Quantal transmitter release by glioma cells: quantification of intramembrane particle changes.

Glial cells in situ are able to release neurotransmitters such as glutamate or acetylcholine (ACh). Glioma C6BU-1 cells were used to determine whether the mechanisms of ACh release by a glial cell line are similar or not to quantal release from neurones. Individual C6BU-1 cells, pre-filled with ACh, were moved into contact with a Xenopus myocyte that was used as a real-time ACh detector. Upon electrical stimulation, C6BU-1 cells generated evoked ACh impulses which were Ca(2+)-dependent and quantal (quantal steps of ca. 100 pA). Changes in plasma membrane ultrastructure were investigated by using a freeze-fracture technique designed for obtaining large and flat replicas from monolayer cell cultures. A transient increase in the density of medium and large size intramembrane particles--and a corresponding decrease of small particles--occurred in the plasma membrane of C6BU-1 cells stimulated for ACh release. Changes in interaction forces between adjacent medium and large particles were investigated by computing the radial distribution function and the interaction potential. In resting cells, the radial distribution function revealed a significant increase in the probability to find two particles separated by an interval of 24 nm; the interaction potential suggested repulsive forces for intervals shorter than 24 nm and attractive forces between 24 and 26 nm. In stimulated cells, this interaction was displaced to 21 nm and made weaker, despite of the fact that the overall particle density increased. The nature of this transient change in intramembrane particles is discussed, particularly with regard to the mediatophore proteolipid which is abundant in the membranes C6-BU-1 like in those of cholinergic neurones. In conclusion, evoked ACh release from pre-filled C6-BU-1 glioma cells is quantal and Ca(2+)-dependent. It is accompanied by a transient changes in the size distribution and the organisation of intramembrane particles in the plasma membrane. Thus, for the release characteristics, glioma cells do not differ fundamentally from neurones.

Morphological changes related to reconstituted acetylcholine release in a release-deficient cell line.

The membrane changes accompanying Ca(2+)-dependent acetylcholine release were investigated by comparing release-competent and release-incompetent clones of mouse neuroblastoma N18TG-2 cells. No release could be elicited in native N18 cells or in a N18-choline acetyltransferase clone in which acetylcholine synthesis was induced by transfection with the gene for rat choline acetyltransferase. However, acetylcholine release was operative in a To/9 clone which was co-transfected with complementary DNAs from rat choline acetyltransferase and Torpedo mediatophore 16,000 mol. wt subunit. In thin sections, the aspect of resting N18 and To/9 cells was identical: a very dense cytoplasm with practically no vesicle-like organelles. Cells were chemically fixed at different times during a stimulation using A-23187 and Ca2+, and examined following both freeze-fracture and thin section. Stimulation of To/9 cells induced a marked change affecting the intramembrane particles. The number of medium-sized particles (9.9-12.38 nm) increased, while that of the small particles decreased. This change was not observed in control, release-incompetent cell lines. In the To/9 clone (but not in control clones), this was followed by occurrence of a large new population of pits which initially had a large diameter, but subsequently became smaller as their number decreased. Coated depressions and invaginations became abundant after stimulation, suggesting an endocytosis process. By considering the succession of events and by comparison with data from experiments performed on synapses in situ, it is proposed that a particle alteration was the counterpart of acetylcholine release in co-transfected To/9 cells; this was followed by a massive endocytosis.

Binding of gold-protein ligand complexes to neurokinin receptors. visualization with the electron microscope on pig coronary artery and rat portal vein.

Active neurokinin (NK) receptors were visualized with 5-nm colloidal gold-protein-substance P (GPSP) and -senktide (GPSenk) complexes on vascular tissues. Electron micrographs of pig coronary strips incubated with GPSP showed gold particles either bound to the plasmalemma or inside intracytoplasmic vesicles of endothelial cells. Preincubation with SP or the NK1 receptor antagonist L-703606 prevented GPSP marking. No gold particles were seen after incubation with GPSenk. On coronary strips in vitro, which had been precontracted with U46619, GPSP induced relaxations similar to those produced by equimolar concentrations of SP, both relaxations being inhibited by L-703606. Analogous to senktide, GPSenk was totally inactive on arterial strips. Incubation of rat portal veins with GPSenk showed gold particles bound to the plasmalemma or inside intracytoplasmic vesicles of smooth muscle cells. Preincubation with senktide or the NK3 receptor antagonist R-820 prevented GPSenk marking. On portal veins in vitro GPSenk induced contractions similar to those induced by equimolar concentrations of senktide or NKB; these effects were inhibited by R-820. Our results show that colloidal gold-protein complexes present biological activity and selectivity similar to those of their respective native ligand and detect the presence of active receptors; in addition, they suggest the presence of NK-receptor-mediated endocytosis in endothelial cells of coronary artery and in smooth muscle cells of the portal vein.

