31
J. Phyaiol. (1976), 261, pp. 31-48 With 6 text-figurem Printed in Great Britain
ACTION OF VINBLASTINE ON THE SPONTANEOUS RELEASE OF ACETYLCHOLINE AT THE FROG NEUROMUSCULAR JUNCTION
BY MONIQUE PRCOT-DECHAVASSINE From the Laboratoire de Cytologie, Universite Pierre et Marie Curie, 7, quai Saint Bernard, 75230 Paris Cedex 05 France
(Received 29 December 1975) SUMMARY
1. Vinblastine induces reversible changes of the spontaneous release of acetylcholine (ACh) at the frog neuromuscular junction as characterized by the appearance of giant potentials. These large potentials occur soon after soaking the muscle in vinblastine and are not consequent to a large increase in the frequency of spontaneous release. Their number, relative to the total number of spontaneous potentials, increases with the duration of soaking. 2. Large potentials appear even in the presence of tetrodotoxin or in low Ca2+-high Mg2+ Ringer solution. 3. Following vinblastine treatment, the amplitude histogram of spontaneous potentials recorded from a number of fibres display an evident periodicity with peaks-occurring regularly at simple multiples of the model amplitude of the unitary potentials. It is suggested that giant potentials are produced by the release of preformed pluriquantal packets of ACh. 4. Comparison of the amplitude distribution of spontaneous potentials and end-plates potentials show that only an insignificant number of large quanta are released by nerve stimuli. The absolute frequency of giant potentials does not markedly change when spontaneous discharge is accelerated by hypertonic solution. 5. The mechanism by which vinblastine induces the appearance of giant potentials is discussed. INTRODUCTION
It has already been shown that the large spontaneous potentials occurring at the normal neuromuscular junction of the rat (Liley, 1957; Hubbard & Jones, 1973) or provoked experimentally in frog (Katz & Miledi, 1969; Pecot-Dechavassine, 1970; Pecot-Dechavassine & Couteaux, 1972 a; Benoit, Audibert-Benoit & Peyrot, 1973; Heuser, 1974) are not caused by the random coincident release of unitary miniature potentials (min.e.p.p:s). 2
PlH *'
26i
M. PICOT-DECHA VASSINE 32 The analysis of their occurrence led to the postulate that they might be produced by the release of large pre-formed packets of ACh. Thus the question arises whether these large spontaneous potentials are related to some structural changes inside the nerve terminals, specially at the level of the synaptic vesicles. Till now, no definite conclusion could be drawn from the researches on giant potentials occurrring at rat neuromuscular junction because of their low relative frequency. A coincident appearance of giant potentials and oversized vesicles was clearly observed at frog endplates after various stimulations (P6cot-Dechavassine & Couteaux, 1972 a, b; Heuser, 1974). However, the procedures used in these experiments were often drastic and irregular in their effects. Moreover, the morphological results led to two different assumptions on the possible origin of large vesicles: They could be formed by coalescence of synaptic vesicles (P6cot-Dechavasine & Couteaux, 1972a); or they could be derived from irregular cisternae resulting from membrane re-cycling (Heuser, 1974). Vinblastine, which had been shown by Turkanis (1973) to give giant potentials at frog neuromuscular junctions, was found suitable for further investigations of the properties and the origin of these abnormal potentials. Its action on spontaneous release is obtained at low concentrations, and it is reversible. In a previous report (PNcot-Dechavassine, 1975; P6cot-Dechavasine &
Couteaux, 1975), it has been shown that the spontaneous giant potentials elicited by vinblastine are probably produced by the release of large packets of ACh. The present investigation deals with a detailed study of the effects of vinblastine on both spontaneous and evoked release of transmitter. An attempt has been made here to answer the following two questions: (i) are the giant potentials composed of multiples of the min.e.p.p.s, and thus produced by the release of pluriquantal packets of ACh? (ii) are the large packets of ACh also available for release by nerve stimuli? The specific mode of action of vinblastine has also been discussed in order to provide additional information concerning the possible origin of the giant spontaneous potentials. A preliminary account has been previously reported in abstracts (P6cotDechavassine & Couteaux, 1976). METHODS The experiments were performed on sartorius and rectus internus major of Rana eaculenta or Rana temporaria. Conventional methods were used to record potentials
intracellularly from single muscle fibres. The composition of the standard physiological solution was (mM): NaCl, 111; KC1, 2; CaCl2, 2; NaHCO3, 2; pH was 7-7-2.
VINBLASTINE ON SPONTANEOUS RELEASE
33
Some modified solutions were used according to the nature of the experiment: solutions with a different concentration of Ca2+ (with or without 0 5 mM-EDTA), with tetrodotoxin (TTX) (2 x 10-7 g/ml.) or with neostigmine (1-10-4 M). Low Ca2+ (0.4 mm)- high Mg2+ (5-10 mM) solutions (blocking-Ringer solutions) were used in experiments requiring end-plate potentials (e.p.p.s.) of low mean quantal content. Mean quantal content was estimated from the number of 'failures' in response to nerve stimuli and from the ratio of the mean size of evoked e.p.p.s. to the mean size of the unitary min e.p.p.s. The concentrations of vinblastine sulphate (gift from Eli Lilly, France) ranged from 0.1 to 03 mei. In a few experiments, we have used colchicine and cytochalasin B (Calbiochem) as control substances. Colchicine, like vinblastine,is an antimitotic substance; its action is associated with microtubules, while cytochalasin B probably inhibits the contractile apparatus of microfilaments (Carter, 1967; Wessels, Spooner, Ash, Bradley, Luduena, Wrenn & Yamada, 1971). Experiments were done at room temperature of 20-24o C in both summer and winter. RESULTS
EffeciB of vinbladtine on the 8pontaneow8 transmitter release on Effect8 min.e.p mi. 40
300]n -h-Failures a. I I
.
End-plate potentials
II I
C
0 L.
4s 0
.0 0 L.
.0
Spontaneous potentials
E
z
t 0-5
t
4t
3 2 Amplitude (mV) Fig. 5. Histograms of end-plate potential and spontaneous potential amplitudes from an end-plate displaying a high proportion of giant spontaneous potentials (211/605) in low Ca2+-high Mg2+ Ringer solution. ml (from the number of failures) and m2 (from the ratio mean amplitude of the e.p.p.s/ mean amplitude of the unitary min.e.p.p.s) are 0-55 and 0-65 respectively. Only one e.p.p. is found outside the theoretical distribution according to Poisson's law. 1
(ii) The distribution of amplitudes of e.p.p.s calculated according to Poissons' law, from the number of failures, should differ from the observed distribution. This was tested in fibres with a high proportion of large spontaneous potentials (from about 10 to 50 %) and e.p.p.s of low mean quantal content. Only e.p.p.s which were well outside the range of the predicted distribution were considered as abnormal.
