Uhrastructural Changes in the Motor Nerve Terminals Caused by 13-Bungarotoxin*

Transcription

1 Virchows Arch. Abt. B Zellpath. 6, (1970) 9 by Springer-Verlag 1970 Uhrastructural Changes in the Motor Nerve Terminals Caused by 13-Bungarotoxin* I-LI CHEN and C. Y. LEE Department of Anatomy and Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China Received July 22, 1970 Summary. fl-bungarotoxin, isolated from Buuyarus.raulticinctus (banded krait) venom, induced increased profiles of opened synaptic vesicles at the axolemma, accompanied with decreased numbers of synaptic vesicles and subsequently, almost complete depletion of the vesicles in the axon terminal of motor endplates. The toxin also caused progressive swelling and vacuolization of the mitochondria in the motor nerve endings. Two kinds of neurotoxin, called ~- and fl-bungarotoxin respectively, have been isolated from the venom of Bungarus multicinctus {banded krait) by means of zone electrophoresis on starch. ~-Bungarotoxin produces a "non-depolarizing" type of neuromuscular block by acting on the postsynaptie membrane of the motor endplate, whereas fl-bungarotoxin acts presynaptically, depressing acetylcholine release from the motor nerve endings and leaving the sensitivity of the endplate to acctylcholine unaffected (Chang and Lee, 1963). Subsequent studies have revealed that fl-bungarotoxin causes complete disappearance of miniature endplate potentials (MEPPs) after a period of initial increase in the frequency of MEPPs (Lee and Chang, 1966). The MEPPs are supposed to result from spontaneous quantal release of acetylcholine from the axon terminals in the motor endplate (Fatt and Katz, 1952; del Casti]lo and Katz, 1956). The presence of synaptic vesicles in the axon terminals has been postulated to link with the quantal packets of acetylcholine release (Katz, 1959; de Robertis, 1964). The present work was started in the hope of obtaining any correlating morphological changes in the motor nerve terminals with the observed electrophysiological effects of fi-bungarotoxin. Materials and Methods The bungarotoxins (~ and fl) were isolated from the venom of Bungarus raulticinctu8 by column chromatography on CM-Sephadex (C-50) and further purified by rechromatography on CM-cellulose column. Both of them have been shown to be free from any known enzyme activities, such as phospholipase A, acetylcholinesterase, NADase, and phosphomonoesterase, which are present in the crude venom {Lee et al., in preparation). Sixteen adult albino mice (NIH strain) were injected intraperitoneally with ~g/g of fl-bungarotoxin and were killed randomly at 30 min, hrs, and 2-4 hrs after envenomation. Four mice were injected with 0.4 tzg/g of ~-bungarotoxin and killed immediately * We wish to thank Professor B. Katz, Department of Biophysics, University College, London, for his kindness in revising the manuscript.

2 fl-bungarotoxin and Motor Nerve Terminals 319 Fig. 1. Motor endplate of a control mouse. The axon terminal is filled with numerous synaptic vesicles and a few elongated mitochondria before respiratory arrest. Some other mice served as saline-injected controls. All mice were perfused with 3% glutaraldehyde in 0.1 M phosphate buffer (ph, 7.4) through the left ventricle. After perfusion the endplate zone of the diaphragm was cut into small pieces and further fixed with 3% glutaraldehyde followed by osmication. The tissues were then dehydrated and embedded in Epon. Thin sections showing gold interference colors were stained with lead citrate or uranyl acetate followed by lead citrate and examined with a Hitachi HU ll electron microscope. Results The fine structure of diaphragmatic endplates in mice is similar to that of intercostal ones (Zacks, 1964). The axon terminal of motor fibers lies in a small groove in the surface of the muscle fiber and is filled with numerous synaptic vesicles, a few elongated mitochondria as well as occasional bundles of fine neurofilaments (Fig. 1). One or two profiles of small invaginations in the axolemma, which have been suggested as the opening of synaptic vesicles into the primary synaptic cleft (de Robertis, 1959, 1964), are encountered in some sections of axon terminals. Similarly to the results of Zacks (1964) with cobra (Naja haje) venom, no decrease in the number of synaptic vesicles nor any alteration in the ultrastructure of the endplates was observed when ~-bungarotoxin was used, whose mode of action is similar to that of cobra neurotoxin (Lee and Chang, 1966). After fl-bungarotoxin administration some definite changes were observed in the axon terminals in the endplates. In general, 30 min after envenomation profiles of opened synaptic vesicles at the axolemma of the nerve terminal seemed to

