Brain Research, 104 (1976) 171-175
© Elsevier Scientific Publishing Company, Amsterdam
171
Printed in The Netherlands
Effect of cytochalasin B on neuromuscular transmission in tissue culture
LEE L. RUBIN, A L F R E D O G O R I O AND A L E X A N D E R M A U R O
The Rockefeller University, New York, N. Y. 10021 (U.S.A.) (Accepted November 24th, 1975)
A m o n g the many actions of cytochalasin B (CB) are modifications in the cellular release of secretory products 1. CB partially inhibits release of acetylcholine in the sympathetic ganglion 15 and of noradrenaline fccm sympathetic nerve endings in the vas deferens ~4 and in the atrium 21. The studies reported here show, for the first time, that low doses of CB produce a striking, often total, decrease in end-plate activity recorded electrophysiologically at individual neuromuscular junctions in tissue culture. These results establish the direct action of CB on the neurotransmitter release mechanism in culture. In contrast, CB seems to have no observable effect on neuromuscular transmission in the adult frog or mouse. The possible loci of action of CB are discussed, as are the differences between the adult and cultured neuromuscular junctions. Thigh muscles of 12-day-old chick embryos were trypsinized, suspended in culture medium containing 8 0 ~ Eagle's Minimum Essential Medium with Earle's Salts (MEM) and antibiotics, 10~o horse serum and 10 ~ chick embryo extract, and then plated in collagen-coated Falcon 35 m m tissue culture dishes. After 2 or 3 days, these cultures were often treated with 10-5 M cytosine arabanoside for 48 h to reduce fibroblast growth 7. Three or 4 days following the muscle dissociation, explants from spinal cords of 5-7-day-old chick embryos were added to the dishes. Synapses formed rapidly, and nerve-muscle contacts were often studied after an additional 24-48 h. For electrophysiological experiments, cells were bathed in 90~o M E M (with 15 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) instead of bicarbonate) at pH 7.4, and 10 ~ horse serum. The tissue culture dish was placed on the heated stage of a Leitz (Diavert) inverted phase microscope; temperature was maintained at 36 °C. Myotubes were impaled with glass micropipettes filled with 3 M KCI and having resistances of 30-100 M~). Synaptic contacts were identified by extracellular stimulation of fine processes near myotubes through 2-20 M ~ micropipettes filled with 1 M NaC1. Stimulating pulses were 2 msec in duration and 10-50 V in amplitude. Myotubes often contracted due to spontaneous, neurally evoked activity; in such cases both miniature end-plate potentials (MEPPs) and end-plate potentials (EPPs) were recorde&. The adult preparations used were frog cutaneous pectoris (at room temperature)
172
and mouse phrenic nerve hemidiaphragm (at 37 °C). Standard intracellular electrophysiological techniques were employed. Stock solutions (10 mg/ml) of cytochalasin B (Aldrich Chemical Co.) were made in dimethyl sulfoxide (DMSO). Control experiments demonstrated that D M S O at the concentrations used with the CB (0.01-0.04 ~ ) had no effect on neuromuscular transmission in tissue-cultured myoneural junctions. In 12 experiments on tissue-cultured junctions, concentrations of CB ranged from 0.8 #g/ml to 4.0 #g/ml ; the dose most commonly used was 2.0/~g/ml. Following CB application, the frequency of M EPPs and of spontaneous EPPs decreased suddenly and dramatically. This occurred with a latency of 4-20 min in 11 experiments and of 60 min in one experiment. Small changes in the amplitude of these end-plate events during onset of the CB effect cannot be excluded. A recording of spontaneous endplate activity before and after treatment with CB is shown in Fig. 1A and B: EPPs produced by external nerve stimulation were also blocked with approximately the same latency (Fig. 1A and B, inset). The time course of this effect was not examined closely enough to decide whether it was necessarily preceded by a decrement in EPP amplitude. The extent of inhibition of the frequency of spontaneous end-plate events (MEPPs and EPPs) varied from 67 7{; and 82 % in two experiments to 95 %-100 30 in the remainder. The time to onset and the degree of CB inhibition were both somewhat variable; such variability has been seen in a variety of studies using CB22, 26. When the CB was washed away, preparations eventually recovered full activity. Recovery, as measured in two experiments by nerve fiber stimulation, was complete in 5-10 min
(Fig. lC).
