501

Journal of Physiology (1990), 425, pp. 501-526 With 10 figures Printed in Great Britain

CORRELATION BETWEEN QUANTAL SECRETION AND VESICLE LOSS AT THE FROG NEUROMUSCULAR JUNCTION BY W. P. HURLBUT*, N. IEZZI, R. FESCE AND THE LATE B. CECCARELLI From the Department of Medical Pharmacology, CNR Center of Cytopharmacology and Center for the Experimental Study of Peripheral Neuropathies and Neuromuscular Diseases, Via Vanvitelli 32, 20129 Milano, Italy, and *The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA

(Received 29 September 1989) SUMMARY

1. We measured the rate of occurrence of miniature endplate potentials (MEPPs) at identified endplates in frog cutaneus pectoris muscles treated with crude black widow spider venom (BWSV) or purified ac-latrotoxin (a-LTX) in calcium-free solutions, and we examined the relationship between the length of the nerve terminal and the total number of quanta secreted, and the relationship between the number of quanta secreted and the number of vesicles remaining at different times. 2. The venom, or toxin, was applied in a modified Ringer solution with tetrodotoxin, 1 mM-EGTA and no divalent cations, and quantal secretion was started by applying Ca2+-free solutions with Mg2+. This was done to synchronize the quantal discharge at the various junctions in a muscle. Ringer solution was applied after the MEPP rate had declined to low levels, and then the muscle fibre was injected with Lucifer Yellow, the endplate stained for acetylcholinesterase and the length of the nerve terminal and the length of a sarcomere were measured on the fluorescent fibre. 3. The total number of quanta secreted by a terminal was measured under a wide variety of experimental conditions: the weights of the frogs ranged from 13 to 68 g, the temperature from 9 to 28 0C, and the concentration of Mg2+ from 2 to 10 mM. In one series of experiments the Mg2+ was withdrawn after 3-4 min and reapplied 35-40 min later in order to divide the total output of quanta into two approximately equal bouts of secretion that were well separated in time. 4. The total number of MEPPs recorded at a junction was loosely correlated with the length of its nerve terminal, but it was not affected by the temperature, the concentration of Mg2+ or the division of secretion into well-separated bouts of quantal release. The average total secretion per unit length was about 3700 quanta/sarcomere or about 1200 quanta/,#m. 5. The average time course of quantal secretion per micrometre of terminal was determined at single junctions in muscles held at 22-23 0C or at 9-10 TC. Other muscles were fixed at various times during the course of secretion at each temperature and the number of synaptic vesicles remaining in cross-sections of the terminals were counted on electron micrographs. The number of vesicles remaining MS 7979

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per micrometre of terminal was determined from the number per cross-section and the section thickness. 6. The average number of vesicles remaining per micrometre of terminal was negatively correlated with the average number of quanta secreted per micrometre; the slope of the regression line was - 14 to -12 vesicles/quantum and its correlation coefficient was -0-91. It is concluded that a quantum of transmitter is derived from a single vesicle. INTRODUCTION

Several attempts have been made to measure the number of quanta of acetylcholine (ACh) released by frog motor nerve terminals during periods of intense secretion and to compare it to the number of synaptic vesicles they lose (Ceccarelli, Hurlbut & Mauro, 1972, 1973; Ceccarelli & Hurlbut, 1975; Haimann, Torri-Tarelli, Fesce & Ceccarelli, 1985; Segal, Ceccarelli, Fesce & Hurlbut, 1985; Fesce, Segal, Ceccarelli & Hurlbut, 1986a; Ceccarelli, Hurlbut & Iezzi, 1988; Dekhuijzen, Iezzi & Hurlbut, 1989), or to examine the relationship between the amplitude of the endplate potential (EPP) and the number of synaptic vesicles remaining in nerve terminals (Koenig, Saito & Ikeda, 1983; Koenig, Kosaka & Ikeda, 1989). When the terminals are stimulated under conditions that permit vesicles to recycle, as indicated by their uptake of extracellular tracers such as horseradish peroxidase (HRP), then the number of quanta secreted can be severalfold greater than the number of vesicles lost and even greater than the number estimated to reside in unstimulated control terminals. However, when terminals are stimulated under conditions that interfere with vesicle recycling, then the number of quanta secreted approximately equals the number of vesicles lost and it approaches the number of vesicles stored in control terminals. Agents or procedures that stimulate secretion and permit extensive recycling include electric stimulation of the nerve (Ceccarelli et al. 1972, 1973; Heuser & Reese, 1973; Rose, Pappas & Kriebel, 1978; Lynch, 1982; Koenig et al. 1983), La3+ (041-2 mm) applied at room temperature (Heuser & Miledi, 1971; Segal et al. 1985) or at 3-5 0C (Heuser & Miledi, 1971; Florey & Kriebel, 1983; Dekhuijzen et al. 1989), and low doses of black widow spider venom (BWSV), or a-latrotoxin (az-LTX), applied at room temperature in solutions with normal levels of Ca2` (Ceccarelli & Hurlbut, 1980; Fesce et al. 1986a; Valtorta, Jahn, Fesce, Greengard & Ceccarelli, 1988). Agents or conditions that stimulate secretion and interfere with recycling include electric stimulation of motor nerves in a Drosophila mutant with defective vesicle recycling (Koenig et al. 1983, 1989), solutions with 0-05-041 mM-ouabain (Haimann et al. 1985), K+-rich, ClP-deficient solutions (Gennaro, Nastuk, & Rutherford, 1978; Ceccarelli, Molenaar, Oen, Polak, Torri-Tarelli & van Kempen, 1989), high doses of a-LTX applied at 1-3 0C (Ceccarelli et al. 1988), and low doses of BWSV or a-LTX applied at room temperature in Ca2+-free solutions with 4 mmMg2+ (Ceccarelli & Hurlbut, 1980; Fesce et al. 1986a; Valtorta et al. 1988). Synaptophysin, a specific integral protein of the vesicle membrane, is permanently incorporated into the nerve terminal axolemma under the latter conditions, but it is not incorporated in detectable amounts when Ca21 is present and vesicles are recycled (Valtorta et al. 1988).

