Addition of dimethylsulfoxide at concentrations of 1% and 2% (vol/vol) to cells of mouse neuroblastoma clone NIE-115 in the confluent phase of growth resulted in the production of morphologicalfir differentiated cultures with extensive process formation. Cells maintained in 2% dimethylsulfoxide remained in a stable nondividing condition for periods of up to 4 weeks. A high degree of electrical excitability was found in these cells, but there was no clear correlation of this property with the level of induction of either acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) or tyrosine hydroxylase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2]. In addition, intracellular levels of cyclic 3':5'-AMP were not elevated in fully morphologically and electrically differentiated cells. While cell division was markedly inhibited by 2% or higher concentrations of dimethylsulfoxide, at 1% growth continued at a somewhat slowed rate and such cultures exhibited enhanced process formation and electrical activity for a relatively short period. High concentrations (3% or 4%) of dimethylsulfoxide totally suppressed process formation and did not result in increased excitability, but cells maintained high resting potentials. The results suggest that the development of the excitable membrane in neuroblastoma cells maybe expressed independently of neurospecific enzyme induction, and does not require a sustained elevation of cyclic 3'i5'-AMP levels.Nerve cells are characterized by their unique morphological appearance, the possession of an excitable membrane, and a specialized biochemical machinery. In order to study the expression of specific neuronal properties during the maturation process it is necessary to obtain sufficient quantities of a relatively homogeneous differentiating population. Cloned cell lines isolated from the C-1300 mouse neuroblastoma may serve as a useful system for exploring certain aspects of nerve cell differentiation. The transition of a culture of neuroblastoma cells from the actively dividing state to the confluent one is characterized by the synthesis of various enzymes involved in neurotransmitter metabolism (1), an enhancement of electrical excitability (2, 22), and some degree of process formation. To achieve a further expression of these properties, cells have been treated with various agents such as aminopterin (3, 4) or dibutyryl cAMP (5-7).In the present study, we show that in the presence of dimethylsulfoxide (Me2SO) neuroblastoma cells will extend neurites and develop a highly excitable membrane. It appears that this method offers certain advantages over those most commonly used, especially in that cells appear to reach a higher level of electrical differentiation and can be maintained in this state for extended periods. In experiments where logarithmically growing cells were used, cultures were trypsinized before attaining confluency and replated in 60 mm dishes at a density of 1 X 105 cells per dish. Two days later, the medium was replaced w...

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Sequential removal of surface glycopeptides was achieved by subjection of mouse neuroblastoma cells to a two-step trypsin treatment under different conditions. The glycopetides released by each trypsinization step were digested by Pronase and examined on columns of Sephadex G-50. Different chromatographic patterns were found for the two digests. Thus, several groups of glycopeptides can be distinguished by the trypsinization procedure. One group is readily removed and appears to be at a more accessible location on the cell surface. Among the four neuroblastoma clones examined, the glycopeptide patterns from axon-forming cells differed from those of axon-minus cells.It is generally accepted that the conductance of impulse in nerve cells depends on specific changes in the ionic permeability of the surface membrane. These membranes are involved in the formation of axons and dendrites, the specificity of neuron junctions, and the formation of the synaptic structure.Glycoproteins appear to be on the outermost surface of all cells examined (1-3) and have been implicated in cell recognition (4, 5) and contact (6, 7). It has been suggested that sialic acid, a terminal component of glycoproteins and glycolipids may be fundamental in processes occurring during excitation of nerve cells (8).As part of an approach to these problems, we have examined the glycoproteins from the cell surface of clones isolated from a mouse neuroblastoma, C-1300. These clones differ in their capacity to extend neurites, to synthesize various neurotransmitters, and to respond to electrical stimuli (9). In this study a trypsinization procedure has been developed by which several groups of glycopeptides are distinguished. One group is readily removed and appears to be at a more accessible location on the cell surface. A preliminary report has been published (10). Cell Cultures. Clones N-18, NS-20, N-1, and NlA-103 derived from a mouse neuroblastoma C-1300 (9), were obtained from Dr. M. Nirenberg, NIH, Bethesda, Md. Clone N-18, which is inactive with respect to tyrosine hydroxylase (EC 1.14.3a) and choline acetyltransferase (EC 2.3.1.6), contains acetylcholinesterase (EC 3.1.1.7) and is able to extend neurites in the absence of serum. Clone NS-20, a cholinergenic clone, contains choline acetyltransferase and acetylcholinesterase and is able to form neurites but does not contain detectable activity of tyrosine hydroxylase. Clone N-1 contains low activity of tyrosine hydroxylase, choline acetyltransferase, and acetylcholinesterase and does not form neurites. Clone NlA-103, a subelone of N-1, is almost devo'd of tyrosine hydroxylase and choline acetyltransferase activities, has a low acetylcholinesterase activity, and is an axon-minus line (9).The cells were grown in Dulbecco modified Eagle's medium (Grand Island Biological Co., Grand Island, N.Y.) containing 1.2 g of NaHCO3 per liter and supplemented with 10% fetalcalf serum (Colorado Serum Co.), penicillin (100 U/ml), and streptomycin (100 ,ug/ml), in an atmosphere of 5% C02-95% air with 100...

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