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An electron microscope study of synaptic terminals of the spino-olivary fibers in the cat
Brain Research, 104 (1976) 303-308
303
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Short Communications
An electron microscope study of synaptic terminals of the spino-olivary fibers in the cat
NOBORU MIZUNO, AKIRA KONISHI AND YASUHISA NAKAMURA Department of Anatomy, Faculty of Medicine, Kyoto University, Kyoto 606 (Japan.)
(Accepted November 26th, 1975)
The inferior olivary nucleus (IO) has been well known as one of the important precerebeUar nuclei and reported to receive afferent fibers from various sources in the central nervous systemL The distribution pattern of terminals of spino-olivary fibers has been described in the cat on the basis of light microscopical findings6,S, 17. On the other hand, electron microscope studies on the mode of termination of spinal afferent fibers to the IO in the cat are rather sporadic 1, although the ultrastructures of the IO of the cat are relatively well known23, 27,2s. In view of these circumstances the present study was undertaken in an attempt to analyze quantitatively the synaptic terminals of spinal fibers terminating within the IO areas. Of 12 cats used in the present study, 9 were allowed to survive for 2-7 days following hemicordotomy at the level of the second or third cervical cord segment; the remaining 3 served as controls. Surgical procedures were carried out under deep general anesthesia (sodium pentobarbital, 35-40 mg/kg i.p.), and all animals were treated postoperatively with penicillin. After a survival period the cats were deeply anesthetized with an overdose of pentobarbital and perfused intravitally through the ascending aorta with 1.5-2 1 of a mixture composed of 4 70 paraformaldehyde, 0.5 70 glutaraldehyde and 0.002 70 calcium chloride in Millonig's phosphate buffer adjusted to pH 7.2-7.4. Perfusion was extended over a period of 15 min from a height of about 1 m. After perfusion, thin tissue slices containing the caudal portions of the IO ipsilateral to the lesion were postfixed for 90-120 min in a chilled 2 ~ solution of osmium tetroxide in the same buffer used for preparing the perfusion fixative. Embedding was performed in an Epoxy resin after dehydration in a graded series of ethanol. The location of the IO areas was verified in semithin sections which were cut from each block and stained with 0.5 70 toluidine blue in 1 700borax. Of the various subdivisions of the IO, the caudolateral portions of the medial (MO) and dorsal (DO) accessory olivary nuclei were selected for ultrastructural study of thin sections, because the spino-olivary fibers were reported to terminate most numerously in these portions of the 10 6,8,17. Ultrathin sections were mounted on uncoated or collodion-coated copper grid and
304 stained with lead acetatO 6 or lead citrate zS, and viewed with a Hitachi HU-12 electron microscope. The location and extent of the lesion in each of the operated cats were verified histologically as described elsewhere is. In the central parts of each grid hole (120-130 Hm diameter), 2l rectangular fields (8 #m x 9 #m) were photographed serially without reference to their contents at an initial magnification of x 10,000, and subsequently enlarged on paper to a final magnification of x 25,000. On these prints, both axosomatic (AS) and axodendritic (AD) synaptic knobs were categorized into knobs with round vesicles and those with pleomorphic ones (mixture of varying ratios of round and elongated vesicles), and counts were made of the knobs of each category. The density of AS synapses (the number of AS knobs with synaptic active zones per 100/~m length of the cell membrane) was also calculated on each of the somatic profiles cut through the nucleolar plane. Details pertaining to the procedures for these quantitative analyses have been described elsewhere 18,19. In the 10 areas examined in the normal cats, about 20')0 of the AS knobs were filled with round synaptic vesicles, whereas the AD knobs with round vesicles constituted approximately 60 ~/o of the total A D synaptic population. Although the association between the shape of synaptic vesicle and the type of synaptic active zone was not consistent, the A D knobs filled with round vesicles were associated usually with the asymmetrical active zone of the synapses; the vast majority of the A D knobs containing pleomorphic vesicles and the AS knobs were associated with the symmetrical active zones. In all of 9 cats subjected to hemicordotomy, degenerated changes of synaptic knobs were observed in the spinal areas of the IO: the caudolateral portions of the MO and DO ipsilateral to the lesion. Electron-dense degenerated synaptic knobs with discernible active zones were encountered most numerously in the cats with survival period of 3-4 days. Electron-dense knobs and dense bodies engulfed by glial profiles were seen even in a cat with survival period of 2 days, but these were observed most numerously in the cats with survival period of 5-7 days. In a total of 274 electrondense knobs encountered in the MO and DO, 269 were found upon dendritic profiles (Fig. 1) and 5 upon somatic membranes (Fig. 2); the synaptic active zones could be identified in 43 of the former and in 2 of the latter. In addition to these electron-dense knobs, other features of possible degeneration of synaptic knobs, such as the increase of neurofilaments, the paleness of the matrix, the accumulation of glycogen granules and the enlargement of synaptic vesicles were observed. Similar features, however, were also seen in normal cats, although only infrequently and in lower grades. In the present study, therefore, these features of the synaptic knobs were not considered as definitely reliable criteria of degeneration of axon terminals. in the grid holes where electron-dense degenerated synaptic knobs were encountered most frequently, the ratio between degenerated and normal synaptic knobs was calculated. Within a DO area of about 1200 sq. ~m in a cat with survival period of 4 days, of 355 synaptic knobs upon dendritic profiles, 24 (6.8 ~o) were electrondense; one electron-dense knob was also found in contact with a somatic profile as well as 30 dense bodies enveloped by glial profiles. Within a MO area of the same cat,
305
Fig. 1. An electron-dense degenerated axodendritic synaptic knob encountered in the MO of a cat with a spinal lesion in the second cervical cord segment. An arrow indicates the synaptic active zone. 3 days survival, x 76,000. Fig. 2. A degenerated axosomatic synaptic knob found in the DO following hemisection of the third cervical cord segment. An arrow points to the synaptic active zone. 4 days survival, x 63,000.
