin expression in cultured hippocampal neurons using antisense oligonucleotides

Kinesin, a microtubule-based force-generating molecule, is thought to translocate organelles along microtubules. To examine the function of kinesin in neurons, we sought to suppress kinesin heavy chain (KHC) expression in cultured hippocampal neurons using antisense oligonucleotides and study the phenotype of these KHC "null" cells. Two different antisense oligonucleotides complementary to the KHC sequence reduced the protein levels of the heavy chain by greater than 95% within 24 h after application and produced identical phenotypes. After inhibition of KHC expression for 24 or 48 h, neurons extended an array of neurites often with one neurite longer than the others; however, the length of all these neurites was significantly reduced. Inhibition of KHC expression also altered the distribution of GAP-43 and synapsin I, two proteins thought to be transported in association with membranous organelles. These proteins, which are normally localized at the tips of growing neurites, were confined to the cell body in antisense-treated cells. Treatment of the cells with the corresponding sense oligonucleotides affected neither the distribution of GAP-43 and synapsin I, nor the length of neurites. A full recovery of neurite length occurred after removal of the antisense oligonucleotides from the medium. These data indicate that KHC plays a role in the anterograde translocation of vesicles containing GAP-43 and synapsin I. A deficiency in vesicle delivery may also explain the inhibition of neurite outgrowth. Despite the inhibition of KHC and the failure of GAP-43 and synapsin I to move out of the cell body, hippocampal neurons can extend processes and acquire as asymmetric morphology.     

Early differentiation of vertebrate spinal neurons in the absence of voltage-dependent Ca2+ and Na+ influx

The development of the action potential and responses to neurotransmitters have been described for a population of embryonic spinal neurons developing in vivo. A comparable pattern is seen for spinal neurons developing in dissociated cell culture. The impulse appears very early in this developmental sequence, and the action potential involves a large inward Ca2+ current. Since Ca2+ is a ubiquitous intracellular regulator, we questioned whether a large influx of Ca2+ is necessary for the subsequent differentiation of membrane properties. Embryonic Xenopus neurons grown in normal culture medium do not make Ca2+- or Na+-dependent action potentials in their cell bodies in a Ca2+-free saline containing tetrodotoxin (TTX). To achieve a chronic blockade of impulse activity, neurons were grown in a medium in which Ca2+ was replaced by Mg2+, and to which 1 mM EGTA was added. In some instances TTX was present. Neurons grown in these experimental culture media extend neurites more rapidly than controls. Action potentials cannot be elicited from neurons when examined in experimental medium. However, examination in saline reveals that the change in the ionic dependence of the impulse is indistinguishable from that observed in neurons grown in normal medium. Furthermore, the time of onset of responses to GABA is unaffected by this experimental treatment. Thus the expression of Ca2+- and Na+-dependent action potentials seems not to play a part in the early differentiation of these membrane properties. However, the later development of GABA sensitivity is reduced.     

Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule, N-cadherin.

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2 mM Ca++ or 0.2 mM Ca++, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca++, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca++ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca++ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca++ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca++ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca++, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca++. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca++ levels had similar effects as lowering the Ca++ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.     

Specific asparagine-linked oligosaccharides are not required for certain neuron-neuron and neuron-Schwann cell interactions

To determine whether specific asparagine-linked (N-linked) oligosaccharides present in cell surface glycoproteins are required for cell-cell interactions within the peripheral nervous system, we have used castanospermine to inhibit maturation of N-linked sugars in cell cultures of neurons or neurons plus Schwann cells. Maximally 10-15% of the N-linked oligosaccharides on neuronal proteins have normal structure when cells are cultured in the presence of 250 micrograms/ml castanospermine; the remaining oligosaccharides are present as immature carbohydrate chains not normally found in these glycoproteins. Although cultures were treated for 2 wk with castanospermine, cells always remained viable and appeared healthy. We have analyzed several biological responses of embryonic dorsal root ganglion neurons, with or without added purified populations of Schwann cells, in the presence of castanospermine. We have observed that a normal complement of mature, N-linked sugars are not required for neurite outgrowth, neuron-Schwann cell adhesion, neuron-induced Schwann cell proliferation, or ensheathment of neurites by Schwann cells. Treatment of neuronal cultures with castanospermine increases the propensity of neurites to fasciculate. Extracellular matrix deposition by Schwann cells and myelination of neurons by Schwann cells are greatly diminished in the presence of castanospermine as assayed by electron microscopy and immunocytochemistry, suggesting that specific N-linked oligosaccharides are required for the expression of these cellular functions.     