A
IL
L
[11!115
Fi
L
i
i
l.j"/
I
!111 [I
Ill I
I
L_ I
_.I] I0 mV I 0 0 ms
Fig. 1. Effect of CB on spontaneous and evoked end-plate activity. A: muscle synaptic potentials evoked by spontaneous firing of spinal cord neurons before CB application. Fast potentials associated with muscle twitch (see ref. 7) are at the peak of many of the synaptic potentials, The prolonged time course of the synaptic potentials may indicate that this myotube is multiply innervated (see ref. 7)~ MEPPs in this preparation, chosen to illustrate the reversibility of the CB effect, were low in amplitude and frequency and are not visible in this record. B: absence of end-plate activity 20 rain after treatment with CB. C: recovery of activity 10 rain after rinsing with CB-free medium. Inset: End,plate response produced by extracellular stimulation of nerve process. Arrow indicates shock artifact. Calibration for inset is 10 mV and 25 msec.
173 ~ ~ - ~
x L_ L LL tl J~X~X ,uala_ I
L
~ ~SmV I00 ms
Fig. 2. Miniature end-plate potentials 3 rain after addition of black widow spider venom (0.01 ffg/ml)
in a preparation initially blocked (as in Fig. IB) by CB treatment. CB appears to induce presynaptic modifications leading to a decrease in the frequency of end-plate activity. Since MEPPs in addition to EPPs are sensitive to CB, effects on the release mechanism itself, rather than on nerve propagation, are likely to be responsible for the inhibition. Axon retraction from the muscle was demonstrated not to be the primary cause for decreased end-plate activity since black widow spider venom evoked a massive discharge in end-plates blocked with CB (Fig. 2). The venomstimulated discharge confirms the presence of postsynaptic acetylcholine sensitivity (which has also been seen in preliminary iontophoresis experiments) after CB application. Inhibition of glucose uptake is thought to be the underlying mechanism of CB in some cases 1~. In two experiments, dishes were rinsed and refilled with medium made without glucose and containing 10 ~ dialyzed horse serum. End-plate activity was not inhibited after 1-2 h in glucose-free medium, and the full effect of CB was subsequently observed. To confirm and extend previously reported results 10, CB was applied to adult neuromuscular junctions. No effect of CB in doses of up to 80 #g/ml was observed on the frog cutaneous pectoris after 4 h. Similarly, no change in end-plate activity was produced by 50 #g/ml of CB for 30 min or by 25 #g/ml of CB for 60 rain in the mouse diaphragm. ]n this latter case, however, 0.25 ~o D M S O itself in 60 rain, and 0.5 ~o in 30 min, effected a reversible block of the end-plate potential and a somewhat increased M EPP rate. The explanations for the diverse cellular alterations caused by application of the cytochalasins are usually limited to three general categories: (a) interference with uptake of metabolic precursorsl4,15, (b) disorganization of a microfilament network present in many cell types 25, and (c) association with plasma membrane sites other than those related to the uptake system 19. Localization studies using [aH]cytochalasin D 23 and [3H]CB la support the feasibility of each alternative. As described above, inhibition of sugar uptake does not appear to be the primary cause of CB's inhibition of neuromuscular transmission. The two remaining possibilities must be considered. Actin- and myosin-like molecules may be structural components of microfilamentous networks found in many non-muscle cells 16. Such molecules have been isolated from neurons6,18 and appear to be concentrated in the synaptic region TM. In addition, association of CB with actin 22 and of cytochalasin D with myosin 17 has been reported. Although CB apparently does not disrupt actin-myosin interactions in striated muscle, these molecules or their regulatory factors may be different in nonmuscle cellst6, 22. Gray's ultrastructural studies 9 and, more recently, those of LeBeux and Willemot using heavy meromyosin labeling 12 have suggested the existence in the synaptic region of an organized, actin-like system of filaments which may correspond
