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Biomedical subjects

Y Dunant

Publications and source records attributed to Y Dunant.

At least 73 records · Page 4Linked to original sources
PubMed1986

Increase in the number of presynaptic large intramembrane particles during synaptic transmission at the Torpedo nerve-electroplaque junction.

Small pieces of Torpedo electric organ were treated with 4-aminopyridine, a drug which greatly increases the duration of transmitter release in a single nerve impulse, transforming the normally brief electroplaque potential to a giant discharge. Specimens of tissue were cryofixed by rapid freezing using liquid coolants at precise time intervals during transmission of a single giant discharge, and then examined by freeze fracture. In each experiment, we monitored the electrical response of one specimen during the freezing run to check the physiological responsiveness of the tissue and to determine the precise time of contact with the cryogenic liquid. The general appearance of nerve terminals after cryofixation was similar to that of terminals from chemically fixed and cryoprotected tissue. The major morphological change observed during the time course of the giant discharge was a marked increase in the density of intramembrane particles larger than 10 nm on both the protoplasmic and external faces of the presynaptic membrane. This change appeared in specimens frozen within the first few milliseconds after the stimulus, that is, at a time corresponding to the onset of the rising phase of the potential (3 ms). At the end of the giant discharge, the particle density returned to control values with the same time course as the potential trace. Pits of 20 nm or larger, probably due to vesicle-membrane interaction, were found in a small proportion of nerve terminals. Their occurrence increased only at 120-150 ms after the stimulus, that is, a long time after the beginning of the giant potential and of the change in intramembrane particles. The size distribution of particles was also determined in the membrane of synaptic vesicles exposed by cross fracture of terminal boutons; it was found to be similar to that of the unstimulated presynaptic membrane and it did not change during the giant discharge. Stimulation experiments were also carried out in a modified solution containing no added calcium, 20 mM magnesium and 4-aminopyridine. The propagation of impulses along the nerves to the electric organ was not inhibited in the modified solution but acetylcholine release was prevented and no increase in particle density was found on the presynaptic membrane. These and previous biochemical experiments on this tissue suggest that the release of the neuro-transmitter acetylcholine is associated with a transient occurrence of large intramembrane particles on the two fracture faces of the presynaptic membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Aminopyridine↗
PubMed1986

Release of ethanolamine, but not of serine or choline, in rat pontine nuclei on stimulation of afferents from the cortex, in vivo.

Release of ethanolamine, serine, and choline in rat pontine nuclei on electrical stimulation of afferents from the cortex was investigated using in vivo push-pull cannula techniques. Ethanolamine was determined by using gas chromatographic techniques; serine was measured with a HPLC system; and choline was assayed with a luminescence method. Resting elution rates of ethanolamine, serine, and choline were 50.8 +/- 8.4, 34.8 +/- 12.6, and 1.16 +/- 0.20 pmol/5 min, respectively. Stimulation of the cortico-pontine tract evoked a highly significant 3.4-fold increase in release of ethanolamine, whereas serine and choline release was unaffected. Reactions in membrane phospholipids are most likely involved in the stimulation-dependent release of ethanolamine and special consideration was given to base-exchange reactions. Alternatively, a release from intracellular, possibly synaptic stores cannot be excluded.

Afferent Pathways↗
PubMed1986

Quantal release of acetylcholine evoked by focal depolarization at the Torpedo nerve-electroplaque junction.

To analyse evoked acetylcholine (ACh) release in the electric organ of Torpedo marmorata, a loose patch-clamp technique was used that allowed with a single extracellular electrode both focal depolarization of nerve endings and recording of the post-synaptic currents produced by the released transmitter. Two different types of post-synaptic response could be evoked by depolarizing pulses of increasing intensity: a graded response appearing with a delay of 0.6 ms (pulses of 0.2 ms duration), and an all-or-none response characterized by a mean delay of 1.4 ms. Both responses had a similar maximal amplitude and a similar rise time of 0.6 ms. The graded response was evoked in all places where spontaneous miniature electroplaque currents (m.e.e.s) could be recorded. It was not modified by 1 microM-tetrodotoxin (TTX), but was Ca2+ dependent and was abolished by Cd2+ (0.2 mM) or Mg2+ (10 mM). The all-or-none response could be evoked in only 30% of places where m.e.c.s. were recorded, it was highly TTX sensitive, Ca2+ dependent, and abolished by Cd2+ (0.2 mM) or Mg2+ (10 mM). K+ channel blocking agents, such as 4-aminopyridine (4-AP) or tetraethylammonium (TEA), which are known to prolong the duration of action potentials, prolonged the delay of the all-or-none response, but not that of the graded response. At low strength stimulation, the graded response was clearly evoked in a quantal way, with the quantum corresponding to the amplitude of spontaneous m.e.c.s. The amplitude distribution of the evoked responses closely followed a Poisson distribution. The maximum synchronous release of transmitter was found to be approximately 1.3 quanta/micron2 of presynaptic membrane and a mean quantal size of about 7000 ACh molecules was estimated from the charge transfer of m.e.c.s. The nerve terminal time constant was calculated from strength-duration curves obtained with depolarizing pulses just able to evoke either the all-or-none response or the first few quanta of the graded response. Respective mean values of 0.22 and 0.40 ms were found. Increasing the duration of the depolarizing pulse had two consequences: it differently affected the delay of the all-or-none response and that of the graded response; it increased the mean quantal content of the graded response. Both effects could not simply be accounted for by the influence of the nerve terminal time constant.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1985