41 VINBLASTINE ON SPONTANEOUS RELEASE The results are summarized in Table 2. Generally m2 is a little higher than m1 (thirteen experiments out of fourteen). The paired t test performed on data, shows that the difference between the two means (ml = 0 95 + 0 14; m2 =1-05 + 0-17) is significant at the 0 001 level of significance (mean difference = 0 103 + 0-024; t = 4-29 for 13 degrees of freedom; critical value of t = 4-22 for P = 0 001). The more crude Wilcoxon's pair signed rank test also shows that there is evidence for the reality of the difference. However, the difference between ml and m2 is small, which suggests that very few large quanta are released by nerve stimuli. The analysis of the distribution of the e.p.p.s (see above in (ii)) shows, indeed, that in most series only one or two e.p.p.s could be considered outside the predicted distribution (Table 2). Fig. 5 illustrates the distribution of both e.p.p.s and min.e.p.p.s obtained in an experiment in which only one e.p.p (out of 217) is outside the theoretical distribution. The impulse-evoked release of transmitter in known to be a Ca-dependent synchronous release of a large number of ACh quanta (del Castillo & Katz, 1954). It can be regarded as an intense transient acceleration of the spontaneous release due to depolarization of terminals (Liley, 1956b). The effect of an acceleration of spontaneous release on the proportion of giant potentials has been examined by changing the osmotic pressure. The osmolarity of the Ringer was increased by adding sucrose, and decreased by adding H20. This has several advantages over other procedures; in particular, the changes in the frequency of min.e.p.p.s occur very rapidly and are quickly reversible. Thus, many tests could be made within a short period, interspersed with controls, during the persistence of Vinblastine action. Moreover, the membrane potential of the muscle fibre does not change markedly during these osmotic alterations. The amplitude histograms of Fig. 6 illustrate the changes in the frequency of min.e.p.p.s, and in proportion of large potentials, when tonicity was alternatively changed. The hypotonic solution (Fig. 6B, D, E) was made by diluting the Ringer to one third; the hypertonic solution (Fig. 6C, F) by adding sucrose (0 1 M) to the Ringer. The proportion of giant potentials decreases considerably when the min.e.p.p. fequency is increased by hypertonic Ringer solution. It decreases from 18 and 35 % (Fig. 6B, E) to 5 % and 4 % (Fig. 6B, E) while the total min.e.p.p. frequency increases from 4-5 sec-' and 2 sec-' to 16 sec-' and 13 sec-' respectively. This figure indicates that the absolute frequency of giant potentials did not change when the total spontaneous discharge rate was increased.
Action of colchicine and cytochalasin B Colchicine, which, like vinblastine, is an antimitotic drug whose action is apparently associated with microtubules (Borisy & Taylor, 1967a, b;
42 M. P1COT-DECHA VA SSINE Wilson & Friedkin, 1967; Malawista, Sato & Bensch, 1968) has been used as control. Five experiments were performed on isolated frog muscle with concentrations of colchicine ranging from 0 5 to 2 mm. A
D
6 sec-'
3 sec-'
100
0.
17%
50
so
Jk I B
C
0 "
rbk~33
I
E
45 sec-'
50
0 0
E
go
__
z
1 ~ ~ ~ ~ ~ ~18%
_
__*_
--
35%
l
PI
F
16 sec'
No-
s0
0*5
5%
/ Oim~~
--ow-
I
C
I
2 sec-'
100-
.00
-
_P
13 sec-'
..
--
4%
2 0*5 1 2 Amplitude (mV) Fig. 6. Changes in proportion of giant potentials concomitant with changes in min.e.p.p. frequency. Changes in min.e.p.p. frequency were obtained by alternate immersion in hypo-(B, D, E) and hyper- (C, F) osmotic Ringer solution. Same soaking in D and E at different times (10 and 15 min). In A, isotonic Ringer solution. Sartorius muscle of Rana temporaria had been soaked, before this experiment, in vinblastine (0-2 mM) in blocking-Ringer solution for 75 min. As
previously
I
shown
by
Turkanis
(1973), colchicine had
no
consistent
effect on the distribution of amplitudes of min.e.p.p.s. Negative results were also obtained when colchicine was added in Ca2+ rich Ringer solution (two to three times the normal concentration).
43 VINBLASTINE ON SPONTANEOUS RELEASE Cytochalasin B was used in order to check whether the action of vinblastine on spontaneous release might be mediated by a possible action on microfilaments (Albert, Norton & Shelanski, 1970; Schlaepfer, 1971). Owing to the low solubility of cytochalasin B, it was dissolved in DMSO (dimethylsulphoxide) before adding it to Ringer solution. Four experiments were done with final concentrations of cytochalasin B ranging from 30 to 50 ,sg/ml. A few min.e.p.p.s with slightly larger amplitude than normal were observed in two experiments. However, they were very rare and their amplitude did not extend beyond two to three times the modal value of the control. Moreover, the very low solubility of cytochalasin B did not allow the use of higher concentrations in order to obtain more clear-cut effects. DISCUSSION
Characteri8tic8 of giant potentials The properties of giant potentials induced by vinblastine are similar to those of giant potentials induced by other procedures (Katz & Miledi, 1969; Pecot-Dechavassine, 1970; Heuser, 1974): They occur when the min.e.p.p frequency is low and in nerve-muscle preparations treated with TTX or blocked by Mg2+. This suggests that they cannot be attributed to random coincidence of release of several independent quanta, nor to spontaneous action potentials or local depolarization of nerve terminals. Moreover, their site of release is probably the same as that of ordinary min.e.p.p.s for their time courses are very similar. All these findings suggest that the large spontaneous potentials are due to pre-formed large packets of ACh released from the nerve terminals. Although a periodicity is not always evident in the amplitude histograms, this could be explained by the large spread of unitary amplitudes. It seems more significant that many histograms do display a clear multi-modal periodicity with peaks occurring at integral multiples of the unitary min.e.p.p. size. These results strengthen the idea that giant potentials at normal rat end-plates (Liley, 1957), and at frog end-plates after prolonged transmitter release (Heuser, 1974) may arise from pluriquantal packets of ACh, which in turn could be derived from the fusion of synaptic vesicles. Such a view has been proposed previously: large size and scalloped outline of many vesicles, suggestive of coalescing synaptic vesicles, were observed in muscles treated with vinblastine and displaying giant potentials at the time of fixation (Pecot-Dechavassine & Couteaux, 1975). Pictures of such oversized vesicles were also seen with other procedures (e.g. P6cot-Dechavassine & Couteaux, 1972a, b; Heuser, 1974). Thus, there is a converging physiological and morphological evidence in favour of a relation between giant potentials and large vesicles. However, the