503 QUANTA AND SYNAPTIC VESICLES All these results are consistent with the vesicle hypothesis of quantal secretion, and they suggest that a quantum of transmitter is derived from a single vesicle, rather than 7-10 vesicles as some have suggested (Kriebel & Gross, 1974; Wernig & Stirner, 1977; Wernig & Motelica-Heino, 1978; reviewed by Tremblay, Laurie & Colonnier, 1983). However, in many of the works quoted above secretion was allowed to proceed until the terminals appeared to be exhausted of quanta, and no attempt was made to examine the time course of vesicle loss. Furthermore, the estimates of the number of vesicles contained in resting terminals were based upon the average length of terminals measured in unrelated experiments. The objective of the experiments described here is to measure, at two different temperatures, the time courses of vesicle loss and quantal secretion at junctions in frog cutaneus pectoris muscles stimulated by applying BWSV or x-LTX in Ca2+-free solution with Mg2+, and to measure the length of the nerve terminals whose secretion had been recorded. A good correlation is obtained between the average number of vesicles lost per micrometre of terminal and the average number of quanta secreted per micrometre, with a ratio close to 1 vesicle/quantum. This result supports the hypothesis that a quantum of ACh is stored within a single synaptic vesicle and is released when the vesicle fuses with the presynaptic membrane. METHODS

General. Frogs, R. pipiens, of either sex and ranging in weight from 13 to 68 g, were used in order to obtain a wide range of nerve terminal lengths (Jans, Salzmann & Wernig, 1986). The animals were anaesthetized by immersing them for 10-15 min in tap water with 1-2% ethyl-3aminobenzoate (MS-222) (Aldrich Chem. Co, Milwaukee, WI, USA), and they were decapitated and pithed; their cutaneus pectoris muscles were dissected out and mounted in Lucite recording chambers containing 2-5-2-8 ml of Ringer solution whose composition (in mM) was: Na+, 116; K+, 2-1; Ca2 , 1P8; Cl-, 117; HP042-, 1; H2PO4-, 0 5; pH, 6-9. The chamber was flushed with 10-15 ml of a modified Ringer solution with 1 mM-ethyleneglycol-bis(fl-aminoethylether)N,N'tetraacetic acid (EGTA), no Ca2+ or Mg2+ and tetrodotoxin (TTX, 2-20 x 10" g/ml; Sigma Chem. Co., St Louis, MO, USA) to prevent the spontaneous twitching that develops in muscles soaked in solutions without divalent cations (Builbring, Holman & Luillmann, 1956). This solution was renewed once 10 min, then BWSV (0 5 glands from Italian spiders or 1 gland from American or twice during spiders) or a-LTX (0 5-1 ag) was added to the chamber, the bath gently stirred and the preparation incubated for 25-35 min. Quantal secretion is not stimulated under this condition (Misler & Hurlbut, 1979; Misler & Falke, 1987), and the toxin can be allowed to penetrate the muscle completely and bind to its receptor (Valtorta, Madeddu, Meldolesi & Ceccarelli, 1984). A wellsynchronized discharge of quanta from the various junctions in the muscle was started by applying Mg2+ and stopped by withdrawing it, and the rate of discharge was regulated by controlling the Mg2+ concentration (Misler & Hurlbut, 1979; Misler & Falke, 1987) or the temperature (Ceccarelli et al. 1988). The chambers were mounted on a Peltier device (Cambion, Cambridge, MA, USA) when we wanted to control the temperature, and the solutions were cooled in an ice bath for the experiments at 9-10 °C. When these ice-cooled solutions were flushed through the chambers, the temperature changed by less than + 2 °C, and it returned to the regulated value within a few minutes. The miniature endplate potentials (MEPPs) were recorded throughout the experiments, and when their rate of occurrence had fallen to low values (- 30 s-1 after 10-20 min at room temperature or 60 s-1 after 50-70 min at 9-10 °C), Ringer solution with TTX was applied to determine whether the terminals still contained a large residual store of quanta. Then the junction was impaled by a second micropipette filled with Lucifer Yellow (Stewart, 1978), and the fluorescent dye was injected ionophoretically for 5-10 min. The chamber was flushed with Ringer solution, filled at room temperature with a solution of 1-2 % formaldehyde (freshly prepared from

