amplitude of spontaneous miniature end-plate potentials (m.e.p.p.s).

Transcription

1 J. Physiol. (1984), 348, pp With 9 text-figures i Printed in Great Britain A STUDY OF THE ACTION OF TETANUS TOXIN AT RAT SOLEUS NEUROMUSCULAR JUNCTIONS BY STUART BEVAN AND LINDA M. B. WENDON From the Department of Zoology, University College London, Gower Street, London WCJE 6BT; Department of Physiology, The Hebrew University, Hadassah Medical School, P.O. Box 1172 Jerusalem 91, Israel and Cold Spring Harbor Laboratory, P.O. Box 1, Cold Spring Harbor, NY 11724, U.S.A. (Received 4 February 1983) SUMMARY 1. Tetanus toxin (TeTX) inhibits the evoked release of acetylcholine (ACh) at rat soleus end-plates. The effects of various procedures which evoke ACh release by raising the level of free intracellular calcium have been investigated at various stages of tetanus intoxication. 2. At all stages studied TeTX has little or no effect on either the frequency or the amplitude of spontaneous miniature end-plate potentials (m.e.p.p.s). 3. After TeTX poisoning, e.p.p. latency is more variable than normal and the slope of the relationship between ln m (quantal content) and in [Ca]. is reduced from the control value of about Plots of m-i/n against 1/[Ca]o, for n = 1-4, suggest that mmax, the maximum number of quanta releasable by nerve stimulation, is reduced at intoxicated end-plates. 5. Blocking delayed rectification with 3-aminopyridine (1-5 mm) increases m, but has little or no effect on either the slope of ln m - ln [Ca]. plots or estimates of mmax. 6. Several treatments which raise m.e.p.p. rate (high [Ca]., hyperosmotic medium, addition of lanthanum) are less effective after TeTX poisoning. Some of the tested agents increase m.e.p.p. frequency by a mechanism which is thought to involve a mobilization of calcium from intracellular stores. 7. The decline in m.e.p.p. rate after a period of high-frequency nerve stimulation is different at normal and TeTX-treated end-plates. At tetanus-intoxicated end-plates, the decline differs from that expected if TeTX acted simply to block calcium entry into the terminal. In addition, the increase in m.e.p.p. frequency observed with a high rate of nerve stimulation suggests that considerable amounts of calcium can enter the terminal with each action potential. 8. It is concluded that TeTX blocks transmitter release by acting at a step between calcium influx to the terminal and transmitter release such that the mechanism for ACh release shows a reduced sensitivity to intracellular calcium. The possibility of an additional effect on the presynaptic calcium conductance cannot be excluded. Some differences between the properties of end-plates poisoned with TeTX and botulinum toxin are discussed. 1 PHY 348

2 2 S. BEVAN AND L. M. B. WENDON INTRODUCTION Tetanus toxin (TeTX), a protein of molecular weight 15 produced by Clostridium tetanii, blocks synaptic transmission by a presynaptic action (see Mellanby & Green, 1981). In the spinal cord TeTX acts to block inhibitory inputs to motoneurones (Curtis & de Groat, 1968; Brooks, Curtis & Eccles, 1975), and the increased activity of motoneurones causes the typical spastic paralysis of tetanus intoxication. In addition to its action on central synapses, TeTX can also block neuromuscular transmission, and clinical reports of flaccid paralysis have been noted (Kaeser, Muller & Friedrich, 1968). When TeTX is administered experimentally, end-plates in mammalian slow-twitch muscle fibres are more severely affected than end-plates in fast-twitch muscle fibres (Duchen & Tonge, 1973; Kretzschmar, Kirchner & Takano, 198). Neuromuscular junctions on slow muscle fibres therefore provide a convenient system for looking at the mechanism by which TeTX acts. From previous studies it appears that the action of TeTX resembles that of botulinum toxin (BoTX), a presynaptically acting toxin produced by another strain of Clostridium. Neither toxin blocks action potential invasion of the nerve terminal and both act at a step between membrane depolarization and quantal acetylcholine (ACh) release. However, for both toxins the exact site of action remains unclear. For botulinum poisoning, Hirokawa & Heuser (1981) concluded from an ultrastructural study that BoTX blocks neuromuscular transmission primarily by impairing the calcium influx normally associated with nerve terminal depolarization. In contrast, electrophysiological studies of BoTX-poisoned muscles have shown that the mechanism for ACh release shows a reduced sensitivity to intracellular calcium (Cull-Candy, Lundh & Thesleff, 1976) and suggest that the voltage-activated calcium current in motor nerve endings is not significantly reduced (Gundersen, Katz & Miledi, 1982). We have examined the effects of TeTX on the calcium sensitivity of ACh release, using procedures which should allow us to assess whether TeTX acts by simply interfering with the influx of calcium to the nerve terminal or by blocking a subsequent step in the release process. A preliminary account of some of these results has been communicated to the Physiological Society (Wendon, 198). METHODS All experiments were made on the soleus nerve-muscle preparation from Wistar rats of g weight. TeTX was injected intramuscularly into one leg in the region of the soleus muscle. Lethal doses were determined for each batch of toxin used and -5-5 lethal doses were administered in -2 ml of sterile saline. Animals to be used for experiments more than 48 h after the initial toxin injection were given sublethal doses at 48 h intervals. For electrophysiological examination, nerve-muscle preparations were removed from rats under ether anaesthesia and perfused with a solution of the following composition: 138 mm-nacl, 5 mm-kcl,2 mm-cacl2, 1 mm-mgcl2, 15 mm-nahco3, 1 mm-nah2po, 11 mm-glucose. Thissolution was bubbled with a 5% C-95% 2 mixture and the final ph adjusted to between 7-15 and In experiments with added lanthanum and in some experiments with high CaCl2 concentrations, a 2 mm-tris buffer (ph 7-2) replaced the NaHCO, and NaH2PO and the solution was equilibrated with 12%- When necessary the MgCl, and CaCl2 concentrations were varied to give end-plate potentials of an amplitude suitable for analysis. With the exception of experiments investigating the effects of hyperosmotic solutions, the osmolarity of all recording solutions was kept constant by varying the NaCl concentration.