M. PtCOT-DECHA VASSINE origin of these large vesicles remains uncertain. With the procedures previously used, giant potentials appeared only after drastic release of ACh and after a long delay. These features led Heuser (1974) to suggest that ' ... oversized vesicles are intermediates in membrane re-cycling which form by retrieval of vesicle membrane from the plasmalemma during stimulation. . . '. In this case, the delay would represent '.. .the time required for cisternae to accumulate sufficient transmitter to discharge. . . '. The results presented here suggest a different mode of origin for the induction of giant potentials by vinblastine, for they appear quickly and, are not preceded by a period of massive spontaneous release. It would, therefore, seem more probable that these potentials are derived from large packets of ACh formed from fusion of synaptic vesicles shortly before release. Though the present observations indicate that giant potentials resemble min.e.p.p.s in many ways, it appears that they do not contribute in a significant manner to the responses evoked by the nerve impulses. Menrath & Blackman (1970) have obtained similar results concerning giant potentials at normal rat end-plates. They conclude that a non-neuronal source of large quanta (which might be the Schwann cell) could be responsible for giant potentials. The present experiments provide little support for such an assumption. The time course of min.e.p.p.s and giant potentials is similar and no- change in morphological relations between Schwann cells and post-synaptic membrane was detected in electron micrographs (P6cotDechavassine & Couteaux, 1975). The fact that the relative proportion of large spontaneous potentials decreases considerably when the spontaneous release is highly accelerated by hyperosmotic solution might provide a possible explanation. It may be that the mobility of large quanta is less than that of single quanta. Under conditions of high rates of quantal release (e.g. during acceleration of spontaneous release), the access of large quanta to release sites might be limited, on account of their large size; therefore the probability of their release may be lower. This would also explain their low probability of release by nerve impulse. Finally, another possibility is that two different ACh stores might independently participate in spontaneous and evoked release. Such an alternative explanation has been proposed elsewhere (Harris &; Miledi, 1971; Dennis &; Miledi, 1974) to explain a difference in the transmitter quanta released spontaneously and those during nerve stimulation at some recently re-innervated frog neuromuscular junctions and at mature junctions intoxicated with botulinum toxin. 44
VINBLASTINE ON SPONTANEOUS RELEASE
45
Mode of action of vinblastine Vinblastine like colchicine is considered to exert its antimitotic effects through an action on microtubules. It is also through this action that these drugs are thought to interfere in the mechanism of various glandular secretions and in the release of neurotransmitters at synapses (Rasmussen, 1970) as well as in the maintainance of synaptic transmission (Perisic & Cu6nod, 1972; Felder, 1975). Chronic application of colchicine, vinblastine and vincristine on peripheral nerves produces at the neuromuscular junction both physiological changes (Hofmann & Thesleff, 1972; Hofmann, Struppler, Weindl & Velho, 1973; Albuquerque, Warnick, Sansone & Ohur, 1974; L0mo, 1974) and morphological changes (Anderson, Song & Slotwiner, 1967; Hsu & Lentz, 1972; Wuerker & Bodley, 1973) similar to those occurring after denervation. They could result from an action on microtubules, interfering either with the maintenance of elongated axonal process (Wessels et al. 1971) or with the transport of trophic substances (Hofmann & Thesleff, 1972; Hofmann et al. 1973; Albuquerque et al. 1974). However, the effect of vinblastine on spontaneous release described in the present work cannot be attributed to an action on axonal transport through microtubules because this effect occurs very rapidly (within 20 min). Moreover it has been shown that colchicine and vinblastine have markedly different effects on transmitter release (Katz, 1972; Turkanis, 1973). Many arguments agree well with an alternative hypothesis supported by findings which provide evidence that vinblastine precipitates many proteins in addition to colchicine-binding proteins. (1) Vinblastine might combine with neurofilaments which have been shown in some mammalian nerves and brains to contain, in addition to tubulin, several protein components precipitated by vincristine (Albert et al. 1970; Schlaepfer, 1971). In support of this hypothesis is the fact that many crystal-like structures have been observed close to neurofilaments in nerve terminals of nerve muscle preparations soaked for a short time in vinblastine (P6cot-Dechavassine & Couteaux, 1975). The lack of effect of cytochalasin B on the spontaneous release could plausibly be attributed to its low solubility and slow penetration rate. (ii) Vinblastine could combine also with membrane proteins either of the presynaptic terminal or of synaptic vesicles. This hypothesis is supported by the results of Wilson, Bryan, Ruby & Mazia (1970) who observed that vinblastine (but not colchicine), presumably acting as a cation, precipitates proteins derived from erythrocyte membranes, by combining with sites which can also bind Ca2+ ions. These biochemical effects could explain
M. PICOT-DECHA VASSINE our observations concerning the interference of vinblastine and calcium at sites involved in inducing giant potentials. One might assume that both vinblastine and Ca2+, by reacting with certain membrane proteins, could alter the membrane properties so as to modify the permeability or facilitate the phenomenon of fusion. High concentrations of vinblastine were, indeed, reported to increase cell Na+ and make the cells more fragile (Seeman, Chau-Wong & Moyyen, 1973). Such an action of Ca2+ on membranes could also explain the occurrence of giant spontaneous potentials at end-plates of muscles exposed for several hours to an isotonic Ca-Ringer solution (Katz & Miledi, 1969). 46
I wish to thank Professor B. Katz for helpful criticism of the manuscript and Professor R. Couteaux for his constant interest during the course of the work.
REFERENCES ALBERT, S., NORTON, W. T. & SHELNTsi, M. L. (1970). Isolation of mammalian axonal neurofflaments. J. ceU Biol. 47, 4A. ALBUiQUERQuE, E. X., WARNICK, J. E., SsoNE, F. M. & ONE, R. (1974). The effects of vinblastine and colchicine on neural regulation of muscle. Ann. N.Y. Acad. Sci. 228, 224-243. ANDERsoN, P. J., SONG, S. K. & SLOTWUTER, P. (1967). The fine structure of spheromembranous degeneration of skeletal muscle induced by vincristine. J. Neuropath. exp. Neurol. 26, 15-23. BENOIT, P. H., AUDIBERT-BENOIT, M. L. & PEYROT, M. (1973). Importance des effects pr5synaptiques dans le blockage de la jonction neuromusculaire de grenouille par substitution du lithium au sodium dans le milieu de survie. Arch. ital. Biol. 111, 323-335. BENOIT, P. R. & MAMBRN, J. (1969). Correlation entre la liberation spontanee et la liberation 6voqu6e du m6diateur ^ la jonction neuro-musculaire de Grenouille. J. Physiol., Pari 61, 214. BORIsY, G. G. & TAYLOR, E. W. (1967 a). The mechanism of action of colchicine. Binding of colchine"H to cellular protein. J. ceU Biol. 34, 525-534. BoRisy, G. G. & TAYLOR, E. W. (1967b). The mechanism of action of colchicine. Colchicine binding to sea urchin eggs and the mitotic apparatus. J. cell Biol. 34, 535-548. BoYD, I. A. & MA RTIN, A. R. (1956). The end-plate potential in mammalian muscle. J. Physiol. 132, 74-91. CARTER, S. B. (1967). Effects of cytochalasins on mlian cells. Nature, Lond. 213, 261-264. DEL CAsTILLo, J. & KATZ, B. (1954). Quantal components of the end-plate potential. J. Phy8iol. 124, 560-573. DENNs, M. J. & MILEDI, R. (1974). Characteristics of transmitter release at regenerating frog neuromuscular junctions. J. Physiol. 239, 571-594. FELDER, M. (1975). Vinblastine: Influence on nerve conduction and synaptic transmission. J. Neuroeci. Rem. 1, 121-130. HuAusI, A. J. & MEIEDI, R. (1971). The effect of type D botulinum toxin on frog neuromuscular junctions. J. Physiol. 217, 497-515. HEusER, J. E. (1974). A possible origin of the 'giant' spontaneous potentials that occur after prolonged transmitter release at frog neuromuscular junctions.
