Biomedical subjects
Publications and source records attributed to Y Dunant.
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.
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.
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].
Several neuronal and non-neuronal cell lines express a Ca(2+)-dependent mechanism of transmitter release that can be demonstrated after loading the cells with acetylcholine during culture. In contrast, a particular cell line, the neuroblastoma N18TG-2, was found to be deficient for release. We transfected N18TG-2 cells with a plasmid encoding Torpedo mediatophore, a protein able to translocate acetylcholine in response to calcium. The N18TG-2 cells expressed the Torpedo protein which reached their plasma membrane. At the same time, these cells acquired a Ca(2+)-dependent quantal release mechanism similar to the one naturally expressed by other cell lines. Hence, the presence of mediatophore in the plasma membrane seems essential for quantal release.
Mediatophore is a protein of approximately 200 kDa able to translocate acetylcholine in response to calcium. It was purified from the presynaptic plasma membranes of the electric organ nerve terminals. Mediatophore is a homooligomer of a 16-kDa subunit, homologous to the proteolipid of V-ATPase. Cells of the N18TG-2 neuronal line are not able to produce quantal acetylcholine release. We show here that transfection of N18TG-2 cells with a plasmid encoding the mediatophore subunit restored calcium-dependent release. The essential feature of such a release was its quantal nature, similar to what is observed in situ in cholinergic synapses from which mediatophore was purified.
Treatment with 100 or 250 microM ZnCl2 irreversibly blocked neurotransmission in the Torpedo electric organ by inhibiting acetylcholine (ACh) release. In Zn2+-treated tissue, release failure did not result from impairment of Ca2+ entry since stimulation still provoked an accumulation of Ca2+. Also pretreatment of isolated synaptosomes with Zn2+ inhibited to the same extent the release elicited by KCl-evoked depolarisation and the release elicited by using the Ca2+ ionophore A23187. On the other hand, after application of A23187, Zn2+ by itself efficiently triggered ACh release from synaptosomes. This dual effect of Zn2+ was also observed to occur in proteoliposomes equipped with mediatophore (a protein of the presynaptic membrane characterised by its capability to support Ca2+-dependent transmitter release). Hence, Zn2+ mimicked two fundamental actions of Ca2+ on nerve terminals, which are: (1) the immediate activation of release, and (2) a more slowly developing desensitisation of release. Zn2+ was more powerful than Ca2+ for both actions. It is concluded that the dual action of Zn2+ on the mediatophore protein accounts at least in part for its complex effects on neurotransmission.
The effects of zinc (Zn2+) on quantal acetylcholine release at the Torpedo nerve-electroplate junction were analysed by using loose patch electrodes designed to record evoked and spontaneous electroplate currents in a delimited area (electrode diameter of 10-15 microm) of the synaptic region. Zn2+ reduced the amplitude, prolonged the synaptic delay and slowed down the rising phase of all-or-none electroplate currents (EPCs) generated in response to activation of Na+ channels in a preterminal nerve branch. In graded EPCs (generated in response to direct activation of terminal Ca2+ channels), Zn2+ caused a reduction of quantal content but no change in the quantal size or in the minimum synaptic delay. The rise time of graded EPCs was prolonged but their half-decay time was not affected. Miniature EPCs (MEPCs) in control preparations had a widely distributed amplitude distribution but a homogeneous and rapid time course. Conversely, MEPCs in Zn2+-treated tissue exhibited a homogeneous and small amplitude, but a prolonged and more variable time course. Zn2+ at 1 mM caused, by itself, a high occurrence of MEPCs under conditions (flat-edged electrodes) when MEPCs are normally very infrequent. It is concluded that Zn2+ can both activate and inhibit the release mechanism and Zn2+-induced quanta exhibit an abnormal time course. The activation of the release process by Zn2+ or by Ca2+ may result in the production of quanta with different kinetics.
Mediatophore is the only nerve terminal membrane protein known to translocate acetylcholine upon calcium action. It is localized at the active zone. In this review we attempted to describe its role in relation to the vesicular and membrane protein complexes that are formed at the active zone. The model pictures a possible set of sequential steps that lead to exocytosis. The smallest quantal events are attributed to mediatophore opening momentarily, while synaptic vesicles synchronize release by controlling the calcium microdomain. A clear distinction is made between sub-quantal ACh release preserved after Botulinum toxin action, and exocytosis of vesicular contents. A cybernetic model for release and exocytosis related to protein interactions is presented for future works.
