A factor from neurons induces partial immobilization of nonclustered acetylcholine receptors on cultured muscle cells.

A factor or factors released by cultured NG108-15 neuroblastoma X glioma hybrid cells and added to the medium of rat myotube primary cultures was found to immobilize some of the previously mobile acetylcholine receptors in the myotube membrane. Partial receptor immobilization occurred within 3 h after the beginning of treatment with the NG108-15-conditioned medium factor and persisted for at least 24 h of continuous treatment. A similarly derived conditioned medium concentrate from the non-neuronal parent glioma cell line did not immobilize receptors, relative to untreated controls. Acetylcholine receptors were visualized by fluorescent alpha-bungarotoxin and their lateral motion was observed by the technique of fluorescence photobleaching recovery.

Computer analysis of organelle translocation in primary neuronal cultures and continuous cell lines.

Organelle translocation in a number of cell types in tissue culture as seen by high-resolution Zeiss-Nomarski differential interference contrast optics was filmed and analyzed by computer. Principal cell types studied included primary chick spinal cord, chick dorsal root ganglion, ratbrain, and various clones of continuous cell lines. Organelle translocations in all cell types studied exhibited frequent, large changes in velocity during any one translocation. The appearance of particles as seen with Nomarski optics was correlated with their fine structures in one dorsal root ganglion neurite by fixing the cell as it was being filmed and obtaining electron micrographs of the region filmed. This revealed the identity of several organelles as well as the presence of abundant neurotubules but no neurofilaments. Primary cell cultures exhibited more high-velocity organelle movements than continuous cell lines. The net progress of an organelle in a given direction was greater in primary neuronal cells than in fibroblasts or continuous cell lines. These findings are correlated with the literature on organelle translocation and axoplasmic transport.

Interaction of the cytoskeletal framework with acetylcholine receptor on th surface of embryonic muscle cells in culture.

To monitor the interaction of cell surface acetylcholine (AcCho) receptors with the cytoskeleton, cultured muscle cells were labeled with radioactive or fluorescent alpha-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 skeleton organization responsible for restricting AcCho receptors to a patched region may also result in their retention after detergent extraction.

Synapse formation between two clonal cell lines.

Clonal neuroblastoma x glioma hybrid cells frequently formed synapses with clonal mouse striated muscle cells. Clonal myotubes were similar to cultured mouse embryo myotubes with respect to acetylcholine sensitivity and other membrane properties examined. However, acetylcholine sensitivity measurements indicate that acetylcholine receptors of clonal myotubes are distributed more uniformly over the cell surface than the receptors of cultured mouse embryo myotubes.

Pharmacologic responses of cells of a neuroblastoma X glioma hybrid clone and modulation of synapses between hybrid cells and mouse myotubes.

Cells of the hybrid clone NG108-15 responded to 5-hydroxytryptamine (5-HT), dopamine or acetylcholine with graded depolarizations involving membrane conductance increases. Responses desensitized during continuous application of the neurotransmitters, and responses to 5-HT and dopamine cross-desensitized: a desensitizing application of one neurotransmitter also desensitized the hybrd cell to the other neurotransmitter. 5-HT and acetylcholine did not cross-desensitize. The hybrid cell 5-HT response was not attenuated by D-LSD, and was blocked by 10(-5) M morphine, although not via binding to naloxone-sensitive opiate receptors. 5-HT or the prostaglandin PGF2alpha caused the release of acetylcholine at the synapses of hybrid cells with mouse myotubes. Application of 5-HT or PGF2alpha also facilitated the synaptic release elicited by hybrid cell action potentials. Following treatment with the antimitotic agent cytosine arabinoside, co-cultures of hybrid cells and mouse myotubes exhibited plentiful synaptic connections only if maintained in medium containing 1 mM dibutyryl cAMP (dBcAMP). After X-irradiation, co-cultures were synaptically active even in the absence of dBcAMP. Thus, methods have been found to regulate both the short-term and long-term synaptic activity of NG108-15 hybrid cells.

Characterization and partial purification of a neuronal factor which increases acetylcholine receptor aggregation on cultured muscle cells.

