709
J. Anat. (1979), 128, 4, pp. 709-720 With 1O figures Printed in Great Britain
Experimental degeneration of primary afferent terminals in the cuneate nucleus of the monkey (Macaca fascicularis) C. Y. WEN, C. K. TAN AND W. C. WONG
Department of Anatomy, Faculty of Medicine, University of Singapore, Sepoy Lines, Singapore 3
(Accepted 8 May 1978) INTRODUCTION
The projection of primary afferent fibres to the mammalian cuneate nucleus has been studied by light microscopy in the rat (Basbaum & Hand, 1973), cat (Keller & Hand, 1970; Rustioni & Macchi, 1968; Kuypers & Tuerk, 1964) and monkey (Dunn & Matzke, 1967; Shriver, Stein & Carpenter, 1968) by means of Nauta and Fink-Heimer methods after cervical dorsal rhizotomy, and under the electron microscope in the cat after transection of the dorsal funiculus (Walberg, 1965, 1966). However, there has been no report of the ultrastructural features of these primary afferent terminals and their patterns of degeneration in the cuneate nucleus of the monkey following dorsal rhizotomy. The present study is a sequel to our earlier study of the normal ultrastructure of the cuneate nucleus of the monkey (Wen, Wong & Tan, 1978) and reports the types of degenerative changes observed in the terminals of cervicothoracic dorsal roots in the cuneate nucleus after multiple rhizotomies. MATERIALS AND METHODS
Six adult monkeys of both sexes, weighing 1-4-1 9 kg, were used for this study. All animals were anaesthetized with an intraperitoneal injection of 0 5 ml of Sagatal (sodium pentobarbital 60 mg/ml) per kg body weight. All surgical procedures were performed under aseptic conditions. Multiple laminectomies, extending from the fifth cervical to the first thoracic vertebrae to expose the relevant parts of the spinal cord, were made on the right side. The dura mater was slit longitudinally along the lateral side of the cord in order to expose the fifth cervical to the first thoracic dorsal roots. The dorsal roots to be sectioned (Table 1) were then identified, lifted gently with a fine glass hook, and cut with a fine blade (No. 12) under a Zeiss Operation Microscope. Care was taken to avoid any damage to the spinal cord or the adjacent radicular vessels. After each operation, penicillin and sulphanilamide powder was sprinkled on the wound. The skin was sutured with silk and sprayed with Neomycin aerosol. The animals were killed after various post-operative survival periods between 2 and 6 days (Table 1). All animals were anaesthetized with Sagatal and artificially respired with oxygen given through a tracheostomy. After the thorax was opened, 1000 units of heparin and 2 ml of 1 % sodium nitrite per kg body weight were given by intracardiac injection. Perfusion was initiated 3-5 minutes later through the left cardiac ventricle with 200 ml of Ringer's solution at pH 7-3-7 4, followed by 500 ml of a dilute fixative (1 % paraformaldehyde+ 1 25 % glutaraldehyde) and then
710
C. Y. WEN, C. K. TAN AND W. C. WONG
Table 1. Data concerning animals and operations Body weight
Dorsal roots
Animal no.
Sex
(kg)
cut
Post-operative survival period
M18 M22 M42 M30 M24 M32
Male Male Male Male Male Female
1-7 1.9 1-4 1-4 1-9 1-6
C8, TI, 2 C5, 6, 7 C6, 7, 8 C7, 8 C7, 8 C6, 7
2 days 3 days 4 days 5 days 6 days 6 days
1000 ml of a concentrated fixative (4 % paraformaldehyde +5 % glutaraldehyde). After decapitation the head was immersed in fresh concentrated fixative and kept overnight at 4 °C before the caudal part of the medulla was dissected out. Transverse slices about 1 mm thick were cut with a razor blade. From these slices portions of the cuneate nucleus were trimmed under a stereomicroscope and placed in ice cold 0* 1 M cacodylate buffer at pH 7-4 containing 5 % sucrose. After four changes of the buffer at intervals of 10 minutes, the slices were post-fixed with 1 % ice cold osmium tetroxide for 2 hours. The tissue slices were dehydrated through an ascending series of acetone and embedded in an Epon mixture. 1 ,tm thick sections were stained with I % methylene blue. Ultrathin sections were doubly stained with uranyl acetate and lead citrate and examined in a Hitachi HS-8 electron microscope. RESULTS
2 days' survival Very few degenerating axon terminals were observed in this animal. Degenerating profiles of terminals contained round synaptic vesicles, had an electron-dense axoplasm (Fig. 1), and their mitochondria were swollen. Some large axon terminals were found within synaptic complexes (Szentagothai, 1970) and formed asymmetrical axodendritic synapses, while others formed isolated axodendritic synapses with small dendritic profiles outside the synaptic complexes. None of the degenerating terminals formed axosomatic synapses. 3 days' survival More degenerating axon terminals were observed in the neuropil at this stage, and at least four types of degenerative changes could be identified. Most were of the electron-dense type; they appeared to be shrunken, with crenated margins, closely packed swollen mitochondria, and round synaptic vesicles. The axoplasm was more electron-dense than previously and some of the profiles contained vacuoles (Fig. 2). Synaptic contact between the degenerating axon terminal and the postsynaptic dendrite was still maintained, although some axon terminals were partially surrounded by glial processes. Other electron-dense terminals showed a less advanced degree of degeneration and resembled those observed after 2 days' survival. The second commonest type of degenerating axon terminal showed neurofilamentous changes. Such terminals were filled with neurofilaments, which were sectioned in various planes (Fig. 3). Small clusters of round synaptic vesicles were usually observed near the synaptic site. Occasionally, clumps of synaptic vesicles were seen among the neurofilaments, but none of the clumps was observed to be surrounded by neurofilamentous whorls (Fig. 4). The axoplasm showed varying
Primary afferent terminals in monkey cuneate nucleus
Fig. 1 A degenerating axon terminal in a synaptic complex showing increased electron densitv and containing swollen mitochondria (AM). 2 days after dorsal rhizotomy. Fig. 2. A distorted electron-dense degenerating axon terminal containing vacuoles (V), forming asymmetrical synapses with two dendrites. A glial process (GP) invaginates the axon terminal. 3 days after dorsal rhizotomy.
71
712
C. Y. WEN, C. K. TAN AND W. C. WONG
Fig. 3. A degenerating axon terminal with neurofilamentous hyperplasia. The neurofilaments (NF) are arranged in loose bundles which show no special orientation and hence some filaments are longitudinally sectioned while others are cut obliquely and transversely. Synaptic vesicles are clustered near the synaptic site (arrow). 3 days after dorsal rhizotomy. Fig. 4. A degenerating axon terminal with neurofilamentous hyperplasia (NF) in a synaptic complex. It is presynaptic to three dendrites (D). Synaptic vesicles were found near the periphery of the axon terminal and also near the synaptic sites 3 days after dorsal rhizotomy.
