Disappearance of small vesicles from adrenergic nerve endings in the rat vas deferens caused by red back spider venom

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1 Journal of Neurocytology z, (I973) Disappearance of small vesicles from adrenergic nerve endings in the rat vas deferens caused by red back spider venom R. C. HAMILTON 1 and P. M. ROBINSON~ 1Commonwealth Serum Laboratories, Parkville, Victoria, Australia 2Anatomy Department, University of Melbourne, Parkville, Victoria, Australia Received 22 June I973; revised and accepted 2 October I973 Summary As well as causing the release of acetylcholine and the disappearance of synaptic vesicles from somatic nerve endings Latrodectus venom also causes the release of noradrenaline and the disappearance of small vesicles from adrenergic nerve endings. The large granular vesicles of the adrenergic endings are less sensitive to the action of the venom. Introduction The venoms of the black widow spider (Latrodectus mactans tredecimguttatus), and the Australian red back spider (Latrodectus mactans hasseltii), two closely related spiders, cause the disappearance of synaptic vesicles from somatic motor nerve endings in amphibia, (Clark et al., I97o; Clark et al., I972), and mammals, (Okamoto et al., I972; Hamilton, I972). The fluorescent histochemical studies of Frontali (I972), showed that the venom of the black widow spider depleted catecholamines from adrenergic nerve endings in the rat iris. In the present experiments, red back spider venom was applied to the isolated rat vas deferens to determine the effects, if any, of Latrodectus venom on the small vesicles and large granular vesicles of adrenergic nerve endings. Methods The two vasa deferentia were dissected from each of eight rats. The vasa were placed in Kreb's solution in separate organ baths. One vas of each pair was treated with venom and the other left untreated as a control. In some experiments, the vasa were stimulated transmurally with pulses of supramaximal voltage, o.i ms in duration, at a frequency of iohz for 5 s every 2 min. Contractions were recorded on a kymograph. Venom in the form of freshly ground venom glands was added to one 9 I973 Chapman and Hall Ltd. Printed in Great Britain

2 466 HAMILTON and ROBINSON organ bath to make a final concentration of o.i gland per ml. Upon addition of the venom, the vas shortened and after several minutes developed spontaneous activity. Its response to stimulation diminished, and after about 3o min, disappeared altogether. When the venom-treated vas no longer responded to stimulation, the tissues were removed from the organ baths and cut into transverse slices I mm in thickness. Some slices from each vas were frozen in liquid propane, freeze-dried and exposed to formaldehyde vapour for the demonstration of catecholamines by fluorescence microscopy. Other transverse slices were fixed in 2.5% glutaraldehyde buffered to ph 7.2 with o.i M cacodylate buffer. The pre-fixed slices were cut into small blocks, post-fixed with 1% osmium tetroxide in cacodylate buffer, dehydrated in an acetone series and embedded in Durcupan so that thin sections could be examined with the electron microscope. In other experiments, vasa deferentia were not stimulated but incubated with the venom for 3 ~ min and then processed for electron microscopy and fluorescence microscopy as above. Observations Although electron microscopy and fluorescence histochemistry were not both performed on all pairs of vasa, all the figures presented here are from the one pair of stimulated vasa which were examined by both methods. Stimulated and unstimulated vasa were of similar appearance. When transverse sections of the control vas were examined with the fluorescence microscope, spots of specific noradrenaline fluorescence were observed throughout the musclelayers (Fig. i). In the venom-treated vas, specific fluorescence had disappeared from the outermost and innermost muscle layers, i.e. the area immediately under the serosa and the area surrounding the mucosa (Fig. 2). Thus red back spider venom has a similar action to black widow spider venom, namely, when tissues are incubated in vitro in the presence of the venom they are unable to maintain their normal store of catecholamines (Frontali, 1972). During the electron microscopic examination of the control vas, adrenergic nerve endings of normal appearance were observed. The endings contained small vesicles, some of which were granular, some large granular vesicles, and a few small mkochondria (Fig. 3). When sections from the middle layers of the venom-treated vas (areas in which specific fluorescence was still present in the fluorescence specimens) were examined in the electron microscope, nerve endings of normal appearance were found. On the other hand, in the outermost and innermost muscle layers, (i.e. areas where catecholamine depletion had Fig. I. Cross-section of control rat vas deferens processed for catecholamine fluorescence, a, adventitia; ram, Mucosa-muscle junction (bar = IOO am). Fig. 2. Cross-section of venom-treated rat vas deferens. Note loss of fluorescence from outer muscle layer (o) and inner muscle layer (i). Fluorescence remains in middle muscle layer (m). Because this section of vas is larger than the control, the mucosa lies outside this field, a, adventitia (bar = IOO ~.m). Fig. 3. Axons from control rat vas deferens. These axons contain small vesicles (s), large granular vesicles (l), and mitochondria (m) (bar = I ~zm). Fig. 4. Axon from innermost muscle layer of venom-treated rat vas deferens. This axon contains very few small vesicles (bar = I am). Fig. 5. Axons from innermost muscle layer of venom-treated rat vas deferens. One axon contains only large granular vesicles (bar = I ~m). Fig. 6. 'Empty' axon from innermost muscle layer of venom-treated rat vas deferens (bar = I am).