174 to microfilaments seen in the nerve growth cone. Interestingly, the immobility of secretory vesicles in mast, chromaffin, and adenohypophyseal cells 5 may provide additional indication of such a structural lattice. Berl et al. have already proposed that an actomyosin-like complex may be involved in neurotransmitter releaseL The present results are consistent with, although certainly no proof for, a disruptive influence of CB on an actomyosin-like system responsible for maintaining the orientation of synaptic vesicles and for introducing vesicles into their release sites in the synaptic cleft. Plasma membrane modifications cannot be dismissed. CB may be involved in an inhibition of membrane-membrane interaction (as has been seen with fusing myoblasts2°), thus preventing discharge of neurotransmitter from synaptic vesicles. Alternatively, CB might depress transmitter release by altering the highly organized presynaptic plasma membrane structure (see ref. 26). The difference in sensitivity to CB between cultured chick neuromuscular junctions and junctions in adult frog and mouse cannot be completely accounted for. The release process in chicken appears, electrophysiologically, to be quite similar to that in frog and mouse s. Moreover, it is unlikely that the embryonic release system differs substantially from that in the adult 7. Penetration of CB into the adult junction might be limited by an extensive extracellular matrix not present in the cultured junction. This is not probable, however, since black widow spider venom, whose active component has a molecular weight of 100,000, seems to have unimpaired access to the adult terminal a. Since CB appears to act either at the plasma membrane or intracellularly at a microfilamentous array, the sensitivity difference may exist not in the release mechanism itself, but in membrane binding of CB or in resistance to structural changes induced by the bound drug. One remaining explanation is that the adult synaptic membrane is not permeable to CB, and thus CB never reaches its intracellular binding sites. The mechanisms responsible for secretion in neurons and in other cell types, such as pituitary cells and mast cells, may be similar 4. CB alters the secretion in a variety of these cells and may therefore effect similar changes in each. The tissuecultured neuromuscular junction should be a suitable system for using immunofluorescence 11 and other techniques to explore the role of actin-tike networks in secretion and to establish the basis for action of the cytochalasins. Supported, in part, by Research Grant NS07080 from the United States Department of Health, Education and Welfare (to Bruce S. McEwen), by a special grant from the Muscular Dystrophy Associations of America, and by a National Science Foundation Predoctoral Fellowship (to Lee L. Rubin). The authors wish to thank Drs. H. Holtzer, W. P. Hurlbut and B. S. McEwen for helpful discussions.
1 ALLISON,A, C., The role of microfilaments and microtubules in cell movement, endocytosis and exocytosis. In R. PORTERAND D. W. FITZSIMONS(Eds.), Locomotion of Tissue Cells, Elsevier, Amsterdam, 1973, pp. 109--143.