J. Phy8iol. 239, 106-108P.
47 VINBLASTINE ON SPONTANEOUS RELEASE HoF1NNw, W. W. & STRUIPLER, A., WEINDL, A. & VELHO, F. (1973). Neuromuscular transmission with colchicine-treated nerves. Brain Re8 49, 208-213.
HoFMANw, W. W. & THESLEFF, S. (1972). Studies on the trophic influence of nerve on skeletal muscle. Eur. J. Pharmacol. 20, 256-260. Hsu, L. & LENTZ, T. L. (1972). Effect of colchicine in the fine structure of the neuromuscular junction. Z. ZeUfor8ch. mikro8k. Anat. 135, 439-448. HUBBARD, J. I. & JoNEs, S. F. (1973). Spontaneous quantal transmitter release: statistical analysis and some implications. J. Phyriol. 232, 1-21. KATZ, N. L. (1972). The effects on frog neuromuscular transmission of agents which act upon microtubules and microfilaments. Eur. J. Pharmacol. 19, 88-93. KArz, B. & MILEDI, R. (1969). Spontaneous and evoked activity of motor nerve endings in calcium Ringer. J. Phy8iol. 203, 689-706. LiAxy, A. W. (1956a). The quantal components of the mammalian end-plate potential. J. Physiol. 133, 571-587. LuILY, A. W. (1956b). The effects of presynaptic polarization on the spontaneous
activity of the mammalian neuromuscular junction. J. Phyeiol. 134, 427443. TLTTIY, A. W. (1957). Spontaneous release of transmitter substance in multiquantal units. J. Phy8iol. 136, 595-605. LoMo, T. (1974). Neurotrophic control of colchicine effects on muscle. Nature, Lond. 249, 473-474. MATAWISTA, S. E., SATO, H. & BENSCH, K. G. (1968). Vinblastine and griseofulvin reversibly disrupt the living mitotic spindle. Science, N.Y. 160, 770-772. MENRATH, R. L. E. & BLACKMANN, J. G. (1970). Observations on the large spontaneous potentials which occur at end-plates of the rat diaphragm. Proc. Univ.
Otago med. Sch. 48, 72-73. PPOOT-DEcHAvAssnNE, M. (1970). Effects conjugu6s du pH et des cations divalents sur la liberation spontanee d'ac6tylcholine au niveau de la plaque motrice de la Grenouille. C. r. hebd. S&nc. Acad. Sci., Pari8 D 271, 674-677. PACOT-DECHAVASsNE, M. (1975). Modalites d'apparition des potentials spontan6s giantss' provoques par la vinbiastine sur la preparation isol6e du muscle de grenouille. C. r. hebd. Seanc. Acad. Sci., Parin D 280, 303-306. PACOT-DECHAVASSINE, M. & COUTEAUX, R. (1972 a). Potentials miniatures d'amplitude anormale obtenus dans des conditions experimentales et changements concomitants des structures presynaptiques. In International Sympoaium on Cholinergic Tranemie8ion of the Nerve Impulse, pp. 177-185. Paris: INSERM. PECOT-DECHAVASSINE, M. & COUTEAUX, R. (1972b). Potentials minatures d'amplitude anormale obtenus dans des conditions exp6rimentales et changements concomitants des structures pr6synaptiques. C. r. hebd. S&anc. Acad. Sci., Paris D 275, 983-986. PECOT-DECHAvAssInE, M. & COUTEAux, R. (1975). Modifications structurales des terminaisons motrices de muscle de grenouille soumis * l'action de la vinblastine. C. r. hebd. Sganc. Sci., Pari8 D 280, 1099-1101. PtCOT-DEcHAvAssuNE, M. & COUTEAux, R. (1976). Action of vinblastine on the spontaneous release of ACh at the frog neuromuscular junction. Abstracts of the Scottish Electrophysiological Society, Symposium on The Synap8e, St Andrews, Scotland, 29 March-2 April. PERIsIC, M. & CUtNoD, M. (1972). Synaptic transmission depressed by colchicine blockade of axoplasmic flow. Science, N.Y. 175, 1140-1142. RAsMUsSEN, H. (1970). Cell communication, calcium ion, and cyclic adenosine monosphate. Science, N. Y. 170, 404-412. SCHLAEPFER, W. W. (1971). Stabilisation of neurofilaments by vincristine sulfate in low ionic strength media. J. Ultrastr. Re8. 36, 367-374.
48
M. PJCOT-DECHA VASSINE
SEEMAN, P., CHAU-WONG, M. & MOYYEN, S. (1973). Membrane expansion by vinblastine and strychnine. Nature, New Biol. 241, 22. TuIJR Is, S. A. (1973). Some effects of vinblastine and colchicine on neuromuscular transmission. Brain Be8. 54, 324-329. WEssELs, N. K., SPOONER, B. S., ASH, J. F., BRADixrY, M. A., LUDUIENA, M. A., WRENN, J. T. & YAMADA, K. M. (1971). Microfilament in cellular and developmental processes. Science, N.Y. 171, 135-143. WnsoN, L. BKYAN, J. RUBy, A. & MARYi, D. (1970). Precipitation of proteins by vinblastine and calcium ions. Proc. natn. Acad. Sci. U.S.A. 66, 807-814. WILsoN, L. & FRIDEDN, M. (1967). The biochemical events of mitosis. The in vivo and in vitro binding of colchicine in grasshopper embryos and its possible relation to inhibition of mitosis. Biochemistry 6, 3126-3135. WuK=KER, R. B. & BODLEY, H. D. (1973). Changes in muscle morphology and histochemistry produced by denervation, 3.3'-iminodipropionitrile and epineurial vinblastine. Am. J. Anat. 135, 221-234.