W. P. HURLBUT AND OTHERS 504 paraformaldehyde) in 0-1 M-phosphate buffer, pH 7-4, and the muscle was fixed for 15 min. The

chamber was then put on ice and the muscle was washed for 15 min with cold phosphate buffer (two rinses), treated for 15 min with an ice-cold stain for acetylcholinesterase (Karnovsky, 1964; Karnovsky & Roots, 1964), then warmed to room temperature; fresh staining solution was added and the reaction was developed for 30 min more. The preparation was then washed for - 5 min with phosphate buffer and infiltrated with 50 % glycerol in 0-05 M-phosphate buffer. In most of the early experiments, the central portion of the muscle was cut out, mounted in the glycerol phosphate solution on a glass slide, covered gently with a cover-slip, sealed with clear nail polish and kept in a cold room overnight. The slide was examined the next morning in a fluorescence microscope (Zeiss, Photomicroscope III, Oberkochen, FRG), and the outlines of the cholinesterase stain on (or under) the fluorescent muscle fibre were sketched onto graph paper with the aid of a Zeiss drawing tube (Zeiss, Oberkochen, FRG) (final magnification, x 480-540). The fluorescent fibre was also photographed, and its diameter, D, and the average length of its sarcomeres, S, or the sarcomere length of an adjacent fibre, were measured from the prints. In other experiments the fluorescent muscle fibre was isolated and photographed on the day of the experiment, and D, S and the length of the junction were measured on the prints. The diameters of the isolated fibres may have been overestimated since these fibres may have been flattened when the cover-slips were pressed against them. Electric recording. Junctions in the superficial layer of muscle fibres were impaled with glass micropipettes filled with 3 M-KCl (resistances 15-30 MCI), and the membrane potential was recorded by a conventional high-input-impedance amplifier. A high-gain AC-coupled record (1-5 s time constant) and a low-grain DC-coupled record of the membrane potential were stored on FM magnetic tape (A. R. Vetter, Rebersburg, PA, bandwidth = DC- I100 Hz or Racal 4DS, bandwidth = DC-1250 Hz) or on a pulse-code-modulated video cassette recorder (A. R. Vetter Co., model 200T, bandwidth = DC-20 kHz) for later analysis by computer (PDP 11/73. Digital Equipment Corp., Maynard, MA, USA). We tried to record from a single neuromuscular junction in each muscle throughout the entire sequence of solution changes, but usually we were not successful since the muscle fibres often depolarized rapidly after the divalent cations were removed, especially the small fibres in muscles from small frogs. Many of the recordings were obtained from junctions impaled after the toxin had been added, but 5-10 min before Mg2+ was applied, and the membrane potentials were generally low; we recorded MEPPs as long as the potential did not fall much below 40 mV. In each experiment we tried to get several minutes of control record in the divalent-cation-free solution before Mg2+ was applied, but in seven experiments the MEPPs rate rose abruptly to 10-70 s-1 before the control record had been obtained. The Mg2+ was applied immediately whenever this happened, and the control record was taken at the end of the experiment 5-10 min after Ringer solution had been applied and the MEPP rate was low. Lucifer Yellow. Micropipettes were filled with a solution of 5 % Lucifer Yellow CH (di-lithium salt) (Aldrich Chem. Co., Sigma Chem. Co. or Molecular Probes, Eugene, OR, USA) in distilled water, and their resistances were 80-200 MQ (i.e. 5-10 x the resistances of the KCl micropipettes). A micropipette was prepared and tested at the beginning of each experiment; at the end of the experiment it was inserted into the junction whose MEPPs had been recorded, and hyperpolarizing current pulses of 10-20 nA, 50 ms in duration, 10 s-1 repetition rate (i.e. 50% duty cycle) were applied for 5-10 min while the electrotonic response of the muscle fibre was monitored. The ionophoretic currents were measured by a virtual ground amplifier inserted into the ground circuit of the membrane potential amplifier. The resistance of the Lucifer Yellow micropipettes usually increased greatly when we attempted to inject the dye at 9-10 °C, and most of the markings we tried at this temperature were unsuccessful. We were more successful when we warmed the preparations to 20 °C before trying to inject them with Lucifer Yellow. The warming was begun several minutes after Ringer solution had been applied, and few MEPPs occurred during the warming period. Computation of MEPP rate, amplitude, waveform and number of quanta secreted. The MEPP is closely approximated by the difference between two exponentials (Segal et al. 1985; Fesce, Segal & Hurlbut, 1986b; Ceccarelli et al. 1988): MEPP = h[w(t)] = h[exp (t/01) -exp (t/02)], where: h = MEPP amplitude factor (the peak amplitude of the MEPP is about 70 % of h), w(t) =

QUANTA AND SYNAPTIC VESICLES

505

MEPP waveform, 01 = time constant of MEPP decay and 02 = time constant of MEPP rise. The time constants were determined by fitting the product of two Lorentzians to the power spectrum of the fluctuations when the MEPP rate was high (> 20 so1) or, when it was low (< 10 s-l), by fitting exponentials to the waveform obtained by superimposing five to twenty-five individual MEPPs (Fesce et al. 1986a). The mean MEPP rate (r) and mean amplitude factor (h) were computed from the variance (VAR), skew (SK), and fourth cumulant (C4) of the fluctuations of the high-pass-filtered, ACcoupled record of the membrane potential (V), after the moments had been corrected for non-linear summation (Segal et al. 1985; Fesce et al. 1986b): r = (VAR/I2)3/(SK/I )2, h = (SK/I3)/(VAR/I'), where

I= J[w'(t)]ndt

and w'(t) = waveform of the high-pass-filtered MEPP. The number of quanta secreted, Q, was obtained by integrating r over time. With adequate high-pass filtering, these values of r and h are valid in the face of changes in the mean membrane potential that are unrelated to the summation of MEPPs, changes in the rate of non-quantal secretion, non-stationary MEPP rates and non-linear summation of MEPPs. The dispersion of MEPP amplitudes introduces errors, but these can be corrected by multiplying r or h by the factors (Fescue et al. 1986b):