175
2 BERL, S., PUSZKIN, S., AND NICKLAS, W. J., Actomyosin-like protein in brain, Science, 179 (1973) 441-446. 3 CLARK, A. W., HURLBUT, W. P., AND MAURO, A., Changes in the fine structure of the neuromuscular junction of the frog caused by black widow spider venom, J. Cell Biol., 52 (1972) 1-14. 4 DOUGLAS, W. W., Stimulus-secretion coupling: the concept and clues from chromaffin and other cells, Brit. J. Pharmacol., 34 (1968) 451-474. 5 DOUGLAS, W. W., Involvement of calcium in exocytosis and the exocytosis-vesiculation sequence. In R. M. S. SMELLIE (Ed.), Calcium and Cell Regulation, Biochemical Society Symposium No. 39, The Biochemical Society, London, 1974, pp. 1-28. 6 FINE, R. E., AND BRAY, D., Actin in growing nerve cells, Nature New Biok, 234 (1971) ll5-118. 7 FISCHBACH, G. D., Synapse formation between dissociated nerve and muscle cells in low density cell cultures, Develop. Biol., 28 (1972) 407-429. 8 GINSBORG, B. L., Spontaneous activity in muscle fibres of the chick, J. Physiol. (Lond.), 150 (1960) 707-717. 9 GRAY, E. G., The cytonet, plain and coated vesicles, reticulosomes, multivesicular bodies and nuclear pores, Brain Research, 62 (1973) 329-335. l0 KATZ, N. L., The effects of frog neuromuscular transmission of agents which act upon microtubules and microfilaments, Europ. J. Pharmacol., 19 (1972) 88 93. 11 LAZARIDES,E., AND WEBER, K., Actin antibody: the specific visualization of actin filaments in nonmuscle cells, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 2268-2272. 12 LEBEUX, Y. J., AND WILLEMOT,J., An ultrastructural study of the microfilaments in rat brain by means of E PTA staining and heavy meromyosin labelling, lI. The synapses, Cell Tiss. Res., 160 (1975) 37-68. 13 LIN, S., SANTI, D. V., AND SPUDICH, J. A., Biochemical studies on the mode of action of cytochalasin B. Preparation of [3H]cytochalasin B and studies on its binding to cells, J. biol. Chem., 249 (1974) 2268-2274. 14 M1ZEL, S. B., AND WILSON, L., Inhibition of the transport of several hexoses in mammalian cells by cytochalasin B, J. biol. Chem., 247 (1972) 4102-4105. 15 NAKAZATO, Y., AND DOUGLAS, W. W., Cytochalasin blocks sympathetic ganglionic transmission: a presynaptic effect antagonized by pyruvate, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 1730-1733. 16 POLLARD, T. D., AND WEIHING, R. R., Actin and myosin and cell movement, CRC Critical Reviews in Biochemistry, 2 (1972) 1-65. 17 PUSZKIN, E., PUSZKIN, S., Lo, L. W., AND TANNENBAUM, S. W., Binding of cytochalasin D to platelet and muscle myosin, J. biol. Chem., 248 (1973) 7754-7761. 18 PUSZKIN, S., NICKLAS, W. J., AND BERL, S., Actomyosin-like protein in brain: subcellular distribution, J. Neurochem., 19 (1972) 1319-1333. 19 SANGER, J. W., AND HOLTZER, H., Cytochalasin B: effects on cell morphology, cell adhesion, and mucopolysaccharide synthesis, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 253-257. 20 SANGER,J. W., HOLTZER, S., AND HOLTZER,H., Effects of cytochalasin B on muscle cells in tissue culture, Nature New Biol., 229 (1971) 121-123. 21 SORIMACHI,M., OESCH, F., AND THOENEN, H., Effects of colchicine and cytoehalasin B on the release of aH-norepinephrine from guinea-pig atria evoked by high potassium, nicotine and tyramine, Naunyn-Schmiedebergs Arch. exp. Path. Pharmak., 276 (1973) 1-12. 22 SPUDICH, J. A., Effects of cytochalasin B on actin flaments, Cold Spr. Harb. Syrup. quant. Biol., 37 (1973) 585-593. 23 TANNENBAUM,J., TANNENBAUM,S. W., LO, L. W., GODMAN,G. C., AND MIRANDA,A. F., Binding and subcellular localization of tritiated cytochalasin D, Exp. Cell Res., 91 (1975) 47-56. 24 THOA, N. B., WOOTEN, G. F., AXELROD,J., AND KOPIN, I. J., Inhibition of release of dopamine-/:lhydroxylase and norepinephrine from sympathetic nerves by colchicine, vinblastine, or cytochalasin-B, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 520-522. 25 WESSELLS,N. K., SPOONER, B. S., ASH, J. F., BRADLEY,M. O., LUDUENA,M. A., TAYLOR, E. L., WRENN, J. J., AND YAMADA, K. M., Microfilaments in cellular and developmental processes, Science, 171 (1971) 135-143. 26 YAHARA, 1., In B. D. KAHAN AND R. A. REISFELD (Eds.), The Cell Surface. Immunological and Chemical Approaches, Plenum Press, New York, 1974, pp. 113-121.