J. Phyaiol. (1976), 261, pp. 31-48 With 6 text-figurem Printed in Great Britain
ACTION OF VINBLASTINE ON THE SPONTANEOUS RELEASE OF ACETYLCHOLINE AT THE FROG NEUROMUSCULAR JUNCTION
BY MONIQUE PRCOT-DECHAVASSINE From the Laboratoire de Cytologie, Universite Pierre et Marie Curie, 7, quai Saint Bernard, 75230 Paris Cedex 05 France
(Received 29 December 1975) SUMMARY
1. Vinblastine induces reversible changes of the spontaneous release of acetylcholine (ACh) at the frog neuromuscular junction as characterized by the appearance of giant potentials. These large potentials occur soon after soaking the muscle in vinblastine and are not consequent to a large increase in the frequency of spontaneous release. Their number, relative to the total number of spontaneous potentials, increases with the duration of soaking. 2. Large potentials appear even in the presence of tetrodotoxin or in low Ca2+-high Mg2+ Ringer solution. 3. Following vinblastine treatment, the amplitude histogram of spontaneous potentials recorded from a number of fibres display an evident periodicity with peaks-occurring regularly at simple multiples of the model amplitude of the unitary potentials. It is suggested that giant potentials are produced by the release of preformed pluriquantal packets of ACh. 4. Comparison of the amplitude distribution of spontaneous potentials and end-plates potentials show that only an insignificant number of large quanta are released by nerve stimuli. The absolute frequency of giant potentials does not markedly change when spontaneous discharge is accelerated by hypertonic solution. 5. The mechanism by which vinblastine induces the appearance of giant potentials is discussed. INTRODUCTION
It has already been shown that the large spontaneous potentials occurring at the normal neuromuscular junction of the rat (Liley, 1957; Hubbard & Jones, 1973) or provoked experimentally in frog (Katz & Miledi, 1969; Pecot-Dechavassine, 1970; Pecot-Dechavassine & Couteaux, 1972 a; Benoit, Audibert-Benoit & Peyrot, 1973; Heuser, 1974) are not caused by the random coincident release of unitary miniature potentials (min.e.p.p:s). 2
PlH *'
26i
M. PICOT-DECHA VASSINE 32 The analysis of their occurrence led to the postulate that they might be produced by the release of large pre-formed packets of ACh. Thus the question arises whether these large spontaneous potentials are related to some structural changes inside the nerve terminals, specially at the level of the synaptic vesicles. Till now, no definite conclusion could be drawn from the researches on giant potentials occurrring at rat neuromuscular junction because of their low relative frequency. A coincident appearance of giant potentials and oversized vesicles was clearly observed at frog endplates after various stimulations (P6cot-Dechavassine & Couteaux, 1972 a, b; Heuser, 1974). However, the procedures used in these experiments were often drastic and irregular in their effects. Moreover, the morphological results led to two different assumptions on the possible origin of large vesicles: They could be formed by coalescence of synaptic vesicles (P6cot-Dechavasine & Couteaux, 1972a); or they could be derived from irregular cisternae resulting from membrane re-cycling (Heuser, 1974). Vinblastine, which had been shown by Turkanis (1973) to give giant potentials at frog neuromuscular junctions, was found suitable for further investigations of the properties and the origin of these abnormal potentials. Its action on spontaneous release is obtained at low concentrations, and it is reversible. In a previous report (PNcot-Dechavassine, 1975; P6cot-Dechavasine &
Couteaux, 1975), it has been shown that the spontaneous giant potentials elicited by vinblastine are probably produced by the release of large packets of ACh. The present investigation deals with a detailed study of the effects of vinblastine on both spontaneous and evoked release of transmitter. An attempt has been made here to answer the following two questions: (i) are the giant potentials composed of multiples of the min.e.p.p.s, and thus produced by the release of pluriquantal packets of ACh? (ii) are the large packets of ACh also available for release by nerve stimuli? The specific mode of action of vinblastine has also been discussed in order to provide additional information concerning the possible origin of the giant spontaneous potentials. A preliminary account has been previously reported in abstracts (P6cotDechavassine & Couteaux, 1976). METHODS The experiments were performed on sartorius and rectus internus major of Rana eaculenta or Rana temporaria. Conventional methods were used to record potentials
intracellularly from single muscle fibres. The composition of the standard physiological solution was (mM): NaCl, 111; KC1, 2; CaCl2, 2; NaHCO3, 2; pH was 7-7-2.
VINBLASTINE ON SPONTANEOUS RELEASE
33
Some modified solutions were used according to the nature of the experiment: solutions with a different concentration of Ca2+ (with or without 0 5 mM-EDTA), with tetrodotoxin (TTX) (2 x 10-7 g/ml.) or with neostigmine (1-10-4 M). Low Ca2+ (0.4 mm)- high Mg2+ (5-10 mM) solutions (blocking-Ringer solutions) were used in experiments requiring end-plate potentials (e.p.p.s.) of low mean quantal content. Mean quantal content was estimated from the number of 'failures' in response to nerve stimuli and from the ratio of the mean size of evoked e.p.p.s. to the mean size of the unitary min e.p.p.s. The concentrations of vinblastine sulphate (gift from Eli Lilly, France) ranged from 0.1 to 03 mei. In a few experiments, we have used colchicine and cytochalasin B (Calbiochem) as control substances. Colchicine, like vinblastine,is an antimitotic substance; its action is associated with microtubules, while cytochalasin B probably inhibits the contractile apparatus of microfilaments (Carter, 1967; Wessels, Spooner, Ash, Bradley, Luduena, Wrenn & Yamada, 1971). Experiments were done at room temperature of 20-24o C in both summer and winter. RESULTS
EffeciB of vinbladtine on the 8pontaneow8 transmitter release on Effect8 min.e.p mi. 40
300]n -h-Failures a. I I
.
End-plate potentials
II I
C
0 L.
4s 0
.0 0 L.
.0
Spontaneous potentials
E
z
t 0-5
t
4t
3 2 Amplitude (mV) Fig. 5. Histograms of end-plate potential and spontaneous potential amplitudes from an end-plate displaying a high proportion of giant spontaneous potentials (211/605) in low Ca2+-high Mg2+ Ringer solution. ml (from the number of failures) and m2 (from the ratio mean amplitude of the e.p.p.s/ mean amplitude of the unitary min.e.p.p.s) are 0-55 and 0-65 respectively. Only one e.p.p. is found outside the theoretical distribution according to Poisson's law. 1
(ii) The distribution of amplitudes of e.p.p.s calculated according to Poissons' law, from the number of failures, should differ from the observed distribution. This was tested in fibres with a high proportion of large spontaneous potentials (from about 10 to 50 %) and e.p.p.s of low mean quantal content. Only e.p.p.s which were well outside the range of the predicted distribution were considered as abnormal.