Cr =R2/(3R-2)/(2R- 1), and Ch = (3R -2)/R, where R = (SK/I3)2/(VAR/I')/(C4/I4). The ratio R was continually evaluated throughout each experiment, but since it is a noisy parameter, especially during the late stages of the experiments when the moments of the fluctuations are often barely significant, it was not used to correct the results obtained at individual junctions. Instead, we used the average values of R determined for each class of experiments to correct the average values of r and h for that class. The rate-weighted averages of R were used to correct the average values of Qt. the total number of quanta secreted by a terminal. Electron microscopy. Muscles were fixed in the recording chambers with a cold solution of 1 % OS04 in 041 M-phosphate buffer, pH 7 4, for a total of 1 h. Small bits of muscle rich in endplates were cut out, dehydrated, embedded in Epon and silver-grey sections (- 40 nm thick) were cut with a diamond knife (Diatome, Ltd, Bienne, Switzerland) on a Reichert-Jung ultramicrotome (Reichert, Wien, Austria). The sections were mounted on coated grids, double-stained with uranyl acetate and lead citrate and examined in a Hitachi H-7000 electron microscope (Hitachi Ltd, Tokyo, Japan). Cross-sections of nerve terminals were photographed (x 10000), and prints were prepared (final magnification x 40000) and analysed with a Zeiss MOP I digitizing image analyzer (Zeiss, Oberkochen, FRG). The following parameters were measured on each cross-section: area of axoplasm, perimeter of nerve terminal, number of synaptic vesicles, number of coated vesicles, number of large dense-core vesicles and number of large vesicular structures, i.e. irregular closed structures with diameters > 60 nm. The sectioning and the electron microscopy were done in Milan, Italy. Venom and toxin. Whole spiders (Latrodectus mactans tredicimguttatus), or their cephalothoraxes, were kept frozen at -80 TC and thawed as needed. The glands were plucked out and homogenized in 120 mM-NaCl (1 gland/100 14l). The homogenate was stored at 3 TC and used for up to 1 week. The toxin was purified as described previously (Meldolesi, 1982), and stored at -80 0C in aliquots of -1 jug at a concentration 20 ,sg/ml. An aliquot was used within a day after it had been thawed. Experimental programme. The full time course of quantal secretion was recorded at forty-nine junctions in six groups of muscles (A-F) subjected to a wide variety of experimental conditions and dissected from several frogs obtained between January 1987 and October 1988. The weights of the frogs ranged from 13 to 68 g, the temperature from 9 to 28 0C, and the concentration of Mg2+ from 2 to 10 mm. a-Latrotoxin from several different purifications, or crude BWSV from Italian or from -

-

506

W. P. HURLBUT AND OTHERS

American spiders, were used, and secretion was interrupted in some experiments (Table 1). This was done to conserve toxin, to maximize the range of nerve terminal lengths included in the correlation and to check that the total number of quanta secreted was independent of the rate or

temporal pattern of secretion, as is expected if recycling is totally blocked and the initial store of quanta in the terminal is completely exhausted. The thirteen muscles in group A were used from January to April 1987 in New York. They were dissected from several batches of small frogs (similar in size to those we had used in the past), were pre-treated with crude BWSV from Italian spiders, were held at room temperature (21-25 TC), and were exposed to 2 (two muscles), 4 (eight muscles) or 10 (three muscles) mM-Mg2+. Ten of these junctions were marked with Lucifer Yellow, but S was measured only at nine of these. No muscles from this group of frogs were fixed for electron microscopy. The thirteen muscles in group B were used during June-July 1987 in Milan. They were dissected from a single batch of relatively large frogs, were pre-treated with a single batch of a-LTX, were held either at 28 0C (two muscles) or at 22-23 0C (eleven muscles), and were exposed to 4 mM-Mg2+; twelve of these fibres were injected with Lucifer Yellow (ten at 22-23 TC). Four additional muscles from this same batch of frogs were pre-treated with toxin, exposed to 4 mM-Mg2+ for 0, 5, 10 or 15 min at 22-23 TC and fixed for electron microscopy. The three muscles in group C were used during November 1987 in New York. They were dissected from three very large frogs, were pre-treated with a second batch of a-LTX and were exposed to 4 mM-Mg2+ at room temperature (21-23 TC). All were marked with Lucifer Yellow, but none was fixed for electron microscopy. The toxin was inadvertently applied at 0 1 x the usual dose in one of these experiments, but its results were used since secretion seemed normal. The six experiments in group D were performed from November 1987 to January 1988 in New York. Their objective was to split the secretion from a single junction into two approximately equal bouts of release that were well separated in time. The muscles were dissected from the batch of relatively large frogs used in C. They were pre-treated with the same batch of a-LTX, were held at room temperature (21-24 TC) and were exposed to 4 mM-Mg2+. However, the Mg2+ was withdrawn from these muscles after 3-4 min, and it was reapplied 35-40 min later. The timing in these experiments was not fixed, since the onset of secretion varied slightly from junction to junction, and the Mg2+ was withdrawn when the membrane potential in the fibre we were recording from seemed to have reached a minimum. None of these junctions was marked with Lucifer Yellow and none was fixed for electron microscopy. The electron microscopy was done about a year later using a pair of muscles from a frog in group E; its main purpose was to determine whether the number of synaptic vesicles changed during the interval between the times the two muscles were fixed (- 33 min with low values of r). The fourteen muscles in group E and F were used from November 1988 to January 1989 in New York. They were dissected from a single batch of smaller frogs, were held at 9-10 °C, were pretreated with a different batch of c-LTX (E) or with crude BWSV from American spiders (F), and were exposed to 4 mM-Mg2+; five of these fibres were injected with Lucifer Yellow (four from group F). Four additional muscles from this batch of frogs were held at 9-10 °C, were pre-treated with toxin and were fixed for electron microscopy 10, 20, 39 or 75 min after 4 mM-Mg2' had been applied; the last muscle in this series was also treated with Ringer solution during the last 10 min of the experiment. One other muscle from this batch of frogs was fixed at room temperature in Ringer solution, and it was not treated with toxin. It serves as a control for the initial content of synaptic vesicles in terminals from these frogs. The average time courses of quantal secretion and vesicle loss are compared only for muscles exposed to 4 mM-Mg2+ ai.L held either at 22-23 'C (group B) or at 9-10 'C (groups E and F). In both cases the muscles that were fixed for electron microscopy were dissected from the same batch of frogs, were treated with the same batch of toxin, and were used in the same time period as were the muscles used for electrophysiology. The results obtained from three additional junctions were rejected: two (from groups A and E) because they did not secrete rapidly when 4 mM-Mg2+ was applied, and one from group F because its cholinesterase reaction was spotty (Jans et al. 1986). All numerical results are reported as mean values+s.D., unless stated otherwise.

