- Email: [email protected]
Granular coated vesicles in the goldfish Mauthner cell following axotomy
Brain Research, 273 (1983) 341-346 Elsevier
341
Short Communications
Granular coated vesicles in the goldfish Mauthner cell following axotomy MALCOLM R. WOOD and DONALD S. FABER Division of Neurobiology, Department of Physiology, SUNY at Buffalo, Buffalo, NY 14214 (U.S.A.) (Accepted April 26th, 1983) Key words: granular coated vesicles- - Mauthner cell - - axotomy
Axotomy of the goldfish Mauthner cell induces a marked increase in the occurrence of granular coated vesicles in close association with axosomatic synapses. This phenomenon, which persists for over 200 days, is discussed in relation to other morphological and physiologicalchanges which occur over the same time period. Coated vesicles are widely encountered in the nervous systems of a variety of vertebrate preparations 17. Numerous functions have been attributed to them, including receptor membrane insertion during developmental synaptogenesis 1,13,15 and during synapse formation following deafferentation 2,11, membrane recycling 4.s, and the removal of specialized regions of contact such as gap junctions 10 and puncta adherentialL In addition their presence was originally considered to be a sign of degeneration in general 16. Less widely encountered are coated vesicles containing an electron dense core or granule, the socalled 'granular coated vesicle '1 or 'double-walled coated vesicle '4. These structures have been implicated in the process of membrane retrieval or the internalization of apposed cell surface membranes. The results from many studies suggest a common denominator: that granular coated vesicles are frequently present in systems undergoing radical changes, e.g. during synapse formation 1.2,11.15, neuronal maturation ~2 or remodelling 4 during development. Axotomy of the teleost Mauthner (M-) cell induces a typical chromatolytic response and a persistent shift in membrane organization as evidenced by a spread of electrical excitability from the initial segment and axon hillock only into the soma and proximal dendrites. Both changes appear at about 15-20 days 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
postaxotomy and last at least 200 daysS. In addition axotomy triggers slow ongoing reactive deafferentation of excitatory terminals which normally make synaptic contact onto the soma 19. These long-term changes make this preparation a model system for the study of neuronal membrane regulation and intercellular communication. This is particularly relevant in view of the appearance of granular coated vesicles in the M-cell soma 22-207 days postaxotomy as described in this communication. Goldfish (Carassius auratus), 5-6 in. in body length, were anesthetized in MS-222 (0.3 g/l) and their spinal cords were completely transected by inserting a no. 11 scalpel blade into the lateral body wall dorsal to the operculum. The distance between the M-cell soma and transection point was 8-10 mm. Operated fish were maintained at a constant temperature (15 °C) and sacrificed at intervals up to 207 days post-axotomy. Fixation for light and electron microscopy consisted of intracardial perfusion of fixative containing 1% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M Na cacodylate with 1.5 mM CaCI2. Although initial preparations were perfused first with fish saline~8, this step was subsequently omitted, in order to get improved ultrastructural preservation (Triller, personal communication). Following the fixative perfusion, the fish brain and proximal part of the spinal cord including the lesion, were exposed
342
Fig. 1. Electron micrograph illustrating part of the initial segment (IS) of a control M-cell. Within the cytoplasm, two coated vesicles (solid arrows) are evident close to the plasmalemma, together with two other coated stuctures (arrowheads) apparently budding off from elements of the smooth endoplasmic reticulum. Scale: 0.5/~m. and immersed in situ in the same fixative for 3 h; subsequently, the tissue was dissected out and fixed for a further 2 h. T r e a t m e n t continued as follows: the brain was buffer washed in 0.1 M Na cacodylate for 2 h; then it was t r i m m e d , osmicated overnight in 2% OsO 4 in 0.1 M N a cacodylate, d e h y d r a t e d in a g r a d e d acetone series and e m b e d d e d in Spurr. Serial thick (3 ~ m ) sections were cut to locate (a) the distal and (b) the proximal regions of the lateral d e n d r i t e and (c) the M-cell soma. A t each of these 3 identified regions, serial thin (60 nm) sections were p r e p a r e d for examination on either a Jeol 100B or Siemens 1 electron microscope. F o r goldfish 5-6 in. body length, the mean M-cell area20 is estimated at 2800 ~tm 2. F o r each p r e p a r a t i o n in this study, the M-cell has been examined ultrastructurally in a m i n i m u m of 3 separate areas of the lateral dendrite and through areas of the soma which include the nucleus. In 4 control fish, ordinary c o a t e d pits and vesicles 11.17 are apparently restricted to the axon hillock/ initial segment region as 29 out of a total of 31 were observed in that region, the remaining 2 being in the soma and lateral dendrite. W h e n present in these regions, they were subjacent to an afferent terminal,