Neuronal properties such as neurotransmitter uptake and release can be expressed in non-neuronal cells. We show here that fibroblasts-mouse cell line L-M(TK-)-are able to take up acetylcholine from the external medium and to release it in response to a calcium influx. Release was assessed biochemically by a luminescence method, but it was also elicited from individual fibroblasts and recorded in real-time using a Xenopus myocyte as an acetylcholine detector. After treatment for three to six days with dibutyryl-cyclic AMP, the cells changed their shape and acetylcholine release was greatly enhanced. Surprisingly, in differentiated fibroblasts the time-course transmitter release exhibited a high degree of variability even for the successive responses evoked from the same cell; many currents recorded in myocytes on electrical stimulation of fibroblasts had an extremely long duration (up to 1 s or more). This suggested that the release sites were kept open for a very long time. Cyclic AMP treatment also caused a marked increase in the expression of mediatophore 16,000 mol. wt proteolipid in fibroblast membranes. Mediatophore is an acetylcholine-translocating protein which is abundant in cholinergic presynaptic plasma membranes. It is concluded that cyclic AMP differentiation of fibroblasts prolongs the duration of acetylcholine release at individual sites and enhances the expression of the 16,000 mol. wt proteolipid-forming mediatophore.
Transmitter release was elicited in two ways from cultured cells filled with acetylcholine: (a) in a biochemical assay by successive addition of a calcium ionophore and calcium and (b) electrophysiologically, by electrical stimulation of individual cells and real-time recording with an embryonic Xenopus myocyte. Glioma C6-Bu-1 cells were found to be competent for Ca(2+)-dependent and quantal release. In contrast, no release could be elicited from mouse neuroblastoma N18TG-2 cells. However, acetylcholine release could be restored when N18TG-2 cells were transfected with a plasmid coding for mediatophore. Mediatophore is a protein of nerve terminal membranes purified from the Torpedo electric organ on the basis of its acetylcholine-releasing capacity. The transfected N18TG-2 cells expressed Torpedo mediatophore in their plasma membrane. In response to an electrical stimulus, they generated in the myocyte evoked currents that were curare sensitive and calcium dependent and displayed, discrete amplitude levels, like in naturally occurring synapses.
In rapidly transmitting synapses, the mediatophore, a protein located in the presynaptic membrane, seems to play a key role in the last step of transmitter release. Reconstituted either in proteoliposomes or in Xenopus oocytes, or transfected in particular cell lines, the mediatophore is able to release acetylcholine with characteristics which meet several typical features of transmitter release in natural synapses. Good correspondence between the two conditions was found for: i) the dependency of release upon calcium concentration; ii) the desensitisation of release by persistence of internal calcium; iii) the effect of several drugs; iv) the fleeting formation of a population of large intramembrane particles during the precise time of release; and v) the pulsatile or quantal nature of transmitter release. All these features therefore could well be ascribed to intrinsic properties of the mediatophore molecule. How is the mediatophore integrated in the whole presynaptic apparatus? To what extent is its function regulated by the other proteins of the active zone? These questions are far from being solved. We want nevertheless to propose here a general view in which characteristic presynaptic functions such as transmitter release, calcium entry, sequestration and extrusion, regulation of short- and long-term changes in release efficiency, are supported by an ordered succession of molecular events involving the proteins of the active zone. It will be seen that some proteins compete for a common binding site. It is thus expected that they will occupy this site in a regulated succession, according to simple cybernetic rules.
Distinct mechanisms operate in different secreting systems, they are 1) free diffusion through the plasma membrane; 2) exocytosis resulting from fusion of a secretory granule with the plasma membrane; 3) fleeting release from a granule through a transient pore without full fusion; 4) release through a specialised plasmalemmal molecule such as the mediatophore. The latter mechanism is proposed to operate in rapid synapses in which the neurotransmitter is emitted as an abrupt chemical impulse of quantal composition. There, release is momentarily signalled in the plasma membrane by large intramembrane particles. Synaptic vesicles are also essential for regulation of this type of release. They fuse with the plasma membrane only late after activity and seem to be involved in calcium sequestration and extrusion.
Synaptic transmission, metabolism of calcium and ultrastructural changes were investigated at the nerve-electroplaque synapse of Torpedo marmorata during and after a brief tetanic stimulation. Calcium was found to accumulate in stimulated tissue as a function of the number of stimuli; it was subsequently expelled during the recovery period. This period was also accompanied by a marked hydrolysis of energy-rich phosphates (ATP and creatine phosphate). Histochemical localization combined with electron spectroscopic imaging showed calcium deposits in synaptic vesicles and in other substructures. The number of synaptic vesicles containing a calcium deposit transiently increased at the end of activity and declined later during the recovery phase. Rapid cryofixation of the tissue followed by freeze-fracturing revealed membrane openings (pits) in the presynaptic membrane. The density of pits was low in resting tissue; it did not rise during the tetanic stimulation. In contrast, the number of presynaptic pits increased significantly soon after, reaching a maximum value at 1 min after tetanus. These results are discussed in the light of current hypotheses. They suggest that synaptic vesicles play an important role in intraterminal calcium homeostasis. The vesicles might sequester calcium ions in synaptic terminals during activity and expel them afterwards by exocytosis.