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507

RESULTS

Electrophy8iology Figure 1 illustrates MEPPs recorded at a single neuromuscular junction throughout the entire sequence of solution changes during an experiment from group D at 22 'C. This junction was selected for illustration because its membrane potential was unusually stable, probably because the muscle fibre was very large, but its general behaviour is representative of most fibres. The MEPP rate fell after the A

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Fig. 1. MEPPs recorded from a single neuromuscular junction during an experiment at 22 'C. All records are AC coupled: the time constant was 100 ms in panels A and B and 1 s in panels C-H. The traces were selected to show more than one MEPP. A, MEPPs recorded in Ringer solution at the beginning of the experiment; V = 88 mV. B, MEPPs recorded about 10 min after the divalent cations were removed; V = 84 mV; the MEPPs are smaller and briefer. a-LTX (0-33 ,ug/ml) was applied for 21 min and removed by flushing the chamber with a divalent-cation-free solution. C, MEPPs recorded about 3 min later during the control period before Mg2+ was applied; V= 85 mV; r is low. D, MEPPs recorded about 1 min after 4 mM-Mg2+ had been applied; V = 85 mV; r is beginning to rise. E, MEPPs recorded about 3 min later when r was near its peak; V = 79 mV; the gain was reduced 50 %. F, MEPPs recorded about 30 min after Mg2+ had been added; V = 81 mV; r is low, and the dispersion of MEPP amplitudes has increased. 0, MEPPs recorded a few minutes after Ringer solution had been reapplied; V = 83 mV; both r and h have increased. H, MEPPs recorded about 6 min after Ringer solution had been applied; V = 84 mV; both r and h have decreased. Calibration 50 ms and 1-0 mV for panel E; 0 5 mV for all other panels. -

W. P. HURLBUT AND OTHERS 508 divalent cations were removed, and the MEPPs became smaller and briefer even though the membrane potential changed only slightly (B). The MEPP rate had not changed 25 min after a-LTX had been added (C), but it increased noticeably a few minutes after 4 mM-Mg2+ was applied (D), rose to high levels after about 4 min (E) and then declined to low levels 15-20 min later (F). Some small MEPPs, and some large ones, occurred at this time, and the dispersion of MEPP amplitudes had clearly increased (Fesce et al. 1986a). Both MEPP rate and amplitude increased when Ringer solution was applied (0), and both decreased again during the next few minutes (H). The time constants of the MEPPs did not change much during the experiments. The average values of 01 and 02 measured during the control period before Mg2+ was added were: 3-08 + 0-86 ms and 044 + 0-09 ms (n = 35), respectively, at 21-28 0C, or 11 + 3-3 ms and 1-39 + 0-26 ms (n = 17), respectively, at 9-10 0C. The time constants increased slightly after Mg2+ was added, but changed by less than + 30 % during the course of secretion. The average value of 01 at room temperature is about half that measured previously at junctions in Ringer solution or Ca2+-free solutions (Fesce et al. 1986 a), and this low value probably results from increases in membrane conductance due to the exposure to solutions with no divalent cations. The power spectra computed during secretion were well-fitted by double Lorentzians, as observed previously (Fesce et al. 1986 a). Figure 2 shows the time courses of r, Q, h and V, at three neuromuscular junctions, each subjected to one of our primary experimental conditions (groups B, D and E-F). The junctions were pre-treated with z-LTX in a solution without divalent cations and then exposed to 4 mM-Mg2+ at 22 'C (A-B and C-D), or at 9 0C (E and F). The Mg2+ was withdrawn after about 4 min in C and D and reapplied about 35 min later. The results shown have not been corrected for the dispersion in MEPP amplitudes since R, the parameter that monitors that dispersion, is quite noisy, especially at late times when the fluctuations are small and the moments barely significant. Figure 3 illustrates how R, and the resulting correction factors for r (cr) and h (Ch), changed during the course of secretion. For clarity, the results of all the experiments have been combined. Although the final values of the correction factors can be quite large, about 2-3, the major changes occur after most of the quanta have been secreted. We have used the values of R for each group of experiments to correct the average values of r and h for that group, and Table 1 summarizes the results obtained. The two groups at 9-10 0C have been combined because they are not significantly different, and the grand average of all the results at room temperature (21-28 0C) are presented in the last column. At room temperature r increased significantly within 1-2 min after Mg2+ had been applied; it reached peak values of 1-5 x 103 s-1 after 4-8 min and fell to < 100 s-1 (except at one junction in group C) after 15-20 min. When Ringer solution was applied at the end of the experiment, r approximately doubled before it fell again to low values (Fig. 2). A total of about 106 quanta were secreted and half of these were released during the first 3-8 min in 4 mM-Mg2+. If Mg2+ was removed when the rate of secretion was near its peak (1804+ 307 s-1, n = 6), then r fell to < 10 s-1 within 3-4 min and it remained low until Mg2+ was reapplied 30-35 min later, whereupon it rose to a slightly lower peak value (1411+391 -1, n = 6) and then fell more slowly,