elements of glia, extracellular spaces or attached to elements of the SER. Their m e a n d i a m e t e r m e a s u r e d 112 + 16.6 nm (range 67-149 nm; n = 31) including the fuzzy coat of 20.7 + 3.5 nm thickness (range 15-29 nm; n = 27). Some of these vesicles were associated with the p l a s m a l e m m a of the initial segment, forming a flask-shaped structure or coated pit there. As such, the pits o p e n e d into the extracellular space frequently in close proximity to an afferent terminal immediately adjacent to, and in continuity with, the postsynaptic m e m b r a n e . Less frequently, the coated pits were a p p o s e d to elements of glia or expanses of extracellular space between a d j a c e n t terminals and/ or glia. In Fig. 1, two coated vesicles are evident close to the initial segment (IS) m e m b r a n e together with two o t h e r coated structures a p p a r e n t l y budding off from elements of the smooth e n d o p l a s m i c reticulum (SER). The size, shape and clear separation of this S E R from the p l a s m a l e m m a suggest that it is not a subsurface cistern. All but 2 of the coated vesicles observed in control p r e p a r a t i o n s lacked any contents d e m o n s t r a b l e by conventional uranium/lead staining procedures. Following a x o t o m y , ultrastructural examination of
Fig. 2. A composite series of electron micrographs (a-e) which illustrate a variety of coated structures in the axotomized M-cell and a proposed schema for production of granular coated vesicles (f). a: an element of smooth ER in the cytoplasm of the M-cell soma 64 days after axotomy. The lateral margins of the ER are slightly distended and possess a fuzzy coat (open arrows). Note the close proximity to a junctional complex, b-e have been grouped to represent what we believe is a sequence progressing from a granular coated pit at a zone of synaptic contact (b and c; open arrows) to formation of a granular coated vesicle (d and e; open arrows), b is from the lateral dendrite (*); while a, c, d and e, are from the M-cell soma (*). In b, c and d, the M-axon was lesioned 64 days previously whereas e is from a preparation 22 days after axotomy. Scale bars: 0.5/zm. f: presumably a coated vesicle formed in the Golgi (Go) is transported via the smooth ER to the plasmalemma of the postsynaptic cell (Po). The formation of a coated pit is accompanied by the evagination of an element of the presynaptic (Pr) membrane which provides the granular core to the coated vesicle. The fate of the latter is unclear (?L
343 the M-cell soma and lateral dendrite has revealed a marked increase in the numbers of coated pits and
vesicles. In 5 axotomized preparations (sacrificed at 22, 56, 64, 112 and 207 days) a total of 48 examples of
344 coated pits and vesicles were encountered. However, it is clear that the increased numbers of coated pits and vesicles are found in association with terminals which make synaptic contact onto the soma. The feature of many of these structures which distinguishes them from most of the coated vesicles in the control animals is the presence of a single, roughly spherical dense body or granule within the coated vesicles. The mean diameter of these granules is 57.04 + 14.65 nm (range 45-102 nm, n = 18). In total, of 48 coated pits/vesicles observed in the experimental preparations, 18 (37.5%) possessed a granule. The structural features of these granules were identical with the 2 'exceptions' noted above in the control preparations. Granules were not found in vesicles associated with SER (Fig. 2a), but rather were associated with or close to the plasmalemma such as the coated pits. Indeed, the profiles of the coated pits suggest that granulation may occur as coated membrane is pinched off from the plasma membrane. A total of 24 coated pits were observed in the experimental preparations; of these, 12 (50%) were associated with evaginations of the presynaptic terminal membrane. In these instances invaginations of the soma membrane (coated pits) were accompanied by projections of the presynaptic membrane bearing an electron dense tip and a small 'stalk' of presynaptic cytoplasm. In several instances, the presynaptic component appeared to have broken away from the terminal and seemed to be suspended as a spherical dense granule within the still open coated pit. In Fig. 2b-e, we have grouped a series of these structures to represent what we believe to be a sequence of