Antipsychotic drugs depress acetylcholine release in the Torpedo electric organ, a purely cholinergic system.

We used the electric organ of Torpedo, a modified neuromuscular system, to investigate the direct effects of antipsychotic drugs on cholinergic transmission. All the antipsychotic drugs tested inhibited transmission by decreasing the amount of ACh released by nerve impulses. Their potency for this action was as follows: trifluoperazine less than clozapine less than thiethylperazine less than droperidol less than haloperidol less than chlorpromazine = beta-flupentixol less than alpha-flupentixol. Depression of ACh release by antipsychotics was poorly reversible, and was not mediated by dopamine receptors in this system since neither dopamine nor apomorphine had any effect on transmission. Antipsychotics did not act through presynaptic cholinergic receptors since the effect was not antagonized by atropine or quinuclidinyl benzilate. Trifluoperazine had no effect on the total ACh content of the tissue, on the compartmentation of ACh inside and outside synaptic vesicles, or on the rate of ACh turnover or the accumulation of 45Ca observed after repetitive stimulation. We conclude that antipsychotic drugs depress the neurally evoked release of ACh by acting directly on the releasing mechanism.

Acetylcholine↗
PubMed1985

Incorporation of acetate into acetylcholine, acetylcarnitine, and amino acids in the Torpedo electric organ.

The metabolism of acetate was investigated in the nerve-electroplaque system of Torpedo marmorata. In intact fragments of electric organ, radiolabeled acetate was incorporated into acetylcholine (ACh), acetylcarnitine (ACar), and three amino acids: aspartate, glutamate, and glutamine. These compounds were identified by TLC, high-voltage electrophoresis, column chromatography, and enzymic tests. The system responsible for acetate transport and incorporation into ACh displayed a higher affinity but a lower Vmax than that involved in the synthesis of ACar and amino acids. Choline, when added to the medium, increased the rate of acetate incorporation into ACh but decreased (at concentrations greater than 10(-5) M) that into ACar and amino acids. Monofluoroacetate slightly depressed ACh and ACar synthesis from external acetate but inhibited much more the synthesis of amino acids. During repetitive nerve stimulation, the level of the newly synthetized [14C]ACh was found to oscillate together with that of endogenous ACh, but the level of neither [14C]ACar nor the 14C-labeled amino acids exhibited any significant change as a function of time. This means that there is probably no periodic transfer of acetyl groups between ACh and the investigated metabolites in the course of activity. Acetate metabolism was also tested in the electric lobe (which contains the cell bodies of the neurons innervating the electric organ) and in Torpedo synaptosomes (which are nerve terminals isolated from the same neurons). Radioactive pyruvate and glutamine were also assayed in some experiments for comparison with acetate. These observations are discussed in connection with ACh metabolism under resting and active conditions in tissues where acetate is the preferred precursor of the neurotransmitter.

Acetates↗
PubMed1982

Cholinergic inhibition of acetylcholine release in the electric organ of Torpedo.