3 ACTION OF TETANUS TOXIN AT N.M.J. 3 Experiments were made at room temperature (21-27 ) using standard electrophysiological techniques. The nerve was stimulated with a suction electrode using a pulse of 4-5 Fss duration. For normal and lightly intoxicated muscles, the pulse amplitude was set at 3-5 times the voltage necessary to evoke muscle contraction. For highly poisoned muscles, in which no muscle contraction could be evoked in the above standard solution, the pulse amplitude was set initially at 15 V, which was always sufficient to cause contraction of untreated muscles. In many instances the amplitude was subsequently reduced from this high level and reset at 5 times the voltage necessary to evoke end-plate potentials in the muscle fibre under study. TABLE 1. Summary of various agents and procedures at normal and TeTX-poisoned end-plates at different stages of intoxication Relative m.e.p.p. frequency Time Quantal content after [Ca]. High-frequency Hypertonic High injection (mm) -3-AP + 3-AP stimulation solutions [Ca]o 2 * 42-4 (3) 5-2 (4) 6-6 (4) ±12 (8) * 1-1 (4) 1-4 (4) 1-2 (5) (5) 7-6±5-8 (6) 3.4 (5) 1*1 (5) -8 (4) ±13 (4) (6) 1.5 (5) -9 (2) 7 (4) Data based on population samples. E.p.p. quantal content (m) estimated in solutions containing 1 mm-mgcl2. Asterisk (*) indicates the presence ofevoked action potentials in fibres under test. 3-AP tested at a concentration of 1 mm. Effect of high-frequency nerve stimulation examined after 1 s of 1 Hz stimulation (15 mm-cac12, 1 mm-mgcl2); relative m.e.p.p. frequency expressed as multiple of pre-stimulus frequency. Other relative frequencies refer to effects of either raising [Ca]o from 1 to 1 mm or adding 1 mm-sucrose. Numbers in parentheses represent the number of fibres examined. Intracellular recordings were made from surface muscle fibres with 3 M-KCl-filled micro-electrodes (5-15 MCI). Records of end-plate potentials (e.p.p.s) and miniature end-plate potentials (m.e.p.p.s) were usually stored on magnetic recording tape for subsequent analysis, although for some experiments on4ine computer analysis was used. E.p.p. quantal content (m) was assessed either from the equation m = In (number of impulses/number of failures) or from the ratio (mean e.p.p. amplitude/mean m.e.p.p. amplitude). In only a few experiments did e.p.p. amplitudes exceed 5 mv; in these cases m was calculated using an amended version of Martin's correction (Martin, 1955; MacLachlan & Martin, 1981). When m was low, special care was taken to correct for the number of m.e.p.p.s expected in the time bin used to classify potentials as e.p.p.s (Andreu & Barrett, 198). Time-dependent changes in m.e.p.p. frequency were examined using the 'moving bin' method of Erulkar & Rahamimoff (1978). RESULTS General ob8ervation8. The development of neuromuscular block with tetanus intoxication was followed for up to 15 h after the initial TeTX injection. The first signs of spastic paralysis were usually observed by 2 h. By this time evoked quantal ACh release was already impaired at soleus neuromuscular junctions and some muscles showed no contraction with suprathreshold nerve stimulation. For other animals functional soleus neuromuscular transmission was usually lost by 48 h. Some variation in the time course and extent of TeTX's action was noted; nevertheless, in general a progressive impairment of evoked ACh release was observed. This is illustrated in Table 1, which shows the results of a series of recordings made from 1-2

4 4 S. BEVAN AND L. M. B. WENDON muscles of sibling rats at varying times after TeTX injection. Several features of TeTX action can be seen. Neuromuscular transmission was already depressed at 25 h, when the muscle showed only a very weak twitch to 5 Hz nerve stimulation. At longer times after TeTX injection m was further reduced and by 15 h few end-plates showed obvious e.p.p.s to nerve stimuli delivered at 5 Hz. In all muscles studied m could be raised, by a variable amount, either by increasing the external A B 5-5- w C o e. Ị,. --- I *5 I -I 1-5 E z 5 - C 5- D 1-1* Amplitude (mv) Fig. 1. Distribution of m.e.p.p. amplitudes at normal (A) and TeTX-poisoned (B, C and D) end-plates. Time after initial TeTX injection: B, 5 h; C and D, 15 h. For C and D, neostigmine methylsulphate (1 jag ml-') present. calcium concentration ([Ca]o) or by adding 1-5 mm-3-aminopyridine (3-AP), a blocker of delayed rectification. Table 1 also illustrates that several procedures which can increase m.e.p.p. frequency at normal end-plates are less effective in TeTX-treated muscles. Thus high-frequency nerve stimulation, hypertonic solutions and raised [Ca]o progressively lose their effectiveness with increasing time of intoxication. In contrast to the effect of TeTX on evoked ACh release, little or no change was noted in the normal m.e.p.p. frequency in toxin-treated muscles at any stage of block. For example, in our standard recording solution, m.e.p.p. frequency in muscles examined 48-5 h after injection ( sol, mean + S.D., twenty-one end-plates, five muscles) was not significantly different (P > -2, t test) from that seen in untreated muscles ( s-1, twenty end-plates, four muscles). Similarly, m.e.p.p. amplitudes appeared unaltered by the toxin treatment. The mean m.e.p.p. amplitude at thirty-one end-plates in seven muscles h after TeTX application ( mv) was not significantly different (P> 1) from that at normal