Antisense oligodesoxynucleotides were used to determine whether the mediatophore proteolipid is necessary for the Ca(2+)-dependent release of the neurotransmitter acetylcholine. Xenopus laevis oocytes were injected with poly(A)+ mRNAs extracted from the electric lobes of Torpedo marmorata. The electric lobes contain an homogeneous population of cholinergic neurons homologous to motoneurons. Addition of antisense probes hybridizing to the mediatophore 15 kDa subunit inhibited the expression of both the mediatophore proteolipid in oocyte membranes and the Ca(2+)-dependent acetylcholine release. Expression of other neuronal functions such as synthesis of [14C]acetylcholine from [14C]acetate was not inhibited. Another antisense probe specific for the sequence of a related proteolipid cDNA (the 15 kDa subunit of the chromaffin granule protonophore) was used as a control. It did not hybridize with the Torpedo mediatophore mRNA and, injected in addition to electric lobe mRNAs, it did not inhibit either mediatophore expression or acetylcholine release. We showed in addition that the mRNA primed oocytes did not contain a vesicular pool of acetylcholine. It was concluded (i) that the mediatophore proteolipid is essential for Ca(2+)-dependent acetylcholine release and (ii) that the cytosolic pool of neurotransmitter seems to be preferentially used in this system.
Function-dependent changes of calcium distribution were studied in the nerve-electroplaque synapses of Torpedo marmorata before and after the transmission of a nerve impulse. For the cytochemical demonstration of calcium at the ultrastructural level the oxalate-pyroantimonate technique was combined with electron spectroscopic imaging. Cholinergic synapses of the electric organ were stimulated in the presence of 4-aminopyridine, a drug which powerfully potentiates transmitter release. A single stimulus evoked a giant electrical discharge, which was followed by a long refractory period. Calcium cytochemistry was performed by fixing the tissue at four well defined functional states: (i) before and (ii) immediately after the giant discharge, and (iii) at 1 min or (iv) at 30 min of subsequent rest, corresponding to partial and complete functional recovery, respectively. In the non-stimulated synapses about 20% of synaptic vesicles contained small electron-dense precipitates. The element specific mapping by electron spectroscopic imaging clearly showed that calcium was present in the vesicular granules. The volume density of synaptic vesicles did not change among the four experimental states, but we detected a significant increase in the proportion of calcium containing vesicles at 1 min after the giant discharge. The vesicular calcium accumulation was transient: it returned to the control value at the end of the recovery period. Our data suggest that the synaptic vesicles play a role in sequestering the excess calcium which enters the nerve terminal during stimulation.
We would like to review here some of the acquisitions gained by recent work in our two laboratories. Our approaches and results were intermingled and complementary. Thus we found it appropriate, for clarity and intelligibility, to merge them into a single chapter.
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1. A loose patch electrode was used to stimulate axon terminals and to record evoked electroplaque currents (EPCs) in a limited area of innervated membrane of the electric organ of Torpedo marmorata. Electrophysiological signals were compared to the predictions of a semi-quantitative model of synaptic transmission which was designed to simulate the release of several packets of neurotransmitter molecules, at the same or at different sites of the synapse, synchronously or with various temporal patterns. 2. The amplitude distribution of EPCs evoked by activation of nerve terminals showed quantal steps. The time to peak of EPCs was in most cases independent of amplitude, but in their decaying phase a positive correlation was seen between half-decay time and amplitude. Comparison with the model suggested that (i) a dynamic interaction occurred at the end of the EPC between the fields of postsynaptic membrane activated by individual quanta, and (ii) the sites of quantal release in the electric organ are separated from each other by 600-1000 nm. 3. Spontaneous miniature electroplaque potentials (MEPPs) were recorded externally with the same type of loose patch electrode. The majority (75%) of external MEPPs displayed a homogeneous and rapid time course. This fast MEPP population had a mean time to peak of 0.43 ms, a half-decay time of 0.45 ms and a time constant of decay of 0.35 ms. 4. Despite homogeneous characteristics of time course, fast MEPPs exhibited a wide amplitude distribution with a main population which could be fitted by a Gaussian curve around 1 mV, and another population of small amplitude. Both the time-to-peak and the half-decay time of fast MEPPs showed a positive correlation with the amplitude from the smallest to the largest events. Acetylcholinesterase was not blocked. 5. In addition to the fast MEPPs, spontaneous signals exhibiting a slow rate of rise, or a slow rate of decay, or both were observed. They occurred at any time during the experiment, independently of the overall frequency. Approximately 15% of the total number of events had a slow rise but their decay phase was nevertheless rapid and could be ascribed to the kinetics of receptors. These slow-rising MEPPs exhibited a variety of conformations: slow but smooth rise, sudden change of slope and sometimes several bumps or inflexions. Their average amplitude was significantly smaller than that of the main population of fast MEPPs. 6. Composite MEPPs with multiple peaks as well as bursts of small MEPPs were often encountered, even during periods of low frequency.(ABSTRACT TRUNCATED AT 400 WORDS)