We have developed a series of mouse-mouse neural hybrid cell lines by fusing the aminopterin-sensitive neuroblastoma N18TG2 with motor neuron-enriched embryonic day 12-14 spinal cord cells. Of 30 neuroblastoma-spinal cord (NSC) hybrids displaying a multipolar neuron-like phenotype, 10 express choline acetyltransferase, and 4 induce twitching in cocultured mouse myotubules. NSC-19, NSC-34, and their subclones express additional properties expected of motor neurons, including generation of action potentials, expression of neurofilament triplet proteins, and acetylcholine synthesis, storage, and release. In addition, NSC-34 cells induce acetylcholine receptor clusters on cocultured myotubes, and undergo a vimentin-neurofilament switch with maturation in culture, similar to that occurring in neuronal development. NSC cell lines appear to model selected aspects of motor neuron development in an immortalized clonal system.

https://anatomypubs.onlinelibrary.wiley.com/doi/pdf/10.1002/aja.1001940306

Neuroblastoma x spinal cord (NSC) hybrid cell lines resemble developing motor neurons.

Capsaicin is a pungent pain-producing compound found in plants of the capsicum family; it exerts excitatory, desensitizing, and toxic effects on a subset of sensory neurons, including the polymodal nociceptor population. We have carried out a quantitative study of capsaicin- induced fluxes of sodium, guanidine, calcium, rubidium, and chloride ions in cultures of neonatal and adult rat DRG neurons, in conjunction with the use of a histochemical stain that identifies capsaicin- sensitive neurons by means of cobalt uptake. Those cells that take up cobalt in a capsaicin-dependent manner (EC50 = 0.2 microM) represent about 50% of the total neuronal population derived from neonatal DRGs on short-term culture. Overnight treatment of cultures with 2 microM capsaicin leads to the loss of the cobalt-staining subpopulation. The capsaicin-insensitive neurons contain immunoreactive neurofilament epitopes that are present in fewer than 10% of capsaicin-sensitive neurons. This observation provides indirect evidence that the sensitive cells correspond to the small, dark B-type neurons, which are negative for neurofilament immunoreactivity in vivo. A capsaicin-dependent calcium uptake (EC50 = 0.2 microM), as measured by 45Ca incorporation, is shown by a DRG neuronal subpopulation that, like the cobalt-staining population of DRG neurons, is lost after overnight capsaicin treatment (2 microM). Capsaicin application leads to the accumulation of millimolar levels of calcium within a few minutes. Cadmium and other divalent cations block capsaicin-induced calcium uptake, but little or no inhibition is seen with organic calcium channel antagonists. Mitochondria, rather than the endoplasmic reticulum, are the probable destination of the internalized calcium, because ruthenium red inhibits calcium uptake (IC50 = 0.05 microM), whereas methylxanthines are inactive. The subset of sensory neurons that takes up calcium also releases 86Rb when exposed to capsaicin (EC50 = 0.06 microM). No efflux of 36Cl ions could be induced by capsaicin. These cells also show a capsaicin-induced uptake of 22Na or 14C guanidine (EC50 = 0.06 microM). In contrast, chick DRG cells in culture showed no capsaicin-induced calcium or cobalt uptake. Primary cultures of rat superior cervical ganglion neurons and Schwann cells, and a number of neuronal cell lines, also failed to respond to capsaicin, as judged by the calcium, cobalt, or guanidine uptake assays.

https://www.jneurosci.org/content/jneuro/8/9/3208.full.pdf

Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture.

Opioid receptor-coupled second messenger systems

The site of plasminogen activator release by differentiated neuroblastoma clonal cell lines was determined with a fibrin overlay assay. Release of plasminogen activator was seen at the growth cone in 72 percent of the cells bearing neurites. For 21 percent of these cells the growth cone was the predominant or exclusive site of this enzyme activity. Selective release of protease at the "trailblazing" tip of the neurite may be important in neuron migration and neurite growth in vivo.

https://science.sciencemag.org/content/sci/213/4515/1532.full.pdf

Plasminogen activator release at the neuronal growth cone

Publisher Summary
Alpha-1 (α1) receptors by and large appear to occur in a single-affinity state with respect to both agonists and antagonists. By comparison, α2 receptors may exist in multiple-affinity states reflecting the ability of the α2 binding-site protein to form a complex with additional membrane proteins that themselves are receptors for the physiological substrates guanosine 5'-triphosphate (GTP), Na+, Mg2+, and possibly Ca2+-calmodulin. Binding studies have also strongly indicated that α2 receptors in most, if not all, tissues are probably coupled in an inhibitory manner to adenylate cyclase, as has been demonstrated in platelets, adipocytes, and NG 108-15 cells. Nothing is known about the mechanisms of regulation of α1 receptors, although both up- and down-regulations of α1 receptor have been demonstrated. In this regard, the ability to label and study α1 receptors on cells in culture would be particularly useful. With regard to α2 receptors, the number of affinity states that exist and their role in terms of the kinetics of α2-receptor coupling to adenylate cyclase are still not clear.

Characterization of alpha 1- and alpha 2-adrenergic receptors.

Publisher Summary This chapter discusses the structural, electrophysiological, biochemical and pharmacological properties of neuroblastoma-glioma cell hybrids in cell culture. Hybrid cells are formed by the fusion of two different cell types. The cells fused may be as different as those of humans and of mosquitos, as similar as cells from two strains of mice, or only clearly different in one gene or in the kind of tissue of the same animal they were derived from. Hybrids are prepared by fusion, e.g., of a mutant hamster cell line and a wild-type human cell line. If one selects for wild-type cells, the segregation of human chromosomes will provide hybrid cells that retain a single human chromosome, or part of one, carrying the gene that complements the hamster cell to a wild-type cell. This and similar methods are also used for the analysis of gene linkage. Other important applications of cell hybrids are the rescue of latent viruses and the genetic analysis of malignancy and of viral gene expression.

B. Hamprecht

Papers

Structural, electrophysiological, biochemical, and pharmacological properties of neuroblastoma-glioma cell hybrids in cell culture.