© Elsevier Scientific Publishing Company, Amsterdam
171
Printed in The Netherlands
Effect of cytochalasin B on neuromuscular transmission in tissue culture
LEE L. RUBIN, A L F R E D O G O R I O AND A L E X A N D E R M A U R O
The Rockefeller University, New York, N. Y. 10021 (U.S.A.) (Accepted November 24th, 1975)
A m o n g the many actions of cytochalasin B (CB) are modifications in the cellular release of secretory products 1. CB partially inhibits release of acetylcholine in the sympathetic ganglion 15 and of noradrenaline fccm sympathetic nerve endings in the vas deferens ~4 and in the atrium 21. The studies reported here show, for the first time, that low doses of CB produce a striking, often total, decrease in end-plate activity recorded electrophysiologically at individual neuromuscular junctions in tissue culture. These results establish the direct action of CB on the neurotransmitter release mechanism in culture. In contrast, CB seems to have no observable effect on neuromuscular transmission in the adult frog or mouse. The possible loci of action of CB are discussed, as are the differences between the adult and cultured neuromuscular junctions. Thigh muscles of 12-day-old chick embryos were trypsinized, suspended in culture medium containing 8 0 ~ Eagle's Minimum Essential Medium with Earle's Salts (MEM) and antibiotics, 10~o horse serum and 10 ~ chick embryo extract, and then plated in collagen-coated Falcon 35 m m tissue culture dishes. After 2 or 3 days, these cultures were often treated with 10-5 M cytosine arabanoside for 48 h to reduce fibroblast growth 7. Three or 4 days following the muscle dissociation, explants from spinal cords of 5-7-day-old chick embryos were added to the dishes. Synapses formed rapidly, and nerve-muscle contacts were often studied after an additional 24-48 h. For electrophysiological experiments, cells were bathed in 90~o M E M (with 15 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) instead of bicarbonate) at pH 7.4, and 10 ~ horse serum. The tissue culture dish was placed on the heated stage of a Leitz (Diavert) inverted phase microscope; temperature was maintained at 36 °C. Myotubes were impaled with glass micropipettes filled with 3 M KCI and having resistances of 30-100 M~). Synaptic contacts were identified by extracellular stimulation of fine processes near myotubes through 2-20 M ~ micropipettes filled with 1 M NaC1. Stimulating pulses were 2 msec in duration and 10-50 V in amplitude. Myotubes often contracted due to spontaneous, neurally evoked activity; in such cases both miniature end-plate potentials (MEPPs) and end-plate potentials (EPPs) were recorde&. The adult preparations used were frog cutaneous pectoris (at room temperature)
172
and mouse phrenic nerve hemidiaphragm (at 37 °C). Standard intracellular electrophysiological techniques were employed. Stock solutions (10 mg/ml) of cytochalasin B (Aldrich Chemical Co.) were made in dimethyl sulfoxide (DMSO). Control experiments demonstrated that D M S O at the concentrations used with the CB (0.01-0.04 ~ ) had no effect on neuromuscular transmission in tissue-cultured myoneural junctions. In 12 experiments on tissue-cultured junctions, concentrations of CB ranged from 0.8 #g/ml to 4.0 #g/ml ; the dose most commonly used was 2.0/~g/ml. Following CB application, the frequency of M EPPs and of spontaneous EPPs decreased suddenly and dramatically. This occurred with a latency of 4-20 min in 11 experiments and of 60 min in one experiment. Small changes in the amplitude of these end-plate events during onset of the CB effect cannot be excluded. A recording of spontaneous endplate activity before and after treatment with CB is shown in Fig. 1A and B: EPPs produced by external nerve stimulation were also blocked with approximately the same latency (Fig. 1A and B, inset). The time course of this effect was not examined closely enough to decide whether it was necessarily preceded by a decrement in EPP amplitude. The extent of inhibition of the frequency of spontaneous end-plate events (MEPPs and EPPs) varied from 67 7{; and 82 % in two experiments to 95 %-100 30 in the remainder. The time to onset and the degree of CB inhibition were both somewhat variable; such variability has been seen in a variety of studies using CB22, 26. When the CB was washed away, preparations eventually recovered full activity. Recovery, as measured in two experiments by nerve fiber stimulation, was complete in 5-10 min
(Fig. lC).
