Biomedical subjects
Publications and source records attributed to R Manaranche.
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A neurotoxin able to increase the spontaneous release of transmitter was found in the venom glands of the polychaete annelid Glycera convoluta. We studied the effect of this venom on the frog cutaneous pectoris muscle, where its application produced a prolonged (20-h), high-frequency discharge of miniature potentials. After 5 h of action, the initial store was renewed several times but no detectable ultrastructural changes were observed. After 19 h of sustained activity, nerve terminals with their normal vesicular contents were infrequent; others were fragmented and contained swollen mitochondria, abnormal inclusions, and vesicles of various sizes. In the noncholinergic crayfish neuromuscular preparation, the venom triggered an important increase in spontaneous quantal release that subsided in 1 h. An activity higher than that in resting conditions then persisted for many hours. This high electrical activity was not accompanied by any detectable structural modifications after 3 h. In the torpedo electric organ preparation, the venom elicited a burst of activity that returned to control levels in 1 h. The release of ACh (evaluated by the efflux of radioactive acetate) paralleled the high electrical activity. No morphological changes or significant depletion of tissue stores were detected. The venom of Glycera convoluta appears to enhance considerably the release of transmitter without impairing its turnover. The venom effect is Ca++ dependent and reversible by washing, at least during the first hour of action. Because the high rate of transmitter release appears dissociated from the later-occurring structural modifications, it is possible that the venom mimics one component of the double mode of action proposed for black widow spider venom.
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Pure synaptic vesicles have been isolated in sucrose-KCl media. They are able to take up calcium in the presence of ATP and Mg. This is based on the following evidence. First, the synaptic vesicle fraction is the gradient peak for calcium uptake. Second, it was not possible to separate ACh and ATP from the uptake peak after refractionation of synaptic vesicles. Third, the fraction appears very pure on morphological and biochemical grounds. The physiological significance of the calcium uptake by synaptic vesicles is discussed.
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1. When the electric organ of Torpedo is stimulated a large number of synchronized cholinergic synapses are activated. This permits the measurement of changes in the tissue level of ACh associated with the release process, usually recorded as an electric discharge. 2. At 5 Hz stimulation the output per impulse and the amount of cytoplasmic (free ACh) declines for about 30 s. The output then remains constant while ACh is synthesized for about 90 s. Finally, the output and cytoplasmic ACh are exhausted after 120 s. These 'slow wave' changes in ACh represent about 50% of the total. 3. Superimposed on the 'slow wave' are rapid oscillations of 5 s period, which represent about 30% of the total ACh. 4. The amount of ATP oscillates in phase with ACh. These oscillations might result from regulation of enzymes involved in the synthesis of transmitter. 5. The amplitude of electrical discharge does not normally oscillate. Transmitter output is therefore not directly related to ACh concentration changes. The mechanism releasing ACh is a saturable process.
A model, considering cytoplasmic ACh as the difference between cumulated outputs per impulse, and synthesized ACh leads to simple equations correlating biochemical and electrical measurables. Intrinsic properties of the release mechanism are deduced.
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The amount of transmitter in the electric organ of Torpedo was measured with a time resolution of 1 sec in the course of stimulation. In parallel, the modifications of the electrophysiological response were analysed by determining the conductance increase (deltaG) and the electromotive force of electroplaques. Large changes in the level of total acetylcholine (ACh) were seen during stimulation. These changes were two-fold: a slow wave and, superimposed on it, a rapid oscillation. The slow wave raised total ACh to the initial level, or even higher. It was probably related to modifications in the amount of ACh released since it corresponded to characteristic inflections in the evolution of the deltaG curve. The slow wave and this physiological parameter were similarly affected when the experiments were performed at a reduced temperature. The rapid oscillation had an amplitude of about 20-40% of the total ACh. It was undamped and its period was 4-5 sec. In contrast to the slow wave, no clear physiological change associated with the rapid oscillation has been observed. The slow wave and rapid oscillation occurred in the 'free pool' of ACh, whereas bound ACh, the fraction associated with synaptic vesicles, was not affected by these changes. A dynamic description of synaptic activity is proposed. The content of 'free' ACh is used and renewed completely after a few tens of impulses, so that transmission seems to imply the continual recycling of the same pool of transmitter rather than utilization of a large preloaded store. The release process must then be integrated in rapid metabolic loops.
Pure cholinergic nerve endings (synaptosomes) were isolated from the electric organ of Torpedo by a rapid procedure. These synaptosomes are approximately 3 micron in diameter. They contain an occasional mitochondrion, numerous synaptic vesicles, and sometimes an active zone is observed. No postynaptic membrane attachment is found. This nerve ending fraction is extremely pure as shown by morphological controls and biochemical data. It is rich in choline acetyltransferase (450 nmol/h per mg protein) and acetylcholine (ACh) (130 nmol/mg protein). The isolated endings retain their cytoplasmic components and they synthesize ACh and are stable in vitro for several hours, as shown by biochemical measurements and morphological analysis.
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A rapid method for the preparation of highly purified cholinergic nerve endings from the electric organ of Torpedo is described. The endings retain their cytoplasmic components, as shown by biochemical and morphological observations. The homogeneity of these synaptosomes make them a useful tool for further studies.
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When tissue samples of the electric organ of the Torpedo fish, are taken every second during a period of stimulation at 5/s, it is noticed that their acetylcholine level oscillates in a quasi-sinusoidal shape. The amplitude of the oscillation is of about 350 nmoles/g and its period of 4 to 6 seconds. The speed of acetylcholine formation during such a process is extremely high.
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