Previous analysis of single-channel current records has shown that both the opening and closing transitions of chemically activated ion channels are operated by fast and slow kinetic processes. The fast component in the kinetics of channel opening has been interpreted as the reopening of a channel that has just closed. The fast component in the kinetics of channel closure has many possible explanations and is therefore more difficult to interpret. We can gain insight into the closing process by asking whether the lifetimes of successive openings of an acetylcholine receptor channel are correlated in open-state lifetime. Five kinetic models of channel closure are considered. Two of these models predict uncorrelated open-state lifetimes, one predicts correlated open-state lifetimes, and for two others a range of behavior is possible. Acetylcholine receptor channel data from cultured rat muscle are analyzed to show that open-state lifetimes are correlated, eliminating two models of channel gating.

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...

To monitor the interaction of cell surface acetylcholine (AcCho) receptors with the cytoskeleton, cultured muscle cells were labeled with radioactive or fluorescent a-bungarotoxin and extracted with Triton X-100, using conditions that preserve internal structure. A significant population of the AcCho receptors is retained on the skeletal framework remaining after detergent extraction . The proportion of nonextracted AcCho receptors increases during myotube development. Both photographic images and quantitative fluorescence measurements indicate that AcCho receptors in patched or aggregated areas are retained on the cytoskeleton while the diffuse receptors are partially extracted by detergent. The skeleton organization responsible for restricting AcCho receptors to a patched region may also result in their retention after detergent extraction .

There is an increase in the number of acetylcholine (AcCho) receptor aggregates on striated embryonic mouse myotubes when they are cocultured with clonal neuroblastoma-glioma hybrid cells. Medium conditioned by hybrid cells contains a factor which increases the number of AcCho receptor aggregates on myotubes cultured from mouse, rat or chick muscle. AcCho receptor-aggregating activity was present in medium conditioned by the neuroblastoma parent clone but was not detected in medium conditioned by cells of the parent glioma clone, fibroblasts, or HeLa cells. The factor increased the aggregation of AcCho receptors within 24 hr without a significant increase in the total number of AcCho receptors, and its action did not depend on myotube protein synthesis. The factor appears to rearrange the distribution of myotube AcCho receptors either by aggregating mobile AcCho receptors or by stabilizing labile receptor aggregates. Acetylcholine (AcCho) receptors of the innervated skeletal muscle are localized at the neuromuscular junction (1) seemingly by two processes: (i) accumulation of AcCho receptors at the neuromuscular contact region, and (ii) elimination of AcCho receptors from noncontact regions (2). Activation of the muscle fiber by electrical stimulation or synaptic activity can decrease the number of extrajunctional AcCho receptors (3); however, there is only speculation concerning the mechanism of AcCho receptor accumulation at muscle regions aligned with presynaptic acetylcholine release sites (4, 5). In various cells, randomly dispersed surface receptors form patches or caps when the cells are treated with specific immunoglobulins or lectins (6, 7). We here report a similar phenomenon: a factor produced by the cholinergic neuroblastoma-glioma hybrid cell line NG108-15 increases the number of AcCho receptor aggregates on cultured myotubes. We propose that such a factor may be involved in the aggregation of AcCho receptors at the site of nerve contact during synapse formation. MATERIALS AND METHODSCell Culture. Mouse muscle cultures were prepared from the hindlimbs of 18-20-day-old C57B/6N mouse embryos, as described (8), except that cell proliferation was suppressed by the addition of 10-5 M fluorodeoxyuridine on the fifth and sixth days of culture. Cultures were established by adding a suspension of 5 X 105 single cells to 35-mm plastic plates, or 1.5 X 105 cells to the 16-mm wells of plastic multiwell plates (Costar). Conditioned medium produced as described below was added to muscle cultures 10-21 days after plating.Rat muscle cultures were prepared from Fischer strain rat embryos by using the same methods.Chick muscle cultures were prepared from the pectoral muscle of 11-day-old chick embryos (9). A suspension of mechanically dissociated single cells was prepared in a medium of 85% Eagle's minimal essential medium, 10% horse serum, and 5% chick embryo extract, and 2,0 X 104 cells in 40ul 'of medium were added to the 5-mm round wells in Teflon-covered glass slides (Roboz Surgical Instrument Co.). On ...

Neuroblastoma X glioma NGIOS-15 hybrid cells exposed to N6,02'-dibutyryladenosine 3':5'-cyclic mono- (1), and the fetal bovine serum concentration was decreased from 10% to 5%. Cells to be used for experiments were dissociated and transferred to 200-Ml disposable glass capillary pipettes (total volume, 300 gl) bent in the form of a "U" and connected in series (1.5-2.0 X 105 cells per capillary) and incubated for 3 hr at 370 to promote cell attachment to the glass. The tubes then were perfused with 2 ml of medium per hr for 2 days unless indicated otherwise. and other 3H-labeled metabolites of choline were to be determined, the tubes were perfused for 1.5 min (0.7 ml/min) with medium A, a small air bubble was introduced into the line, and the capillary was filled with acetone/1 M formic acid, 85:15 (vol/vol) The 3H-labeled compounds extracted from cells were fractionated by high-voltage paper electrophoresis with 1.5 M acetic Abbreviations: Bt2cAMP, N6,02'-dibutyryladenosine 3':5'-cyclic monophosphate; ACh, acetylcholine; PG, prostaglandin; DMEM, Dulbecco's modification of Eagle's minimum essential medium; 5-HT, 5-hydroxytrypamine (serotonin).