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Y Dunant

Publications and source records attributed to Y Dunant.

At least 19 recordsLinked to original sources
PubMed1999

Acetylcholine synthesis and quantal release reconstituted by transfection of mediatophore and choline acetyltranferase cDNAs.

Neuroblastoma N18TG-2 cells cannot synthesize or release acetylcholine (ACh), and do not express proteins involved in transmitter storage and vesicle fusion. We restored some of these functions by transfecting N18TG-2 cells with cDNAs of either rat choline acetyltransferase (ChAT), or Torpedo mediatophore 16-kDa subunit, or both. Cells transfected only with ChAT synthesized but did not release ACh. Cells transfected only with mediatophore expressed Ca2+-dependent ACh release provided they were previously filled with the transmitter. Cell lines produced after cotransfection of ChAT and mediatophore cDNAs released the ACh that was endogenously synthesized. Synaptic-like vesicles were found neither in native N18TG-2 cells nor in ChAT-mediatophore cotransfected clones, where all the ACh content was apparently cytosolic. Furthermore, restoration of release did not result from enhanced ACh accumulation in intracellular organelles consecutive to enhanced acidification by V-ATPase, as Torpedo 16 kDa transfection did not increase, but decreased the V-ATPase-driven proton transport. Using ACh-sensitive Xenopus myocytes for real-time recording of evoked release, we found that cotransfected cells released ACh in a quantal manner. We compared the quanta produced by ChAT-mediatophore cotransfected clones to those produced by clones transfected with mediatophore alone (artificially filled with ACh). The time characteristics and quantal size of currents generated in the myocyte were the same in both conditions. However, cotransfected cells released a larger proportion of their initial ACh store. Hence, expression of mediatophore at the plasma membrane seems to be necessary for quantal ACh release; the process works more efficiently when ChAT is operating as well, suggesting a functional coupling between ACh synthesis and release.

Acetylcholine↗
PubMed1998

A simple, low-cost and fast Peltier thermoregulation set-up for electrophysiology.

Most of the parameters recorded in electrophysiology are strongly temperature dependent. In order to control temperature fluctuations we have built a system that ensures an accurate thermoregulation of the recording chamber. Temperature of physiological preparations can be changed relatively quickly (about 8 degrees C/min) and with a good accuracy (+/- 0.5 degrees C) without inducing thermal oscillations. Contrary to other thermoregulating devices, the temperature regulation is not carried out through the perfused medium but directly at the bottom of the chamber where a 3-cm2 Peltier element has been placed. The element is driven by a dedicated electronic device which controls the amount and the direction of the current flowing across the Peltier thermocouple. All construction details and the appropriate electrical circuits are provided. Using this home-made device, the steady-state chamber temperature could be precisely monitored with a resolution of +/- 0.1 degrees C in a range of 0-40 degrees C. This set-up was tested in experiments designed to evaluate the temperature dependence of synaptic transmission in the Torpedo nerve electroplate synapses and of calcium currents recorded from isolated nerve cells. This low-cost method is suitable for a wide range of applications.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Acetylcholine release and the cholinergic genomic locus.

Choline acetyltransferase and vesicular acetylcholine-transporter genes are adjacent and coregulated. They define a cholinergic locus that can be turned on under the control of several factors, including the neurotrophins and the cytokines. Hirschprung's disease, or congenital megacolon, is characterized by agenesis of intramural cholinergic ganglia in the colorectal region. It results from mutations of the RET (GDNF-activated) and the endothelin-receptor genes, causing a disregulation in the cholinergic locus. Using cultured cells, it was shown that the cholinergic locus and the proteins involved in acetylcholine (ACh) release can be expressed separately ACh release could be demonstrated by means of biochemical and electrophysiological assays even in noncholinergic cells following preloading with the transmitter. Some noncholinergic or even nonneuronal cell types were found to be capable of releasing ACh quanta. In contrast, other cells were incompetent for ACh release. Among them, neuroblastoma N18TG-2 cells were rendered release-competent by transfection with the mediatophore gene. Mediatophore is an ACh-translocating protein that has been purified from plasma membranes of Torpedo nerve terminal; it confers a specificity for ACh to the release process. The mediatophores are activated by Ca2+; but with a slower time course, they can be desensitized by Ca2+. A strictly regulated calcium microdomain controls the synchronized release of ACh quanta at the active zone. In addition to ACh and ATP, synaptic vesicles have an ATP-dependent Ca2+ uptake system; they transiently accumulate Ca2+ after a brief period of stimulation. Those vesicles that are docked close to Ca2+ channels are therefore in the best position to control the profile and dynamics of the Ca2+ microdomains. Thus, vesicles and their whole set of associated proteins (SNAREs and others) are essential for the regulation of the release mechanism in which the mediatophore seems to play a key role.