713 Primary afferent terminals in monkey cuneate nucleus degrees of electron density. Most of these terminals were large and were found in synaptic complexes (Fig. 5) where they formed the central element. They were presynaptic to dendrites and postsynaptic to small axon terminals containing flattened vesicles. Again, no degenerating axon terminals were seen to form axosomatic synapses. The third type of degenerating axon terminal was electron-lucent. Such terminals were large and were characterized by clear axoplasm in which only a few round synaptic vesicles were found, these being clustered together at the synaptic site (Fig. 6). Mitochondria were still present, but some were swollen and showed loss of cristae. The fourth type of degenerating axon terminal was characterized by its content of flocculent material. Synaptic vesicles were few in number and were clustered near the synaptic site. Mitochondria were swollen, and some of them showed loss of cristae. These terminals formed axodendritic synapses. Occasionally this type of degenerating axon terminal synapsed on a dendrite which was also synaptically contacted by another electron-dense degenerating axon terminal with neurofilamentous change (Fig. 7). 4-6 days' survival All profiles of degenerating axon terminals in these animals were of the electrondense type (Figs. 8-10). Most of them were found within synaptic complexes, in which they were usually the central element, and they were presynaptic to dendrites and postsynaptic to axon terminals containing flattened vesicles. None was observed to form axosomatic synapses (Figs. 9, 10). At this stage, most of the degenerating and shrunken profiles showed some degree of distortion owing to the glial reaction (Figs. 8-10). Vacuoles of different sizes were present in many of the degenerating terminals. Synaptic vesicles, which were tightly packed because of shrinkage of the axon terminals, were still visible, while virtually all the mitochondria were now electron-dense and their cristae and membranes were indistinct. DISCUSSION
Types of degenerative changes in primary afferent terminals following dorsal rhizotomy least four types of degenerating axon terminal (electronat study In the present and floccular) could be distinguished in the neurofilamentous dense, electron-lucent, dorsal rhizotomy. 2-6 after of days cuneate nucleus monkeys was found. After 3 days all four degeneration After 2 days only electron-dense electron-dense type was the The were present. types of degenerative change axon terminals containing the second commonest; neurofilamentous commonest, the the fourth least common. post-operative day all the were By flocculent material of the electron-dense type. were terminals degenerating axon
Electron-dense type of degeneration This type of degeneration was first described by Colonnier & Gray (1962) in the cerebral cortex and regarded by Alksne, Blackstad, Walberg & White (1966) as a pathognomic feature of anterograde degeneration. Subsequently Cohen & Pappas (1969) questioned the validity of using this criterion for identifying degenerating axon terminals. They observed electron-dense axon profiles and darkened neurons
714
C. Y. WEN, C. K. TAN AND W. C. WONG
Primary afferent terminals in monkey cuneate nucleus 715 in normal animals and suggested that they might be attributable to post-mortem changes. However, in our previous study of the normal cuneate nucleus, which was well fixed, no such profiles were seen (Wen et al. 1978). Although 'altered' axon terminals which resembled some of those described by Sotelo & Palay (1968) in the vestibular nucleus of the rat were present, none corresponded to the electron-dense type of degenerating axon terminal of the present study. Similar electron-dense degenerating axon terminals were reported earlier in the cuneate nucleus of the cat following dorsal funiculotomy (Walberg, 1965, 1966) and in the gracile nucleus of the cat after lumbar dorsal rhizotomy (Blomqvist & Westman, 1970; Rustioni & Sotelo, 1974). Electron-lucent type of degeneration The electron-lucent, or clear, type of degeneration was first reported by De Robertis (1956), and has since been demonstrated in several areas of the mammalian central nervous system. McMahan (1967) suggested that such profiles might have resulted from inadequate fixation. While this might have been the case in early electron microscopic studies, the greatly improved quality of fixation in more recent ultrastructural studies seems to rule out this explanation. Concerning the fate of such degenerating axon terminals, O'Neal & Westrum (1973) have suggested that they may change rapidly to the dark form, but our present studies provided no evidence to support this.
Neurofilamentous hyperplasia In the present study, neurofilamentous hyperplasia in degenerating axon terminals was most frequently met with in the 3 day material. Such terminals were rarely seen in the normal monkey cuneate nucleus (Wen et al. 1978). The phenomenon has been demonstrated in the lateral geniculate nucleus of the cat (Szentagothai, Hamori & Tombol, 1966) and monkey (Colonnier & Guillery, 1964; Glees, Mehler & Eschner, 1966; Guillery, 1965) after eye enucleation, and in the ventral part of the dorsal horn of the spinal cord after rhizotomy (Ralston, 1968). On the other hand, no neurofilamentous changes were observed after experimental degeneration in the cuneate (Walberg, 1965, 1966) and gracile nuclei (Blomqvist & Westman, 1970) of the cat or in the cuneate nucleus of the rat (Tan, unpublished observations). The reasons for such species differences are not known. There was some evidence in the present study that degenerating axon terminals which showed neurofilamentous hyperplasia might have progressed to the electron-dense type because there were some intermediate forms between the purely neurofilamentous and the electron-dense types. Such degenerating terminals were shrunken, and in addition to their content of neurofilaments they showed varying degrees of electron density. This suggests that there may be a progressive change from neurofilamentous hyperplasia to the electron-dense type of degeneration. This would agree with the views of many authors Fig. 5. A degenerating axon terminal forming the central element in a synaptic complex. Its matrix shows increased electron density and contains neurofilaments (NF). This axon terminal is surrounded by several dendrites (D) to which it is presynaptic. In addition it is also postsynaptic to small axon terminals containing flattened vesicles (AF). 3 days after dorsal rhizotomy. Fig. 6. A swollen electron-lucent degenerating axon terminal characterized by the clear appearance of its axoplasm. The mitochondria are swollen and their cristae show signs of disintegration. The synaptic vesicles are few in number and are clustered near the synaptic site (arrow). 3 days after dorsal rhizotomy.
716
C. Y. WEN, C. K. TAN AND W. C. WONG `A *.
_
Fig. 7. A swollen degenerating axon terminal (FL) with flocculent matrix was presynaptic to a large dendrite. The mitochondria were swollen and disintegrated. Synaptic vesicles were clustered near the synaptic site. 3 days after dorsal rhizotomy. Fig. 8. A shrunken electron-dense degenerating axon terminal partially surrounded by glial processes (GP). Its synaptic contact with a dendrite (D), and an axon terminal containing flattened vesicles (Af) are still maintained. 4 days after dorsal rhizotomy.
