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Effect of Alpha-Latrotoxin on the Frog Neuromuscular Junction at Low

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Effect of Alpha-Latrotoxin on the Frog Neuromuscular Junction at Low Journal of Physiology (1988), 402, pp. 195-217 195 With 4 plate8 and 5 text-figures Printed in Great Britain EFFECT OF ae-LATROTOXIN ON THE FROG NEUROMUSCULAR JUNCTION AT LOW TEMPERATURE BY B. CECCARELLI, W. P. HURLBUT* AND N. IEZZI From the Department of Medical Pharmacology, CNR Center of Cytopharmacology and Center for the Study of Peripheral Neuropathies and Neuromuscular Diseases, Via Vanvitelli 32, 20129 Milano, Italy, and the Rockefeller University*, 1230 York Avenue, New York, NY 10021, U.S.A. (Received 13 July 1987) SUMMARY 1. a-Latrotoxin (a-LTx) was applied to frog cutaneus pectoris muscles bathed at 1-3 °C in either Ringer solution, Ca2"-free Ringer solution with 1 mM-EGTA and 4 mM-Mg2+ or Ringer solution plus 4 mM-Mg2+, and its effects on miniature end-plate potential (MEPP) frequency, nerve terminal ultrastructure and uptake of horse- radish peroxidase (HRP) were studied. 2. Large concentrations (2 ,ug/ml) of a-LTx increased MEPP rates to levels above 100/s at all junctions, but the time course of the increases depended upon the divalent cation content of the bathing solution. However, similar numbers ofMEPPs (0-3-407 x 106) were recorded at all junctions during 2 h of secretion. 3. Nerve terminals exposed to a-LTx for 2 h lost 60-75 % oftheir synaptic vesicles and were swollen; their presynaptic membranes were deeply infolded and they often contained many large vesicular structures. Terminals in Ringer solution retained the largest number of synaptic vesicles; terminals in Ringer solution plus Mg2+ swelled the least and contained the largest number of coated vesicles. The average number of synaptic vesicles lost was approximately equal to the average number of MEPPs recorded. 4. Few vesicles became loaded with HRP when this extracellular tracer was present in the bathing solution and the muscles were fixed near the peak of secretion. 5. When the terminals were warmed to 20 °C, those in the Caa2+-free solution with Mg2+ secreted additional quanta and lost almost all their residual vesicles; those in Ringer solution without Mg2+ secreted few additional quanta and retained most of their residual vesicles. 6. These results suggest that recycling was blocked at these terminals and that for each quantum secreted a vesicle became permanently incorporated into the axolemma. INTRODUCTION The acetylcholine (ACh) in vertebrate motor nerve terminals is stored in two major compartments: the axoplasm and the synaptic vesicles (Whittaker, Michaelson & Kirkland, 1964; Dunant, Gautron, Israel, Lesbats & Manaranche, 1972). ACh is 7-2 196 B. CECCARELLI, W. P. HURLBUT AND N. IEZZI released from a terminal both in a steady 'leak' (Katz & Miledi, 1977; Vyscocil & Illes, 1979; Edwards, Dolezal, Tucek, Zemkova & Vyskocil, 1985), and in a pulsatile manner as multimolecular packets, or quanta (del Castillo & Katz, 1954). The total release, i.e. the sum of the 'leak' and the quantal components, is measurable by biochemical means (Gorio, Hurlbut & Ceccarelli, 1978; Israel & Lesbats, 1981). The 'leak', or non-quantal component of release, is difficult to measure directly since its major known effect is to produce a small, steady depolarization at an end-plate when acetylcholinesterase has been thoroughly inhibited. Therefore, neither its function or origin within the terminal are known (Edwards et al. 1985). Quantal release, on the other hand, is easily detected by electrophysiological means, and its function is well known since it generates the transient end-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) that mediate neuromuscular transmission (Fatt & Katz, 1952; del Castillo & Katz, 1954; Boyd & Martin, 1956). Although recent quick-freeze studies of frog terminals treated with 4-amino- pyridine have demonstrated that vesicles fuse with the presynaptic membrane a few milliseconds after the arrival of a single nerve impulse (Heuser, Reese, Dennis, Jan, Jan & Evans, 1979; Torri-Tarelli, Grohovaz, Fesce & Ceccarelli, 1985), controversy still exists over whether the secreted quanta represent ACh that is released from the interiors of the fused vesicles (Tauc, 1982). An alternative hypothesis is that the quanta represent axoplasmic ACh which diffuses in pulses through channels in the presynaptic membrane that become transiently permeable to it (Tauc, 1982; Israel & Manaranche, 1985). If quanta were released by exocytosis from the interiors of vesicles which have fused with the presynaptic membrane, then one might expect a vesicle to be lost whenever a quantum is secreted. However, most terminals can secrete large numbers of quanta before they lose significant numbers of vesicles (reviewed by Ceccarelli & Hurlbut, 1980a). On the other hand, vesicles are formed from the axolemma of rapidly secreting terminals, and it has been suggested that these newly formed vesicles are recycled vermions of the original ones and can reaccumulate and secrete additional