A
IL
L
[11!115
Fi
L
i
i
l.j"/
I
!111 [I
Ill I
I
L_ I
_.I] I0 mV I 0 0 ms
Fig. 1. Effect of CB on spontaneous and evoked end-plate activity. A: muscle synaptic potentials evoked by spontaneous firing of spinal cord neurons before CB application. Fast potentials associated with muscle twitch (see ref. 7) are at the peak of many of the synaptic potentials, The prolonged time course of the synaptic potentials may indicate that this myotube is multiply innervated (see ref. 7)~ MEPPs in this preparation, chosen to illustrate the reversibility of the CB effect, were low in amplitude and frequency and are not visible in this record. B: absence of end-plate activity 20 rain after treatment with CB. C: recovery of activity 10 rain after rinsing with CB-free medium. Inset: End,plate response produced by extracellular stimulation of nerve process. Arrow indicates shock artifact. Calibration for inset is 10 mV and 25 msec.
173 ~ ~ - ~
x L_ L LL tl J~X~X ,uala_ I
L
~ ~SmV I00 ms
Fig. 2. Miniature end-plate potentials 3 rain after addition of black widow spider venom (0.01 ffg/ml)
in a preparation initially blocked (as in Fig. IB) by CB treatment. CB appears to induce presynaptic modifications leading to a decrease in the frequency of end-plate activity. Since MEPPs in addition to EPPs are sensitive to CB, effects on the release mechanism itself, rather than on nerve propagation, are likely to be responsible for the inhibition. Axon retraction from the muscle was demonstrated not to be the primary cause for decreased end-plate activity since black widow spider venom evoked a massive discharge in end-plates blocked with CB (Fig. 2). The venomstimulated discharge confirms the presence of postsynaptic acetylcholine sensitivity (which has also been seen in preliminary iontophoresis experiments) after CB application. Inhibition of glucose uptake is thought to be the underlying mechanism of CB in some cases 1~. In two experiments, dishes were rinsed and refilled with medium made without glucose and containing 10 ~ dialyzed horse serum. End-plate activity was not inhibited after 1-2 h in glucose-free medium, and the full effect of CB was subsequently observed. To confirm and extend previously reported results 10, CB was applied to adult neuromuscular junctions. No effect of CB in doses of up to 80 #g/ml was observed on the frog cutaneous pectoris after 4 h. Similarly, no change in end-plate activity was produced by 50 #g/ml of CB for 30 min or by 25 #g/ml of CB for 60 rain in the mouse diaphragm. ]n this latter case, however, 0.25 ~o D M S O itself in 60 rain, and 0.5 ~o in 30 min, effected a reversible block of the end-plate potential and a somewhat increased M EPP rate. The explanations for the diverse cellular alterations caused by application of the cytochalasins are usually limited to three general categories: (a) interference with uptake of metabolic precursorsl4,15, (b) disorganization of a microfilament network present in many cell types 25, and (c) association with plasma membrane sites other than those related to the uptake system 19. Localization studies using [aH]cytochalasin D 23 and [3H]CB la support the feasibility of each alternative. As described above, inhibition of sugar uptake does not appear to be the primary cause of CB's inhibition of neuromuscular transmission. The two remaining possibilities must be considered. Actin- and myosin-like molecules may be structural components of microfilamentous networks found in many non-muscle cells 16. Such molecules have been isolated from neurons6,18 and appear to be concentrated in the synaptic region TM. In addition, association of CB with actin 22 and of cytochalasin D with myosin 17 has been reported. Although CB apparently does not disrupt actin-myosin interactions in striated muscle, these molecules or their regulatory factors may be different in nonmuscle cellst6, 22. Gray's ultrastructural studies 9 and, more recently, those of LeBeux and Willemot using heavy meromyosin labeling 12 have suggested the existence in the synaptic region of an organized, actin-like system of filaments which may correspond