formation from granular coated pit to granular coated vesicle. In each instance, it is noteworthy that an element of SER is in very close proximity to the coated pit/vesicle, and, with very few exceptions, this is the case in all the other examples observed. The dense granule lacks a discernible substructure except that it is membrane bound. However, there is evidence of a filamentous coat lining the interior of the coated vesicle and bridging the space between the vesicle and the granule (see Fig. 2c and e). This would suggest that the granule is suspended within the coated vesicle. In only two instances was a granule observed in contact with the membrane of the coated vesicle, and then it was only limited contact. Coated pits (with or without granules) were asso-
ciated with terminals containing spherical or flattened synaptic vesicles in the ratio of 2:1. Whereas all coated pits with granules, in varying stages of formation, were associated with terminals, 'empty' coated pits were also apposed to elements of glia of extracellular spaces between terminals. The relationship of the apposed terminal to coated vesicles found in the vicinity with or without granules, remains equivocal since such vesicles have, by definition, separated from the plasmalemma and are suspended in the cytoplasm of the M-cell. The principal finding in this study is the substantial increase in frequency of granular and agranular coated pits and vesicles in the M-cell soma of the goldfish following axotomy. Previous studies (e.g. ref. 17) have demonstrated the wide occurrence of coated vesicles in neurons. Post-synaptic coated vesicles per se have been implicated in receptor endocytosis 16 in dendrites during degeneration of apposed terminals and in micropinocytosis of ferritin TM. Structures resembling granular coated pits have also been implicated in (a) the dissolution (endocytosis) of puncta adherentia which disappear during maturation of the rat cortex12; (b) the dissolution of gap junctions 10 in cultured mouse melanoma cells, human adenocarcinoma and rabbit granuiosa cells; (c) the formation of neuromuscular junctions in tissue culture whereby the coated vesicles in the myoblast membrane appear to 'sample' the axon, in what may represent part of a recognition process 9. In contrast, coated vesicles have also been implicated in the process of receptor membrane insertion during synaptogenesis TM and synapse formation as a result of injury induced plasticity 2 in rat Purkinje cell dendrites. A proposed sequence of events regarding coated vesicles in the axotomized M-cell is diagrammed in Fig. 2f. Coated vesicles, probably originating from the Golgi apparatus, are transported via the SER to the vicinity of the postsynaptic membrane 3. They bud off from the SER and fuse with the postsynaptic membrane adjacent to the active site, forming coated pits. A small evagination of the presynaptic membrane is formed which projects or is drawn into and engulfed by the coated pit. The granular coated pit subsequently breaks away from the plasmalemma as a granular coated vesicle. The fate of granular coated vesicles remains unclear from our observations, although 'granular vesicles' without any coating have
345 been e n c o u n t e r e d d e e p within the soma cytoplasm well away from the p l a s m a l e m m a . These structures may well be returned to the Golgi for m e m b r a n e recycling and a concomitant transfer of information 6 which subsequently could modify cellular morphology and physiology. Alternatively, they may be targeted for d e g e n e r a t i o n by lysosomes. In the goldfish M-cell, reactive deafferentation seems to be a slow ongoing process and involves loss of only those terminals that contain spherical vesicles 19. In contrast, granular coated vesicles have been observed a p p o s e d to some terminals containing flattened vesicles. W h e r e a s we cannot preclude the possibility that M-cell axotomy and the consequent deafferentation induce presynaptic sprouting, our observations showed no structures resembling growth cones which would have signaled the formation of new synapses onto the M-cell. The fact that most of the coated and granular coated vesicles appear to be associated with normal looking terminal contacts leads us to believe that such structures do not reflect synaptogenesis but are rather a sign of continuous m e m b r a n e modifications which may also maintain the integrity of the synaptic junctions following injury. I n d e e d , if the coated vesicles represent p r e a s s e m b l e d plasma m e m b r a n e for insertion, existing receptor-rich regions of the postsynaptic m e m b r a n e s may be enlarging. Nevertheless we do not rule out the possibilities that the process of granular coated vesicle formation represents either a 'sampling' of afferent terminal m e m b r a n e and axoplasm or a manifestation of more rapid m e m b r a n e turn-
