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Cerebral neuroblastoma is often difficult to differentiate by clinical criteria from other central nervous system neoplasm. This report is of a young child with cerebral neuroblastoma and elevated nerve growth factor. This case illustrates the need for further studies into the usefulness of nerve growth factor in differentiating cerebral neuroblastoma from other central nervous system tumors.

The survival of neural tissues depends in part on the balance between the formation of free radicals due to oxidative metabolism and the transformation of the free radicals to non-toxic compounds. Serial subculture of rat glial cells as described here resulted in a decrease of the specific activities of several antioxidant enzymes and a glial specific marker for astrocytes. Thus, there was an increased susceptibility to oxidative stress in cultures by the third passage. These subcultured glial cell cultures may represent a useful model for the study of free radical induced neural damage that may be relevant to CNS trauma and aging.

Increased cellular adhesion has been postulated to be an early event required of neuroblasts undergoing neurite extension during differentiation. Nerve growth factor (NGF) induces neurite extension in a variety of cell types of neural crest origin. In the PC12 rat pheochromocytoma cell line, which has been proposed as a model for precursor cells to sympathetic neurons, NGF and dibutyryl cyclic AMP (dBcAMP) promote both neurite extension and an increased rate of cell-substrate adhesion. Since dBcAMP can substitute for NGF in this enhancement of adhesion rate in the PC12 cell line, cAMP has been suggested as a second messenger for NGF. We have shown that in two nearly diploid adrenergic like human neuroblastoma clones, the KA and SY5Y cell lines, which also extend neurites in response to both NGF and dBcAMP, only NGF enhances cellular adhesion, as defined by an increase in the number of cells cells prelabeled with 35S-methionine which attach to culture dishes at a given time. Incubation with monospecific antibodies directed against murine beta-NGF abolishes the NGF effect on adhesion. The NGF effect on human neuroblastoma is specific insofar as NGF does not facilitate adhesion of two glioma lines. Unlike the results obtained for PC12, in both SY5Y and KA lines, 1 mM dBcAMP decreases the rate of adhesion to levels significantly below those of controls. Adhesiveness of neuroblastoma cells treated with both NGF and dBcAMP is intermediate between that of cells treated with either agent alone. While theophylline mimics the dBcAMP effect, sodium butyrate has no such effect. At 22 degrees C, the effect of NGF on neuroblastoma substrate adhesion is observable within 5 minutes and persists for 2 hours; treatment of the KA line with NGF at 37 degrees C for 24 hours results in a more persistent enhancement of cell adhesion. Furthermore, both SY5Y and KA exhibit different morphologies when challenged with NGF, dBcAMP, or sodium butyrate. This study suggests that NGF and cyclic AMP do not share a common mechanism of action, but can in fact interact antagonistically in an adrenergic like neuroblastoma model system. Furthermore, the results suggest that increased cellular adhesiveness may not be an obligatory prerequisite for neurite extension by neuroblasts in development.

The electrical properties of the SK-N-SH human neuroblastoma cell were studied by standard intracellular recording techniques; the average resting membrane potential was -21 +/- 11 mV, with a few cells showing mebrane potentials greater than - 40 mV. Under standard tissue culture conditions, as used in these experiments, less than 1% of these cells show morphological differentiation (process formation). In response to current injection, a variety of graded responses with a relatively slow rise time were observed. In some cells only delayed rectification was observed. In no instance did current injection result in a characteristic action potential. An analogous method for determining electrical excitability was to measure (22)Na influx in the presence and absence of a depolarizing agent, veratridine (0.1 mM). In such experiments, the influx of (22)Na in SK-N-SH cells was only slightly altered by veratridine. Taken together, these data suggest that the morphologically undifferentiated human neuroblastoma cells are relatively inexcitable electrically. The iontophoretic application of acetylcholine to the cell body produced depolarizing responses whose amplitudes were dependent on the membrane potential.

The nerve growth factor (NGF) subunit of 7S NGF was isolated by chromatography at high pH on QAE-Sephadex. It has the same specific NGF activity as betaNGF isolated at acid pH, showing that this activity is an intrinsic property of the subunit and is independent of the pathway of dissociation. Continued exposure of the NGF subunit to high pH resulted in an increase in the amount of the minor species beta2NGF and the formation of a new species, beta3NGF, of even lower isoelectric point. These two species and the original major species of the preparation, beta1, were isolated by isoelectric focusing. All three species had the same specific NGF activity, but differed in their ability to reform 7S NGF. The beta2 species was one-fifth as competent as beta1, while beta3 was unable to regenerate 7S NGF. Addition of alpha- and gamma-subunits to beta1NGF decreased the amount of NGF protein required to produce one Biological Unit of activity in the bioassay, but had no effect when added to beta3NGF. The interactions between the subunits in 7S NGF therefore determine, in part, the specific activity of the NGF subunit.