174 to microfilaments seen in the nerve growth cone. Interestingly, the immobility of secretory vesicles in mast, chromaffin, and adenohypophyseal cells 5 may provide additional indication of such a structural lattice. Berl et al. have already proposed that an actomyosin-like complex may be involved in neurotransmitter releaseL The present results are consistent with, although certainly no proof for, a disruptive influence of CB on an actomyosin-like system responsible for maintaining the orientation of synaptic vesicles and for introducing vesicles into their release sites in the synaptic cleft. Plasma membrane modifications cannot be dismissed. CB may be involved in an inhibition of membrane-membrane interaction (as has been seen with fusing myoblasts2°), thus preventing discharge of neurotransmitter from synaptic vesicles. Alternatively, CB might depress transmitter release by altering the highly organized presynaptic plasma membrane structure (see ref. 26). The difference in sensitivity to CB between cultured chick neuromuscular junctions and junctions in adult frog and mouse cannot be completely accounted for. The release process in chicken appears, electrophysiologically, to be quite similar to that in frog and mouse s. Moreover, it is unlikely that the embryonic release system differs substantially from that in the adult 7. Penetration of CB into the adult junction might be limited by an extensive extracellular matrix not present in the cultured junction. This is not probable, however, since black widow spider venom, whose active component has a molecular weight of 100,000, seems to have unimpaired access to the adult terminal a. Since CB appears to act either at the plasma membrane or intracellularly at a microfilamentous array, the sensitivity difference may exist not in the release mechanism itself, but in membrane binding of CB or in resistance to structural changes induced by the bound drug. One remaining explanation is that the adult synaptic membrane is not permeable to CB, and thus CB never reaches its intracellular binding sites. The mechanisms responsible for secretion in neurons and in other cell types, such as pituitary cells and mast cells, may be similar 4. CB alters the secretion in a variety of these cells and may therefore effect similar changes in each. The tissuecultured neuromuscular junction should be a suitable system for using immunofluorescence 11 and other techniques to explore the role of actin-tike networks in secretion and to establish the basis for action of the cytochalasins. Supported, in part, by Research Grant NS07080 from the United States Department of Health, Education and Welfare (to Bruce S. McEwen), by a special grant from the Muscular Dystrophy Associations of America, and by a National Science Foundation Predoctoral Fellowship (to Lee L. Rubin). The authors wish to thank Drs. H. Holtzer, W. P. Hurlbut and B. S. McEwen for helpful discussions.
1 ALLISON,A, C., The role of microfilaments and microtubules in cell movement, endocytosis and exocytosis. In R. PORTERAND D. W. FITZSIMONS(Eds.), Locomotion of Tissue Cells, Elsevier, Amsterdam, 1973, pp. 109--143.
