In amyotrophic lateral sclerosis, down‐regulation of the astrocyte‐specific glutamate excitatory amino acid transporter 2 is hypothesized to increase extracellular glutamate, thereby leading to excitotoxic motor neuron death. The antibiotic ceftriaxone was recently reported to induce excitatory amino acid transporter 2 and to prolong the survival of mutant superoxide dismutase 1 transgenic mice. Here we show that ceftriaxone also protects fibroblasts and the hippocampal cell line HT22, which are not sensitive to excitotoxicity, against oxidative glutamate toxicity, where extracellular glutamate blocks cystine import via the glutamate/cystine‐antiporter system xc−. Lack of intracellular cystine leads to glutathione depletion and cell death because of oxidative stress. Ceftriaxone increased system xc− and glutathione levels independently of its effect on excitatory amino acid transporters by induction of the transcription factor Nrf2 (nuclear factor erythroid 2‐related factor 2), a known inducer of system xc−, and the specific xc− subunit xCT. No significant effect was apparent in fibroblasts deficient in Nrf2 or xCT. Similar ceftriaxone‐stimulated changes in Nrf2, system xc−, and glutathione were observed in rat cortical and spinal astrocytes. In addition, ceftriaxone induced xCT mRNA expression in stem cell‐derived human motor neurons. We conclude that ceftriaxone‐mediated neuroprotection might relate more strongly to activation of the antioxidant defense system including Nrf2 and system xc− than to excitatory amino acid transporter induction.

The PC12 clone of a rat pheochromocytoma is able to synthesize acetylcholine. The amount of acetylcholine synthesized, and the specific activity of choline O-acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6), varies as a function of the culture growth curve and is dependent on cell density. The specific activity of choline acetyltransferase is increased by nerve growth factor, growth-conditioned medium from a variety of cell types, and adenosine 3':5'-monophosphate. Finally, the PC12 cell line is able to form cholinergic synapses with a clonal cell line of skeletal muscle origin.

The nerve cell line PC12, in its morphologically undifferentiated state, synthesizes, stores, and secretes catecholamines and acetylcholine. At least 60% of the basal level of neurotransmitter release is due to a calcium-dependent mechanism, and the rate of secretion is enhanced by increasing external potassium. A minimum of 80% of the intracellular acetylcholine and catecholamines are stored in particulate structures. The storage site for acetylcholine is dense core vesicles that can be distinguished from those containing catecholamines on the basis of vesicle density on sucrose gradients, vesicle size, and reserpine sensitivity. These results are discussed in relation to what are thought to be the early stages of synapse formation in cell culture. A clonal cell line was recently isolated which shares many properties with primary cultures of sympathetic ganglion neurons. This clone, designated PC12, responds to exogenous nerve growth factor (NGF) by the extension of neurites (1), and synthesizes catecholamines (1) and acetylcholine (ACh) (2). In addition, the cells form cholinergic synapses with a clonal cell line of skeletal muscle (2), and miniature endplate potentials (mepps) have been detected within 1 hr after mixing PC12 and skeletal muscle cells in the absence of exogenous NGF (Y. Kidokoro, personal communication). Due to the rapidity of synapse formation and the large amounts of both ACh and catecholamines synthesized by the PC12 clone, these cells are ideally suited for study of the storage and release of the neurotransmitters and the relationship of these processes to the early stages of synapse formation. The following experiments show that neurotransmitters are continually released by morphologically undifferentiated PC12 cells and that ACh is stored in a dense core vesicle distinct from the 30-to 50-nm clear vesicles normally associated with cholinergic synapses. MATERIALS AND METHODSThe clonal rat cell line PC12 was obtained from Lloyd Greene (1). The cells were grown on plastic tissue culture dishes in 10% fetal calf and 5% horse serum as described (2) lease buffer (0.01 M N-2-hydroxyethylpiperazine-N'-1-ethylsulfonic acid, pH 7.1/0.15 M NaCI/5 mM KCI/0.01 M glucose/2 mM CaCl2/1 mM MgCl2, 0.1 mM eserine) and treated as indicated. To assay neurotransmitter release, cells were incubated in release buffer with or without increased concentrations of potassium at 370 for the indicated times, the cells were pelleted by centrifugation, and aliquots of the supernatant were analyzed for ACh, norepinephrine (NE), choline, dopamine (DA), and tyrosine by high-voltage electrophoresis (3). For density gradient analysis, isotopically labeled washed cells were homogenized in 0.25 M sucrose/i mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.0/0.1 mM eserine, and centrifuged on one of the following gradients in a Beckman SW 41 rotor: (i) 0.6-1.8 M sucrose linear gradient for 2 hr at 35,000 rpm (4); and (ii) a step gradient of 0.4, 0.8, 1.2, and 1.6 M sucrose at 25,000 rpm for 90 min (5). Isoto...

Acquisition of acetylcholine receptors during differentiation of a clonal myoblast cell line was monitored with a neurotoxin isolated from venom of the Indian Cobra Naja naja. Toxin bound specifically and reversibly to acetylcholine receptors of the differentiated cells. Specificity of the binding reaction was assayed by measurement of the ability of various cholinergic agonists and antagonists to compete with neurotoxin for its binding site. The rate of toxin binding paralleled the rate of inactivation of functional acetylcholine receptors, as measured by iontophoretic application of acetylcholine. Bound toxin was released from the cells with a half-life of about 7 hr. This release was not associated with a decrease in the total number of toxin-binding sites. A slow hyperpolarizing response to acetylcholine seen in myoblasts was insensitive to toxin; the appearance of toxin-binding sites parallels the appearance of fused fibers during differentiation of the muscle cells in tissue culture.

A primary system has been developed in which it is possible to study the production of electrically excitable neuron-like cells from a precursor population of olfactory epithelial cells. Rat nasal epithelium was dissociated and placed in culture. The initial surviving cells are flat and ciliated and contain glial fibrillary acidic protein (GFAP). After 3-5 days electrically excitable cells appear that contain neuronspecific enolase but not GFAP. These round cells originate by means of the differentiation of the GFAP-positive flat cell to a round cell, followed by the division of the round cell. Therefore, neuron-like cells can be derived from cells that synthesize GFAP. Immunohistochemistry. Rabbit anti-glial fibrillary acidic protein (GFAP) was obtained from A. Bignami, mouse monoclonal anti-rat T200 protein was obtained from R. Hyman, and monoclonal anti-S100 and anti-neuron-specific enolase was from B. Boss. For staining with anti-T200, cultures were incubated with the primary antibody for 30 min at room temperature. The cultures were then washed and exposed for an additional 30 min to a rhodamine-labeled goat antibody against mouse immunoglobulin (Tago, Burlingame, CA). Finally, the cultures were washed, air dried, and fixed with 95% ethanol for 10 min at -20°C. For labeling with anti-GFAP, cultures were first fixed with 95% ethanol for 10 min at -20°C, dried, and then incubated for 30 min with the primary antiserum. After washing, the cultures were exposed to a fluorescein-labeled goat antibody against rabbit immunoglobulin for an additional 30 min, followed by more washing and a final air drying. For double-labeling the cells were first stained with anti-T200, fixed, and then stained with anti-GFAP. Staining with anti-S100 and anti-nerve-specific enolase was done according to published procedures (8).