Synapse formation between clonal neuroblastoma X glioma hybrid cells and striated muscle cells.

Abstract

Clonal neuroblastoma X glioma hybrid cells were shown to form synapses with cultured, striated muscle cells. The properties of the synapses between hybrid and muscle cells were similar to those of the normal, neuromuscular synapse at an early stage of development. The number of synapses formed and the efficiency of transmission across synapses were found to be regulated, apparently independently, by components in the culture medium. Under appropriate conditions synapses were found with 20% of the hybrid-muscle cell pairs examined; thus, the hybrid cells form synapses with relatively high frequency.Little is known at the molecular level about the process of synapse formation and the basis for the specificity of synaptic connections between cells. Some of the difficulties in studying this problem stem from the many cell types that are present in the nervous system and the lack of adequate criteria that can be used to distinguish one type of neuron from another. Clonal lines of neuroblastoma cells (1, 2) and somatic cell hybrids (3-6) derived from neuroblastoma cells have proved experimentally advantageous for the study of some neural properties. Such cells continue to proliferate; thus populations of cells that appear to be fairly homogeneous can be obtained, yet cells retain the ability to express various properties of differentiated neurons. One of the primary objectives in generating and characterizing these cell lines has been to obtain cell lines that are able to form synapses in vitro with high frequency. In this report, we show that clonal neuroblastoma X glioma hybrid cells which are known to synthesize, store, and excrete acetylcholine* form synapses with striated muscle cells in vitro. MATERIALS AND METHODSCell Cultures. NG108-15 hybrid cells were derived by fusion of mouse neuroblastoma clone N18TG-2 resistant to 6-thioguanine (4), with rat glioma clone C6BU-1, resistant to 5-bromodeoxyuridine (5). NG108-15 cells routinely were cultured at 37°in medium D (90% Dulbecco-Vogt modification of Eagle's minimal essential medium, GIBCO H-21; 10% fetal bovine serum; 1 X 10-M hypoxanthine; 1 X 10-6 M aminopterin; and 1.6 X 10-5 M thymidine) in a humidified atmosphere of 10% C02-90% air. NG108-15 cells were cultured for 1-3 weeks in the presence of 1 mM N6, 02-dibutyryl-adenosine-3':5'-cyclic monophosphate (dibutyryl cAMP) to shift the hybrid cells to a more differentiated state*. All experiments were performed with NG108-15 cells that had been subcultured 14 to 20 times.Abbreviations: DMEM, the Dulbecco-Vogt modification of Eagle's minimal essential medium; MEM, Eagle's minimal essential medium; dibutyryl cAMP, N6,02-dibutyryl-adenosine-3':5'-cyclic monophosphate; dibutyryl cGMP, N2,02-dibutyryl guanosine-3':5'-cyclic monophosphate.It was necessary to remove butyric acid and perhaps other contaminants from dibutyryl cAMP prior to use. The following method was used. Dibutyryl cAMP dissolved in H20 (1 g/20 ml) was adjusted to pH 3.0 with 0.1 M HCI and then extracted vigorously (and sometimes repeatedly) wit...

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“…As ACh and serotonin can be released in the same fashion, gene and cell therapies for other diseases related to ACh and serotonin, such as Alzheimer's disease, depression, schizophrenia, and syndromic autism, may utilize ATP6V0C in the future. This approach is applicable for iPSC therapy for similar categories of diseases [72][73][74] because better recovery would be expected if iPSC contains transmitter synthesizing genes and ATP6V0C, because most of iPS cells drived from the skin fibroblasts are likely silent [27]. subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila.…”

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