over. A x o t o m y may induce a change in the quality of the soma p l a s m a l e m m a ; that change being achieved by the resorption and reorganization of m e m b r a n e (cf. ref. 4). Such changes may ultimately d e t e r m i n e the rejection and subsequent disjunction of specific somatic inputs which characterize the slow ongoing process of deafferentation of this cell. Finally, the changes in soma m e m b r a n e excitability may be a product of increased m e m b r a n e insertion elsewhere in the M-cell, particularly at the IS, a region notable for its population of coated vesicles in the normal preparation and a region considered to be the site of action potential generation 7. Recent evidence has shown that axotomy of the goldfish M-cell a p p r o x i m a t e l y 2-5 m m distal to the soma frequently results in sprouting ~l. Since our studies have been at 8-10 m m caudal to the soma, it is reasonable to suggest that a p r o p o r t i o n of our animals are likely to have shown axon sprouting. It would therefore be of interest to establish whether granular coated vesicles occur p r e d o m i n a n t l y in preparations where the M-cells are actively involved in cellular reorganization leading to regenerative sprouting,
1 Altman, J.. Coated vesicles and synaptogenesis. A developmental study in the cerebellar cortex of the rat. Brain Research, 30 ( 1971) 311-322. 2 Chen, S. and Hillman. D. E.. Plasticity of the parallel fiberPurkinje cell synapse by spine takeover and new synapse formation in the adult rat, Brain Res., 240 (1982) 205-220. 3 Chen, S. and Hillman, D. E., Spatio-temporal occurrence of coated vesicles during Purkinje cell development and synaptogenesis, Soc. Neurosci. Abstr., 8 (1982) 863. 4 Eckenhoff. M. F. and Pysch. J. J.. Double-walled coated vesicle formation: evidence for massive and transient conjugate internalization of plasma membranes during cerebellar development, J. Neurocytol., 8 (1979) 623-638. 5 Faber. D. S. and Zottoli, S. J., Axotomy induced changes in cell structure and membrane excitability are sustained in a vertebrate central neuron. Brain Research, 223 (1981) 436--443. 6 Farquhar. M. G. and Palade, G. E.. The Golgi apparatus (complex) - - (1954-1981) - - from artifact to center stage, J. Cell Biol., 91 (1981)77s-103s.
7 Furshpan, E. J. and Furukawa, T., Intracellular and extracellular responses of the several regions of the Mauthner cell of the goldfish, J. Neurophysiol., 25 (1962) 732-771. 8 Heuser, J. R. and Reese, T. S., Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction, J. Cell Biol., 57 (1973) 315-344. 9 James, D. W. and Tresman, R. L., An electron-microscopic study of the de novo formation of neuromuscular junctions in tissue culture, Z. Zellforsch., 100 (1969) 126-140. 10 Larsen, W. J., Tung, H.-N., Murray, S. A. and Swenson, C. A., Evidence for the participation of actin microfilaments and bristle coats in the internalization of gap junction membrane, J. Cell Biol., 83 (1979) 576-587. 11 McWilliams, J. R. and Lynch, G., Sprouting in the hippocampus is accompanied by an increase in coated vesicles, Brain Research, 211 (1981) 158-164. 12 Privat, A., A possible mechanism for the resorption of attachment plates in the growing rat brain, Brain Research, 67 (1974) 125-129.
S u p p o r t e d in part by N I H grants NS 15335 and B R S G 2S07RR0540021 to D . S . F . W e are grateful to the Dept. of A n a t o m y and to Dr. Peter Nickerson, Dept. of Pathology, for use of their electron microscopes. Special thanks to Jan Jordan and Jean Seiler for their efficient secretarial assistance.
346 13 Rees. R. P., Bunge, M. B. and Bunge, R. P., Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture, J. Cell Biol.. 68 (1976) 240-263. 14 Rosenbluth. T. and Wissig, S. L., The distribution of exogenous ferritin in toad spinal ganglia and the mechanism of its uptake by neurons, J. Cell Biol.. 23 (1964) 307-325. 15 Stelzner. D. J.. Martin. A. H. and Scott, G. L.. Early stages of synaptogenesis in the cervical spinal cord of the chick embryo. Z. Zellforsch.. 138 (1973) 475-488. t6 Walberg. F., Role of normal dendrites in removal of degenerating terminal boutons, Exp. Neurol., 8 (1963) 112-124. 17 Waxman, S. G. and Pappas, G. D.. Pinocytosis at post-
18 19
20 21
synaptic membranes: electron microscopic evidence, Brain Research, 14 (1969) 240-244. Wolf, K., Physiological salines for freshwater teleosts, Progressive Fish Culturist, July (1963) 135-140. Wood, M. R. and Faber. D. S., Reactive deafferentation in the teleost Mauthner cell following axotomy, Soc. Neurosci., 8 (1982) 749. Zottoli, S. J., Comparison of Mauthner cell size in Teleosts, J. Comp. NeuroL, 178 (1978) 741-757. Zottoli, S. J., Regeneration following selective axotomy of an identified vertebrate central neuron, Soc. Neurosci., 7 (1981) 259.