Primary afferent terminals in monkey cuneate nucleus Al
E
afWx
.-
X,
717
.ti w. k
Fig. 9. A shrunken and distorted electron-dense degenerating axon terminal in a synaptic complex. The axon terminal is invaded on one side by glial processes (GP). Swollen synaptic vesicles and degenerating mitochondria are tightly packed in the terminal. 6 days after dorsal rhizotomy. Fig. 10. An electron-dense degenerating axon terminal in a synaptic complex. The axon terminal is grossly distorted by glial processes (GP). At this stage the synaptic vesicles are still clearly visible within the axon terminal. 6 days after dorsal rhizotomy. L6
ANA I28
C. Y. WEN, C. K. TAN AND W. C. WONG (Glees & Hasan, 1968; Jones & Rockel, 1973; Mugnaini & Walberg, 1967; Mugnaini, Walberg & Brodal, 1967; Pecci-Saavedra, Vaccarezza, Reader & Pasqualini, 1970; Ralston, 1969; Szentagothai et al. 1966; Vaccarezza, Reader, Pasqualini & Pecci-Saavedra, 1970; Wong-Riley, 1972).
718
Floccular change In recent years, a fourth
type of degenerative change has been observed in the nervous mammalian central system. The content of such degenerating terminals material' by Jones & Rockel (1973) in the electron-dense been 'flocculent has called material' inferior colliculus, and 'fuzzy by Rosenstein, Page & Leure-DuPree (1977) in the external cuneate nucleus. In the present study, axon terminals containing to granular material were observed after rhizotomy. Such changes have not floccular been described in previous studies of the dorsal column nuclei (Walberg, 1965, 1966; Valverde, 1966; Rustioni & Sotelo, 1974). While the genesis of such terminals was not clear in the present study, there have been suggestions from Jones & Rockel (1973) that the flocculent material arose from empty 'shells' of complex vesicles, and that such axon terminals finally became electron-dense. Terminals which showed flocculent change in the present study had varying degrees of electron density and possibly they could have changed into the electron-dense type. It was not certain whether the floccular and granular changes represented two distinct phenomena, or transitional stages in a spectrum of changes which led to electron density. But a progressive change from floccular to granular and finally to electron-dense remained a possibility.
of primary afferent terminals The results of studies in the rat (Valverde, 1966; Tan, unpublished observations), cat (Walberg, 1965, 1966; Rustioni & Sotelo, 1974) and in the monkey (present study) have shown a remarkable similarity in the mode of termination of primary Mode of termination
afferent fibres in the mammalian dorsal column nuclei. There is general agreement that primary afferent terminals are mostly of large diameter and that they degenerate after dorsal rhizotomy. Profiles of degenerating primary afferent terminals in the cuneate nucleus of the monkey, however, have shown great variability in size. Prominent features of the neuropil of the dorsal column nuclei are the clusters of neural elements, called synaptic glomeruli, in the cuneate nucleus of the rat (Tan & Lieberman, 1974) and the gracile nucleus of the cat (Rustioni & Sotelo, 1974) and termed synaptic complexes in the monkey (Wen et al. 1978). The primary afferent fibres, which contain round synaptic vesicles, terminate within these complexes, where they form asymmetrical synapses with dendrites. Some of these dendrites are organelle-rich, while others are of smaller diameter and have dendritic excrescences. Some of the degenerating axon terminals appear to establish isolated axodendritic synapses. Some of the organelle-rich dendritic profiles are presumably derived from projection cells, but their identity has not yet been clearly determined; it is also possible that some of the postsynaptic dendritic profiles belong to interneurons, since electrophysiological evidence has been produced that both cuneothalamic projection cells and cuneate interneurons can be excited by stimulation of peripheral nerves (Anderson, Eccles, Schmidt & Yokota, 1964). It has been noted previously (Walberg, 1966; Rustioni & Sotelo, 1974) that primary afferent terminals rarely form axosomatic synapses with cuneate neurons. In the present study none of the degenerating axon terminals synapsed directly with any cuneate neuronal somata. On the other hand, axo-axonal synapses in which the
Primary afferent terminals in monkey cuneate nucleus
719
primary afferent terminal is postsynaptic to a small diameter axon terminal containing flattened vesicles were frequently observed. This was first reported in the cuneate nucleus of the cat by Walberg (1965), who suggested that this might be the anatomical substrate for presynaptic inhibition in the cuneate nucleus previously demonstrated electrophysiologically by Andersen, Eccles & Schmidt (1962). SUMMARY
Six monkeys (Macacafascicularis) were used for the present study. In animals which survived for 2-6 days after section of C5 to TI dorsal roots, at least four types of degenerating afferent terminal were observed - electron-dense, electron-lucent, neurofilamentous and flocculent. The electron-dense degeneration was the most common and was seen as early as 2 days after rhizotomy. The neurofilamentous type was the second commonest and was found predominantly in the 3 days' survival material. The electron-lucent and flocculent types were less commonly encountered. Since the profiles exhibiting neurofilamentous hyperplasia showed varying degrees of electron density it is suggested that this type of degeneration progresses to the electron-dense type with time. The present study also showed that the primary afferent terminals in the cuneate nucleus of the monkey are mostly large and that they contain round vesicles. They are commonly found within synaptic complexes in which they are presynaptic to dendrites of various sizes, and are themselves postsynaptic to smaller axon terminals containing flattened vesicles. Degenerating terminals forming isolated synapses were less commonly seen. No dorsal root axon terminals formed axosomatic synapses. The excellent technical assistance of Mr H. L. Chan and Staff of the E.M. Unit is gratefully acknowledged. The authors also wish to thank Professor R. Kanagasuntheram for his interest in the project. One of us (C.Y.W.), who is on leave from the National Taiwan University, is supported by a China Medical Board Faculty Scholarship. REFERENCES ALKSNE, J. F., BLACKSTAD, T. W., WALBERG, F. & WHITE, L. E., JR. (1966). Electron microscopy of axon degeneration: a valuable tool in experimental neuroanatomy. Ergebnisse der Anatomie und