3 Fig. 2. Motor endplate of the mouse sacrificed an hour after fl-bungarotoxin injection. A marked decrease in number of synaptic vesicles is seen in the axon terminal. Most of the remaining vesicles accumulate at the periphery of the axolemma facing the primary synaptic gap and there are a few profiles suggesting exteriorization of the content of the vesicles, (opened synaptic vesicles) (arrows). Mitochondria in the axon terminal are swollen and spheroid in shape but those in myclinated portion of the axon, the muscle fiber and the Schwann cell appear to be morphologically normal. Note numerous neurofilaments in the nerve terminal, x22000

4 I-Li Chen and C. Y. Lee: fl-bungarotoxin and Motor Nerve Terminals 321 Fig. 3. Motor endplate of the mouse sacrificed an hour after fl-bungarotoxin injection. In this nerve terminal, almost all the synaptic vesicles are in contact with the axolemma or open into the primary synaptic gap. Mitochondria in the axon terminal are swollen and spherical. Numerous neurofilaments are present in the axon terminal

5 322 I-Li Chen and C. Y. Lee: be increased. However, the structure and number of synaptic vesicles and most of the mitoehondria in the axon terminals appeared to be unchanged. During 1 to 1.5 hrs after injection the number of synaptic vesicles decreased considerably, accompanied by a further increase in the number of opened synaptic vesicles at the axolemma and the size of the synaptic vesie]es appeared to be slightly larger than that of the controls (Figs. 2 and 3). Early in this stage the mitoehondria in the axon terminals began to swell and later showed vaeuolization. Two to 4 hrs after injection the synaptic vesicles in many axon terminals were almost completely depleted, and profiles of opened vesicles were encountered on]y occasionally. Vaeuolization of the mitochondria in the axon terminals became prominent and small mitochondrial vacuoles fused to form larger ones. The terminal axons now showed, apart from swollen and vacuolated mitochondria and a few rather large vesicles, a fine granular or floceulent matrix surrounded by an intact axolemma (Figs. 4 and 5). Throughout all the phases following fl-bungarotoxin injection the fine structure of muscle fibers, fibroeytes, endothelial cells and myelinated axons in the diaphragm appeared to be normal. Discussion Attempts have been made to deplete synaptic vesicles in the nerve endings by various means. De Robertis and Ferreira (1957) reported that high frequency electrical stimulation of the sp]anchnic nerve caused a depletion of synaptic vesicles within the cholinergic nerve endings of the adrenal medulla. Birks et al. (1960), however, were not able to obtain conclusive findings from muscles which had been subjected to even more drastic synaptic stimulation (i.e., exposure to high potassium concentration and hypertonic media). On the other hand, Hubbard and Kwanbungumpen (1968) reported that hypertonie KC1 solution caused depletion of the whole population of synaptic vesicles without a change in population of "fusing vesicles" in the motor nerve terminals. Experiments with botulinum toxin, which is known to prevent the release of acetylcholine from the cholinergic nerve endings, also revealed no abnormalities in the ultrastructure of motor endplates (Thesleff, 1960; Zacks et al., 1962). Although Hubbard and Kwanbungumpen (1968) doubted that the "fusing vesicles" (corresponding to opened synaptic vesicles in our materials) represented a releasing process of the vesicle content, the marked increase in profiles of opened synaptic vesicles at the axolemma accompanied by a considerable depletion of the synaptic vesicles within the axon terminals observed in the present studies suggests that the opened synaptic vesicles at the axolemma represent a releasing rather than an absorbing process (Birks et al., 1960; Andres, 1964; Westrum, 1965) in the axon terminals. De Robertis and his co-worker (1956, 1964) have reached a similar conclusion in Figs. 4 and 5. Motor endplates of the mice sacrificed two hours after fl-bungarotoxin administration. Synaptic vesicles in the nerve terminals have been almost completely depleted and swelling and vacuolization of mitochondria in the nerve terminals are apparent. The nerve terminals are filled with a finely granular or flocculent matrix. Note that the mitochondria in the muscle fibers are morphologically normal. Fig. 4, 22000; Fig. 5, 15000

