Detection of depolarization-induced coated vesicles within presynaptic terminals in cat sympathetic ganglia maintained under a low temperature

Brain Research, 151 (1978)201-205 © Elsevier/North-HollandBiomedicalPress

201

Detection, of depolarization-induced coated vesicles within presynaptic terminals in cat sympathetic ganglia maintained under a low temperature

KEN KADOTA and TOMOKO KADOTA Departments of Pharmacology and Anatomy, Osaka University Medical School, Kita-ku, Osaka 530 (Japan)

(Accepted February 16th, 1978)

Endocytosis has been suggested to be an important property of nerve terminal membrane either for synaptic vesicle formation or for retrograde axonal flow for macromoleculesS,11,12,15-1s, and is often mediated by coated vesicles with a unique coat structure. The present authors and Gray and Willis have shown that in the process of formation of synaptic vesicles the coated vesicles are first built from the presynaptic terminal membrane. After they shed the shell structure of proteinaceous nature, they become indistinguishable from the ordinary synaptic vesicles 4,7-1°. Douglas et al. 2, who identified coated vesicles in the rat hypophysis, proposed that formation of coated vesicles was linked to retrieval of the neurosecretory granule membrane that was incorporated into the terminal surface during exocytosis. In frog nerve-muscle preparations, Heuser and Reese6 observed that, after continuous nerve stimulation, the nerve terminal expanded with a decrease of synaptic vesicles and an increase of coated vesicles, as well as non-vesicular membrane-delimited compartments. They suggested that the synaptic vesicle membrane was retrieved via the coated vesicle and the non-vesicular compartment from the enlarged terminal surface membrane. Thus it seems likely that the function of the coated vesicles is essential in the recovery process of presynaptic changes after stimulation. However, this is not consistent with a report of Pysh and Wiley 14. Studying effects of electrical stimulation upon the cat sympathetic ganglia in situ, these investigators did not observe coated vesicles within presynaptic terminals, though other effects of stimulation such as synaptic vesicle depletion and terminal membrane increase were evident immediately after stimulation and disappeared after a period of rest. The purpose of this investigation was to find a possible explanation for Pysh and Wiley's 14 failure to observe coated vesicles in the stimulated presynaptic terminals. In view of the fact that Heuser and Reese6, who identified coated vesicles, carried out their experiments under low temperature, it seemed to us that temperature would be critical for revealing coated vesicles. We therefore attempted to repeat Pysh and Wiley's experiment with isolated preparations under two different temperatures: at 37 °C and at 10 °C. The results show that coated vesicles did appear within the presynaptic terminals of the cat sympathetic ganglia stimulated at 10 °C, and in those

202 stimulated at 37 °C and then rested at 10 °C. A preliminary report o f the present findings has appeared elsewhere 1°. The superior cervical sympathetic ganglia of both sides were dissected from cats anesthetized by an intraperitoneal injection of pentobarbital (30-50 mg/kg) and incubated in Krebs solution with a supply o f COz/O2 (5: 95) gas mixture. The temperature of the bathing fluid was kept either at l0 °C or at 37 °C with a thermomodule, which was placed in the b o t t o m of the fluid chamber. The preganglionic nerve of either side served as control, while the other was electrically stimulated with a bipolar platinum electrode after lifting it into air f r o m the bathing solution. The stimulus parameters were the same as in Pysh and Wiley's experiment14: 1 msec square pulses o f 20 V were applied at 10 H z for 30 min. The stimulus pulses were supplied from a N i h o n K o h d e n M S E - 3 R stimulator. To examine effects of stimulation and recovery processes, 4 types of specimen were prepared (I, II, III and IV): ~I) Specimen I : after having been stimulated for 30 min at 37 °C, the ganglia were fixed; (2) Specimen 11: same as Specimen I, except that stimulation was made at 10 °C; (3) Specimen I I I : after having been stimulated for 30 min at 37 °C, the ganglia were allowed to recover for 10 min at 37 °C and then fixed; (4) Specimen IV: the same as Specimen II1, except that the poststimulation recovery was allowed to proceed at 10 °C. The materials were processed for electron microscopy with conventional procedures consisting of 2 ~ osmium fixation in 0.1 M phosphate buffer at p H 7.3 (1 h), block staining with 4 ~ uranyl acetate solution (30 min), dehydration in ascending concentrations of ethanol, and embedding in E p o n 812. Thin sections were cut with a Porter-Blum MT-I ultramicrotome in the transverse plane of ganglia and double-stained with uranyl acetate and lead citrate. Specimens were examined under Hitachi 11DS and 12 electron microscopes. In transvetse sections synapses between preganglionic nerve fibers and principal cell dendrites were located clearly. Synaptic organizations of the unstimulated superior cervical ganglia are as follows (Figs. 1 and 2). The fine structure of synapses in the unstimulated ganglia maintained at 37 °C was essentially similar to that seen in the control ganglia incubated at 10 °C. At contact regions both pre- and postsynaptic membrane are seen to be associated with dense cytoplasmic material. On the presynaptic terminal membrane this material forms dense projections. M a n y presynaptic bags, usually oval in shape, have a diameter ranging from 0.4 to 3/~m. Two kinds of synaptic vesicles are