Entwicklungsgeschichte 39, 1-31. ANDERSEN, P., ECCLES, J. C. & SCHMIDT, R. F. (1962). Presynaptic inhibition in the cuneate nucleus. Nature 194, 741-743. ANDERSEN, P., ECCLES J. C. SCHMIDT, R. F. & YOKOTA, T. (1964). Identification of relay cells and interneurones in the cuneate nucleus. Journol of Neurophysiology 27, 1080-1095. BASBAUM, A. I. & HAND, P. J. (1973). Projections of cervicothoracic dorsal roots to the cuneate nucleus of the rat, with observations on cellular 'bricks'. Journal of Comparative Neurology 148, 347-360. BLoMQvIST, A. & WESTMAN, J. (1970). An electron microscopical study of the gracile nucleus in the cat. Acta Societatis medicorum upsaliensis 75, 241-252. COHEN, E. B. & PAPPAS, G. D. (1969). Dark profiles in the apparently-normal central nervous system: a problem in the electron microscopic identification of early antegrade axonal degeneration. Journal of Comparative Neurology 136, 375-396. COLONNIER, M. & GRAY, E. G. (1962). Degeneration in the cerebral cortex. In Electron Microscopy, Fifth Intemational Congress for Electron Microscopy (ed. S. S. Breese, Jr.), vol. 2 New York: Academic Press. COLONNIER, M. & GUILLERY, R. W. (1964). Synaptic organization in the lateral geniculate nucleus of the monkey. Zeitschrift fur Zellforschung und mikroskopische Anatomie 62, 333-355. DE ROBERTIS, E. (1956). Submicroscopic changes of the synapse after nerve section in the acoustic ganglion of the guinea pig. An electron microscope study. Journal of Biophysical and Biochemical Cytology 2, 503-519. 46.2
C. Y. WEN, C. K. TAN AND W. C. WONG 720 DUNN, J. & MATZKE, H. (1967). Central distribution of the dorsal root of the first cervical nerve of the marmoset monkey. Anatomical Record 157, 357. GLEES, P. & HASAN, M. (1968). The signs of synaptic degeneration - a critical appraisal. Acta anatomica 69, 153-167. GLEES, P., MEHLER, K. & ESCHNER, J. (1966). Terminal degeneration in the lateral geniculate body of the monkey: an electron microscopic study. Zeitschrift fir Zellforscfung und mikroskopische Anatomie 71, 29-40. GUILLERY, R. W. (1965). Some electron microscopical observations of degenerative changes in central nervous synapses. In Degeneration Patterns in the Nervous System (ed. M. Singer and J. P. Schade). Progress in Brain Research 14, 57-76. JONES, E. G. & ROCKEL, A. J. (1973). Observations on complex vesicles, neurofilamentous hyperplasia and increased electron density during terminal degeneration in the inferior colliculus. Journal of Comparative Neurology 147, 93-118. KELLER, J. H. & HAND, P. J. (1970). Dorsal roots projections to nucleus cuneatus of the cat. Brain Research 20, 1-17. KuYPERS, H. G. J. M. & TUERK, J. D. (1964). The distribution of the cortical fibres within the nuclei cuneatus and gracilis in the cat. Journal of Anatomy 98, 143-162. MCMAHAN, U. J. (1967). Fine structure of synapses in the dorsal nucleus of the lateral geniculate body of normal and blinded rats. Zeitschrift fiir Zellforschung und mikroskopische Anatomie 76, 116-146. MUGNAINI, E. & WALBERG, F. (1967). An experimental electron microscopical study on the mode of termination of cerebellar corticovestibular fibres in the cat lateral vestibular nucleus (Deiter's nucleus). Experimental Brain Research 4, 212-236. MUGNAINI, E., WALBERG, F. & BRODAL, A. (1967). Mode of termination of primary vestibular fibers in the lateral vestibular nucleus: an experimental electron microscopical study in the cat. Experimental Brain Research 4, 187-211. O'NEAL, J. T. & WESTRUM, L. E. (1973). The fine structural synaptic organization of the cat lateral cuneate nucleus. A study of sequential alterations in degeneration. Brain Research 51, 97-124. PECCI-SAAVEDRA, J., VACCAREZZA, 0. L., READER, T. A. & PASQUALINI, E. (1970). Synaptic transmission in the degenerating lateral geniculate nucleus. An ultrastructural and electrophysiological study. Experimental Neurology 26, 607-620. RALSTON, H. J. III. (1968). Dorsal root projections to dorsal horn neurons in the cat spinal cord. Journal of Comparative Neurology 132, 303-330. RALSTON, H. J. III. (1969). The synaptic organisation of lemniscal projections to the ventrobasal thalamus of the cat. Brain Research 14, 99-115. ROSENSTEIN, J. M., PAGE, R. B. & LEURE-DUPREE, A. E. (1977). Patterns of degeneration in the external cuneate nucleus after multiple dorsal rhizotomies. Journal of Comparative Neurology 175, 181-206. RUSTIONI, A. & MACCHI, G. (1968). Distribution of dorsal root fibers in the medulla oblongata of the cat. Journal of Comparative Neurology 134, 113-126. RUSTIONI, A., & SOTELO, C. (1974). Synaptic organization of the nucleus gracilis of cat. Experimental identification of dorsal root fibers and cortical afferents. Journal of Comparative Neurology 155, 441 467. SHRIVER, J. E., STEIN, B. M. & CARPENTER, M. B. (1968). Central projections of spinal dorsal roots in the monkey. I. Cervical and upper thoracic dorsal roots. American Journal of Anatomy 123, 27-74. SOTELO, C. & PALAY, S. L. (1968). The fine structure of the lateral vestibular nucleus in the cat. I. Neurons and neuroglial cells. Journal of Cell Biology 36, 151-180. SZENTAGOTHAI, J. (1970). Glomerular synapses, complex synaptic arrangements and their operational significance. In Neuroscience: Second Study Program (ed. F. 0. Schmitt), pp. 427443. New York: The Rockefeller University Press. SZENTAGOTHAI, J., HAMORI, J. & TOMBOL, T. (1966). Degeneration and electron microscope analysis of the synaptic glomeruli in the lateral geniculate body. Experimental Brain Research 2, 283-301. TAN, C. K. & LIEBERMAN, A. R. (1974). The glomerular synaptic complexes of the rat cuneate nucleus: some ultrastructural observations. Journal of Anatomy 118, 374. VACCAREZZA, 0. L., READER, T. A., PASQUALINI, E. & PEccI-SAAVEDRA, J. (1970). Temporal course of synaptic degeneration in the lateral geniculate nucleus. Its dependence on axonal stump length. Experimental Neurology 28, 277-285. VALVERDE, F. (1966). The pyramidal tract in rodents. A study of its relations with the posterior column nuclei, dorsolateral reticular formation of the medulla oblongata and cervical spinal cord (Golgi and electron microscopic observations). Zeitschrift fuir Zellforschung und mikroskopische Anatomie 71, 297-363.
WALBERG, F. (1965). Axo-axonal contacts in the cuneate nucleus, a probable basis for presynaptic depolarization. Experimental Neurology 13, 218-231. WALBERG, F. (1966). The fine structure of the cuneate nucleus in normal cats and following interruption of afferent fibers. An electron microscopical study with particular reference to findings made in Glees and Nauta sections. Experimental Brain Research 2, 107-128. WEN, C. Y., WONG, W. C. & TAN, C. K. (1978). The fine structural organization of the cuneate nucleus in the monkey (Macaca fascicularis). Journal of Anatomy 127, 169-180. WONG-RILEY, M. T. T. (1972). Terminal degeneration and glial reactions in the lateral geniculate nucleus of the squirrel monkey after eye removal. Journal of Comparative Neurology 144, 61-92.