6 fl-bungarotoxm and Motor Nerve Terminals 323 Figs. 4 and 5

7 324 I-Li Chen and C. Y. Lee : the retina of dark-adapted animals submitted to an intense light stimulation. These morphological findings, coupled with the pharmacological data that fibungarotoxin markedly increases the frequency of MEPPs in the early stage of intoxication (Lee and Chang, 1966), provide strong support for the hypothesis that acetylcholine is stored in synaptic vesicles and released from the axon terminals in quantal packets (Katz, 1959: de Robertis, 1964). This view is further strengthened by the findings that the synaptic vesicles in many axon terminals are almost completely depleted during the later stage of poisoning with consequent depression of the release of acetylcholine from the motor nerve endings and almost complete disappearance of the MEPPs (Lee and Chang, 1966). The exact mechanism by which fi-bungarotoxin exerts its effect on the motor nerve endings is not well understood. The swelling and vacuolization of the axoplasmic mitochondria in the axon terminals does not seem to be due to fixation and embedding artifacts as was implied in the studies with botulinum toxin (Zacks, 1964), since it was consistently observed after administration of flbungarotoxin. The change is not due to phospholipase A, a mitochondrial poison contained in the crude venom, since /~-bungarotoxin is free from such enzyme activity. It is also unlikely that the acetylcholine synthesis is directly interfered with by fi-bungarotoxin, since it does not inhibit acetylation of choline by brain extracts in the presence of ATP (Lee and Chiou, umpublished). However, it is possible that fl-bungarotoxin selectively affects the membranes of the motor nerve endings, accelerating the release of vesicle content and only secondarily causes changes in the axonal mitochondria, which may lead to a blockage of the energy supply required for resynthesis of acetylcholine (de Robertis, 1964). As a result, neuromuscular blockage takes place due to exhaustion of acetylcholine stores in the motor nerve endings. References Andres. K. H. : Mikropinozytose im Zentralnervensystem. Z. Zellforsch. 64, (1964). Birks, R., Huxley, H. E., Katz, B.: The fine structure of the neuromuscular junction of the frog. J. Physiol. (Lond.) 150, (1960). Castillo, J. del, Katz, B. : Biophysical aspects of neuromuscular transmission. Progr. Biophys. 6, (1956). Chang, C. C., Lee, C. Y.: Isolation of neurotoxins from the venom of Bungarus multicinctus and their modes of neuromuscular blocking action. Arch. int. Pharmacodyn. 144, (1963). De Robertis, E.: Submicroscopic morphology of the synapse. Int. Rev. Cytol. 8, (1959). -- Histophysiology of synapses and neurosecretion. Oxford-London: Pergamon Press Vaz Ferreira, A. V. : Submicroscopic changes of the nerve endings in the adrenal medulla after stimulation of the splanchnic nerve. J. biophys, biochem. Cytol. 3, (1957). -- Franchi, C. M. : Electron microscopic observations on synaptic vesicles in synapses of the retinal rods and cones. J. biophys, biochem. Cytol. 2, (1956). Fatt, P., Katz, B. : Spontaneous subthreshold activity at motor nerve endings. J. Physiol. (Lond.) llt, (1952). Hubbard, J. I., Kwanbunbumpen, S. : Evidence for the vesicle hypothesis. J. Physiol. (Lond.) 194, (1968). Katz, B. : Mechanism of synaptic transmission. Rev. rood. Phys. 3], (1959). Lee, C. Y., Chang, C. C. : Modes of actions of purified toxins from elapid venoms on neuromuscular transmission. Mem. Inst. Butantan, Simp. Internac. 33 (2), (1966).

8 - - Metzfer, fl-bungarotoxin and Motor Nerve Terminals 325 Thesleff, S. : Supersensitivity of skeletal muscle produced by botulinum toxin. J. Physiol. (Lond.) 151, (1960). Westrum, L. E. : On the origin of synaptic vesicles in cerebral cortex. J. Physiol. (Lond.) 179, 4p-6p (1965). Zacks, S. I. : The motor endplate. Philadelphia: W. B. Saunder Co J.F., Smith, J. F., Blumber, J. M. : Localization of ferritin-labelled botulinus toxin in the neuromuscular junction of the mouse. J. Neuropath. exp. Neurol. 21, (1962). I-Li Chen, Ph.D. Department of Anatomy College of Medicine National Taiwan University Taipei, Taiwan Republic of China