341
Short Communications
Granular coated vesicles in the goldfish Mauthner cell following axotomy MALCOLM R. WOOD and DONALD S. FABER Division of Neurobiology, Department of Physiology, SUNY at Buffalo, Buffalo, NY 14214 (U.S.A.) (Accepted April 26th, 1983) Key words: granular coated vesicles- - Mauthner cell - - axotomy
Axotomy of the goldfish Mauthner cell induces a marked increase in the occurrence of granular coated vesicles in close association with axosomatic synapses. This phenomenon, which persists for over 200 days, is discussed in relation to other morphological and physiologicalchanges which occur over the same time period. Coated vesicles are widely encountered in the nervous systems of a variety of vertebrate preparations 17. Numerous functions have been attributed to them, including receptor membrane insertion during developmental synaptogenesis 1,13,15 and during synapse formation following deafferentation 2,11, membrane recycling 4.s, and the removal of specialized regions of contact such as gap junctions 10 and puncta adherentialL In addition their presence was originally considered to be a sign of degeneration in general 16. Less widely encountered are coated vesicles containing an electron dense core or granule, the socalled 'granular coated vesicle '1 or 'double-walled coated vesicle '4. These structures have been implicated in the process of membrane retrieval or the internalization of apposed cell surface membranes. The results from many studies suggest a common denominator: that granular coated vesicles are frequently present in systems undergoing radical changes, e.g. during synapse formation 1.2,11.15, neuronal maturation ~2 or remodelling 4 during development. Axotomy of the teleost Mauthner (M-) cell induces a typical chromatolytic response and a persistent shift in membrane organization as evidenced by a spread of electrical excitability from the initial segment and axon hillock only into the soma and proximal dendrites. Both changes appear at about 15-20 days 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
postaxotomy and last at least 200 daysS. In addition axotomy triggers slow ongoing reactive deafferentation of excitatory terminals which normally make synaptic contact onto the soma 19. These long-term changes make this preparation a model system for the study of neuronal membrane regulation and intercellular communication. This is particularly relevant in view of the appearance of granular coated vesicles in the M-cell soma 22-207 days postaxotomy as described in this communication. Goldfish (Carassius auratus), 5-6 in. in body length, were anesthetized in MS-222 (0.3 g/l) and their spinal cords were completely transected by inserting a no. 11 scalpel blade into the lateral body wall dorsal to the operculum. The distance between the M-cell soma and transection point was 8-10 mm. Operated fish were maintained at a constant temperature (15 °C) and sacrificed at intervals up to 207 days post-axotomy. Fixation for light and electron microscopy consisted of intracardial perfusion of fixative containing 1% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M Na cacodylate with 1.5 mM CaCI2. Although initial preparations were perfused first with fish saline~8, this step was subsequently omitted, in order to get improved ultrastructural preservation (Triller, personal communication). Following the fixative perfusion, the fish brain and proximal part of the spinal cord including the lesion, were exposed
342
Fig. 1. Electron micrograph illustrating part of the initial segment (IS) of a control M-cell. Within the cytoplasm, two coated vesicles (solid arrows) are evident close to the plasmalemma, together with two other coated stuctures (arrowheads) apparently budding off from elements of the smooth endoplasmic reticulum. Scale: 0.5/~m. and immersed in situ in the same fixative for 3 h; subsequently, the tissue was dissected out and fixed for a further 2 h. T r e a t m e n t continued as follows: the brain was buffer washed in 0.1 M Na cacodylate for 2 h; then it was t r i m m e d , osmicated overnight in 2% OsO 4 in 0.1 M N a cacodylate, d e h y d r a t e d in a g r a d e d acetone series and e m b e d d e d in Spurr. Serial thick (3 ~ m ) sections were cut to locate (a) the distal and (b) the proximal regions of the lateral d e n d r i t e and (c) the M-cell soma. A t each of these 3 identified regions, serial thin (60 nm) sections were p r e p a r e d for examination on either a Jeol 100B or Siemens 1 electron microscope. F o r goldfish 5-6 in. body length, the mean M-cell area20 is estimated at 2800 ~tm 2. F o r each p r e p a r a t i o n in this study, the M-cell has been examined ultrastructurally in a m i n i m u m of 3 separate areas of the lateral dendrite and through areas of the soma which include the nucleus. In 4 control fish, ordinary c o a t e d pits and vesicles 11.17 are apparently restricted to the axon hillock/ initial segment region as 29 out of a total of 31 were observed in that region, the remaining 2 being in the soma and lateral dendrite. W h e n present in these regions, they were subjacent to an afferent terminal,