J. Anat. (1979), 128, 4, pp. 709-720 With 1O figures Printed in Great Britain
Experimental degeneration of primary afferent terminals in the cuneate nucleus of the monkey (Macaca fascicularis) C. Y. WEN, C. K. TAN AND W. C. WONG
Department of Anatomy, Faculty of Medicine, University of Singapore, Sepoy Lines, Singapore 3
(Accepted 8 May 1978) INTRODUCTION
The projection of primary afferent fibres to the mammalian cuneate nucleus has been studied by light microscopy in the rat (Basbaum & Hand, 1973), cat (Keller & Hand, 1970; Rustioni & Macchi, 1968; Kuypers & Tuerk, 1964) and monkey (Dunn & Matzke, 1967; Shriver, Stein & Carpenter, 1968) by means of Nauta and Fink-Heimer methods after cervical dorsal rhizotomy, and under the electron microscope in the cat after transection of the dorsal funiculus (Walberg, 1965, 1966). However, there has been no report of the ultrastructural features of these primary afferent terminals and their patterns of degeneration in the cuneate nucleus of the monkey following dorsal rhizotomy. The present study is a sequel to our earlier study of the normal ultrastructure of the cuneate nucleus of the monkey (Wen, Wong & Tan, 1978) and reports the types of degenerative changes observed in the terminals of cervicothoracic dorsal roots in the cuneate nucleus after multiple rhizotomies. MATERIALS AND METHODS
Six adult monkeys of both sexes, weighing 1-4-1 9 kg, were used for this study. All animals were anaesthetized with an intraperitoneal injection of 0 5 ml of Sagatal (sodium pentobarbital 60 mg/ml) per kg body weight. All surgical procedures were performed under aseptic conditions. Multiple laminectomies, extending from the fifth cervical to the first thoracic vertebrae to expose the relevant parts of the spinal cord, were made on the right side. The dura mater was slit longitudinally along the lateral side of the cord in order to expose the fifth cervical to the first thoracic dorsal roots. The dorsal roots to be sectioned (Table 1) were then identified, lifted gently with a fine glass hook, and cut with a fine blade (No. 12) under a Zeiss Operation Microscope. Care was taken to avoid any damage to the spinal cord or the adjacent radicular vessels. After each operation, penicillin and sulphanilamide powder was sprinkled on the wound. The skin was sutured with silk and sprayed with Neomycin aerosol. The animals were killed after various post-operative survival periods between 2 and 6 days (Table 1). All animals were anaesthetized with Sagatal and artificially respired with oxygen given through a tracheostomy. After the thorax was opened, 1000 units of heparin and 2 ml of 1 % sodium nitrite per kg body weight were given by intracardiac injection. Perfusion was initiated 3-5 minutes later through the left cardiac ventricle with 200 ml of Ringer's solution at pH 7-3-7 4, followed by 500 ml of a dilute fixative (1 % paraformaldehyde+ 1 25 % glutaraldehyde) and then
710
C. Y. WEN, C. K. TAN AND W. C. WONG
Table 1. Data concerning animals and operations Body weight
Dorsal roots
Animal no.
Sex
(kg)
cut
Post-operative survival period
M18 M22 M42 M30 M24 M32
Male Male Male Male Male Female
1-7 1.9 1-4 1-4 1-9 1-6
C8, TI, 2 C5, 6, 7 C6, 7, 8 C7, 8 C7, 8 C6, 7
2 days 3 days 4 days 5 days 6 days 6 days
1000 ml of a concentrated fixative (4 % paraformaldehyde +5 % glutaraldehyde). After decapitation the head was immersed in fresh concentrated fixative and kept overnight at 4 °C before the caudal part of the medulla was dissected out. Transverse slices about 1 mm thick were cut with a razor blade. From these slices portions of the cuneate nucleus were trimmed under a stereomicroscope and placed in ice cold 0* 1 M cacodylate buffer at pH 7-4 containing 5 % sucrose. After four changes of the buffer at intervals of 10 minutes, the slices were post-fixed with 1 % ice cold osmium tetroxide for 2 hours. The tissue slices were dehydrated through an ascending series of acetone and embedded in an Epon mixture. 1 ,tm thick sections were stained with I % methylene blue. Ultrathin sections were doubly stained with uranyl acetate and lead citrate and examined in a Hitachi HS-8 electron microscope. RESULTS
2 days' survival Very few degenerating axon terminals were observed in this animal. Degenerating profiles of terminals contained round synaptic vesicles, had an electron-dense axoplasm (Fig. 1), and their mitochondria were swollen. Some large axon terminals were found within synaptic complexes (Szentagothai, 1970) and formed asymmetrical axodendritic synapses, while others formed isolated axodendritic synapses with small dendritic profiles outside the synaptic complexes. None of the degenerating terminals formed axosomatic synapses. 3 days' survival More degenerating axon terminals were observed in the neuropil at this stage, and at least four types of degenerative changes could be identified. Most were of the electron-dense type; they appeared to be shrunken, with crenated margins, closely packed swollen mitochondria, and round synaptic vesicles. The axoplasm was more electron-dense than previously and some of the profiles contained vacuoles (Fig. 2). Synaptic contact between the degenerating axon terminal and the postsynaptic dendrite was still maintained, although some axon terminals were partially surrounded by glial processes. Other electron-dense terminals showed a less advanced degree of degeneration and resembled those observed after 2 days' survival. The second commonest type of degenerating axon terminal showed neurofilamentous changes. Such terminals were filled with neurofilaments, which were sectioned in various planes (Fig. 3). Small clusters of round synaptic vesicles were usually observed near the synaptic site. Occasionally, clumps of synaptic vesicles were seen among the neurofilaments, but none of the clumps was observed to be surrounded by neurofilamentous whorls (Fig. 4). The axoplasm showed varying
Primary afferent terminals in monkey cuneate nucleus
Fig. 1 A degenerating axon terminal in a synaptic complex showing increased electron densitv and containing swollen mitochondria (AM). 2 days after dorsal rhizotomy. Fig. 2. A distorted electron-dense degenerating axon terminal containing vacuoles (V), forming asymmetrical synapses with two dendrites. A glial process (GP) invaginates the axon terminal. 3 days after dorsal rhizotomy.
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Fig. 3. A degenerating axon terminal with neurofilamentous hyperplasia. The neurofilaments (NF) are arranged in loose bundles which show no special orientation and hence some filaments are longitudinally sectioned while others are cut obliquely and transversely. Synaptic vesicles are clustered near the synaptic site (arrow). 3 days after dorsal rhizotomy. Fig. 4. A degenerating axon terminal with neurofilamentous hyperplasia (NF) in a synaptic complex. It is presynaptic to three dendrites (D). Synaptic vesicles were found near the periphery of the axon terminal and also near the synaptic sites 3 days after dorsal rhizotomy.