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4 468 HAMILTON and ROBINSON occurred), most nerve endings were devoid of all types of vesicles (Fig. 6). Other nerve endings were similar in appearance but contained one or two large granular vesicles (Fig. 5), whilst a few partially affected nerve endings with a reduced number of small vesicles were also observed (Fig. 4). It was not possible in this series of experiments to determine precisely the order of disappearance of axonal organelles, because most axon profiles were either normal (in the unaffected areas), or totally depleted. However, examination of the full range of axon profiles which were present led us to the view that large granular vesicles were either not depleted by the venom or were the last structures to disappear from the axon profiles. Discussion Depletion of noradrenaline by displacement with ~-methyl-m-tyrosine or inhibition of vesicle uptake by reserpine results in loss of granules from the small granular vesicles, but no discernible change in the number of vesicles present (Van Orden et al., 1966). Consideration of this and other evidence has led to support for the view that a form of exocytosis (in which an empty vesicle remains) occurs in drug-induced noradrenaline depletion (Geffen and Livett, I97I). The disappearance of vesicles after Latrodectus venom treatment, on the other hand, indicates that a mechanism similar to reverse pincoytosis, in which the vesicle membrane is destroyed or becomes incorporated in the plasma membrane, may be taking place in this case. Although the release of noradrenaline and the destruction of granular vesicles can be caused by 6-hydroxydopamine there is a temporal separation of these two effects. Transmitter release precedes vesicle destruction by a few hours (Malmfors and Sachs, I968; Tranzer and Thoenen, 1968). Latrodectus venom causes transmitter release and vesicle destruction to occur simultaneously. No significant changes in vesicle numbers have been detected in adrenergic axons after noradrenaline release under physiological conditions (see Geffen and Livett, 1971). The observation that the large granular vesicles are relatively unaffected by the Latrodectus venom may indicate that these vesicles are not immediately involved in the release of noradrenaline. The present studies have been carried out with unfractionated venom preparations. It is possible, therefore, that the release of transmitter and disappearance of vesicles are caused by different components in the venom. However, ~-bungarotoxin, a pure toxin from the venom of the krait (Bungarus multicinctus), causes both the release of acetylcholine and the disappearance of synaptic vesicles from somatic motor nerve endings, (Chen and Lee, I97O), and it is possible that a similar single molecular species is the active component of Latrodectus venom. Further experiments with purified venom will be necessary to establish which component or components of the crude venom is (are) responsible for the changes described in this study. Acknowledgements We thank Roslyn Perry for help with the fluorescence histochemistry and Rudolfus Sikkes for his ultramicrotomy.

5 Disappearance of small vesicles from adrenergic nerve endings 469 References CHEN, I. L. and LEE, C. Y. (197o) Ultrastructural changes in motor nerve terminals caused by ~3- bungarotoxin. Virchows Archly Abteilung B. Zell Pathologie 6, CLARK, A. W., HURLBUT, W. P. and MAURO, A. (I972) Changes in the fine structure of the neuromuscular junction of the frog caused by black widow spider venom.ffournal of CellBiology 52, x-14. CLARK~ A. W., MAURO~ A., LONGENECKER, H. E. JR. and HURLBUT, W. P. (I97o) Effects of black widow spider venom on the frog neuromuscular junction. Effects on the fine structure of the frog neuromuscular junction. Nature (London) FRONTALI, N. (I972) Catecholamine-depleting effect of black widow spider venom on iris nerve fibres. Brain Research 373 I46-8. G E v r E N, e. S. and e I V E T T, B. G. (1971 ) Synaptic vesicles in sympathetic neurons. Physiological Reviews 51, 98-I57. HAMILTON, R. C. (I972) Ultrastructural studies of the action of Australian spider venoms. In 3orh Annual Proceedings of the Electron-Microscopy Society of America (edited by ARCENEAUX, C. J.) pp. 4o-1. Baton Rouge: Claitor's Publishing Division. MALMFORS, T. and SACHS, C. (1968) Degeneration of adrenergic nerves produced by 6-hydroxy- dopamine. European Journal of Pharmacology 3, OKAMOTO~ N., LONGENECKER, H. E. JR., RIKER, W. F. and SONG, S. K. (1971) Destruction of mammalian motor nerve terminals by black widow spider venom. Science 172, TRANZER, J. P. and THOENEN, H. (1968) An electron microscopic study of selective, acute degeneration of sympathetic nerve terminals after administration of 6-hydroxydopamine. Experientia 24, I55-6. VAN ORDEN~ L. S. III., BLOOM, F. E., BARgNETT, g. J" and GIARMAN, N. j. (~966) Histochemical and functional relationships of catecholamines in adrenergic nerve endings. I. Participation of granular vesicles. Journal of Pharmacology and Experimental Therapeutics 154, I85-99-