306 TABLE I AXODENDRITIC SYNAPTIC KNOBS
Knobs counted
Controls Medial accessory olive Dorsal accessory olive
Knobs with round vesicles ( d: S.E.M.)
Knobs with pleomorphic vesicles ( ± S.E.M.)
1476 1266
859 (58.2 ± 1.28%) 775 (61.2 ± 1.37%)
617 (41.8 :c 1.28%) 491 (38.8 ± 1.37%)
Experimentals (7 days survival) Medial accessory olive* 1513 Dorsal accessory olive** 1671
974 (64.4 ± 1.23%) 1069 (64.0 ± 1.17%)
539 (35.6 ± 1.23 %) 602 (36.0 ± 1.17%)
* Medial accessory olive: Z~ 12.02,n -- 1, 0.01 3> P. ** Dorsal accessory olive: Z2 -- 2.34, n -- 1, 0.13 ~ P ~-- 0.12. out of 273 synaptic k n o b s u p o n dendritic profiles 13 (4.8 %) were electron-dense; 18 dense bodies were also observed in this M O area. Thus, degenerated knobs constituted only a small fraction of the total synaptic k n o b s in the areas examined; they never exceeded more t h a n 7 % even in the area where degenerated k n o b s were observed most numerously. Such unexpected sparseness of degenerated k n o b s at a particular survival time, however, could be a t t r i b u t a b l e to variable time courses of degeneration process. The ratio of the two types of intact A D k n o b s a n d / o r the density of intact AS k n o b s could then be expected to be changed accordingly in the animals with survival period of 7 days; in these cats almost all of the degenerated k n o b s were observed to be enveloped by glial profiles a n d the vast majority of the synaptic knobs of severed axons were considered to be in a detectable stage of degeneration or to have disappeared. The data thus o b t a i n e d are summarized in Tables I a n d II: the ratio of A D k n o b s with r o u n d vesicles to those with pleomorphic vesicles was somewhat increased in the spinal areas of the M O of the operated cats; no other significant differences were n o t e d at this level o f accuracy between the data o b t a i n e d from the operated cats a n d those from the controls. Even in the spinal TABLE lI THE DENSITY OF AXOSOMATIC SYNAPSES (THE NUMBER OF AXOSOMATIC SYNAPTIC KNOBS PER
100 //na
LENGTH OF THE SOMATIC MEMBRANE)
Number of cells
Range o] density
Mean of density ( ± S.E.M.)
Controls Medial accessory olive Dorsal accessory olive
61 45
0-12.0 0- 8. l
2.4 ± 0.34 3.0 ± 0.33
Experimentals (7 days survival) Medial accessory olive* Dorsal accessory olive**
30 32
0- 6.5 0- 8.5
2.2 ± 0.37 3.0 :L 0.42
* Medial accessory olive: F296° 1.698,t 0.173,n 89, 0.9 :-~ P ** Dorsal accessory olive: F3144 1.141,t - 0.025, n := 75, P ~ 0.9.
0.8.
307
areas of the MO of the operated animals, however, the subjective impression was that the number of the intact synaptic knobs did not seem to be decreased, although no further quantitative measurements were undertaken. As regards the numerous knobs remaining intact in the spinal areas of the IO even after deafferentation of spinal afferents, several possible sources for these could be assumed. A considerable number of fibers arising from the gracile nucleus are reported to terminate contralaterally in the spinal areas of the IO (especially of the DO), but the amount of these afferent fibers does not seem to exceed that of the spinal fibersS,6,10, la. The MO and DO areas were also reported to receive afferent fibers from the cerebral cortex24, 26, mesodiencephalic areas15, 29, cerebellar nuclei°, 12 and cuneate nucleus6,1L In the spinal areas of the IO examined in the present study, however, these extra-spinal fibers did not seem to terminate so numerously as the spino-olivary fibers. Other possible sources for the remaining normal synaptic knobs could be the medullary reticular formation4,14, zz and/or fibers of intrinsic origin. The presence of axon collaterals of IO neurons terminating within the confines of the IO was indicated electrophysiologicallyS, 11 as well as histologically 2°. There is no clear evidence suggesting the existence of internuncial neurons within the 10 7,21. In summary, the majority of synaptic knobs in the spinal areas of the IO are tentatively considered to be axon terminals of afferent fibers of extra-spinal origin, and/or terminals of axon collaterals of IO neurons, although precise knowledge concerning the sites of origin of these extra-spinal fibers is still lacking. It is inferred that the spinal areas of the IO may function as more than simple relay areas for spinal inputs.