The possibility that acetylcholine (ACh) may inhibit its own release from nerve terminals by acting on presynaptic receptors has been investigated using the electric organ of Torpedo marmorata. ACh release was analysed by electrophysiological and biochemical methods conjointly. Oxotremorine, at micromolar concentrations, depressed nerve-electroplaque transmission by reducing the amount of ACh released by nerve impulses. This effect was competitively antagonised by nanomolar concentrations of atropine or methylatropine. Other muscarinic agonists, betanechol, pilocarpine and muscarine, however, failed to depress transmission but choline was effective at high concentrations. Anticholinesterase drugs, physostigmine, neostigmine or fluostigmine (diisopropylfluorophosphate, given as pretreatment and subsequently washed out) markedly depressed evoked ACh release. When cholinesterase was inhibited, the addition of oxotremorine or exogenous ACh caused a further depression of ACh release. Atropine was found to be very effective in reversing the depression of transmitter release produced by anticholinesterases. Looking for the mechanism of these presynaptic changes, we found that oxotremorine had little, if any, effect on the size of the ACh store of the tissue, on the compartmentation of ACh inside and outside synaptic vesicles, or on the rate of ACh turnover. The changes induced by oxotremorine cannot be explained by a reduction in calcium entry, since the presence of oxotremorine did not change the uptake of 45Ca observed after repetitive stimulation. Electrophysiological techniques were used to test for an effect of atropine in experiments where transmission of one impulse was expected to depress ACh release by subsequent impulses. This depression was not affected atropine, making it unlikely that the 'muscarinic' inhibition of ACh release has a role as a short-term feedback regulation of transmission. A second possibility is that oxotremorine (and external non-hydrolysed ACh) can enter the presynaptic membrane and interfere with the mechanism of transmitter release.

Acetylcholine↗
PubMed1982

Acetylcholine measured at short time intervals during transmission of nerve impulses in the electric organ of Torpedo.

1. The amounts of total acetylcholine (ACh) and ATP, and of vesicle-bound ACh were measured at short time intervals in the electrogenic tissue of Torpedo marmorata. The aim of this study is to approach with biochemical analysis the speed of electrophysiological phenomena.2. A stimulator coupled to a rapid freezer device was used to quench a number of tissue samples simultaneously, at different time intervals during transmission of a brief train of impulses at 100 Hz.3. The level of total ACh decreased significantly with the first ten impulses. Then a rapid but transient increase in total ACh occurred, reaching a maximum value by the fifteenth to sixteenth impulse.4. Vesicle-bound ACh did not exhibit any changes parallel to those of total ACh, and did not decrease beyond the control level during transmission of twenty impulses at 100 Hz.5. The amount of ATP in the tissue varied in close relation to that of total ACh. No significant phase shift was observed between the transmitter and the nucleotide and the ACh/ATP molar ratio was not significantly different from 1.6. The shortest time interval investigated in this work was 10 ms. The rate at which the pieces of tissue are quenched for biochemical measurements when plunged into a liquid at low temperature has been estimated. It has also been evaluated to what extent the freezing rate may distort measurements of the biochemical changes occurring in the tissue.7. It is concluded that fast freezing appears to be a valuable approach for investigating the rapid biochemical changes underlying cholinergic transmission; a better time resolution might be reached at the price, however, of greatly reducing the size of the samples. The second conclusion is that transmission of a brief train of impulses is accompanied by significant changes in the amount of extravesicular ACh.

Acetylcholine↗
PubMed1982

Acetylcholine changes underlying transmission of a single nerve impulse in the presence of 4-aminopyridine in Torpedo.