713 Primary afferent terminals in monkey cuneate nucleus degrees of electron density. Most of these terminals were large and were found in synaptic complexes (Fig. 5) where they formed the central element. They were presynaptic to dendrites and postsynaptic to small axon terminals containing flattened vesicles. Again, no degenerating axon terminals were seen to form axosomatic synapses. The third type of degenerating axon terminal was electron-lucent. Such terminals were large and were characterized by clear axoplasm in which only a few round synaptic vesicles were found, these being clustered together at the synaptic site (Fig. 6). Mitochondria were still present, but some were swollen and showed loss of cristae. The fourth type of degenerating axon terminal was characterized by its content of flocculent material. Synaptic vesicles were few in number and were clustered near the synaptic site. Mitochondria were swollen, and some of them showed loss of cristae. These terminals formed axodendritic synapses. Occasionally this type of degenerating axon terminal synapsed on a dendrite which was also synaptically contacted by another electron-dense degenerating axon terminal with neurofilamentous change (Fig. 7). 4-6 days' survival All profiles of degenerating axon terminals in these animals were of the electrondense type (Figs. 8-10). Most of them were found within synaptic complexes, in which they were usually the central element, and they were presynaptic to dendrites and postsynaptic to axon terminals containing flattened vesicles. None was observed to form axosomatic synapses (Figs. 9, 10). At this stage, most of the degenerating and shrunken profiles showed some degree of distortion owing to the glial reaction (Figs. 8-10). Vacuoles of different sizes were present in many of the degenerating terminals. Synaptic vesicles, which were tightly packed because of shrinkage of the axon terminals, were still visible, while virtually all the mitochondria were now electron-dense and their cristae and membranes were indistinct. DISCUSSION
Types of degenerative changes in primary afferent terminals following dorsal rhizotomy least four types of degenerating axon terminal (electronat study In the present and floccular) could be distinguished in the neurofilamentous dense, electron-lucent, dorsal rhizotomy. 2-6 after of days cuneate nucleus monkeys was found. After 3 days all four degeneration After 2 days only electron-dense electron-dense type was the The were present. types of degenerative change axon terminals containing the second commonest; neurofilamentous commonest, the the fourth least common. post-operative day all the were By flocculent material of the electron-dense type. were terminals degenerating axon
Electron-dense type of degeneration This type of degeneration was first described by Colonnier & Gray (1962) in the cerebral cortex and regarded by Alksne, Blackstad, Walberg & White (1966) as a pathognomic feature of anterograde degeneration. Subsequently Cohen & Pappas (1969) questioned the validity of using this criterion for identifying degenerating axon terminals. They observed electron-dense axon profiles and darkened neurons
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Primary afferent terminals in monkey cuneate nucleus 715 in normal animals and suggested that they might be attributable to post-mortem changes. However, in our previous study of the normal cuneate nucleus, which was well fixed, no such profiles were seen (Wen et al. 1978). Although 'altered' axon terminals which resembled some of those described by Sotelo & Palay (1968) in the vestibular nucleus of the rat were present, none corresponded to the electron-dense type of degenerating axon terminal of the present study. Similar electron-dense degenerating axon terminals were reported earlier in the cuneate nucleus of the cat following dorsal funiculotomy (Walberg, 1965, 1966) and in the gracile nucleus of the cat after lumbar dorsal rhizotomy (Blomqvist & Westman, 1970; Rustioni & Sotelo, 1974). Electron-lucent type of degeneration The electron-lucent, or clear, type of degeneration was first reported by De Robertis (1956), and has since been demonstrated in several areas of the mammalian central nervous system. McMahan (1967) suggested that such profiles might have resulted from inadequate fixation. While this might have been the case in early electron microscopic studies, the greatly improved quality of fixation in more recent ultrastructural studies seems to rule out this explanation. Concerning the fate of such degenerating axon terminals, O'Neal & Westrum (1973) have suggested that they may change rapidly to the dark form, but our present studies provided no evidence to support this.
Neurofilamentous hyperplasia In the present study, neurofilamentous hyperplasia in degenerating axon terminals was most frequently met with in the 3 day material. Such terminals were rarely seen in the normal monkey cuneate nucleus (Wen et al. 1978). The phenomenon has been demonstrated in the lateral geniculate nucleus of the cat (Szentagothai, Hamori & Tombol, 1966) and monkey (Colonnier & Guillery, 1964; Glees, Mehler & Eschner, 1966; Guillery, 1965) after eye enucleation, and in the ventral part of the dorsal horn of the spinal cord after rhizotomy (Ralston, 1968). On the other hand, no neurofilamentous changes were observed after experimental degeneration in the cuneate (Walberg, 1965, 1966) and gracile nuclei (Blomqvist & Westman, 1970) of the cat or in the cuneate nucleus of the rat (Tan, unpublished observations). The reasons for such species differences are not known. There was some evidence in the present study that degenerating axon terminals which showed neurofilamentous hyperplasia might have progressed to the electron-dense type because there were some intermediate forms between the purely neurofilamentous and the electron-dense types. Such degenerating terminals were shrunken, and in addition to their content of neurofilaments they showed varying degrees of electron density. This suggests that there may be a progressive change from neurofilamentous hyperplasia to the electron-dense type of degeneration. This would agree with the views of many authors Fig. 5. A degenerating axon terminal forming the central element in a synaptic complex. Its matrix shows increased electron density and contains neurofilaments (NF). This axon terminal is surrounded by several dendrites (D) to which it is presynaptic. In addition it is also postsynaptic to small axon terminals containing flattened vesicles (AF). 3 days after dorsal rhizotomy. Fig. 6. A swollen electron-lucent degenerating axon terminal characterized by the clear appearance of its axoplasm. The mitochondria are swollen and their cristae show signs of disintegration. The synaptic vesicles are few in number and are clustered near the synaptic site (arrow). 3 days after dorsal rhizotomy.
716
C. Y. WEN, C. K. TAN AND W. C. WONG `A *.
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Fig. 7. A swollen degenerating axon terminal (FL) with flocculent matrix was presynaptic to a large dendrite. The mitochondria were swollen and disintegrated. Synaptic vesicles were clustered near the synaptic site. 3 days after dorsal rhizotomy. Fig. 8. A shrunken electron-dense degenerating axon terminal partially surrounded by glial processes (GP). Its synaptic contact with a dendrite (D), and an axon terminal containing flattened vesicles (Af) are still maintained. 4 days after dorsal rhizotomy.