Figs. 1 and 2. Unstimulated control synapses. Presynaptic vesicles consist of a large number of agranular synaptic ones and a few dense-cored ones. 1 : a synapse in the ganglion incubated at 37 °C. 2: a synapse in the material maintained at 10 °C. Bar = 0.5/~m. Fig. 3. A synapse stimulated at 37 °C and fixed immediately (Specimen 1). Presynaptic terminals are swollen and undulated. Synaptic vesicles are depleted. A small non-vesicular compartment is seen in place of synaptic vesicles (double-headed arrow). Bar -- 0.5/~m. Fig. 4. A synapse stimulated at 10 °C and fixed immediately (Specimen II). Coated pits and coated vesicles are seen (small and large arrows). Bar = 0.5 ttm. Fig. 5. A synapse stimulated at 37 °C and then rested at the same temperature (Specimen 11I). Surface contour and synaptic vesicle number have partially restored to normal. Coated vesicles are not seen. Double-headed arrow; non-vesicular compartment. Bar -- 0.5/zm. Fig. 6. A synapse stimulated at 37 °C and then rested at 10 °C (Specimen IV). Coated pits and coated vesicles are seen (small and large arrows). Bar = 0.5/tin.

204 seen: one is the small agranular vesicle, and the other, the dense-cored one. The agranular synaptic vesicles, predominant in number, are spheroidal in shape and 40-50 nm in diameter. The dense-cored vesicles are 70-100 nm in diameter. Other presynaptic structures are mitochondria, which scatter among agranular and dense-cored vesicles at some distance from the presynaptic membrane. Coated vesicles are not seen in nerve endings in the ganglia incubated at 37 °C. Though rarely, they are found in the materials maintained at 10 °C. The present findings on isolated and incubated cat superior sympathetic ganglia are consistent with those described by Elfvin z and Pysh and Wiley 14 on the ganglia in vivo. Stimulation of the preganglionic fiber at 37 °C caused marked changes in the fine structure of many presynaptic terminals (Specimen I) (Fig. 3). The terminals were expanded and undulated. Synaptic vesicles were depleted. Rarely, a small number of irregular membrane-delimited compartments (non-vesicular compartments) were seen. Mitochondria were swollen. These findings following stimulation at 37 °C agree in many respects with those of Pysh and Wiley 14 obtained from in vivo preparations. Electrical stimulation of the preganglionic nerve of the ganglia maintained at 10 °C resulted in an increase in the number of coated vesicles and coated pits in nerve endings (Specimen II) (Fig. 4). Reduction of the number of synaptic vesicles was not apparent. The terminals were slightly undulated. Other presynaptic changes were the appearance of non-vesicular compartments which often had coats on their membrane surface. In the ganglia stimulated at 37 °C and then rested at the same temperature (Specimen III), most nerve endings appeared nearly normal in both the surface contour and the synaptic vesicle number, although a slight enlargement of surface membrane, a small number of non-vesicular compartments, and swelling of mitochondria were seen. Coated vesicles were not seen in these nerve endings (Fig. 5). In the ganglia stimulated at 37 °C and then allowed to recover at 10 °C (Specimen IV), many nerve terminals partially returned to normal. The degree of recovery, however, was less marked at 10 °C than at 37 °C. A great difference between i0 °C and 37 °C was that the coated vesicles and coated pits were seen in most nerve endings rested at the lower temperature (Fig. 6). The present findings on the isolated cat sympathetic ganglia, either stimulated at 37 °C or stimulated and rested at 37 °C, agree in many respects with those described by Pysh and Wiley 14. The effectiveness of low temperature on coated vesicle formation can explain why Pysh and Wiley 14were unable to see coated vesicles in presynaptic terminals recovering from the effects of stimulation. As shown in Figs. 3 and 4, coated vesicles are identified in presynaptic bags stimulated at 10 °C, but not in those stimulated at 37 °C. In addition, coated vesicles can be seen in nerve endings stimulated at 37 °C and subsequently rested at 10 °C, but not in those activated and rested at 37 °C (Figs. 5 and 6). The results suggest that the depolarization-induced coated vesicles are short-lived at normal body temperature and have slow turnover rates at temperatures lower than this. This suggestion is given support by a report of Model et al. ~a, who have described a paralyzing effect of cold on coated endocytosis; these investigators have shown that repetitive stimulation of the Mauthner fiber of hatchet fishes cooled to 12-14 °C results in an increase in the number of coated vesicles within presynaptic terminals of the axoaxonic synapses between the Mauthner fibers and the giant fibers. Moreover, Andersson