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Fig. 2. Time course of the changes in r, Q, h and V at three neuromuscular junctions treated for 20-25 min with a-LTX in a solution without divalent cations and then exposed to 4 mM-Mg2+. Results not corrected for the dispersion in MEPP amplitudes; h not corrected for changes in V. Panels A, C and E: r (0) and Q (continuous line). Panels B, D and F: h (0) and V (continuous line). The filled symbols show values of r obtained by counting MEPPs, or the values of h either calculated from the average of the peak amplitudes of twenty to thirty MEPPs or obtained by curve-fitting the waveforms resulting from the superposition of ten to thirty MEPPs. A and B, 23 'C. Ringer solution was applied at about 20 min (vertical arrow); about 0-64 x 10s quanta were secreted by this junction. Most of the late increase in h in the Ca2+-free solution is artifactual, and results from the increase in the dispersion of MEPP amplitudes (see text). C and D, 22 0C: note change in time scale. Mg2+ was removed at about 4 min (first vertical arrow) and reapplied at about 42 min (second vertical arrow); Ringer solution was applied at about 62 min (third vertical arrow). About 0-36 x 101 quanta were released by this junction during the first bout of secretion and about 0 40 x 106 during the second. Most of the late increase in h in the Ca2+-free solution results from the increased dispersion of MEPP amplitudes (see text). E and F, 9 00: Ringer solution was applied at 62 min (vertical arrow); 0-42 x 10t quanta were secreted by this junction.

W P. HURLBUT AND OTHERS reaching levels of 30 sO 10-15 min later; 46 ± 8 % of the MEPPs occurred during the first bout of secretion. At 9-10 TC, r increased significantly within 2-8 min after Mg2` had been applied; it reached peak values of 400-1200 s51 after 14-30 min and fell to 100 s51 after 45-60 min. It rose again to peak values of 200-300 s-1 if Ringer solution was applied, or if the preparation was warmed to 20 0C in the Ca2+-free solution (not shown), and then fell to 10 s-1 about 10 min later. Half the quanta were secreted within 20-30 min at this temperature. 510

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The amplitude factor, h, appeared to increase during secretion at room temperature (Fig. 2), and it increased further a few minutes after Ringer solution was applied (Fig. 1). However, most of the apparent increase in h during secretion in Mg2+ resulted from the increased dispersion of the MEPP amplitudes, and when the results are corrected for that dispersion, using the average value of R measured shortly after Ringer solution had been applied, then the final values of h in the Ca2+-free solution were lower than, or approximately equal to the starting ones, and they increased after Ringer solution was applied (Table 1). At 9-10 0C the final corrected values of h were about 60 % of the starting ones, a significant reduction; the maximum values after Ringer solution had been applied were about equal to the initial ones (Table 1). The total number of quanta secreted, Qt. tended to increase with the size of the frogs in the experiments at room temperature, but for frogs of a given size, it was not affected by withdrawing Mg2+ and reapplying it about 35 min later (Table 1). These results are expected if recycling is blocked and similar sized terminals contain similar numbers of preformed quanta. However, the values of Qt measured at 9-10 0 are large for the sizes of the frogs that were used, and we suspected at first that recycling had not been totally blocked at these junctions. Instead, it turned out that the nerve

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Fig. 9. Electron micrographs of terminals in two muscles from the same frog. Both muscles were treated for 20 min at 20 0C with a-LTX (018 jgg/ml) in a solution without divalent cations and then exposed to 4 mM-Mg2+. The Mg2+ was removed from both muscles after about 4 min when r was near its peak; A was fixed about 10 min later when r was < 10 s1l and B was fixed about 33 min after A, when r was still very low. The terminals contain few vesicles. Scale marker = 1 jum; x 22000.

cations contained similar numbers of vesicles as the controls; they appeared slightly swollen, presumably because the toxin increased the ionic permeability of their axolemmas (Finkelstein, Rubin & Tzeng, 1976; Wanke, Ferroni, Gattanini & Meldolesi, 1986). Figure 7 shows electron micrographs of cross-sections of nerve terminals from muscles that had been treated with a-LTX for about 25 min at 23 C and fixed 5 min (A), 10 min (B) or 15 min (C) after 4 mM-Mg2+ was added. All terminals appear to be swollen: A contains relatively few vesicles, B fewer and C almost none.