Studies with the Golgi method in central gangliogliomas and dysplastic gangliocytoma of the cerebellum (Lhermitte-Duclos disease).

The rapid Golgi method, combined with current optical and electronmicroscopical techniques, was used in three central gangliogliomas and in one dysplastic gangliocytoma of the cerebellum to study the morphology of ganglionic cells. Gangliogliomas were composed of bipolar, fusiform and radiate cells with dense core and clear vesicles in the perikaryon and cellular processes, the number of each cellular type varying from one case to another. These features, together with the fact that isodendritic neurons are considered to be phylogenetically old neurons, suggest that these tumours are composed of "primitive" neurons that are not homogeneous with regard to their morphology. In contrast, ganglionic cells in dysplastic gangliocytoma are huge cells with long, stereotyped neurites that establish unique asymmetric contacts with neighbouring perikarya and neurites by means of claw-shaped processes covered with synaptic buttons. These morphological characteristics are different from those of any other neuron of the CNS.     

Release of pedal peptide from Aplysia neurons in primary culture

Pedal peptide (Pep) is a modulatory neuropeptide that is predominantly synthesized in a group of neurons on the dorsal surfaces of the pedal ganglia of Aplysia. Following the determination that Pep is the major peptide selectively present in these neurons in situ, primary cell culture of single Pep-neurons was used to study the release of this neuropeptide. Individual Pep-neurons were grown in culture where they extended many branched neurites with large varicosities. Immunocytology revealed that these newly grown varicosities were intensely Pep immunoreactive. Cultured Pep-neurons, grown in a medium containing radiolabeled methionine, synthesized labeled Pep and transported it into their regenerated neurites. Finally, these neurons released radiolabeled Pep in a calcium- and stimulation-dependent fashion. These results, taken together with previous findings, strongly support the proposition that Pep is a transmitter in Aplysia.     

The role of endogenous heparan sulfate proteoglycan in adhesion and neurite outgrowth from dorsal root ganglia

Some phases of dorsal root ganglion (DRG) substratum attachment and growth cone morphology are mediated through endogenous cell surface heparan sulfate proteoglycan. The adhesive behavior of intact embryonic chicken DRG (spinal sensory ganglia) is examined on substrata coated with fibronectin, fibronectin treated with antibody to the cell-binding site (anti-CBS), and the heparan sulfate-binding protein platelet factor four. DRG attach to fibronectin, anti-CBS-treated fibronectin, and platelet factor four. The ganglia extend an extensive halo of unfasciculated neurites on fibronectin and produce fasciculated neurite outgrowth on platelet factor four and anti-CBS antibody-treated FN. Treatment with heparinase, but not chondroitinase, abolishes adhesion to fibronectin and platelet factor four. Growth cones of DRG on fibronectin have well-spread lamellae and microspikes. On platelet factor four, and anti-CBS-treated FN, growth cones exhibit microspikes only. Isolated Schwann cells adhere equally well to fibronectin and platelet factor four, spreading more rapidly on fibronectin. Isolated DRG neurons adhere equally well on both substrata, but only 10% of the neurons extend long neurites on platelet factor four. The majority of the isolated neurons on platelet factor four exhibit persistent microspike production resembling that of the early stages of normal neurite extension. Endogenous heparan sulfate proteoglycan supports the adhesion of whole DRG, isolated DRG neurons, and Schwann cells, as well as extensive microspike activity by DRG neurons, one important part of growth cone activity.     

Histochemical demonstration of an increase in acetylcholinesterase in established lines of human and mouse neuroblastomas by nerve growth factor.

Cells of three established lines of human neuroblastoma and an established line of C1300 mouse neuroblastoma were grown in control medium or in experimental medium containing mouse nerve growth factor (NGF). Cultures were stained histochemically for acetylcholinesterase (AChE) during log growth and at confluency. Human neuroblastoma cells grown in medium containing NGF were morphologically more differentiated and they were stained much more intensely for AChE during both phases of growth than were cells in control cultures. The enzyme was distributed over cell bodies and neurites. Neuroblastoma cells of the mouse line were not stimulated to form neurites by NGF, but they were more intensely stained for acetylcholinesterase than cells grown in control medium. These observations support earlier findings that NGF stimulates differentiation of human and mouse neuroblastoma cells in vitro.