Effect of the Venom of Glycera Convoluta

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 lateroccurring structural modifications, it is possible that the venom mimics one component of the double mode of action proposed for black widow spider venom. Much new information about the physiology of synapses has been obtained by studying the effect of neurotoxins. Among these toxins, black widow spider venom (BWSV [8, 25, 7, 23, 9]), /8-bungarotoxin (5, l), and Red Sea flatfish Pardachirus toxin (31) appear to be powerful and valuable tools for the study of presynaptic mechanisms . A common effect of these presynaptic toxins is J. CELL Bioroc .v © The Rockefeller University Press 0021-9525/80/05/0446/13 $1.00 Volume 85 May 1980 446-458 on July 0, 2017 jcb.rress.org D ow nladed fom to induce a tremendous increase in the frequency of miniature potentials and important ultrastructural changes of nerve terminals . The polychaete annelids Glycera dibranchiata (18, 24) and Glycera convoluta (27) have associated with their jaws, four small glands containing a toxic substance . The toxicity of this substance (at least in the case of G. convoluta) is slight for man, an accidental contact with skin producing only a local epidermal reaction . However, when injected into the coelomic cavity of crabs, the venom rapidly kills them . We therefore looked for a possible neurotoxicity of this substance . An important increase in the frequency of miniature potentials was indeed found after its application . The first experiments were carried out on neuromuscular preparations ofcrayfish that are similar in many respects to other crustacean neuromuscular preparations . The effect, however, was not specific for crustacean preparations . Similar effects were obtained with either frog neuromuscular preparations or torpedo electric organs . We report here that venom of Glycera, which triggers an important and sustained increase in the frequency of miniature potentials, does not induce ultrastructural changes after the release of several times the initial store; modifications are observed only after very long incubations . In this respect, it differs from the presynaptic venom previously cited and may be an interesting tool to use for the further characterization of the release process . MATERIALS AND METHODS Preparation ofthe Venom Extract Glycera convoluta (Keferstein) were collected on the seashore near the Marine Biological Station of Roscoff (France) . After section of the prostomium, the anterior part of the digestive tract was pulled out by means of forceps, and the venom gland complex was removed. The glandular regions of five animals (20 glands) were homogenized at 0°C in 1 ml of 10 mM sodium phosphate buffer (pH 7.2) in a small glass-glass handoperated homogenizer. The homogenate was centrifuged at 10,000g for 10 min and the supernate was collected . This solution was generally used immediately after preparation by diluting it with physiological solution . However, no detectable loss of activity was noticed after storage of the concentrated venom solution up to 8 d at 4°C. It was also found that the glands could be kept at -80°C for several months without any loss of activity . Unless otherwise specified, the crude extract was used . In some experiments, the crude extract was gel filtered. We passed 0.5 ml of extract of venom through a 5-ml column of Sephadex G-50 (coarse) equilibrated with Ringer's solution. This permitted collection (in l ml of Ringer's) of the venom that was found in the void volume. Preparations and Solutions FROG : Thecutaneous pectoris muscle ofRana esculenta was dissected and soaked in Ringer's solution (in mM : NaCl, 110; KCI, 2.14; CaC1 2, 1 .14; NaHCO;) , 0.59 ; adjusted to pH 6.7) . Experiments were carried out at room temperature (18°-22°C) . C R A Y F t sFt : Two different neuromuscular preparations of Procambarus clarkii were used . One was the posterior fast abdominal flexor (30), which was dissected from the 2nd or 3rd abdominal segment. The other preparation was the openerof the dactyl from the l st or 2nd walking leg (extensively used since the work of Dudel and Kuffler [10]) . The preparations were soaked in Van-Harreveld solution (in mM : NaCl, 195; KCI, 5.4; CaC1 2 , 13; MgCl, 2.6: Tris-maleate buffer, 10 ; adjusted to pH 7.2) . Experiments were carried out at 15 °C. TORPEDO : Slices of four to six adjacent electrogenic prisms were dissected from the electric organ of Torpedo marmorata previously chilled for 15 min on ice . They were soaked in a solution containing in mM: NaCl, 280; KCI, 3 ; CaC12 , 3 .4; MgCh, 1.8 ; sodium phosphate buffer, 1 .2 (pH 6.8); glucose, 5 .5 ; urea, 300; sucrose, 100. After equilibration with Oz, 4-5 mM NaHC03 was added to adjust the pH to 7-7.1 . Experiments were carried out at room temperature (18°-22°C) . Electrophysiological Recordings The preparations were pinned on elastomere in a small (2-ml) Perspex chamber continuously perfused with fresh physiological solution described in the previous section . Intracellular records were made through 5-10 MSI microelectrodes filled with 3 M KCI. In the case of torpedo electroplaques, extracellular recordings from superficial nerve terminals of the ventral face were also obtained through larger microelectrodes made by blunting the broken tip on a microforge . These electrodes were filled with the torpedo physiological solution and had a resistance of 1-2 MO. After stabilization of the recordings for at least 30 min, the venom (10-200 ,ul of the concentrated solution) was added to 20 ml of the physiological solution, giving a final concentration of 0.01-0.2 gland/ml. This solution, continuously recycled, was then used for perfusion . During prolonged experiments, the total circulating solution was replaced every 3 h with 20 ml of fresh solution containing the same venom concentration, to minimize hyperosmotic effects resulting from evaporation. The electrical activity was monitored on an oscilloscope and on a pen chart recorder (Brush 2400, Gould Inc. . Cleveland, Ohio). Samples of activity were also recorded on a cassette magnetic recorder (Minilog 4, Philips, Eindhoven, The Netherlands) with a band pass of0-5 kHz for further processing. Numerical Processing of Electrical