520

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Figure 8 shows terminals in muscles that had been treated with A-LTX for about 25 min at 10 0C and fixed 10 min (A), 20 min (B) or 75 min (C) after 4 mM-Mg2+ had been added. The muscle in C was also exposed to Ringer solution during the last 10 min of the experiment. Many vesicles are present in A, fewer in B and almost none in C. The partial depletion of vesicles that was induced by a brief exposure to 4 mMMg2+ was not reversed by prolonged soaking in solutions without divalent cations, even though almost no secretion occurs under this condition. Figure 9 shows crosssections of nerve terminals in a pair of muscles from the same frog (one in group E). TABLE 3. Summary of morphometric results

Group B (22-230C) E (9-10 OC)

D (20 OC)

Number of synaptic Area of vesicles Time Length of fixed No. of axoplasm axolemma sections (min) (Um2) (Em) (No./section) (No./Im) 1650+913 0 43 4-0+3-1 7-9+3-1 132+73 838+738 42 6-1+3-9 67+59 5 10-5+3-1 5.1+4-1 31+35 388+438 10 45 8-7+3-1 44 22+20 275+250 15 5-6+3-4 9-4+3-1 44 2-4+1 1 0* 188+88 2350+1100 68+P19 1700+950 10 43 3-7+1-4 9-1+1-7 136+ 76 3-4+1 8 105+49 1313+613 7-7 +2-0 20 43 42 27+14 338+175 39 11.9+2-4 7-2+3-1 41 7+6 88+75 75 4-4+2-6 9-1+2-5 775+563 6-1+3-4 62+45 8 43 11P2+2-7 42 40 1063+650 7-2+3-1 85+52 11-9+2-4 * Muscle soaked only in Ringer solution at room temperature.

They were used in an experiment at 20 0C in which secretion was started by applying 4 mm-Mg2+ to both muscles and stopped by withdrawing the Mg2+ from both muscles about 3 min later. Muscle A was fixed about 11 min after the Mg2+ had been withdrawn, and r had fallen to < 10 s51; B was fixed about 33 min later when r was still low. Both junctions are swollen and partially depleted of vesicles. The results of the morphometric analysis of forty to forty-five cross-sections of nerve terminals in each of these muscles is presented in Table 3. Also included are the results obtained from a control muscle from group E that was soaked only in Ringer solution at room temperature. The terminals at 22-23 'C lost about half their vesicles during 5 min and about 80 % of them during 15 min. The terminals at 9-10 0C lost only about 44% of their vesicles during 20 min and about 95% during 75 min (Ringer solution was applied during the last 10 min of this experiment). About 67 % of the vesicles were lost from terminals that were exposed to 4 mM-Mg2+ for only about 3 min at 20 'C and fixed about 1 min later, and the number of vesicles increased to about 45% of the initial value during an additional 33 min in the divalent-cation-free solution. This difference is significant (t = 2-18, P < 0 05).

QUANTA AND SYNAPTIC VESICLES

521

Correlation between vesicles remaining and quanta secreted The average number of synaptic vesicles remaining per micrometre of terminal, VI' can be calculated from the average number per cross-section, VJ (Weibel & Paumgartner, 1978):

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W. P. HURLBUT AND OTHERS

522

section for a vesicle to be recognized (d - 2H 40 nm at frog neuromuscular junctions, Ceccarelli et al. 1988). These values of V,, are listed in Table 3 and plotted in Fig. 10A together with their standard errors. The average quantal secretion per micrometre was computed as follows: the total secretion of each junction studied was divided by its length, expressed in sarcomeres to obtain a value (Qt/L, in Table 2) independent of stretch; the average total secretion per micrometre (Qt,,, last column in Table 2) was obtained for each group of frogs by dividing the average value of Qt/LS by the average sarcomere length in the same group. Finally, the average time course of secretion per micrometre, Q,. (t), was determined at each temperature by scaling the average time course of secretion to the average total secretion per micrometre (Qtl) at that temperature. These results are also plotted in Fig. 1OA together with their standard errors. It is clear that the loss of vesicles closely parallels the secretion of quanta. Figure lOB shows the correlation between V,, and Q. obtained from the data in Fig. 10A. The results from the experiments with interrupted secretion are also shown. To obtain the latter values, we multiplied the value of Qt., for frogs in groups E and F (the group of frogs used for the morphometric analysis of the experiments with interrupted secretion) by 0-46, the fraction of the total secretion recorded during the first bout of quantal release in the electrophysiological experiments (group D, see p. 510). The correlations are significant although the points at time zero seem high. The equations of the regression lines are:

V = 1510-l1 IQQ c.c. = 0-968 at 22-23 'C; V = 2110-1 21Q/Il, c.c. = 0-982 at 9-10 'C; and V = 1817- 1-14Q,, c.c. = 0-912, for all the data (including interrupted secretion) combined (not shown). These results imply that a quantum of transmitter is derived from a single vesicle. DISCUSSION

The results reported here show that the loss of synaptic vesicles is closely associated with the secretion of quanta of transmitter. About 80% of the vesicles were lost within 10 min at 22-23 °C, the time required for the massive discharge of quanta to subside to a low residual rate of release, and at 9-10 °C the loss of vesicles was slowed to a degree that was commensurate with the reduced rates of release. Furthermore, when secretion was stopped by withdrawing Mg2+ after a few minutes, then the loss of vesicles also ceased. In general, the number of vesicles lost appears to be correlated in a 1: 1 ratio to the number of quanta secreted, which supports the idea that a quantum of transmitter is released upon the fusion of a single vesicle with the presynaptic axolemma. The one aspect of our results which is least consistent with this conclusion is the large difference between the vesicle counts in terminals fixed before Mg2+ was applied and those fixed at the beginning of active secretion (5 min after adding Mg2+ at room temperature, or 10 min at low temperature). Two possible explanations of this difference are: (1) that the fixative itself causes a large discharge of quanta when it is applied to secreting terminals (Ceccarelli, Fesce, Grohovaz & Haimann, 1988), and (2) that the difference arises randomly from the variations in the richness of the -