5 ACTION OF TETANUS TOXIN AT N.M.J. end-plates ( mv, twenty fibres, four muscles). Most TeTX-poisoned endplates showed m.e.p.p.s with the Gaussian amplitude distribution typical of normal end-plates (Fig. 1 A and B). This was confirmed at eighteen end-plates in four intoxicated muscles (48-1 h) when an anticholinesterase agent, neostigmine methylsulphate (1 jug ml-'), was added to the bathing solution to increase m.e.p.p. amplitudes (Fig. 1C) and so facilitate the detection of unusually small m.e.p.p.s. Occasional A B 5-. (A a. 4-. E z 2 c Time (ms) Fig. 2. Distribution of intervals between stimulus artifact and foot of e.p.p.s at normal (A) and TeTX-treated (B and C) end-plates, 5 Hz stimulation. In C, data from three end-plates in one muscle have been pooled to increase sample size. Data grouped into time bins of 5 ms in A and 1 ms in B and C. Dashed lines show the number of m.e.p.p.s expected in each time bin. Solutions contain CaC12, MgCl2, respectively (mm): A, 8, 5; B, 2, 5; C, 1, 1. Quantal contents (3 ms time bin): A, 1-35; B, -15; C, mean value = 19. Time of intoxication: 48 and 5 h in B and C, respectively. end-plates in TeTX-treated muscles showed an obvious excess of 'dwarf' m.e.p.p.s in addition to the Gaussian component of the amplitude distribution. An example of such an end-plate is shown in Fig. 1D. This excess of small m.e.p.p.s was particularly noticeable at end-plates in two muscles intoxicated for 1 and 15 h. Such a high incidence of 'dwarf' potentials may be related to the development of nerve terminal sprouts after prolonged TeTX poisoning (Duchen & Tonge, 1973).

6 6 S. BEVAN AND L. M. B. WENDON E.p.p. latency at tetanus-poisoned end-plates. A striking feature of evoked ACh release in TeTX-treated muscles was that at highly blocked end-plates e.p.p. latency was more variable than at normal end-plates. At normal rat soleus end-plates, bathed in low [Ca]O, high [Mg]. solutions to yield low quantal contents (m = 1-1), e.p.p. latency, measured from intracellular recordings at a given end-plate, varied by less than 3 ms (Fig. 2A). To attain similar values of m in TeTX-treated muscles, solutions 2- A 2- B *5 *5~~~~~~~~~~~~~~O55-1- ; r [Ca]l (mm) Fig. 3. Double-logarithmic plots of m against [Ca]. for three normal (A) and five TeTX-treated (B) end-plates. Slopes are given adjacent to each line, which was fitted by eye. For B, times (ii) after TeTX injection are:, 24; A, 45; other symbols, 5. [Mg].: A, *, 5 mm; A and, 1 mm: B, 1 mm except i, 2 mm. 3 ms time bin used to calculate m, 2-5 nerve stimuli, -5 Hz. Before each recording, at least 3 min was allowed after changing to a new calcium concentration. In some experiments [Ca]o was increased from the lowest to the highest levels while in other cases [Ca]o was decreased during the experiment. containing a far higher ratio of [Ca]./[Mg]. were needed. Under these conditions e.p.p.s were frequently dispersed over tens of milliseconds. Fig. 2B shows the distribution of e.p.p. latencies at a 48 h-poisoned end-plate bathed in a solution containing 2 mm-cacl2 and 1 mm-mgcl2. The probability of an e.p.p. occurring declines monotonically over about 2 ms from a peak value at or near the minimum latency. Some highly poisoned end-plates, exposed either to higher levels of [Ca]o (5-1 mm) or to 3-AP, showed additional peaks in the distribution of e.p.p. latencies. Fig. 2C shows such a latency periodogram in which data from three end-plates in a single muscle have been pooled to increase the sample size; an excess of potentials with a latency of 3-4 ms is clearly seen. We have no explanation for the presence of such additional peaks. The relationship between [Ca]. and m. At normal end-plates m does not vary linearly with [Ca] but instead displays a power relationship, ma[ca]o, where n is usually

7 ACTION OF TETANUS TOXIN AT N.M.J. greater than 1 (see Martin, 1977). Thus from a plot of in m or in (mean e.p.p. amplitude) against in [Ca]., the slope of the line will give a value of n which may be taken to characterize the 'co-operativity' of calcium action. Fig. 3A shows such in m-in [Ca]. plots for three end-plates in untreated soleus muscles. As can be seen, the slope of the relationship is about 4. The value of n obtained at seven normal end-plates was , which is slightly higher than previously published estimates for rat muscle (Hubbard, Jones & Landau, 1968a). 2 A 7 A,, A (C](m1 1-5En 12 1/[Cal. (mm-') Fig. 4. Plots of m-i against 1/[Ca]o for'normal (A) and TeTX-poisoned (B) end-plates. The end-plates, with the appropriate symbols, are the same as in Fig. 3. A linear relationship over the entire [Ca]. range has been assumed and the line fitted by eye. Estimates of mmax are, A: *, 24; A, 15; O. 1; B: O. 95; O., 1x2; *, x8; A, 4-7; A, 3. In contrast to the normal end-plates, after tetanus poisoning the relationship between in m and in [Ca] showed a reduced slope (n < 4). This is illustrated in Fig. 3B, which shows plots for five end-plates using a 3 ms time bin to classify potentials as e.p.p.s. There appeared to be a progressive reduction in n with time over the first 48 h after TeTX injection. Thus little or no reduction was seen at 24 h-poisoned end-plate (n = , four end-plates in two muscles) whereas the slopes were significantly reduced by 48 h (n = , eleven end-plates). Between 48 and