quanta of ACh (Ceccarelli & Hurlbut, 1980a). Thus, the number of vesicles lost should equal the number of quanta secreted only when endocytosis and vesicle recycling are blocked. Endocytosis is inhibited at terminals treated with ouabain, or with black widow spider venom in Ca2+-free Ringer solution, and such terminals secrete spontaneously until they are exhausted of quanta and depleted of vesicles (Clark, Hurlbut & Mauro, 1972; Ceccarelli & Hurlbut, 1980b; Haimann, Torri-Tarelli, Fesce & Ceccarelli, 1985). The total number of MEPPs that occur at such terminals is similar to the number of synaptic vesicles in resting terminals (- 7 x 105) (Haiman et al. 1985; Fesce, Segal, Ceccarelli & Hurlbut, 1986b), but additional comparisons of MEPP counts and vesicle counts need to be made under other conditions which inhibit endocytosis and stimulate quantal secretion. Since low temperature appears to be an effective inhibitor of most endocytotic processes in mammalian cells (Steinman, Mellman, Muller & Cohn, 1983), we have studied the effects of a-latrotoxin (a-LTx), a purified protein component of black widow spider venom (Frontali, Ceccarelli, Gorio, Mauro, Siekevitz, Tzeng & Hurlbut, 1976), on quantal secretion, nerve terminal ultrastructure and uptake of horseradish peroxidase (HRP) at frog neuromuscular junctions at 1-3 'C. We were especially interested in these ac-LATROTOXIN AT LOW TEMPERATURE 197 experiments because Heuser & Miledi (1971) had previously estimated that several million quanta were secreted from terminals treated with La3+ at 4 'C. Evoked and spontaneous quantal release are highly temperature dependent, and the quantal content of the EPP and the rate of occurrence of MEPPs decline to low values at temperatures below 10 'C (see Barrett, Barrett, Botz, Chang & Mahaffey, 1978, for references). Previous studies of the effect of temperature on the action of black widow spider venom or a-LTx showed that binding occurs at temperatures near 0 'C (Valtorta, Madeddu, Meldolesi & Ceccarelli, 1984), but quantal release is small when moderate doses of these agents were used (Rubin, 1976; Pumplin & Reese, 1977; Fritz, 1980). However, we have found that high concentrations of o- LTx (2 ,ug/ml) raise MEPP rates to several hundred per second at temperatures of 1-3 'C and cause a profound depletion of vesicles after 2 h. METHODS General. Cutaneus pectoris muscles were dissected from frogs (Rana pipien8) together with a short length of nerve, a small flap of skin that covered the throat and a short length of rectus abdominis muscle. The preparation was stretched over a lucite lens on the floor of a thin-bottomed lucite recording chamber and pinned into pads of dental wax at each end. The chamber held about 3 ml of bathing solution and could be perfused. The standard bathing solution (Ringer solution) contained (in mM): NaCl, 116; KCl, 2-1; CaCl2, 1-8; phosphate buffer, 3-0; at pH 7-0. The Ca21- free Ringer solution contained no added CaCl2, 1 mm-ethyleneglycol-bis-(,8-aminoethylether)N,N'- tetraacetic acid (EGTA) and 4 mM-MgCl2; the Ringer solution with Mg2+ contained 4 mM-MgCl2 in addition to the other salts. The concentration of NaCl in the modified Ringer solutions was adjusted to keep the tonicity constant. Tetrodotoxin (TTX, 10-7-10-8 g/ml) was added to all solutions to prevent the muscle fibres from twitching. The recording chamber was mounted on a Peltier device and cooled to a temperature of 1-3 'C as measured by a thermistor pressed firmly against the wax pad near the edge of the muscle. Electrophysiology. End-plate regions were impaled by glass micropipettes filled with 3 M-KCI (resistances 10-30 MQ at room temperature), and MEPPs were recorded with a conventional high- input-impedance amplifier and stored on FM magnetic tape (tape speed: 4-75 cm/s, bandwidth 0-600 Hz for experiments at 1-3 'C; speed 9-5 cm/s, bandwidth 0-1250 Hz at room temperature) for later analysis by computer (Digital Equipment Corp., PDP 11/73). One channel of the tape- recorder stored a high-gain AC-coupled record (bandwidth 0-03-600 Hz at 1-3 'C, or 0-15-1250 Hz at room temperature) of the deviations of the membrane potential from its mean, and a second channel stored a low-gain DC record of the absolute value of the membrane potential. A baseline record 3-5 min in duration was collected, then a-LTx was applied and the potentials were recorded continuously for 45-60 min; they were recorded intermittently at later times. In each experiment we used a different muscle and tried to record from a single neuromuscular junction throughout its discharge of quanta. Data were collected at a particular junction as long as the MEPP rate exceeded - 20/s and the individual MEPPs could be distinguished from noise. We were successful in sixteen experiments at low temperature. In two additional experiments reported here the initial impalements were lost after about an hour (i.e. well after the time the rate of secretion had passed through its peak); in these two cases a second junction was impaled and its quantal secretion added to that of the first junction.
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