41 VINBLASTINE ON SPONTANEOUS RELEASE The results are summarized in Table 2. Generally m2 is a little higher than m1 (thirteen experiments out of fourteen). The paired t test performed on data, shows that the difference between the two means (ml = 0 95 + 0 14; m2 =1-05 + 0-17) is significant at the 0 001 level of significance (mean difference = 0 103 + 0-024; t = 4-29 for 13 degrees of freedom; critical value of t = 4-22 for P = 0 001). The more crude Wilcoxon's pair signed rank test also shows that there is evidence for the reality of the difference. However, the difference between ml and m2 is small, which suggests that very few large quanta are released by nerve stimuli. The analysis of the distribution of the e.p.p.s (see above in (ii)) shows, indeed, that in most series only one or two e.p.p.s could be considered outside the predicted distribution (Table 2). Fig. 5 illustrates the distribution of both e.p.p.s and min.e.p.p.s obtained in an experiment in which only one e.p.p (out of 217) is outside the theoretical distribution. The impulse-evoked release of transmitter in known to be a Ca-dependent synchronous release of a large number of ACh quanta (del Castillo & Katz, 1954). It can be regarded as an intense transient acceleration of the spontaneous release due to depolarization of terminals (Liley, 1956b). The effect of an acceleration of spontaneous release on the proportion of giant potentials has been examined by changing the osmotic pressure. The osmolarity of the Ringer was increased by adding sucrose, and decreased by adding H20. This has several advantages over other procedures; in particular, the changes in the frequency of min.e.p.p.s occur very rapidly and are quickly reversible. Thus, many tests could be made within a short period, interspersed with controls, during the persistence of Vinblastine action. Moreover, the membrane potential of the muscle fibre does not change markedly during these osmotic alterations. The amplitude histograms of Fig. 6 illustrate the changes in the frequency of min.e.p.p.s, and in proportion of large potentials, when tonicity was alternatively changed. The hypotonic solution (Fig. 6B, D, E) was made by diluting the Ringer to one third; the hypertonic solution (Fig. 6C, F) by adding sucrose (0 1 M) to the Ringer. The proportion of giant potentials decreases considerably when the min.e.p.p. fequency is increased by hypertonic Ringer solution. It decreases from 18 and 35 % (Fig. 6B, E) to 5 % and 4 % (Fig. 6B, E) while the total min.e.p.p. frequency increases from 4-5 sec-' and 2 sec-' to 16 sec-' and 13 sec-' respectively. This figure indicates that the absolute frequency of giant potentials did not change when the total spontaneous discharge rate was increased.
Action of colchicine and cytochalasin B Colchicine, which, like vinblastine, is an antimitotic drug whose action is apparently associated with microtubules (Borisy & Taylor, 1967a, b;
42 M. P1COT-DECHA VA SSINE Wilson & Friedkin, 1967; Malawista, Sato & Bensch, 1968) has been used as control. Five experiments were performed on isolated frog muscle with concentrations of colchicine ranging from 0 5 to 2 mm. A
D
6 sec-'
3 sec-'
100
0.
17%
50
so
Jk I B
C
0 "
rbk~33
I
E
45 sec-'
50
0 0
E
go
__
z
1 ~ ~ ~ ~ ~ ~18%
_
__*_
--
35%
l
PI
F
16 sec'
No-
s0
0*5
5%
/ Oim~~
--ow-
I
C
I
2 sec-'
100-
.00
-
_P
13 sec-'
..
--
4%
2 0*5 1 2 Amplitude (mV) Fig. 6. Changes in proportion of giant potentials concomitant with changes in min.e.p.p. frequency. Changes in min.e.p.p. frequency were obtained by alternate immersion in hypo-(B, D, E) and hyper- (C, F) osmotic Ringer solution. Same soaking in D and E at different times (10 and 15 min). In A, isotonic Ringer solution. Sartorius muscle of Rana temporaria had been soaked, before this experiment, in vinblastine (0-2 mM) in blocking-Ringer solution for 75 min. As
previously
I
shown
by
Turkanis
(1973), colchicine had
no
consistent
effect on the distribution of amplitudes of min.e.p.p.s. Negative results were also obtained when colchicine was added in Ca2+ rich Ringer solution (two to three times the normal concentration).
43 VINBLASTINE ON SPONTANEOUS RELEASE Cytochalasin B was used in order to check whether the action of vinblastine on spontaneous release might be mediated by a possible action on microfilaments (Albert, Norton & Shelanski, 1970; Schlaepfer, 1971). Owing to the low solubility of cytochalasin B, it was dissolved in DMSO (dimethylsulphoxide) before adding it to Ringer solution. Four experiments were done with final concentrations of cytochalasin B ranging from 30 to 50 ,sg/ml. A few min.e.p.p.s with slightly larger amplitude than normal were observed in two experiments. However, they were very rare and their amplitude did not extend beyond two to three times the modal value of the control. Moreover, the very low solubility of cytochalasin B did not allow the use of higher concentrations in order to obtain more clear-cut effects. DISCUSSION
Characteri8tic8 of giant potentials The properties of giant potentials induced by vinblastine are similar to those of giant potentials induced by other procedures (Katz & Miledi, 1969; Pecot-Dechavassine, 1970; Heuser, 1974): They occur when the min.e.p.p frequency is low and in nerve-muscle preparations treated with TTX or blocked by Mg2+. This suggests that they cannot be attributed to random coincidence of release of several independent quanta, nor to spontaneous action potentials or local depolarization of nerve terminals. Moreover, their site of release is probably the same as that of ordinary min.e.p.p.s for their time courses are very similar. All these findings suggest that the large spontaneous potentials are due to pre-formed large packets of ACh released from the nerve terminals. Although a periodicity is not always evident in the amplitude histograms, this could be explained by the large spread of unitary amplitudes. It seems more significant that many histograms do display a clear multi-modal periodicity with peaks occurring at integral multiples of the unitary min.e.p.p. size. These results strengthen the idea that giant potentials at normal rat end-plates (Liley, 1957), and at frog end-plates after prolonged transmitter release (Heuser, 1974) may arise from pluriquantal packets of ACh, which in turn could be derived from the fusion of synaptic vesicles. Such a view has been proposed previously: large size and scalloped outline of many vesicles, suggestive of coalescing synaptic vesicles, were observed in muscles treated with vinblastine and displaying giant potentials at the time of fixation (Pecot-Dechavassine & Couteaux, 1975). Pictures of such oversized vesicles were also seen with other procedures (e.g. P6cot-Dechavassine & Couteaux, 1972a, b; Heuser, 1974). Thus, there is a converging physiological and morphological evidence in favour of a relation between giant potentials and large vesicles. However, the