elements of glia, extracellular spaces or attached to elements of the SER. Their m e a n d i a m e t e r m e a s u r e d 112 + 16.6 nm (range 67-149 nm; n = 31) including the fuzzy coat of 20.7 + 3.5 nm thickness (range 15-29 nm; n = 27). Some of these vesicles were associated with the p l a s m a l e m m a of the initial segment, forming a flask-shaped structure or coated pit there. As such, the pits o p e n e d into the extracellular space frequently in close proximity to an afferent terminal immediately adjacent to, and in continuity with, the postsynaptic m e m b r a n e . Less frequently, the coated pits were a p p o s e d to elements of glia or expanses of extracellular space between a d j a c e n t terminals and/ or glia. In Fig. 1, two coated vesicles are evident close to the initial segment (IS) m e m b r a n e together with two o t h e r coated structures a p p a r e n t l y budding off from elements of the smooth e n d o p l a s m i c reticulum (SER). The size, shape and clear separation of this S E R from the p l a s m a l e m m a suggest that it is not a subsurface cistern. All but 2 of the coated vesicles observed in control p r e p a r a t i o n s lacked any contents d e m o n s t r a b l e by conventional uranium/lead staining procedures. Following a x o t o m y , ultrastructural examination of
Fig. 2. A composite series of electron micrographs (a-e) which illustrate a variety of coated structures in the axotomized M-cell and a proposed schema for production of granular coated vesicles (f). a: an element of smooth ER in the cytoplasm of the M-cell soma 64 days after axotomy. The lateral margins of the ER are slightly distended and possess a fuzzy coat (open arrows). Note the close proximity to a junctional complex, b-e have been grouped to represent what we believe is a sequence progressing from a granular coated pit at a zone of synaptic contact (b and c; open arrows) to formation of a granular coated vesicle (d and e; open arrows), b is from the lateral dendrite (*); while a, c, d and e, are from the M-cell soma (*). In b, c and d, the M-axon was lesioned 64 days previously whereas e is from a preparation 22 days after axotomy. Scale bars: 0.5/zm. f: presumably a coated vesicle formed in the Golgi (Go) is transported via the smooth ER to the plasmalemma of the postsynaptic cell (Po). The formation of a coated pit is accompanied by the evagination of an element of the presynaptic (Pr) membrane which provides the granular core to the coated vesicle. The fate of the latter is unclear (?L
343 the M-cell soma and lateral dendrite has revealed a marked increase in the numbers of coated pits and
vesicles. In 5 axotomized preparations (sacrificed at 22, 56, 64, 112 and 207 days) a total of 48 examples of
344 coated pits and vesicles were encountered. However, it is clear that the increased numbers of coated pits and vesicles are found in association with terminals which make synaptic contact onto the soma. The feature of many of these structures which distinguishes them from most of the coated vesicles in the control animals is the presence of a single, roughly spherical dense body or granule within the coated vesicles. The mean diameter of these granules is 57.04 + 14.65 nm (range 45-102 nm, n = 18). In total, of 48 coated pits/vesicles observed in the experimental preparations, 18 (37.5%) possessed a granule. The structural features of these granules were identical with the 2 'exceptions' noted above in the control preparations. Granules were not found in vesicles associated with SER (Fig. 2a), but rather were associated with or close to the plasmalemma such as the coated pits. Indeed, the profiles of the coated pits suggest that granulation may occur as coated membrane is pinched off from the plasma membrane. A total of 24 coated pits were observed in the experimental preparations; of these, 12 (50%) were associated with evaginations of the presynaptic terminal membrane. In these instances invaginations of the soma membrane (coated pits) were accompanied by projections of the presynaptic membrane bearing an electron dense tip and a small 'stalk' of presynaptic cytoplasm. In several instances, the presynaptic component appeared to have broken away from the terminal and seemed to be suspended as a spherical dense granule within the still open coated pit. In Fig. 2b-e, we have grouped a series of these structures to represent what we believe to be a sequence of formation from granular coated pit to granular coated vesicle. In each instance, it is noteworthy that an element of SER is in very close proximity to the coated pit/vesicle, and, with very few exceptions, this is the case in all the other examples observed. The dense granule lacks a discernible substructure except that it is membrane bound. However, there is evidence of a filamentous coat lining the interior of the coated vesicle and bridging the space between the vesicle and the granule (see Fig. 2c and e). This would suggest that the granule is suspended within the coated vesicle. In only two instances was a granule observed in contact with the membrane of the coated vesicle, and then it was only limited contact. Coated pits (with or without granules) were asso-