8 8 S. BEVAN AND L. M. B. WENDON 1 h of intoxication, n, while remaining low, varied quite widely (range, ). Since the latency of e.p.p.s was more variable at TeTX-treated end-plates than at normal end-plates, m was also calculated using a 15 ms time bin for classifying potentials as e.p.p.s. The values of n calculated using these new estimates of m did not depart significantly from the values determined using a 3 ms time bin mr1 5- A A ~~ Fig I[CM], (mm-') Plots of m-l against 1/[Ca]. for TeTX-poisoned end-plates in the absence (open symbols) or presence (filled symbols) of 5 mm-3-ap. [Mg]o was 1 mm in all cases. A further analysis of the m-[ca]o relationship can be made by plotting m-l/8 against 1/[Ca]. Unlike the above analysis, interpretation of such plots is dependent on the exact model used to explain the calcium 'co-operativity' of the release process. If we consider the Dodge & Rahamimoff (1967) model of n independent release sites or subunits (X), each of which must be occupied to elicit release of a quantum, then the relationship is linear for all [Ca]o and the intercept on the Y ordinate will give an estimate of mmax, the maximum number of quanta that can be released. Such plots are shown for normal and TeTX-poisoned end-plates in Fig. 4. For both sets of end-plates a fourth-power relationship has been assumed; this fits the experimental points reasonably well. As can be seen from Fig. 4A, the Y intercept for normal end-plates is consistent with a large value for mmax. Fig. 4B shows that, for those TeTX-treated end-plates which have a reduced n, mmax is lowered. Our data are also compatible with a lowered mmax, even if 'calcium co-operativity' is lost after TeTX poisoning (i.e. n = 1). This is illustrated in Fig. 5, where m-1 has been plotted against 1/[Ca]o. The open symbols show that for two TeTX-poisoned end-plates the experimental points deviate from a straight line and suggest Y ordinate intercepts of -4--7, which correspond to values of mmax < 2x5. For some other models

9 ACTION OF TETANUS TOXIN AT N.M.J. advanced to explain the power relationship between m and [Ca]., plots of m-1/n against 1/[Ca]. depart from linearity, showing a reduced slope as the Y ordinate is approached. The linear extrapolation to the Y ordinate shown in Fig. 4 would be inappropriate in such cases. The plots in Fig. 4 and Fig. 5 suggest that, even for models yielding such a non-linear relationship, our data are consistent with the idea that mmax is reduced after TeTX poisoning, irrespective of whether the value of n is 4 (Fig. 4) or 1 (Fig. 5). 9 5 mv 5 ms Fig. 6. Oscilloscope traces ofthe response of an end-plate, 55 h after TeTX injection, before (upper) and after (lower) addition of 1 mm-3-ap; 2 mm-cacl2, 1 mm-mgcl2. Upper trace: ten consecutive stimuli (5 Hz); no e.p.p. evoked although one spontaneous m.e.p.p. can be seen. Lower trace: two e.p.p.s evoked by nerve stimulation after addition of 1 mm-3-ap, with a weak contraction artifact following each e.p.p. Mean quantal contents: -16 (one hundred stimuli), control; 4-1 (twenty-five stimuli), 3-AP. 3-Aminopyridine (3-AP). 3-AP blocks voltage-sensitive potassium channels and thus prolongs nerve action potentials (Llinas, Walton & Bohr, 1976; Yeh, Oxford, Wu & Narahashi, 1976). Since the amount of calcium entering the nerve terminal is increased by lengthening the action potential, 3-AP raises m at normal end-plates (Molgo, Lundh & Thesleff, 198). 3-AP, at concentrations of 1-5 mm, was tested at twenty-five end-plates in nine poisoned muscles between 25 and 15 h after TeTX injection. In lightly blocked muscles, for example the 25 h-treated muscle in Table 1, addition of 3-AP resulted in the restoration of a strong muscle twitch to indirect stimulation. At more severely blocked end-plates, addition of 3-AP also increased m. An example of such an end-plate is shown in Fig. 6, where m was raised about 25-fold by addition of 1 mm-3-ap. Even at the most highly intoxicated end-plates studied, which showed no obvious e.p.p.s to -5 Hz stimulation in normal recording solution (m < 5), m was increased and e.p.p.s reinstated when 3-AP was added. The relationship between ln m and ln [Ca]. after 3-AP addition was investigated at five highly blocked end-plates, three of which can be seen in Fig. 7A. As can be seen, the slope of this relationship remained low (n = -3--7). Plots of m-1 against