Acetylcholine↗
PubMed1998

Acetylcholine release. Reconstitution of the elementary quantal mechanism.

Choline acetyltransferase and vesicular acetylcholine transporter genes are the products of two adjacent genes defining a cholinergic locus. The release mechanism is expressed independently of this locus in some cell lines. A cholinergic neuron will therefore have to coordinate the expression of release with that of the cholinergic locus. Transfection of a plasmid encoding Torpedo mediatophore in cells that are unable to release this transmitter endows them with a Ca2(+)-dependent and quantal release mechanism. The synchronization of mediatophore activation results from a control of calcium microdomains by the synaptic vesicles. It is therefore dependent on the proteins that dock vesicles close to calcium channels.

Acetylcholine↗
PubMed1998

In vitro reconstitution of neurotransmitter release.

The vesicular hypothesis has stimulated fruitful investigations on many secreting systems. In the case of rapid synaptic transmission, however, the hypothesis has been found difficult to reconcile with a number of well established observations. Brief impulses of transmitter molecules (quanta) are emitted from nerve terminals at the arrival of an action potential by a mechanism which is under the control of multiple regulations. It is therefore not surprising that quantal release could be disrupted by experimental manipulation of a variety of cellular processes, such as a) transmitter uptake, synthesis, or transport, b) energy supply, c) calcium entry, sequestration and extrusion, d) exo- or endocytosis, e) expression of vesicular and plasmalemmal proteins, f) modulatory systems and second messengers, g) cytoskeleton integrity, etc. Hence, the approaches by "ablation strategy" do not provide unequivocal information on the final step of the release process since there are so many ways to stop the release. We propose an alternate approach: the "reconstitution strategy". To this end, we developed several preparations for determining the minimal system supporting Ca2+-dependent transmitter release. Release was reconstituted in proteoliposomes, Xenopus oocytes and transfected cell lines. Using these systems, it appears that a presynaptic plasmalemmal proteolipid, that we called mediatophore should be considered as a key molecule for the generation of transmitter quanta in natural synapses.

Animals↗
PubMed1997

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.

Microscopy, Electron↗
PubMed1997

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.

Acetates↗
PubMed1996

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].

Animals↗
PubMed1996

Cell lines expressing an acetylcholine release mechanism; correction of a release-deficient cell by mediatophore transfection.

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.

Acetylcholine↗
PubMed1996

Quantal acetylcholine release induced by mediatophore transfection.

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.

Acetylcholine↗
PubMed1996

Activation and desensitisation of acetylcholine release by zinc at Torpedo nerve terminals.

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.

Acetylcholine↗
PubMed1996

Disorganisation of quantal acetylcholine release by zinc at the Torpedo nerve-electroplate junction.

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.

Acetylcholine↗
PubMed1996

A unifying hypothesis for acetylcholine release.

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.

Acetylcholine↗
PubMed1996

Enhancement of quantal transmitter release and mediatophore expression by cyclic AMP in fibroblasts loaded with acetylcholine.

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.

Acetylcholine↗
PubMed1996

Evoked acetylcholine release expressed in neuroblastoma cells by transfection of mediatophore cDNA.

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.

Acetylcholine↗
PubMed1995

Mediatophore and other presynaptic proteins. A cybernetic linking at the active zone.

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.

Animals↗