M. PtCOT-DECHA VASSINE origin of these large vesicles remains uncertain. With the procedures previously used, giant potentials appeared only after drastic release of ACh and after a long delay. These features led Heuser (1974) to suggest that ' ... oversized vesicles are intermediates in membrane re-cycling which form by retrieval of vesicle membrane from the plasmalemma during stimulation. . . '. In this case, the delay would represent '.. .the time required for cisternae to accumulate sufficient transmitter to discharge. . . '. The results presented here suggest a different mode of origin for the induction of giant potentials by vinblastine, for they appear quickly and, are not preceded by a period of massive spontaneous release. It would, therefore, seem more probable that these potentials are derived from large packets of ACh formed from fusion of synaptic vesicles shortly before release. Though the present observations indicate that giant potentials resemble min.e.p.p.s in many ways, it appears that they do not contribute in a significant manner to the responses evoked by the nerve impulses. Menrath & Blackman (1970) have obtained similar results concerning giant potentials at normal rat end-plates. They conclude that a non-neuronal source of large quanta (which might be the Schwann cell) could be responsible for giant potentials. The present experiments provide little support for such an assumption. The time course of min.e.p.p.s and giant potentials is similar and no- change in morphological relations between Schwann cells and post-synaptic membrane was detected in electron micrographs (P6cotDechavassine & Couteaux, 1975). The fact that the relative proportion of large spontaneous potentials decreases considerably when the spontaneous release is highly accelerated by hyperosmotic solution might provide a possible explanation. It may be that the mobility of large quanta is less than that of single quanta. Under conditions of high rates of quantal release (e.g. during acceleration of spontaneous release), the access of large quanta to release sites might be limited, on account of their large size; therefore the probability of their release may be lower. This would also explain their low probability of release by nerve impulse. Finally, another possibility is that two different ACh stores might independently participate in spontaneous and evoked release. Such an alternative explanation has been proposed elsewhere (Harris &; Miledi, 1971; Dennis &; Miledi, 1974) to explain a difference in the transmitter quanta released spontaneously and those during nerve stimulation at some recently re-innervated frog neuromuscular junctions and at mature junctions intoxicated with botulinum toxin. 44
VINBLASTINE ON SPONTANEOUS RELEASE
45
Mode of action of vinblastine Vinblastine like colchicine is considered to exert its antimitotic effects through an action on microtubules. It is also through this action that these drugs are thought to interfere in the mechanism of various glandular secretions and in the release of neurotransmitters at synapses (Rasmussen, 1970) as well as in the maintainance of synaptic transmission (Perisic & Cu6nod, 1972; Felder, 1975). Chronic application of colchicine, vinblastine and vincristine on peripheral nerves produces at the neuromuscular junction both physiological changes (Hofmann & Thesleff, 1972; Hofmann, Struppler, Weindl & Velho, 1973; Albuquerque, Warnick, Sansone & Ohur, 1974; L0mo, 1974) and morphological changes (Anderson, Song & Slotwiner, 1967; Hsu & Lentz, 1972; Wuerker & Bodley, 1973) similar to those occurring after denervation. They could result from an action on microtubules, interfering either with the maintenance of elongated axonal process (Wessels et al. 1971) or with the transport of trophic substances (Hofmann & Thesleff, 1972; Hofmann et al. 1973; Albuquerque et al. 1974). However, the effect of vinblastine on spontaneous release described in the present work cannot be attributed to an action on axonal transport through microtubules because this effect occurs very rapidly (within 20 min). Moreover it has been shown that colchicine and vinblastine have markedly different effects on transmitter release (Katz, 1972; Turkanis, 1973). Many arguments agree well with an alternative hypothesis supported by findings which provide evidence that vinblastine precipitates many proteins in addition to colchicine-binding proteins. (1) Vinblastine might combine with neurofilaments which have been shown in some mammalian nerves and brains to contain, in addition to tubulin, several protein components precipitated by vincristine (Albert et al. 1970; Schlaepfer, 1971). In support of this hypothesis is the fact that many crystal-like structures have been observed close to neurofilaments in nerve terminals of nerve muscle preparations soaked for a short time in vinblastine (P6cot-Dechavassine & Couteaux, 1975). The lack of effect of cytochalasin B on the spontaneous release could plausibly be attributed to its low solubility and slow penetration rate. (ii) Vinblastine could combine also with membrane proteins either of the presynaptic terminal or of synaptic vesicles. This hypothesis is supported by the results of Wilson, Bryan, Ruby & Mazia (1970) who observed that vinblastine (but not colchicine), presumably acting as a cation, precipitates proteins derived from erythrocyte membranes, by combining with sites which can also bind Ca2+ ions. These biochemical effects could explain
M. PICOT-DECHA VASSINE our observations concerning the interference of vinblastine and calcium at sites involved in inducing giant potentials. One might assume that both vinblastine and Ca2+, by reacting with certain membrane proteins, could alter the membrane properties so as to modify the permeability or facilitate the phenomenon of fusion. High concentrations of vinblastine were, indeed, reported to increase cell Na+ and make the cells more fragile (Seeman, Chau-Wong & Moyyen, 1973). Such an action of Ca2+ on membranes could also explain the occurrence of giant spontaneous potentials at end-plates of muscles exposed for several hours to an isotonic Ca-Ringer solution (Katz & Miledi, 1969). 46
I wish to thank Professor B. Katz for helpful criticism of the manuscript and Professor R. Couteaux for his constant interest during the course of the work.
REFERENCES ALBERT, S., NORTON, W. T. & SHELNTsi, M. L. (1970). Isolation of mammalian axonal neurofflaments. J. ceU Biol. 47, 4A. ALBUiQUERQuE, E. X., WARNICK, J. E., SsoNE, F. M. & ONE, R. (1974). The effects of vinblastine and colchicine on neural regulation of muscle. Ann. N.Y. Acad. Sci. 228, 224-243. ANDERsoN, P. J., SONG, S. K. & SLOTWUTER, P. (1967). The fine structure of spheromembranous degeneration of skeletal muscle induced by vincristine. J. Neuropath. exp. Neurol. 26, 15-23. BENOIT, P. H., AUDIBERT-BENOIT, M. L. & PEYROT, M. (1973). Importance des effects pr5synaptiques dans le blockage de la jonction neuromusculaire de grenouille par substitution du lithium au sodium dans le milieu de survie. Arch. ital. Biol. 111, 323-335. BENOIT, P. R. & MAMBRN, J. (1969). Correlation entre la liberation spontanee et la liberation 6voqu6e du m6diateur ^ la jonction neuro-musculaire de Grenouille. J. Physiol., Pari 61, 214. BORIsY, G. G. & TAYLOR, E. W. (1967 a). The mechanism of action of colchicine. Binding of colchine"H to cellular protein. J. ceU Biol. 34, 525-534. BoRisy, G. G. & TAYLOR, E. W. (1967b). The mechanism of action of colchicine. Colchicine binding to sea urchin eggs and the mitotic apparatus. J. cell Biol. 34, 535-548. BoYD, I. A. & MA RTIN, A. R. (1956). The end-plate potential in mammalian muscle. J. Physiol. 132, 74-91. CARTER, S. B. (1967). Effects of cytochalasins on mlian cells. Nature, Lond. 213, 261-264. DEL CAsTILLo, J. & KATZ, B. (1954). Quantal components of the end-plate potential. J. Phy8iol. 124, 560-573. DENNs, M. J. & MILEDI, R. (1974). Characteristics of transmitter release at regenerating frog neuromuscular junctions. J. Physiol. 239, 571-594. FELDER, M. (1975). Vinblastine: Influence on nerve conduction and synaptic transmission. J. Neuroeci. Rem. 1, 121-130. HuAusI, A. J. & MEIEDI, R. (1971). The effect of type D botulinum toxin on frog neuromuscular junctions. J. Physiol. 217, 497-515. HEusER, J. E. (1974). A possible origin of the 'giant' spontaneous potentials that occur after prolonged transmitter release at frog neuromuscular junctions.