10 1 S. BEVAN AND L. M. B. WENDON 1/[Ca]. shown in Fig. 7B are also consistent with a maintained low level of mmax after 3-AP addition. Similarly, estimates of mmax are much reduced from control values if we assume that n = 1 for TeTX-poisoned end-plates. This is illustrated by the filled symbols in Fig A 5- m / 4~~*7-5J [Ca]o (mm) 2-- o8 m * ~~~~~~ /[Cal] (mm-') Fig. 7. A, double-logarithmic plots of m against [Ca]. for three TeTX-poisoned end-plates in the presence of 5 mm-3-ap and 1 mm-mgcl2. The lines were fitted by eye and the slopes are given adjacent to each line. B, the data from the same end-plates plotted as mi against 1/[Ca]o. If we assume a linear relationship for all [Ca]o, mmax estimates are:, 68; A, -52;, -13. M.e.p.p.s at TeTX-poisoned end-plate8 Although m.e.p.p.s at unchallenged end-plates in TeTX-treated muscles display normal properties of frequency and amplitude, treatments which elevate m.e.p.p. frequency at normal end-plates show a reduced effectiveness. Raised [Ca]o. Raising [Ca]o by isotonic replacement for sodium is known to increase m.e.p.p. frequency at untreated rat end-plates (Hubbard, 1961; Hubbard, Jones & Landau, 1968 b). At four normal end-plates studied in detail an increase in [Ca]o from -5 to 1 mm evoked a 6-7-fold rise in m.e.p.p. frequency. In contrast, at eighteen analysedtetx-poisoned end-plates, examined more than 1& h aftertetx application,

11 ACTION OF TETANUS TOXIN AT N.M.J. no increase in m.e.p.p. frequency was evoked when [Ca]. was varied over the same range (mean rate, times control). Lanthanum. Addition of lanthanum (La3+) to normal nerve-muscle preparations is known to cause an increase in m.e.p.p. frequency (Heuser & Miledi, 1971; DeBassio, Schnitzler & Parsons, 1971). We found that after TeTX treatment the effect of lanthanum was diminished. In initial control experiments the ability of lanthanum to raise m.e.p.p. rate was found to be reduced by the presence of added external calcium, so subsequent experiments were made in a solution containing 2 mm-mgcl2 and no added calcium. As m.e.p.p. frequency increases progressively during the initial stages of lanthanum exposure, the evoked m.e.p.p. frequency was determined at a fixed interval, 2-3 min, after lanthanum addition. At seven normal end-plates (five muscles), 1 /SM-lanthanum raised m.e.p.p. frequency by a factor of , while 1/SM increased the rate more than 15-fold (three end-plates, three muscles) and 1 /M by more than 1-fold (six end-plates, two muscles). After TeTX poisoning for 36-5 h 1 and 1/uM-lanthanum failed to raise the m.e.p.p. rate at nine tested end-plates (mean rate times control). Exposure to 1 /SM-lanthanum did, however, cause a fold rise in frequency at four tested end-plates. This result is consistent with the finding of Mellanby & Thompson (1981) that m.e.p.p. frequency at TeTX-poisoned goldfish end-plates is increased by addition of millimolar concentrations of lanthanum. Hyperosmotic medium. It has long been known that m.e.p.p. frequency can be raised by increasing the osmotic pressure of the bathing solution (Fatt & Katz, 1952; Furshpan, 1956). This hyperosmotic effect is not simply a result of calcium entry to the terminal but probably reflects a mobilization of calcium from intracellular stores (Shimoni, Alnaes & Rahamimoff, 1977). The effect of adding 1 mm-sucrose to the medium was tested at seven normal end-plates (seven muscles). Similar increases in m.e.p.p. frequency were elicited in the presence of 1 mm-cacl2 (4A-9-8-fold increase, four end-plates) and in a 'calcium-free' solution containing 1 mm-egta ( fold increase, three end-plates). When the effect of a challenge with hyperosmotic medium was investigated in five TeTX-poisoned muscles (one end-plate per muscle, h after injection), no increase in m.e.p.p. frequency was seen (mean rate times control). Potentiation of m.e.p.p. frequency during high-frequency nerve stimulation. The ability of high-frequency nerve stimulation to raise m.e.p.p. frequency (see Lev-Tov & Rahamimoff, 198) was progressively lost after TeTX poisoning. Thus little or no change in m.e.p.p. rate was elicited by 5-1 Hz, 1 s, stimulation in the low [Ca]O, high [Mg]. solutions typically used to study evoked ACh release at normal end-plates (see for example Table 1). However, the increase in m.e.p.p. rate during a train of nerve impulses (2-1 Hz) could be compared at untreated and TeTX-poisoned end-plates showing similar low quantal contents (m = 1-- 1) to 5 Hz stimulation. To achieve such comparable values of m, normal soleus muscles were bathed in solutions containing low calcium (-5-{2 mm) and high magnesium (2-5 mm) concentrations, whereas TeTX-treated muscles were exposed to solutions containing 1 mm-magnesium and normal (2 mm) or even elevated (up to 5 mm) levels of calcium. Fig. 8 shows the pattern of m.e.p.p. frequency increase for a normal (A) and a severely affected TeTX-poisoned end-plate (B) during trains of nerve stimuli given at both it

12 12 S. BEVAN AND L. M. B. WENDON 2 and 5 Hz. The increase in m.e.p.p. frequency differs greatly in the two types of preparation. Normal end-plates showed a modest increase in m.e.p.p. rate during the train of nerve impulses. Values of m were also raised during the period of stimulation although by a smaller factor than m.e.p.p. frequency. In contrast, at highly blocked TeTX-poisoned end-plates, m.e.p.p. frequency rose rapidly during the stimulus train to a new high rate which then remained fairly constant. At these severely affected 1- A 5-!!8: C 3-1 X o 2 3 Time (s) Fig. 8. Changes in m.e.p.p. frequency at a normal end-plate (A) and a 4 h-tetx-poisoned end-plate (B) during stimulation at 2 Hz () and 5 Hz (-). Note change in time scale between A and B. Each point represents the estimated rate for either a 5 s period (A) or a I s period (B), plotted as a multiple of the resting m.e.p.p. frequency. For A, potentials with a latency of 2-4 ms were classed as e.p.p.s and eliminated from the analysis. For B, only potentials with a latency of more than 5 ms were included in the analysis. end-plates no obvious increase in m was evoked by the high stimulus rate. This may be due, at least in part, to the difficulty in detecting low levels of stimulus-locked quantal release when the m.e.p.p. rate rises to high levels. At less severely blocked end-plates, obvious increases in m could be evoked by raising the rate of nerve stimulation. For example, in a muscle intoxicated for 24 h m was increased from 2fl3 ( 5 Hz) to 7-6 (l Hz) after only to s of high-frequency stimulation.