205 et al. 1 have d e m o n s t r a t e d i n cultured h u m a n fibroblasts that coated endocytosis is inh i b i t e d at 4 °C, b u t p r o m p t l y proceeds at 37 °C a c c o m p a n y i n g f o r m a t i o n of coated vesicles a n d a s u b s e q u e n t loss of the shell structure of vesicles. We t h a n k Prof. K. I w a m a for c o n t i n u o u s interest in this w o r k a n d for help i n p r e p a r a t i o n of the manuscript. This w o r k was partially supported by a research g r a n t from the Japanese Education Ministry.

1 Andersson, R. G. W., Brown, M. S. and Goldstein, J. H., Role of coated vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts, Cell, 10 (1977) 351-364. 2 Douglas, W. W., Nagasawa, J. and Schultz, R. A., Electron microscopic studies on the mechanism of secretion of posterior pituitary hormones and significance of microvesicles ('synaptic vesicles'): evidence of secretion by exocytosis and reformation of microvesicles as a by-product of this process, Mern. Soc. endocrinol., 19 (1970) 353-378. 3 Elfvin, L.-G., The ultrastructure of the superior cervical sympathetic ganglion of the cat. II. The structure of the preganglionic end fibers and the synapses as studied by serial sections, J. ultrastruct. Res., 8 (1963) 441-476. 4 Gray, E. G. and Willis, R. A., On synaptic vesicles, complex vesicles, and dense projections, Brain Research, 24 (1970) 149-168. 5 Hendry, I. A., St/Sckel, K., Thoenen, H. and Iversen, L. L., The retrograde axonal transport of nerve growth factor, Brain Research, 68 (1974) 103-121. 6 Heuser, J. E. and Reese, T. S., Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscularjunction, J. Cell Biol., 57 (1973) 315-344. 7 Kanaseki, T. and Kadota, K. ,The 'vesicle in a basket'. A morphological study of the coated vesicle isolated from nerve endings of the guinea pig brain, with special reference to the mechanism of membrane movements, J. Cell BioL, 42 (1969) 202-220. 8 Kadota, K. and Kadota, T., Isolation of coated vesicles, plain synaptic vesicles, and flocculent material from a crude synaptosome fraction, J. Cell BioL, 58 (1973) 125-151. 9 Kadota, T., Kadota, K. and Gray, E. G., Coated-vesicle shells, particle/chain material, and tubulin in brain synaptosomes. An electron microscope and biochemical study, J. Cell Biol., 69 (1976) 608~521. 10 Kadota, K., Effects of temperature on the appearance of coated endocytotic vesicles during transmitter release at frog neuromuscular junctions and at cat superior sympathetic ganglia, Jap. J. Pharmacol., 27 (1977) Suppl. 146P. 11 Kristensson, K. and Olsson, Y., Diffusion pathways and retrograde axonal transport of protein tracers in peripheral nerves, Progr. Neurobiol., 1 (1973) 87-109. 12 LaVail, J. H. and LaVail, M. M., The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic study, J. comp. Neurol., 157 (1974) 303358. 13 Model, P. G., Highstein, S. M. and Bennett, M. V. L., Depletion of vesicles and fatigue of transmission at a vertebrate central synapse, Brain Research, 98 (1975) 209-228. 14 Pysh, J. J. and Wiley, R. C., Synaptic vesicle depletion and recovery in cat sympathetic ganglia electrically stimulated in vivo, J. Cell BioL, 60 (1974) 365-374. 15 Teichberg, S., Holtzman, E., Crain, S. M. and Peterson, E. R., Circulation and turnover of synaptic vesicle membrane in cultured and fetal mammalian spinal cord neurons, J. Cell BioL, 67 (1975) 215-230. 16 Turner, P. T. and Harris, A. B., Ultrastructure of synaptic vesicle formation in cerebral cortex, Nature (Lond.), 242 (1973) 57-59. 17 Zacks, S. I. and Saito, A., Uptake of exogenous horseradish peroxidase by coated vesicles in mouse neuromuscularjunctions, J. Histochem. Cytochem., 17 (1969) 161-170. 18 Zimmerman, H. and Whittaker, V. P., Effect of electrical stimulation on the yield and composition of synaptic vesicles from the cholinergic synapses of the electric organ of Torpedo: a combined biochemical, electrophysiological and morphological study, J. Neurochem., 22 (1974) 435-450.