1. Transmission of a single nerve impulse has been investigated at the nerve-electroplaque junction of Torpedo marmorata in the presence of 4-aminopyridine (4-AP), a drug which powerfully potentiates evoked transmitter release.2. Three methodological approaches were used conjointly. These were (i) electrophysiological recording of the compound electroplaque potential (e.p.p.), (ii) radiochemical measurement of evoked acetylcholine (ACh) release and (iii) analysis of the content of ACh and ATP in the tissue at brief time intervals during the course of the e.p.p. and soon after. The last was achieved by using a stimulator coupled to a rapid tissue freezer.3. In the response to a single stimulus, 4-AP enhanced in a dose-dependent manner the size of the e.p.p., increasing the duration much more than the amplitude. At 10(-4) M-4-AP, this resulted in the generation of a characteristic ;giant e.p.p.' whose area (in V x ms) was approximately 120 times greater than that of a normal e.p.p.4. The giant e.p.p. consisted of an initial peak, lasting for some 100 ms, a late rebound at about 300 ms, and finished between 500 and 1000 ms after the stimulus. Temperature changes greatly affected the shape of the giant e.p.p., modifying particularly the amplitude and time course of the late rebound.5. The amount of ACh released in response to a single stimulus was measured radiochemically and was found to greatly increase in the presence of 4-AP, explaining the potentiation of the e.p.p. With 4-AP concentrations ranging from 10(-6) M to 10(-4) M, the augmentation of ACh release showed a close correlation with increase of the e.p.p. area.6. The large potentiation of evoked transmitter release occurred in spite of a reduction of ACh stores. After treatment with 10(-4) M-4-AP, the total ACh content was reduced by 30-40% in the absence of any electrical stimulation. The reduction affected to a similar extent the vesicular and extravesicular compartments of ACh. This was accompanied by a general increase in the resting rate of ACh turnover.7. Synaptic vesicles were isolated from small fragments of electric organ, rapidly frozen with our device. Compartmental analysis was carried out by labelling the transmitter pools with a radioactive precursor and it was confirmed that vesicular ACh has a relatively low metabolic rate, whereas free ACh (most probably cytoplasmic ACh) turns over more rapidly. The same finding was obtained after treatment with 4-AP, but the starting levels of ACh and the yield of synaptic vesicles were lower.8. The total ACh content was measured at 30 and 100 ms intervals during the course of the giant e.p.p., and soon after. We found characteristic and significant changes which were (i) an initial fall of total ACh occurring within 100-150 ms, (ii) a transient ACh increase which occurred later and seemed to correspond to the late rebound of the giant e.p.p. and (iii) a steady 20% lowering of total ACh, observed from the end of the giant e.p.p. and lasting for more than 1 s.9. The ATP content of the tissue, during and after the giant e.p.p., followed a time course which was remarkably similar to that of total ACh. A significant ATP/ACh relationship was found in most experiments separately, and in the pooled results with a higher degree of significance.10. Vesicular ACh did not exhibit any significant change during and after the giant e.p.p. Neither the transient initial variations of total ACh nor its later lowering were reflected in similar changes of vesicular ACh. It was therefore the extravesicular pool of ACh which was concerned in the characteristic pattern of changes of total ACh.11. Compartmental analysis of transmitter stores was performed during the course of transmission, after labelling ACh in the tissue with a radioactive precursor. It was found that no detectable transfer of ACh occurred from cytoplasm to vesicles, either during the giant e.p.p., or within the following second.12. The following conclusions were reached. The effect of 4-AP is to cause a very strong and long-lasting potentiation of ACh release, resulting in a giant and complex electrical discharge. Transmitter release under these conditions was not only due to sudden liberation of the preformed, available ACh but also to a marked contribution of new ACh made during the giant e.p.p. These changes in ACh content were very significant and took place exclusively in the extravesicular pool of transmitter.

4-Aminopyridine↗
PubMed1980

Thiamine and cholinergic transmission in the electric organ of Torpedo. I. Cellular localization and functional changes of thiamine and thiamine phosphate esters.

The electric organ of Torpedo marmorata was found to contain as much as 120 +/- 24 nmol of thiamine per g of fresh tissue. The vitamin was distributed as nonesterified thiamine (32%), thiamine monophosphate (22%), thiamine diphosphate (8%), and an important proportion of thiamine triphosphate (38%). A high level of thiamine triphosphate was found in synaptosomes isolated from the electric organ. In contrast, the synaptic vesicles did not show any enrichment in thiamine, whereas they contained a marked peak of acetylcholine (ACh) and ATP. Thus thiamine seems to be very abundant in cholinergic nerve terminals; its localization is apparently extravesicular, either in the axoplasm or in association with plasma membrane. When calcium was reduced and magnesium increased in the external medium, the efficiency of transmission was diminished, owing to inhibition of ACh release; in a parallel manner the degree of thiamine phosphorylation was found to increase--this condition is known to modify the repartition of ACh between vesicular and extravesicular compartments. Electrical stimulation, which causes periodic variations of the level of ACh and ATP, also caused significant changes in thiamine esters. In addition, related changes of the vitamin and the transmitter were observed under other conditions, suggesting a functional link between the metabolism of thiamine and that of ACh in cholinergic nerve terminals.

Acetylcholine↗
PubMed1980

Thiamine and cholinergic transmission in the electric organ of Torpedo. II. Effects of exogenous thiamine and analogues on acetylcholine release.