J. Phy8iol. 239, 106-108P.
47 VINBLASTINE ON SPONTANEOUS RELEASE HoF1NNw, W. W. & STRUIPLER, A., WEINDL, A. & VELHO, F. (1973). Neuromuscular transmission with colchicine-treated nerves. Brain Re8 49, 208-213.
HoFMANw, W. W. & THESLEFF, S. (1972). Studies on the trophic influence of nerve on skeletal muscle. Eur. J. Pharmacol. 20, 256-260. Hsu, L. & LENTZ, T. L. (1972). Effect of colchicine in the fine structure of the neuromuscular junction. Z. ZeUfor8ch. mikro8k. Anat. 135, 439-448. HUBBARD, J. I. & JoNEs, S. F. (1973). Spontaneous quantal transmitter release: statistical analysis and some implications. J. Phyriol. 232, 1-21. KATZ, N. L. (1972). The effects on frog neuromuscular transmission of agents which act upon microtubules and microfilaments. Eur. J. Pharmacol. 19, 88-93. KArz, B. & MILEDI, R. (1969). Spontaneous and evoked activity of motor nerve endings in calcium Ringer. J. Phy8iol. 203, 689-706. LiAxy, A. W. (1956a). The quantal components of the mammalian end-plate potential. J. Physiol. 133, 571-587. LuILY, A. W. (1956b). The effects of presynaptic polarization on the spontaneous
activity of the mammalian neuromuscular junction. J. Phyeiol. 134, 427443. TLTTIY, A. W. (1957). Spontaneous release of transmitter substance in multiquantal units. J. Phy8iol. 136, 595-605. LoMo, T. (1974). Neurotrophic control of colchicine effects on muscle. Nature, Lond. 249, 473-474. MATAWISTA, S. E., SATO, H. & BENSCH, K. G. (1968). Vinblastine and griseofulvin reversibly disrupt the living mitotic spindle. Science, N.Y. 160, 770-772. MENRATH, R. L. E. & BLACKMANN, J. G. (1970). Observations on the large spontaneous potentials which occur at end-plates of the rat diaphragm. Proc. Univ.
Otago med. Sch. 48, 72-73. PPOOT-DEcHAvAssnNE, M. (1970). Effects conjugu6s du pH et des cations divalents sur la liberation spontanee d'ac6tylcholine au niveau de la plaque motrice de la Grenouille. C. r. hebd. S&nc. Acad. Sci., Pari8 D 271, 674-677. PACOT-DECHAVASsNE, M. (1975). Modalites d'apparition des potentials spontan6s giantss' provoques par la vinbiastine sur la preparation isol6e du muscle de grenouille. C. r. hebd. Seanc. Acad. Sci., Parin D 280, 303-306. PACOT-DECHAVASSINE, M. & COUTEAUX, R. (1972 a). Potentials miniatures d'amplitude anormale obtenus dans des conditions experimentales et changements concomitants des structures presynaptiques. In International Sympoaium on Cholinergic Tranemie8ion of the Nerve Impulse, pp. 177-185. Paris: INSERM. PECOT-DECHAVASSINE, M. & COUTEAUX, R. (1972b). Potentials minatures d'amplitude anormale obtenus dans des conditions exp6rimentales et changements concomitants des structures pr6synaptiques. C. r. hebd. S&anc. Acad. Sci., Paris D 275, 983-986. PECOT-DECHAvAssInE, M. & COUTEAux, R. (1975). Modifications structurales des terminaisons motrices de muscle de grenouille soumis * l'action de la vinblastine. C. r. hebd. Sganc. Sci., Pari8 D 280, 1099-1101. PtCOT-DEcHAvAssuNE, M. & COUTEAux, R. (1976). Action of vinblastine on the spontaneous release of ACh at the frog neuromuscular junction. Abstracts of the Scottish Electrophysiological Society, Symposium on The Synap8e, St Andrews, Scotland, 29 March-2 April. PERIsIC, M. & CUtNoD, M. (1972). Synaptic transmission depressed by colchicine blockade of axoplasmic flow. Science, N.Y. 175, 1140-1142. RAsMUsSEN, H. (1970). Cell communication, calcium ion, and cyclic adenosine monosphate. Science, N. Y. 170, 404-412. SCHLAEPFER, W. W. (1971). Stabilisation of neurofilaments by vincristine sulfate in low ionic strength media. J. Ultrastr. Re8. 36, 367-374.
48
M. PJCOT-DECHA VASSINE
SEEMAN, P., CHAU-WONG, M. & MOYYEN, S. (1973). Membrane expansion by vinblastine and strychnine. Nature, New Biol. 241, 22. TuIJR Is, S. A. (1973). Some effects of vinblastine and colchicine on neuromuscular transmission. Brain Be8. 54, 324-329. WEssELs, N. K., SPOONER, B. S., ASH, J. F., BRADixrY, M. A., LUDUIENA, M. A., WRENN, J. T. & YAMADA, K. M. (1971). Microfilament in cellular and developmental processes. Science, N.Y. 171, 135-143. WnsoN, L. BKYAN, J. RUBy, A. & MARYi, D. (1970). Precipitation of proteins by vinblastine and calcium ions. Proc. natn. Acad. Sci. U.S.A. 66, 807-814. WILsoN, L. & FRIDEDN, M. (1967). The biochemical events of mitosis. The in vivo and in vitro binding of colchicine in grasshopper embryos and its possible relation to inhibition of mitosis. Biochemistry 6, 3126-3135. WuK=KER, R. B. & BODLEY, H. D. (1973). Changes in muscle morphology and histochemistry produced by denervation, 3.3'-iminodipropionitrile and epineurial vinblastine. Am. J. Anat. 135, 221-234.