13 ACTION OF TETANUS TOXIN AT N.M.J. 13 2, A. :.. *f.. *,-H-v :In.x, U c 1 - B C -._ - 33;. 2-f. -. n l C -- I 1- I Time (s) Fig. 9. Decline in m.e.p.p. frequency after a period of high-frequency stimulation (1 Hz, 1 s). A, normal end-plate, 5 mm-cacl2, 6 mm-mgcl2. B, normal end-plate with no added CaCl2, 1 mm-egta. C, TeTX-poisoned end-plate, 1 h after initial injection, 2 mm-cacl2, 1 mm-mgcl2. Frequencies evaluated using moving bin method: bin 1 a, Abin 1 s. M.e.p.p. frequency after cessation of high-frequency nerve stimulation. When a high-frequency stimulus train terminates, m.e.p.p. frequency declines to the resting level (Miledi & Thies, 1971; Erulkar & Rahamimoff, 1978). Fig. 9A and B shows the time course of this decrease for normal soleus end-plates after the end of a train of nerve impulses given for 1 s at 1 Hz. The results are very similar to those obtained at frog end-plates by Lev-Tov & Rahamimoff (198). In calcium-containing solutions (Fig. 9A) the decline in m.e.p.p. frequency shows two obvious components: an initial, relatively rapid phase and a second slower phase which have been termed augmentation and potentiation, respectively (Magleby & Zengel, 1976). When the

14 14 S. BEVAN AND L. M. B. WENDON electrochemical gradient for calcium is reversed by bathing the muscle in a solution containing no added calcium and 1 mm-egta, the augmentation phase is lost, whereas the slower potentiation phase remains (Fig. 9B). These patterns for the decline in m.e.p.p. rate were confirmed at five end-plates in medium containing calcium and four end-plates in a 'calcium-free' solution. The decline in m.e.p.p. rate at the end of high-frequency stimulation trains (1 s, 1 Hz) was examined at sixteen end-plates, in ten TeTX-treated muscles, bathed in solutions containing calcium. The form of the decline was most easily compared to that at untreated end-plates when the [Ca]./[Mg]. ratio was adjusted to give similar initial elevations in m.e.p.p. rate. In general, a progressive depression of the potentiation phase was observed. Thus after only 24 h of poisoning both the augmentation and potentiation phases could be often, though not always, seen. At more severely affected end-plates, examined usually more than 36 h after TeTX injection, little or no potentiation phase was noted and m.e.p.p. frequency declined relatively rapidly to the control level. Fig. 9C illustrates the pattern observed at one such end-plate, poisoned for 5 h, where the initial increase in m.e.p.p. rate was greater than that at the normal end-plate shown in panel A. This pattern clearly differs from that shown in Fig. 9B where calcium influx to the normal terminal is blocked. DISCUSSION Several features of ACh release at TeTX-poisoned end-plates lead us to conclude that the effects of TeTX cannot be explained simply by a block of calcium influx to the terminal and that TeTX must act at a step between calcium entry and exocytosis. First, the action ofa variety of agents and procedures which evoke or enhance quantal ACh release are depressed at TeTX-poisoned end-plates. Some of these procedures, such as raising the osmolarity of the medium, are not dependent on the presence of external calcium and probably act by mobilizing calcium from intracellular stores. Secondly, the decline in m.e.p.p. frequency after a period of intense nerve stimulation is different at TeTX-poisoned and normal end-plates. With TeTX poisoning, the time course of the decrease in m.e.p.p. rate clearly differs from that found at normal end-plates where calcium influx has been blocked. Highly intoxicated end-plates show only the rapidly decaying augmentation phase, which is thought to represent a residual post-stimulus calcium conductance in the nerve terminal (Lev-Tov & Rahamimoff, 198). Thirdly, the finding that m.e.p.p. frequency can rise rapidly to very high levels during a train of high-frequency nerve impulses suggests that appreciable amounts of calcium can enter TeTX-poisoned nerve terminals with each action potential. Our results do not exclude the possibility that TeTX may also act to reduce the amount of calcium which normally enters the terminal via voltageactivated channels. Further experiments will be necessary to investigate this possibility. Despite the reduced calcium sensitivity of the evoked ACh release process it is possible to raise the level of release at moderately blocked end-plates quite markedly and in some cases to restore neuromuscular transmission. The most potent procedure tested was addition of millimolar doses of 3-AP, which appeared to augment transmitter release even at highly blocked end-plates. High-frequency nerve stimu-