175
2 BERL, S., PUSZKIN, S., AND NICKLAS, W. J., Actomyosin-like protein in brain, Science, 179 (1973) 441-446. 3 CLARK, A. W., HURLBUT, W. P., AND MAURO, A., Changes in the fine structure of the neuromuscular junction of the frog caused by black widow spider venom, J. Cell Biol., 52 (1972) 1-14. 4 DOUGLAS, W. W., Stimulus-secretion coupling: the concept and clues from chromaffin and other cells, Brit. J. Pharmacol., 34 (1968) 451-474. 5 DOUGLAS, W. W., Involvement of calcium in exocytosis and the exocytosis-vesiculation sequence. In R. M. S. SMELLIE (Ed.), Calcium and Cell Regulation, Biochemical Society Symposium No. 39, The Biochemical Society, London, 1974, pp. 1-28. 6 FINE, R. E., AND BRAY, D., Actin in growing nerve cells, Nature New Biok, 234 (1971) ll5-118. 7 FISCHBACH, G. D., Synapse formation between dissociated nerve and muscle cells in low density cell cultures, Develop. Biol., 28 (1972) 407-429. 8 GINSBORG, B. L., Spontaneous activity in muscle fibres of the chick, J. Physiol. (Lond.), 150 (1960) 707-717. 9 GRAY, E. G., The cytonet, plain and coated vesicles, reticulosomes, multivesicular bodies and nuclear pores, Brain Research, 62 (1973) 329-335. l0 KATZ, N. L., The effects of frog neuromuscular transmission of agents which act upon microtubules and microfilaments, Europ. J. Pharmacol., 19 (1972) 88 93. 11 LAZARIDES,E., AND WEBER, K., Actin antibody: the specific visualization of actin filaments in nonmuscle cells, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 2268-2272. 12 LEBEUX, Y. J., AND WILLEMOT,J., An ultrastructural study of the microfilaments in rat brain by means of E PTA staining and heavy meromyosin labelling, lI. The synapses, Cell Tiss. Res., 160 (1975) 37-68. 13 LIN, S., SANTI, D. V., AND SPUDICH, J. A., Biochemical studies on the mode of action of cytochalasin B. Preparation of [3H]cytochalasin B and studies on its binding to cells, J. biol. Chem., 249 (1974) 2268-2274. 14 M1ZEL, S. B., AND WILSON, L., Inhibition of the transport of several hexoses in mammalian cells by cytochalasin B, J. biol. Chem., 247 (1972) 4102-4105. 15 NAKAZATO, Y., AND DOUGLAS, W. W., Cytochalasin blocks sympathetic ganglionic transmission: a presynaptic effect antagonized by pyruvate, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 1730-1733. 16 POLLARD, T. D., AND WEIHING, R. R., Actin and myosin and cell movement, CRC Critical Reviews in Biochemistry, 2 (1972) 1-65. 17 PUSZKIN, E., PUSZKIN, S., Lo, L. W., AND TANNENBAUM, S. W., Binding of cytochalasin D to platelet and muscle myosin, J. biol. Chem., 248 (1973) 7754-7761. 18 PUSZKIN, S., NICKLAS, W. J., AND BERL, S., Actomyosin-like protein in brain: subcellular distribution, J. Neurochem., 19 (1972) 1319-1333. 19 SANGER, J. W., AND HOLTZER, H., Cytochalasin B: effects on cell morphology, cell adhesion, and mucopolysaccharide synthesis, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 253-257. 20 SANGER,J. W., HOLTZER, S., AND HOLTZER,H., Effects of cytochalasin B on muscle cells in tissue culture, Nature New Biol., 229 (1971) 121-123. 21 SORIMACHI,M., OESCH, F., AND THOENEN, H., Effects of colchicine and cytoehalasin B on the release of aH-norepinephrine from guinea-pig atria evoked by high potassium, nicotine and tyramine, Naunyn-Schmiedebergs Arch. exp. Path. Pharmak., 276 (1973) 1-12. 22 SPUDICH, J. A., Effects of cytochalasin B on actin flaments, Cold Spr. Harb. Syrup. quant. Biol., 37 (1973) 585-593. 23 TANNENBAUM,J., TANNENBAUM,S. W., LO, L. W., GODMAN,G. C., AND MIRANDA,A. F., Binding and subcellular localization of tritiated cytochalasin D, Exp. Cell Res., 91 (1975) 47-56. 24 THOA, N. B., WOOTEN, G. F., AXELROD,J., AND KOPIN, I. J., Inhibition of release of dopamine-/:lhydroxylase and norepinephrine from sympathetic nerves by colchicine, vinblastine, or cytochalasin-B, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 520-522. 25 WESSELLS,N. K., SPOONER, B. S., ASH, J. F., BRADLEY,M. O., LUDUENA,M. A., TAYLOR, E. L., WRENN, J. J., AND YAMADA, K. M., Microfilaments in cellular and developmental processes, Science, 171 (1971) 135-143. 26 YAHARA, 1., In B. D. KAHAN AND R. A. REISFELD (Eds.), The Cell Surface. Immunological and Chemical Approaches, Plenum Press, New York, 1974, pp. 113-121.