The electrogenic tissue of Torpedo was found to phosphorylate in vitro external [14C]thiamine by a saturable process. The rate of this metabolisaton was increased when acetate, an efficient precursor of acetylcholine (ACh) synthesis in this tissue, was added to the incubation medium, thus increasing the turnover of ACh. Nerve stimulation did not release significant amounts of the previously accumulated [14C]thiamine. Exogenous thiamine modified the size of the electroplaque potential (e.p.p.). At concentrations higher than 10(-3)M the nerve-electroplaque transmission was depressed after a transient increase of the e.p.p. Such a depression was due to a strong decrease of the ACh release. At concentrations equal to or lower than 10(-3)M, thiamine affected transmission in a rather complex fashion. ACh release was decreased or increased depending on concentration, time of application, and mode of stimulation. Oxythiamine, a structural antimetabolite of thiamine, affected the transmission in a very characteristic manner at 10(-5)M and higher concentrations. The amplitude of the e.p.p. was increased and, more strikingly, its duration was prolonged. These changes were not due to an inhibition of cholinesterase activity but to an enhancement of the evoked release of ACh either on single-impulse or repetitive stimulation. Another antimetabolite, pyrithiamine, had no effect on the transmission nor on ACh release. From this and our previous work, it is proposed that thiamine is involved, directly or indirectly, in the process of ACh release. The possible mechanisms of this involvement are discussed.

Acetates↗
PubMed1980

Acetylcholine release evoked by single or a few nerve impulses in the electric organ of Torpedo.

1. The acetylcholine (ACh) store in the Torpedo electric organ was partially labelled with choline and acetate at the same molar concentration but with different isotopes. Under these conditions the two precursors were incorporated into ACh in a ratio 1 to 1. 2. After a single electrical stimulus, or a brief burst of stimuli, the compound electroplaque potential (e.p.p.) was recorded and the radioactive choline and/or acetate counted in the perfusion fluid, providing a sensitive assay for ACh release in the absence of anticholinesterase drugs. 3. The so-called depression of transmission was found to be due to progressive impairment of ACh release in the successive impulses evoked by repeated stimuli. 4. In a pair of impulses separated by 50 ms interval, less ACh was released by the second than by the first impulse; this explained why the size of the second e.p.p. was depressed, using a direct measurement of ACh. 5. In repetitive stimulations of longer duration, the maximum rate of release declined as the activity was prolonged. Thus the tissue progressively lost its ability to ensure release at high frequencies. 6. An unexpected finding was that anticholinesterases like eserine or pre-treatment with fluostigmine (DFP) greatly reduced ACh release even by a single impulse. 7. Evoked ACh release and e.p.p. amplitude were both maximum between 10 and 20 degrees C. At higher temperatures, the evoked release decreased as the spontaneous release increased. 8. Changes in external Ca2+ and Mg2+ produced similar changes in the e.p.p. and evoked ACh release. The dose--response curve for Ca dependency of ACh release was very steep with a Hill's coefficient of 3.2. 9. With a single stimulus in the presence of 4-aminopyridine, there was a dramatic enlargement of the e.p.p. and a still larger potentiation of the evoked ACh release. 10. It has been possible with this approach to avoid the inconveniences often encountered in simliar studies, i.e. repetitive stimulation, low Ca solutions and cholinesterase inhibition. This permitted a good correlation between electrophysiological and biochemical estimates of transmitter release even by a single nerve impulse.

Acetylcholine↗
PubMed1980

Calcium metabolism and acetylcholine release at the nerve-electroplaque junction.

1. Calcium metabolism was investigated in the electric organ of Torpedo at rest and during synaptic activity. When the tissue was incubated in high calcium concentrations, complete exchange was obtained between cellular calcium and that of the external medium. In the presence of low concentrations, the tissue retained its calcium which, in this case, was poorly exchanged. 2. Stimulation of the nerves to the electric organ provoked a net increase in cellular calcium and an acceleration of its exchange with that of the extracellular space. This entry of calcium seemed to involve mainly the presynaptic axon endings since it occurred even when transmission was blocked by curare. 3. The presynaptic accumulation of calcium was accompanied by a decrease in the vesicular pool of acetylcholine (ACh). On the other hand, the level of extracellular Ca2+ seemed to be critical for the intraterminal repartition of ACh between the vesicular pool and the cytoplasmic pool, and also for exchange of high energy phosphate between ATP and thiamine esters. 4. Radioautography of 45Ca at the electron microscope level confirmed that most of the Ca accumulation following stimulation concerned the presynaptic axon endings. The accumulated 45Ca was closely associated with the plasma-lemma region of nerve terminals, mainly in the region of active zones. 5. Taking this into account, and also the fact that synaptic vesicles have a very efficient Ca sequestration mechanism (see Israël et al., this symposium) it is proposed that, after entry, Ca is taken up by the vesicles situated in active zones, exchanged against vesicular ACh, concentrated in the vesicle and finally released by exocytosis.

Acetylcholine↗