523 QUANTA AND SYNAPTIC VESICLES population of vesicles in terminals from different individual frogs, or from different groups of frogs. These variations can be quite large, and we have seen the average number of synaptic vesicles per cross-section range from 71 vesicles/section (Haimann et al. 1985) to the 188 vesicles/section obtained in this work (control for groups E and F). This variation, which results from variations in the area of the terminals ( t5,tm2 vs. 2-4 jum2, respectively) as well as from variations in the concentration of vesicles in the axoplasm (47 vesicles/jtm2 vs. 78 vesicles/jum2, respectively), sets a limit to the accuracy of the correlations between vesicle loss and quantal secretion per unit length of nerve terminal and between total quantal secretion and length of the nerve terminals. Two other possible sources of error in our experiments are incomplete block of recycling, and inappropriate correction for the effects of stretch. Recycling seems to be completely blocked at junctions treated with BWSV or acLTX at room temperature in Ca2+-free solutions (Ceccarelli & Hurlbut, 1980; Fesce et al. 1986a; Valtorta et al. 1988) and at junctions treated with high concentrations of z-LTX (2 jug/ml) at 1-3 0C, independently of whether Ca2+ is present (Ceccarelli et al. 1988). However, the situation is not as clear in the experiments done here at 9-10 0C with low doses of toxin or BWSV from American spiders, even though the terminals eventually lost almost all their vesicles. The total secretion evoked under this condition was slightly high (Tables 1 and 2), and the values of R were a bit low (Table 1), both of which are expected if some vesicles were recycled but only partially refilled with ACh; furthermore, terminals which had been partially depleted of vesicles at room temperature appeared to regain about 12 % of their initial complement of vesicles during 33 min of rest in a solution without divalent cations. However, this apparent recovery is quite small, and the relatively high secretion obtained at 9-10 0C seems to have occurred because these terminals contained an unusually large number of vesicles. Furthermore, the results obtained here with Italian BWSV are very similar to those obtained previously with frogs of similar size (Fesce et al. 1986a). Thus residual recycling does not seem to have been extensive enough to have seriously affected our results. It seems reasonable to correct our measurements of nerve terminal length for the stretch applied to the muscles when they are mounted in the recording chambers, since stretching a muscle fibre should increase the apparent length of its neuromuscular junction and also change the dimensions of cross-sections of nerve terminals, or the number of synaptic vesicles contained in a section. It seems likely that a terminal follows a crooked path along the surface of a muscle fibre, or that its diameter waxes and wanes, and that the path is straightened, or the diameter is made more uniform, as the fibre is stretched. If this were true, then the volume of axoplasm contained in a section would decrease as the terminal was lengthened, while the number of sections of a given thickness that could be cut from a junction would increase. Under these circumstances, the electrophysiological and morphological results should be compared only for muscles stretched to the same extent, and this is what we tried to do by comparing electrophysiological and morphological results obtained from muscles from the same group of frogs for each experimental condition. These muscles should have been stretched roughly to the same extent, on average, since they were all mounted in the recording chambers. Furthermore, the

524

W. P. HURLBUT AND OTHERS

values of Q,. for each group were computed from the mean sarcomere length for that group and from the mean value of Q.. (total secretion per sarcomere), which is independent of muscle fibre stretch. The measurements of vesicles remaining and quanta could be made at the same terminal (Ko & Propst, 1986; Propst & Ko, 1987), and then the results would not need to be corrected for different degrees of stretch. However, the number of vesicles initially present in the terminal and the number of quanta still to be released by it would not be known, and a meaningful correlation between quanta secreted and vesicles remaining would still require that both measurements be made at many different terminals in many different muscles subjected to different stretches and fixed at several different times. Such a correlation would be very tedious to obtain, and would not greatly improve the accuracy of the results. In conclusion, these results show that the loss of synaptic vesicles is highly correlated with the secretion of quanta, in a ratio of approximately 1:1, at terminals treated with BWSV or ac-LTX in Ca2+-free solutions with 4 mM-Mg2+. Since previous results have shown that the vesicles are not destroyed under these conditions, but are incorporated into the axolemma of the nerve terminal (Valtorta et al. 1988), it seems likely that the incorporation occurs when the quanta are secreted (Heuser, Reese, Dennis, Jan, Jan & Evans, 1979; Torri-Tarelli, Grohovaz, Fesce & Ceccarelli, 1985), and unlikely that the loss of vesicles and the secretion of quanta stem from two independent actions of the toxin. Our results may not be accurate enough to exclude the possibility that a quantum is derived from two vesicles, but they are accurate enough to exclude the possibility that a quantum is released by seven to ten vesicles at a single active zone (Wernig & Stirner, 1977). We are indebted to Professor Jacopo Meldolesi for providing the a-latrotoxin and for his interest in and support of this work. We thank Dr N. Kalderon for the use of her fluorescence microscope and Mr A. Dekhuijzen for helping with some of the early experiments and for suggesting that we correct our measurements for stretch. This work was supported in part by USPHS grant NS 18354 (W. P. H.) and by a grant from the Muscular Dystrophy Associations of America (B. C.). This paper is dedicated to the memory of Professor Alexander Mauro, who died on 6 October 1989. REFERENCES

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Correlation between quantal secretion and vesicle loss at the frog neuromuscular junction.

1. We measured the rate of occurrence of miniature endplate potentials (MEPPs) at identified endplates in frog cutaneous pectoris muscles treated with...
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