15 ACTION OF TETANUS TOXIN AT N.M.J. lation was also effective in raising the level of quantal ACh release, even at the most severely poisoned end-plates studied. Thus the block of ACh release induced by TeTX could be overcome, to some extent, by procedures which raise [Ca]i. After the very early stages of poisoning all end-plates studied showed reduced slopes (n) for the ln m-ln [Ca]. plots. One explanation for the reduction in slope is a loss of 'co-operativity' in the release process. For the Dodge & Rahamimoff (1967) model this would mean a failure to form (CaX)4 complexes with release activated by lower-order complexes (e.g. (CaX)). The probability of release for a (CaX) complex must then be lower than that for a (CaX)4 complex. The very low values of n (e.g. '199-5) recorded at some end-plates suggest that this simple idea alone is inadequate to explain the reduced slopes. Plots of m-1/n against 1/[Ca]o, for values of n from 1 to 4, suggest that the maximum number of quanta, mmax, releasable by nerve stimulation is much reduced after TeTX poisoning. A much higher ratio of [Ca]o/[Mg]o is therefore needed to achieve measurable levels of evoked release. One possibility is that such [Ca]. correspond to the saturating portion of the ln m-ln [Ca]o plot, where m is approaching mmax. On this portion of the curve a reduced slope is expected. We have discussed our results in terms of a model in which n release sites need to be activated for release to occur. Other models in which [Ca]i varies non-linearly with [Ca] (Llinas, Steinberg & Walton, 1981; Parnas & Segel, 1981; Nachshen & Drapeau, 1982) will also show a reduction of n at high [Ca], where calcium entry saturates. No obvious increase in the slope of the ln m-ln [Ca] relationship was noted when 3-AP was added to TeTX-poisoned end-plates, even though m was raised. Indeed, the values of n recorded in the presence of 3-AP were among the lowest obtained. This maintained low value of n is consistent with the idea that the maximum number of releasable quanta is reduced by TeTX. A greater variability in e.p.p. latency was noted after TeTX poisoning; this was most noticeable at end-plates which required high [Ca]o to evoke measurable quantal release. This increased variability could reflect altered kinetics of the release mechanism, for example a reduction in the forward rate constant governing the probability of an activated site releasing a quantum. Another possibility is that the rate of intracellular calcium sequestration is altered after TeTX poisoning, thereby prolonging the profile of raised [Ca]i accompanying each action potential. Even at severely affected end-plates, we found that TeTX had little or no effect on either the frequency or the amplitude of m.e.p.p.s. Several other groups have reported that m.e.p.p. rate is reduced at mammalian end-plates with TeTX poisoning (Duchen & Tonge, 1973; Habermann, Dreyer & Bigalke, 198; Mori, Chou & Gutmann, 198), although the most striking reductions have been found where the m.e.p.p. rate at untreated end-plates was rather high (4-1 s-1). An important feature oftetx action is that evoked transmitter release can be severely affected at end-plates which show normal m.e.p.p.s. A simple explanation for this difference is that spontaneous m.e.p.p.s reflect a process which is not strongly dependent on [Ca]i (cf. Andreu & Barrett, 198) and is relatively resistant to the actions of TeTX. Alternatively, m.e.p.p. rate may be unchanged because a reduced [Ca]i sensitivity of the 'spontaneous' release process is offset by a higher basic [Ca]i. The finding of normal m.e.p.p.s after TeTX treatment contrasts with BoTX poisoning, where the inhibition of evoked ACh release is accompanied by a marked reduction in m.e.p.p. 15

16 16 S. BEVAN AND L. M. B. WENDON frequency. Our results have also revealed some other differences in the properties of ACh release at TeTX- and BoTX-poisoned end-plates. With BoTX poisoning, e.p.p. latency is similar to that at normal end-plates and blocking delayed rectification raises the slope of the ln m-ln [Ca]o relationship to near normal levels (Cull-Candy, Lundh & Thesleff, 1976). At TeTX-treated end-plates, e.p.p. latency is more variable than normal and addition of 3-AP does not appear to increase n. The molecular action of TeTX is unclear. Some other proteinaceous toxins, such as cholera toxin and diphtheria toxin, act intracellularly by modifying cellular proteins. To date, however, no obvious modification of a neuronal protein (phosphorylation or ribosylation) has been noted after TeTX treatment (Wendon & Gill, 1982). Various mechanisms can be advanced to explain the main features of ACh release from TeTX-poisoned nerve endings. Of these, two distinct actions involving a site after calcium entry to the terminal are (1) a total inactivation of some, but not all, release sites, and (2) a reduced [Ca]i sensitivity of each release site brought about by a modification of either the calcium binding or some subsequent step. Unfortunately, our knowledge about the mechanism of transmitter release does not allow us to differentiate readily between these possibilities on the basis of the present results. We thank Professor R. Rahamimoff for providing facilities and encouragement to L.W., Dr D. Armstrong for discussion and Dr S. G. Cull-Candy for criticism of the manuscript. L. W. also thanks Itzic Nussinovitch, Hagai Bergman and Ahroni Lev-Tov for their help and Professor M. Raff for his support. Financial support to L. W. was provided by the M.R.C. REFERENCES ANDREAU, R. & BARRETT, E. F. (198). Calcium dependence of evoked transmitter release at very low quantal contents at the frog neuromuscular junction. J. Phy8iol. 38, BROOKS, V. B., CURTIS, D. R. & ECCLES, J. C. (1957). The action of tetanus toxin on the inhibition of motoneurones. J. Physiol. 135, CULL-CANDY, S. G., LUNDH, H. & THESLEFF, S. (1976). Effects of botulinum toxin on neuromuscular transmission in the rat. J. Phy8iol. 26, CURTIS, D. R. & DE GROAT, W. C. (1968). Tetanus toxin and spinal inhibition. Brain Res. 1, DEBASSIO, W. A., SCHNITZLER, R. M. & PARSONS, R. L. (1971). Influence of lanthanum on transmitter release at the neuromuscular junction. J. Neurobiol. 2, DODGE, F. A. & RAHAMIMOFF, R. (1967). Co-operative action of calcium ions in transmitter release at the neuromuscular junction. J. Physiol. 193, DUCHEN, L. W. & TONGE, D. A. (1973). The effects of tetanus toxin on neuromuscular transmission and on morphology of motor end-plates in slow and fast skeletal muscle of the mouse. J. Physiol. 228, ERULKAR, S. D. & RAHAMIMOFF, R. (1978). The role of calcium ions in tetanic and post-tetanic increase of miniature end-plate potential frequency. J. Physiol. 278, FATT, P. & KATZ, B. (1952). Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, FURSHPAN, E. J. (1956). The effects of osmotic pressure changes on the spontaneous activity at motor nerve endings. J. Physiol. 134, GUNDERSEN, C. B., KATZ, B. & MILEDI, R. (1982). The antagonism between botulinum toxin and calcium in motor nerve terminals. Proc. R. Soc. B 216, HABERMANN, E., DREYER, F. & BIGALKE, H. (198). Tetanus toxin blocks neuromuscular transmission in vitro like botulinum A toxin. Naunyn Schmiederberg8 Arch. Pharmacol. 311,33-4. HEUSER, J. E. & MILEDI, R. (1971). Effect of lanthanum ions on function and structure of frog neuromuscular junctions. Proc. R. Soc. B 179,