Primary afferent terminals in monkey cuneate nucleus Al
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Fig. 9. A shrunken and distorted electron-dense degenerating axon terminal in a synaptic complex. The axon terminal is invaded on one side by glial processes (GP). Swollen synaptic vesicles and degenerating mitochondria are tightly packed in the terminal. 6 days after dorsal rhizotomy. Fig. 10. An electron-dense degenerating axon terminal in a synaptic complex. The axon terminal is grossly distorted by glial processes (GP). At this stage the synaptic vesicles are still clearly visible within the axon terminal. 6 days after dorsal rhizotomy. L6
ANA I28
C. Y. WEN, C. K. TAN AND W. C. WONG (Glees & Hasan, 1968; Jones & Rockel, 1973; Mugnaini & Walberg, 1967; Mugnaini, Walberg & Brodal, 1967; Pecci-Saavedra, Vaccarezza, Reader & Pasqualini, 1970; Ralston, 1969; Szentagothai et al. 1966; Vaccarezza, Reader, Pasqualini & Pecci-Saavedra, 1970; Wong-Riley, 1972).
718
Floccular change In recent years, a fourth
type of degenerative change has been observed in the nervous mammalian central system. The content of such degenerating terminals material' by Jones & Rockel (1973) in the electron-dense been 'flocculent has called material' inferior colliculus, and 'fuzzy by Rosenstein, Page & Leure-DuPree (1977) in the external cuneate nucleus. In the present study, axon terminals containing to granular material were observed after rhizotomy. Such changes have not floccular been described in previous studies of the dorsal column nuclei (Walberg, 1965, 1966; Valverde, 1966; Rustioni & Sotelo, 1974). While the genesis of such terminals was not clear in the present study, there have been suggestions from Jones & Rockel (1973) that the flocculent material arose from empty 'shells' of complex vesicles, and that such axon terminals finally became electron-dense. Terminals which showed flocculent change in the present study had varying degrees of electron density and possibly they could have changed into the electron-dense type. It was not certain whether the floccular and granular changes represented two distinct phenomena, or transitional stages in a spectrum of changes which led to electron density. But a progressive change from floccular to granular and finally to electron-dense remained a possibility.
of primary afferent terminals The results of studies in the rat (Valverde, 1966; Tan, unpublished observations), cat (Walberg, 1965, 1966; Rustioni & Sotelo, 1974) and in the monkey (present study) have shown a remarkable similarity in the mode of termination of primary Mode of termination
afferent fibres in the mammalian dorsal column nuclei. There is general agreement that primary afferent terminals are mostly of large diameter and that they degenerate after dorsal rhizotomy. Profiles of degenerating primary afferent terminals in the cuneate nucleus of the monkey, however, have shown great variability in size. Prominent features of the neuropil of the dorsal column nuclei are the clusters of neural elements, called synaptic glomeruli, in the cuneate nucleus of the rat (Tan & Lieberman, 1974) and the gracile nucleus of the cat (Rustioni & Sotelo, 1974) and termed synaptic complexes in the monkey (Wen et al. 1978). The primary afferent fibres, which contain round synaptic vesicles, terminate within these complexes, where they form asymmetrical synapses with dendrites. Some of these dendrites are organelle-rich, while others are of smaller diameter and have dendritic excrescences. Some of the degenerating axon terminals appear to establish isolated axodendritic synapses. Some of the organelle-rich dendritic profiles are presumably derived from projection cells, but their identity has not yet been clearly determined; it is also possible that some of the postsynaptic dendritic profiles belong to interneurons, since electrophysiological evidence has been produced that both cuneothalamic projection cells and cuneate interneurons can be excited by stimulation of peripheral nerves (Anderson, Eccles, Schmidt & Yokota, 1964). It has been noted previously (Walberg, 1966; Rustioni & Sotelo, 1974) that primary afferent terminals rarely form axosomatic synapses with cuneate neurons. In the present study none of the degenerating axon terminals synapsed directly with any cuneate neuronal somata. On the other hand, axo-axonal synapses in which the
Primary afferent terminals in monkey cuneate nucleus
719
primary afferent terminal is postsynaptic to a small diameter axon terminal containing flattened vesicles were frequently observed. This was first reported in the cuneate nucleus of the cat by Walberg (1965), who suggested that this might be the anatomical substrate for presynaptic inhibition in the cuneate nucleus previously demonstrated electrophysiologically by Andersen, Eccles & Schmidt (1962). SUMMARY
Six monkeys (Macacafascicularis) were used for the present study. In animals which survived for 2-6 days after section of C5 to TI dorsal roots, at least four types of degenerating afferent terminal were observed - electron-dense, electron-lucent, neurofilamentous and flocculent. The electron-dense degeneration was the most common and was seen as early as 2 days after rhizotomy. The neurofilamentous type was the second commonest and was found predominantly in the 3 days' survival material. The electron-lucent and flocculent types were less commonly encountered. Since the profiles exhibiting neurofilamentous hyperplasia showed varying degrees of electron density it is suggested that this type of degeneration progresses to the electron-dense type with time. The present study also showed that the primary afferent terminals in the cuneate nucleus of the monkey are mostly large and that they contain round vesicles. They are commonly found within synaptic complexes in which they are presynaptic to dendrites of various sizes, and are themselves postsynaptic to smaller axon terminals containing flattened vesicles. Degenerating terminals forming isolated synapses were less commonly seen. No dorsal root axon terminals formed axosomatic synapses. The excellent technical assistance of Mr H. L. Chan and Staff of the E.M. Unit is gratefully acknowledged. The authors also wish to thank Professor R. Kanagasuntheram for his interest in the project. One of us (C.Y.W.), who is on leave from the National Taiwan University, is supported by a China Medical Board Faculty Scholarship. REFERENCES ALKSNE, J. F., BLACKSTAD, T. W., WALBERG, F. & WHITE, L. E., JR. (1966). Electron microscopy of axon degeneration: a valuable tool in experimental neuroanatomy. Ergebnisse der Anatomie und