An improved approach to freeze-fracture morphology of monolayer cell cultures.

Much work is currently done on cell cultures to elucidate membrane processes associated with different cell functions. We describe here a modified freeze-fracture method to obtain systematically large fractured areas of the plasma membrane from monolayer cell culture in situ. Cells are grown until confluence on a Thermanox coverslip overlaid with poly-L-ornithine. After chemical fixation, the culture is flattened overnight by sandwiching it between the Thermanox coverslip, a Falcon membrane and a glass coverslip, under a 5 g weight. After freeze-fracture, vast pictures of the protoplasmic leaflets are obtained in a reproducible manner. Our approach was applied to cultures which were stimulated to release acetylcholine; it has been found very appropriate for studying modifications affecting intramembrane particles and vesicles openings in the plasmalemma. Accurate quantifications were performed and correlations were established between the membrane changes and the data revealed by thin sections. The present sandwich method can be applied to a variety of cell preparations, allowing for quantitative study of structure-function relationships.

Zinc blocks acetylcholine release but not vesicle fusion at the Torpedo nerve-electroplate junction.

The combined effects of Zn2+ treatment and nerve stimulation were studied on cholinergic synapses of the Torpedo marmorata electric organ. Incubation of small pieces of electric tissue in 250 microM ZnCl2 for 2 h irreversibly blocked synaptic transmission by inhibiting the release of acetylcholine. This treatment, however, did not cause any significant fine structural alteration in the nerve-electroplate junctions. Preparations treated with Zn2+ were submitted to electrical stimulation. In spite of the fact that no transmitter was released, stimulation resulted in the accumulation of calcium in the tissue, and in marked ultrastructural changes. The density of synaptic vesicles was significantly reduced and many of the remaining vesicles were found in close proximity to the presynaptic membrane. Images of vesicles fused with the plasmalemma were abundant, indicating that numerous vesicles were caught in different phases of exocytosis or endocytosis. Freeze-fracture replicas made from quick-frozen or chemically fixed material showed a high number of vesicle openings (pits) in the presynaptic plasmalemma. No recovery occurred even after a prolonged period of rest, indicating that retrieval was impaired by zinc treatment. In conclusion, the present experimental paradigm created an unusual situation where fusion of synaptic vesicles to the plasma membrane could be activated independently from the release of transmitter.

Simple goniometric search for electron microscopy.

We developed a special software to memorize specific points on a grid for later fine analysis. This program is based on a goniometry search, and implemented on the PC coupled microscope. This allows automatic shifting of the grid, but it can also be programmed on a simple pocket calculator.

Flatten-peeled: a new approach to freeze-fracture morphology.

A new way to obtain in a replica vast pictures of membranes situated at the same level in the preparation is described. The tissue specimen is flattened during fixation with a given orientation. It can be fractured afterwards, either along the plane of fixation or, after rotation, perpendicular to this plane. The method utilizes standard freeze-fracture equipment and readily available materials. This approach has a large range of potential applications, especially with tissues displaying a layered organization such as epithelia, nervous system, etc. It was found very appropriate for studying pre- and postsynaptic membranes in the Torpedo electric organ, to reveal specific junctions between cells in organotypic cultures, and to examine photoreceptors and other layers of the mammalian retina. In those tissues we obtained in a fairly reproducible manner freeze fractures at the desired level and orientation. With Torpedo synapses, vast pictures of the nerve terminals were performed in a plane quasi-parallel to the membrane postsynaptic cells (electroplates). Using double or triple flatten-peeled, it has also been possible to render the whole tissue specimen thin enough to perform gold label fracture [Pinto da Silva and Kan (1984), J. Cell Biol., 99:1156-1161].