ciated with terminals containing spherical or flattened synaptic vesicles in the ratio of 2:1. Whereas all coated pits with granules, in varying stages of formation, were associated with terminals, 'empty' coated pits were also apposed to elements of glia of extracellular spaces between terminals. The relationship of the apposed terminal to coated vesicles found in the vicinity with or without granules, remains equivocal since such vesicles have, by definition, separated from the plasmalemma and are suspended in the cytoplasm of the M-cell. The principal finding in this study is the substantial increase in frequency of granular and agranular coated pits and vesicles in the M-cell soma of the goldfish following axotomy. Previous studies (e.g. ref. 17) have demonstrated the wide occurrence of coated vesicles in neurons. Post-synaptic coated vesicles per se have been implicated in receptor endocytosis 16 in dendrites during degeneration of apposed terminals and in micropinocytosis of ferritin TM. Structures resembling granular coated pits have also been implicated in (a) the dissolution (endocytosis) of puncta adherentia which disappear during maturation of the rat cortex12; (b) the dissolution of gap junctions 10 in cultured mouse melanoma cells, human adenocarcinoma and rabbit granuiosa cells; (c) the formation of neuromuscular junctions in tissue culture whereby the coated vesicles in the myoblast membrane appear to 'sample' the axon, in what may represent part of a recognition process 9. In contrast, coated vesicles have also been implicated in the process of receptor membrane insertion during synaptogenesis TM and synapse formation as a result of injury induced plasticity 2 in rat Purkinje cell dendrites. A proposed sequence of events regarding coated vesicles in the axotomized M-cell is diagrammed in Fig. 2f. Coated vesicles, probably originating from the Golgi apparatus, are transported via the SER to the vicinity of the postsynaptic membrane 3. They bud off from the SER and fuse with the postsynaptic membrane adjacent to the active site, forming coated pits. A small evagination of the presynaptic membrane is formed which projects or is drawn into and engulfed by the coated pit. The granular coated pit subsequently breaks away from the plasmalemma as a granular coated vesicle. The fate of granular coated vesicles remains unclear from our observations, although 'granular vesicles' without any coating have
345 been e n c o u n t e r e d d e e p within the soma cytoplasm well away from the p l a s m a l e m m a . These structures may well be returned to the Golgi for m e m b r a n e recycling and a concomitant transfer of information 6 which subsequently could modify cellular morphology and physiology. Alternatively, they may be targeted for d e g e n e r a t i o n by lysosomes. In the goldfish M-cell, reactive deafferentation seems to be a slow ongoing process and involves loss of only those terminals that contain spherical vesicles 19. In contrast, granular coated vesicles have been observed a p p o s e d to some terminals containing flattened vesicles. W h e r e a s we cannot preclude the possibility that M-cell axotomy and the consequent deafferentation induce presynaptic sprouting, our observations showed no structures resembling growth cones which would have signaled the formation of new synapses onto the M-cell. The fact that most of the coated and granular coated vesicles appear to be associated with normal looking terminal contacts leads us to believe that such structures do not reflect synaptogenesis but are rather a sign of continuous m e m b r a n e modifications which may also maintain the integrity of the synaptic junctions following injury. I n d e e d , if the coated vesicles represent p r e a s s e m b l e d plasma m e m b r a n e for insertion, existing receptor-rich regions of the postsynaptic m e m b r a n e s may be enlarging. Nevertheless we do not rule out the possibilities that the process of granular coated vesicle formation represents either a 'sampling' of afferent terminal m e m b r a n e and axoplasm or a manifestation of more rapid m e m b r a n e turn-