Entwicklungsgeschichte 39, 1-31. ANDERSEN, P., ECCLES, J. C. & SCHMIDT, R. F. (1962). Presynaptic inhibition in the cuneate nucleus. Nature 194, 741-743. ANDERSEN, P., ECCLES J. C. SCHMIDT, R. F. & YOKOTA, T. (1964). Identification of relay cells and interneurones in the cuneate nucleus. Journol of Neurophysiology 27, 1080-1095. BASBAUM, A. I. & HAND, P. J. (1973). Projections of cervicothoracic dorsal roots to the cuneate nucleus of the rat, with observations on cellular 'bricks'. Journal of Comparative Neurology 148, 347-360. BLoMQvIST, A. & WESTMAN, J. (1970). An electron microscopical study of the gracile nucleus in the cat. Acta Societatis medicorum upsaliensis 75, 241-252. COHEN, E. B. & PAPPAS, G. D. (1969). Dark profiles in the apparently-normal central nervous system: a problem in the electron microscopic identification of early antegrade axonal degeneration. Journal of Comparative Neurology 136, 375-396. COLONNIER, M. & GRAY, E. G. (1962). Degeneration in the cerebral cortex. In Electron Microscopy, Fifth Intemational Congress for Electron Microscopy (ed. S. S. Breese, Jr.), vol. 2 New York: Academic Press. COLONNIER, M. & GUILLERY, R. W. (1964). Synaptic organization in the lateral geniculate nucleus of the monkey. Zeitschrift fur Zellforschung und mikroskopische Anatomie 62, 333-355. DE ROBERTIS, E. (1956). Submicroscopic changes of the synapse after nerve section in the acoustic ganglion of the guinea pig. An electron microscope study. Journal of Biophysical and Biochemical Cytology 2, 503-519. 46.2
C. Y. WEN, C. K. TAN AND W. C. WONG 720 DUNN, J. & MATZKE, H. (1967). Central distribution of the dorsal root of the first cervical nerve of the marmoset monkey. Anatomical Record 157, 357. GLEES, P. & HASAN, M. (1968). The signs of synaptic degeneration - a critical appraisal. Acta anatomica 69, 153-167. GLEES, P., MEHLER, K. & ESCHNER, J. (1966). Terminal degeneration in the lateral geniculate body of the monkey: an electron microscopic study. Zeitschrift fir Zellforscfung und mikroskopische Anatomie 71, 29-40. GUILLERY, R. W. (1965). Some electron microscopical observations of degenerative changes in central nervous synapses. In Degeneration Patterns in the Nervous System (ed. M. Singer and J. P. Schade). Progress in Brain Research 14, 57-76. JONES, E. G. & ROCKEL, A. J. (1973). Observations on complex vesicles, neurofilamentous hyperplasia and increased electron density during terminal degeneration in the inferior colliculus. Journal of Comparative Neurology 147, 93-118. KELLER, J. H. & HAND, P. J. (1970). Dorsal roots projections to nucleus cuneatus of the cat. Brain Research 20, 1-17. KuYPERS, H. G. J. M. & TUERK, J. D. (1964). The distribution of the cortical fibres within the nuclei cuneatus and gracilis in the cat. Journal of Anatomy 98, 143-162. MCMAHAN, U. J. (1967). Fine structure of synapses in the dorsal nucleus of the lateral geniculate body of normal and blinded rats. Zeitschrift fiir Zellforschung und mikroskopische Anatomie 76, 116-146. MUGNAINI, E. & WALBERG, F. (1967). An experimental electron microscopical study on the mode of termination of cerebellar corticovestibular fibres in the cat lateral vestibular nucleus (Deiter's nucleus). Experimental Brain Research 4, 212-236. MUGNAINI, E., WALBERG, F. & BRODAL, A. (1967). Mode of termination of primary vestibular fibers in the lateral vestibular nucleus: an experimental electron microscopical study in the cat. Experimental Brain Research 4, 187-211. O'NEAL, J. T. & WESTRUM, L. E. (1973). The fine structural synaptic organization of the cat lateral cuneate nucleus. A study of sequential alterations in degeneration. Brain Research 51, 97-124. PECCI-SAAVEDRA, J., VACCAREZZA, 0. L., READER, T. A. & PASQUALINI, E. (1970). Synaptic transmission in the degenerating lateral geniculate nucleus. An ultrastructural and electrophysiological study. Experimental Neurology 26, 607-620. RALSTON, H. J. III. (1968). Dorsal root projections to dorsal horn neurons in the cat spinal cord. Journal of Comparative Neurology 132, 303-330. RALSTON, H. J. III. (1969). The synaptic organisation of lemniscal projections to the ventrobasal thalamus of the cat. Brain Research 14, 99-115. ROSENSTEIN, J. M., PAGE, R. B. & LEURE-DUPREE, A. E. (1977). Patterns of degeneration in the external cuneate nucleus after multiple dorsal rhizotomies. Journal of Comparative Neurology 175, 181-206. RUSTIONI, A. & MACCHI, G. (1968). Distribution of dorsal root fibers in the medulla oblongata of the cat. Journal of Comparative Neurology 134, 113-126. RUSTIONI, A., & SOTELO, C. (1974). Synaptic organization of the nucleus gracilis of cat. Experimental identification of dorsal root fibers and cortical afferents. Journal of Comparative Neurology 155, 441 467. SHRIVER, J. E., STEIN, B. M. & CARPENTER, M. B. (1968). Central projections of spinal dorsal roots in the monkey. I. Cervical and upper thoracic dorsal roots. American Journal of Anatomy 123, 27-74. SOTELO, C. & PALAY, S. L. (1968). The fine structure of the lateral vestibular nucleus in the cat. I. Neurons and neuroglial cells. Journal of Cell Biology 36, 151-180. SZENTAGOTHAI, J. (1970). Glomerular synapses, complex synaptic arrangements and their operational significance. In Neuroscience: Second Study Program (ed. F. 0. Schmitt), pp. 427443. New York: The Rockefeller University Press. SZENTAGOTHAI, J., HAMORI, J. & TOMBOL, T. (1966). Degeneration and electron microscope analysis of the synaptic glomeruli in the lateral geniculate body. Experimental Brain Research 2, 283-301. TAN, C. K. & LIEBERMAN, A. R. (1974). The glomerular synaptic complexes of the rat cuneate nucleus: some ultrastructural observations. Journal of Anatomy 118, 374. VACCAREZZA, 0. L., READER, T. A., PASQUALINI, E. & PEccI-SAAVEDRA, J. (1970). Temporal course of synaptic degeneration in the lateral geniculate nucleus. Its dependence on axonal stump length. Experimental Neurology 28, 277-285. VALVERDE, F. (1966). The pyramidal tract in rodents. A study of its relations with the posterior column nuclei, dorsolateral reticular formation of the medulla oblongata and cervical spinal cord (Golgi and electron microscopic observations). Zeitschrift fuir Zellforschung und mikroskopische Anatomie 71, 297-363.
WALBERG, F. (1965). Axo-axonal contacts in the cuneate nucleus, a probable basis for presynaptic depolarization. Experimental Neurology 13, 218-231. WALBERG, F. (1966). The fine structure of the cuneate nucleus in normal cats and following interruption of afferent fibers. An electron microscopical study with particular reference to findings made in Glees and Nauta sections. Experimental Brain Research 2, 107-128. WEN, C. Y., WONG, W. C. & TAN, C. K. (1978). The fine structural organization of the cuneate nucleus in the monkey (Macaca fascicularis). Journal of Anatomy 127, 169-180. WONG-RILEY, M. T. T. (1972). Terminal degeneration and glial reactions in the lateral geniculate nucleus of the squirrel monkey after eye removal. Journal of Comparative Neurology 144, 61-92.