17 ACTION OF TETANUS TOXIN AT N.M.J. HIROKAWA, N. & HEUSER, J. E. (1981). Structural evidence that botulinum toxin blocks neuromuscular transmission by impairing the calcium influx that normally accompanies nerve depolarization. J. Cell Biol. 88, HUBBARD, J. I. (1961). The effect of calcium and magnesium on the spontaneous release of transmitter from mammalian motor nerve endings. J. Physiol. 159, HUBBARD, J. I., JONES, S. F. & LANDAU, E. M. (1968a). On the mechanisms by which calcium and magnesium affect the release of transmitter by nerve impulse. J. Physiol. 1%, HUBBARD, J. I., JONES, S. F. & LANDAU, E. M. (1968b). On the mechanism by which calcium and magnesium affect the spontaneous release of transmitter from mammalian motor nerve terminals. J. Physiol. 194, KAESER, H. E., MULLER, H. R. & FRIEDRICH, B. (1968). The nature of tetraplegia in infectious tetanus. Eur. Neurol. 1, KRETZSCHMAR, H., KIRCHNER, F. & TAKANO, K. (198). Relations between the effect of tetanus toxin on the neuromuscular transmission and histological functional properties of various muscles of the rat. Exp. Brain Re8. 38, LEV-Tov, A. & RAHAMIMOFF, R. (198). A study of tetanic and post-tetanic potentiation of miniature end-plate potentials at the frog neuromuscular junction. J. Phy8iol. 39, LLINAS, R., STEINBERG, I. Z. & WALTON, K. (1981). Presynaptic calcium currents in squid giant synapse. Biophys. J. 33, LLINAS, R., WALTON, K. & BOHR, V. (1976). Synaptic transmission in squid giant synapse after potassium conductance blockage with external 3- and 4-aminopyridine. Biophys. J. 16, MACLACHLAN, E. M. & MARTIN, A. R. (1981). Non-linear summation of end-plate potentials in frog and mouse. J. Physiol. 311, MAGLEBY, K. L. & ZENGEL, J. E. (1976). Augmentation: a process that acts to increase transmitter release at the frog neuromuscular junction. J. Physiol. 257, MARTIN, A. R. (1955). A further study of the statistical composition of the end-plate potential. J. Physiol. 13, MARTIN, A. R. (1977). Junctional transmission. II. Presynaptic mechanisms. In Handbook of Physiology, Section 1, The Nervous System, pp Bethesda, MD: American Physiological Society. MELLANBY, J. & GREEN, J. (1981). How does tetanus toxin act? Neuroscience 6, MELLANBY, J. & THOMPSON, P. A. (1981). The interaction of tetanus toxin and lanthanum at the neuromuscular junction in the goldfish. Toxicon 19, MILEDI, R. & THIES, R. (1971). Tetanic and post-tetanic rise in frequency of miniature end-plate potentials in low-calcium solutions. J. Physiol. 212, MOLGO, J., LUNDH, H. & THESLEFF, S. (198). Potency of 3,4-diaminopyridine and 4-aminopyridine on mammalian neuromuscular transmission and the effect of ph changes. Eur. J. Pharmac. 61, MORI, M., CHOU, S. M. & GUTMANN, L. (198). Neuromuscular junction blockade in local tetanus. Neurology, Minneap. 3, 387. NACHSHEN, D. A. & DRAPEAU, P. (1982). A buffering model for calcium-dependent neurotransmitter release. Biophys. J. 38, PARNAS, H. & SEGEL, L. A. (1981). A theoretical study of calcium entry in nerve terminals with application to neurotransmitter release. J. theor. Biol. 91, SHIMONI, Y., ALNAES, E. & RAHAMIMOFF, R. (1977). Is hyperosomotic neurosecretion from motor nerve endings a calcium-dependent process? Nature, Lond. 267, WENDON, L. M. B. (198). On the action of tetanus toxin at the rat neuromuscular junction. J. Physiol. 3, 23P. WENDON, L. M. B. & GILL, D. M. (1982). Tetanus toxin action on cultured nerve cells: does it modify a neuronal protein? Brain Res. 238, YEH, J. Z., OXFORD, G. S., WU, C. H. & NARAHASHI, T. (1976). Dynamics of aminopyridine block of potassium channels in squid axon membrane. J. gen. Physiol. 68,