over. A x o t o m y may induce a change in the quality of the soma p l a s m a l e m m a ; that change being achieved by the resorption and reorganization of m e m b r a n e (cf. ref. 4). Such changes may ultimately d e t e r m i n e the rejection and subsequent disjunction of specific somatic inputs which characterize the slow ongoing process of deafferentation of this cell. Finally, the changes in soma m e m b r a n e excitability may be a product of increased m e m b r a n e insertion elsewhere in the M-cell, particularly at the IS, a region notable for its population of coated vesicles in the normal preparation and a region considered to be the site of action potential generation 7. Recent evidence has shown that axotomy of the goldfish M-cell a p p r o x i m a t e l y 2-5 m m distal to the soma frequently results in sprouting ~l. Since our studies have been at 8-10 m m caudal to the soma, it is reasonable to suggest that a p r o p o r t i o n of our animals are likely to have shown axon sprouting. It would therefore be of interest to establish whether granular coated vesicles occur p r e d o m i n a n t l y in preparations where the M-cells are actively involved in cellular reorganization leading to regenerative sprouting,
1 Altman, J.. Coated vesicles and synaptogenesis. A developmental study in the cerebellar cortex of the rat. Brain Research, 30 ( 1971) 311-322. 2 Chen, S. and Hillman. D. E.. Plasticity of the parallel fiberPurkinje cell synapse by spine takeover and new synapse formation in the adult rat, Brain Res., 240 (1982) 205-220. 3 Chen, S. and Hillman, D. E., Spatio-temporal occurrence of coated vesicles during Purkinje cell development and synaptogenesis, Soc. Neurosci. Abstr., 8 (1982) 863. 4 Eckenhoff. M. F. and Pysch. J. J.. Double-walled coated vesicle formation: evidence for massive and transient conjugate internalization of plasma membranes during cerebellar development, J. Neurocytol., 8 (1979) 623-638. 5 Faber. D. S. and Zottoli, S. J., Axotomy induced changes in cell structure and membrane excitability are sustained in a vertebrate central neuron. Brain Research, 223 (1981) 436--443. 6 Farquhar. M. G. and Palade, G. E.. The Golgi apparatus (complex) - - (1954-1981) - - from artifact to center stage, J. Cell Biol., 91 (1981)77s-103s.
7 Furshpan, E. J. and Furukawa, T., Intracellular and extracellular responses of the several regions of the Mauthner cell of the goldfish, J. Neurophysiol., 25 (1962) 732-771. 8 Heuser, J. R. and Reese, T. S., Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction, J. Cell Biol., 57 (1973) 315-344. 9 James, D. W. and Tresman, R. L., An electron-microscopic study of the de novo formation of neuromuscular junctions in tissue culture, Z. Zellforsch., 100 (1969) 126-140. 10 Larsen, W. J., Tung, H.-N., Murray, S. A. and Swenson, C. A., Evidence for the participation of actin microfilaments and bristle coats in the internalization of gap junction membrane, J. Cell Biol., 83 (1979) 576-587. 11 McWilliams, J. R. and Lynch, G., Sprouting in the hippocampus is accompanied by an increase in coated vesicles, Brain Research, 211 (1981) 158-164. 12 Privat, A., A possible mechanism for the resorption of attachment plates in the growing rat brain, Brain Research, 67 (1974) 125-129.
S u p p o r t e d in part by N I H grants NS 15335 and B R S G 2S07RR0540021 to D . S . F . W e are grateful to the Dept. of A n a t o m y and to Dr. Peter Nickerson, Dept. of Pathology, for use of their electron microscopes. Special thanks to Jan Jordan and Jean Seiler for their efficient secretarial assistance.
346 13 Rees. R. P., Bunge, M. B. and Bunge, R. P., Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture, J. Cell Biol.. 68 (1976) 240-263. 14 Rosenbluth. T. and Wissig, S. L., The distribution of exogenous ferritin in toad spinal ganglia and the mechanism of its uptake by neurons, J. Cell Biol.. 23 (1964) 307-325. 15 Stelzner. D. J.. Martin. A. H. and Scott, G. L.. Early stages of synaptogenesis in the cervical spinal cord of the chick embryo. Z. Zellforsch.. 138 (1973) 475-488. t6 Walberg. F., Role of normal dendrites in removal of degenerating terminal boutons, Exp. Neurol., 8 (1963) 112-124. 17 Waxman, S. G. and Pappas, G. D.. Pinocytosis at post-
18 19
20 21
synaptic membranes: electron microscopic evidence, Brain Research, 14 (1969) 240-244. Wolf, K., Physiological salines for freshwater teleosts, Progressive Fish Culturist, July (1963) 135-140. Wood, M. R. and Faber. D. S., Reactive deafferentation in the teleost Mauthner cell following axotomy, Soc. Neurosci., 8 (1982) 749. Zottoli, S. J., Comparison of Mauthner cell size in Teleosts, J. Comp. NeuroL, 178 (1978) 741-757. Zottoli, S. J., Regeneration following selective axotomy of an identified vertebrate central neuron, Soc. Neurosci., 7 (1981) 259.