Medium conditioned by NG108-15 neuroblastoma x glioma hybrid cells contains a factor which increases the number of acetylcholine receptor (AChR) aggregates on cultured myotubes. Protease digestion indicates that the AChR aggregation factor is a protein, and the molecular weight is from 150,000 to 250,000 daltons as estimated by ultrafiltration and gel filtration. Preparative isoelectrofocusing indicates that the aggregation factor has a pI of about 4.7. The factor is found in the soluble cytoplasmic fraction but not in the plasma membrane fraction of NG108-15 cells. Aggregation activity is not detected in the cytoplasm of liver cells or in the cytoplasm of C6BU-1 glioma cells. A possible developmental role for the aggregation factor is suggested by its presence in embryonic rat brain but not in adult rat brain. AChR aggregation factors found in the cytoplasm or conditioned medium of NG108-15 cells or in the cytoplasmic fraction of fetal brain have similar molecular weights and isoelectric points.

Formation of synapses between cells of a neuroblastoma X glioma hybrid clone and mouse myotubes.

Synapses form between cells of a neuroblastoma X glioma hybrid clone and cultured mouse skeletal myotubes. The synapses are cholinergic, and the acetylcholine release mechanism is dependent on calcium ions. The transmitter output of the synapses is low, with considerable variability in the latency and amplitude of the postsynaptic responses to presynaptic action potentials. The fine structure of physiologically identified functional junctions was examined electron microscopically. Small (50 nm) clear vesicles were seen presynaptically and there were areas with a wide (approx. 50 nm) gap containing basement membrane-like material between the pre- and postsynaptic cells. In addition, in some regions there was a densely staining material lining the muscle membrane and some suggestion of infolding of the muscle membrane. In none of the cases, however, have areas been found where small, clear vesicles cluster around pre- and postsynaptic membrane densities. Thus, functional synapses can occur in the absence of the highly organized synaptic structure seen at mature synapses.

Mouse spinal cord in cell culture. II. Synaptic activity and circuit behavior.

1. Neurons in cell cultures of fetal mouse spinal cord (SC) and dorsal root ganglia (DRG) develop extensive synaptic interconnections. 2. No spontaneous synaptic activity was detectable in the presence of tetrodotoxin or an elevated magnsium ion concentration, but statistical analysis of evoked excitatory postsynaptic potentials (EPSPs) indicates that the quantal size was 200-250 muV, which was below the noise level of the recording system used. 3. In a sample of eight RDG-SC and seven SC-SC cell pairs linked by EPSPs, the quantal content of the SC-SC EPSPs was about 3.5-fold larger than for the DRG-SC EPSPs. 4. The extrapolated equilibrium potential for the SC-SC EPSP was about 20 mV positive. The IPSP reversed at a membrane potential of 60-80 mV negative. 5. Some examples of the types of synaptic circuits commonly encountered are given. Only one case of electrical coupling between neurons was found.

Successive openings of the same acetylcholine receptor channel are correlated in open time.

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.

Cell interactions in nerve and muscle cell cultures.

The neurotransmitter synthesized by a given class of neurones is subject to modification and, indeed, a qualitative switchover in transmitter biochemistry recently has been demonstrated (Furshpan, POtter & Landis, 1980; Walicke, Campenot & Patterson, 1977). In conjunction with the specification of transmitter biosynthesis that becomes established in a given neurone, a complementary specification of appropriate receptor production is required in any cell functionally post-synaptic to that neurone. An additional requirement of peculiar force in the nervous system has to do with the spatial organization of the receptors in the surface membrane of the post-synaptic cell once the receptors are synthesized. Inappropriately distributed receptors are useless receptors. The perfect registration of a variety of types of presynaptic release sites with high post-synaptic concentrations of appropriate receptors constitutes one of the outstanding features of nervous-system organization that must be accounted for. We report some experiments directed toward understanding the cell biology of regulation of receptor distribution over the surface membrane of muscle cells. Functional synaptic connexions are formed quite early in development and the stability and maturation of synaptic networks is contingent on a number of factors. One interesting contingency is that related to the functional activity of developing networks. Do only those networks survive and mature which are activated by stimuli impinging from the environment? (Wiesel & Hubel, 1963). Put more simple, are action potentials and synaptic activity essential for neuronal maturation? We address this question in cell culture systems from the mammalian central nervous system.