The numbers of neurons in the rat superior cervical sympathetic ganglion (SCG) differ in males and females, with the males having 30% more SCG neurons than females at 60 days of age. This sex difference arises during the early postnatal period, when testosterone administration increases the numbers of neurons and alters the nerve growth factor (NGF) content of the rat SCG. In contrast, there is no gender difference in number of neurons in the L1 dorsal root ganglion. In both males and females, the amount of NGF bound per ganglion increased between postnatal days 5 and 15 (P5 and P15) in both dorsal root ganglia (DRGs) and the SCG. There is also a gender difference in NGF binding: SCGs and DRGs of female rats at both P5 and P15 bind more NGF per ganglion than do those of males. This effect was more marked in DRGs than in the SCG. Treatment of neonatal females with testosterone reduced NGF binding in both SCGs and DRGs to levels comparable to males at P5, and in DRGs at P15. In contrast, treatment of males with testosterone from birth resulted in a 2-3 fold increase of NGF binding in both SCGs and DRGs as compared to controls at P15. At P15, testosterone treatment of females increased NGF binding in the SCG. Males and females had opposing responses to neonatal exposure to estradiol. Treatment with estradiol from birth increased NGF binding in SCGs and DRGs of females, but had no effect on NGF binding of SCGs, and reduced NGF binding in DRGs of males.(ABSTRACT TRUNCATED AT 250 WORDS)

Newborn rats were given subcutaneous injections of antibodies to mouse beta -NGF (ANTI-NGF) daily for 1 month. The number of neurons in T4-T6 dorsal root ganglia (DRG) and the numbers of myelinated and unmyelinated axons in the dorsal roots of the same segments were counted in the ANTI-NGF animals and in normal littermates. The ANTI-NGF rats had 38% fewer neurons in thoracic ganglia but 17% more myelinated and 40% more unmyelinated fibers than their untreated littermates. Dorsal root ganglion cells also have a larger average size in the ANTI-NGF animals, which we interpret as a disproportionate loss of small cells. These data are interpreted as showing that some dorsal root ganglion cells, principally small ones, die when endogenous NGF is inactivated, and that the remaining cells emit more processes than normal. Thus, removal of NGF has what appears to be a paradoxical effect, a reduction in dorsal root ganglion cell numbers but an increase in dorsal root axon numbers. The relation of myelin thickness to fiber diameter is also altered, with small fibers being more thinly myelinated in the ANTI-NGF group. Thus, Schwann cell-neuronal interactions are also affected by inactivation of NGF.

Dorsal root unmyelinated axons in 30-day-old rats in which the spinal cord has been hemisected at birth are more numerous on the operated side in animals that receive preimmune serum daily from hemisection to sacrifice. This is interpreted as indicating that sprouting of sensory axons occurred on the operated side. In animals with hemisected cords treated daily with antibodies to nerve growth factor (anti-NGF), the same numbers of unmyelinated axons are found in the roots on the operated side, but there are even more unmyelanated axons on the unoperated side. Our interpretation is that anti-NGF results in sensory axon sprouting with more sprouts on the unoperated side. Thus removal of NGF in cord hemisected newborn rats has an effect on sensory axon numbers.

Human neuroblastoma SK-N-SH-SY5Y (5Y) and rat glioma (C6) cells were cultured with supplemental methionine, glycine, or serine for three to six days. Serine hydroxmethyltransferase (SHMT: L-serine: tetrahydrofolate 5, 10-hydroxymethyltransferase, EC 2.12.1) was assayed radiometrically in whole cell homogenates, crude supernatant fractions and crude particulate fractions. No significant changes in specific activity or cellular morphology were noted at methionine, glycine, or serine concentrations up to 16 mM. Serine concentrations of 20 and 40 mM led to significantly lower gliomal enzyme specific activities. This activity was unevenly distributed between soluble and particulate fractions, with 190 and 398 nmoles of HCHO formed per mg of protein per hour, respectively. Growth stage and time of incubation were major determinants of enzyme specific activity. C6 cells' specific activity rose slowly with increasing time in culture until cellular confluence. At this time there was a pronounced elevation in specific activity, occurring more rapidly in cells grown in 1.2 mM methionine. Intracellular amino acid analysis of C6 cells demonstrated a significant rise in methionine after four days in media containing 0.2 mM methionine. No appreciable diminution in the intracellular levels of glycine or serine occurred following incubation in excess methionine. It is concluded that SHMT-specific activity in C6 and 5Y cells is not regulated by glycine, serine, or methionine levels and that high concentrations of these amino acids (> 30 mM) are not detrimental to these cells derived from the CNS.