Laminin induces acetylcholine receptor aggregation on cultured myotubes and enhances the receptor aggregation activity of a neuronal factor.

The effect of several basement membrane components on the aggregation of acetylcholine (ACh) receptors on cultured myotubes was studied. Cultures were incubated for 16 to 24 hr with laminin, a heparan sulfate proteoglycan, collagen types IV and V, or fibronectin, alone, or together with medium conditioned by NG108-15 neuroblastoma X glioma hybrid cells (NCM). The number of ACh receptor aggregates per myotube was assayed by fluorescence microscopy of cultures stained with tetramethylrhodamine-labeled alpha-bungarotoxin. Laminin induced ACh receptor aggregation on primary rat myotubes and on myotubes formed by G8-1 clonal rat muscle cells. Laminin enhanced the receptor-aggregating activity of NCM in a concentration-dependent manner (0.6 to 6.0 micrograms/ml) and the number of aggregates formed in the presence of laminin and NCM together was greater than the sum of the aggregates induced by NCM and laminin separately. The aggregation factor in NCM is probably not laminin, since less than 10 ng/ml of laminin-like immunoreactivity was detected in NCM, and antiserum against laminin blocked the effects of laminin but had little effect on NCM aggregation activity. Collagen type V enhanced the receptor aggregation activity of NCM, but less strongly than laminin, and had little or no effect by itself. The other basement membrane components did not induce receptor aggregation or enhance the effect of NCM. Experiments in which ACh receptors were labeled before exposure of cultures to NCM and laminin indicated that laminin enhanced the rearrangement of receptors at the cell surface. Immunofluorescence microscopy indicated that laminin binds to the myotubes within 30 min and forms patches on the cell surface over a period of hours. Laminin bound to the myotube surface enhanced receptor aggregation as well as laminin continuously present in the culture medium. The results suggest the possibility that laminin could enhance the receptor aggregation activity of a neuronal factor(s) released at the developing neuromuscular junction.

A factor from neurons increases the number of acetylcholine receptor aggregates on cultured muscle cells.

There is an increase in the number of acetylcholine (AcCho) receptor aggregates on striated embryonic mouse myotubules 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.

Synaptic interactions between mammalian central neurons in cell culture. III. Morphophysiological correlates of quantal synaptic transmission.

The statistical properties of excitatory synaptic transmission between neurons in cell cultures of fetal mouse spinal cord were studied and the anatomical extent of these connections demonstrated by horseradish peroxidase (HRP) injection of the presynaptic cell. In conjunction with previous experiments (7), we have correlated the number of boutons involved in a given synaptic connection with the physiologically determined number of release elements. The number of boutons is somewhat greater than the number of release elements, and our results are supportive of the conclusion of others (1, 2) that the physiologically defined release element corresponds to the bouton. We interpret this to suggest that a rate-limiting mechanism may operate at the level of the bouton to allow release of no more than one quantal unit of transmitter with each presynaptic action potential.

Synaptic interactions between mammalian central neurons in cell culture. II. Quantal Analysis of EPSPs.

The presynaptic release mechanism involved in excitatory synaptic connections between neurons in cell cultures of fetal mouse spinal cord were studied by statistical analysis of intracellularly recorded postsynaptic responses. Quantal parameters were determined for the EPSPs evoked in spinal cord (SC) neurons by stimulation of either other SC or dorsal root ganglion (DRG) neurons. Transmitter release was manipulated by varying the Ca2+ and Mg2+ content of the culture medium. The release process was represented better by binomial than by Poisson statistics. A method was derived for obtaining the probability of release and the number of release elements. The quantal content and the number of release elements were substantially higher for the SC-SC connection than for the DRG-SC connection. This was partially compensated for by a larger quantal amplitude for the DRG-SC connection. There was some indication that the probability of release was higher for the SC-SC connection. The relationship between transmitter output and effective external Ca2+ ion concentration was approximately linear.