1 ALKSNE,J. F., BLACKSTAD,TH. W., WALBERG,F., AND WHITE, L. E., JR., Electron microscopy of axon degeneration: a valuable tool in experimental neuroanatomy, Ergebn. Anat. Entwickl.Gesch., 39 (1966) 1-32. 2 ARMSTRONG,D. M., Functional significance of connections of the inferior olive, PhysioL Rev., 54 (1974) 358--417. 3 ARMSTRONG,D. M., AND HARVEY, R. J., Responses in the inferior olive to stimulation of the cerebellar and cerebral cortices in the cat, J. PhysioL (Lond.), 187 (1966) 553-574. 4 BAUMGARTEN,H. G., BJORKLUND,A., LACKENMAYER,t . , RENSCH, A., AND ROSENGREN,E., Deand regeneration of the bulbospinal serotonin neurons in the rat following 5,6- or 5,7-dihydroxytryptamine treatment, Cell Tiss. Res., 152 (1974) 271-281. 5 BERKLEY, K. J., Different targets of different neurons in nucleus gracilis of the cat, J. comp. Neurol., 163 (1975) 285-304. 6 BOESTEN,A. J. P., AND VOOCD, J., Projections of the dorsal column nuclei and the spinal cord on the inferior olive in the cat, J. comp. Neurol., 161 (1975) 215-238. 7 BRODAL, A., Experimentelle Untersuchungen iiber die olivocerebellare Lokalisation, Z. ges. Neurol. Psychiat., 169 (1940) 1-153. 8 BRODAL, A., WALBERG, F., AND BLACKSTAD,TH. W., Termination of spinal afferents to inferior olive in cats, J. NeurophysioL, 13 (1950) 431454. 9 DOM, R., KING, J. S., AND MARTIN, G. F., Evidence for two direct cerebello-olivary connections, Brain Research, 57 (1973) 498-501. 10 EBBESSON,S. O. E., A connection between the dorsal column nuclei and the dorsal accessory olive, Brain Research, 8 (1968) 393-397. 11 ECCLES,J. C., LLIN.~S, R., AND SASAKI,K., The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum, J. Physiol. (Lond.), 182 (1966) 268-296. 12 GRAYBIEL,A. M., NAUTA, H. J. W., LASEK,R. J., AND NAUTA, W. J. H., A cerebello-olivary path-
308 way in the cat : an experimental study using autoradiographic tracing techniques, Brain Research, 58 (1973) 205-211. 13 GROENEWEGEN,H. J., BOESTEN,A. J. P., AND VOOGD,J., The dorsal column nuclear projections to the nucleus ventralis posterior lateralis thalami and the inferior olive in the cat: an autoradiographic study, J. comp. Neurol., 162 (1975) 505-518. 14 HOFFMAN,O. L., AND SLADEK, J. R., JR., The distribution of catecholamines within the inferior olivary complex of the gerbil and rabbit, J. comp. Neurol., 151 (1973) 101-112. 15 MABUCHI,M., AND KUSAMA,T., Mesodiencephatic projections to the inferior olive and the vestibular and perihypoglossal nuclei, Brain Research, 17 (1970) 133-136. 16 MILLONIG,G., A modified procedure for lead staining of thin sections, J. biophys, biochem. Cytol., 11 (1961) 736-739. 17 MIZUNO, N., An experimental study of the spino-olivary fibers in the rabbit and the cat, J. comp. NeuroL, 127 (1966) 267-292. 18 MIZUNO, N., KONISHI,A., AND NAKAMURA,Y., An electron microscope study of synaptic organization in the lateral reticular nucleus of the medulla oblongata in the cat, Brain Research, 94 (1975) 369-381. 19 MIZUNO, N., NAKAMURA,Y., AND |WAHOR1,N., An electron microscope study of the dorsal cap of the inferior olive in the rabbit, with special reference to the pretecto-olivary fibers, Brain Research, 77 (1974) 385-395. 20 RAM6N Y CAJAL, S., Histologie du Syst~me Nerveux de I'Homme et des Vertdbrds, VoL 1, Maloine, Paris, 1909-191 l, reprinted 1952, C.S.I.C., Madrid, pp. 919-933. 21 SEDWICK,E. M., AND WILLIAMS,T. D., Responses of single units in the inferior olive to stimulation of the limb nerves, peripheral skin receptors, cerebellum, caudate nucleus and motor cortex, J. PhysioL (Lond.), 189 (1967) 261-279. 22 SLADEK, J. R., JR., AND HOFFMAN,D. L., Differences in catecholamine distribution in the inferior olivary complex of various mammals, Anat. Ree., 175 (1973) 444-445. (Abstract). 23 SOTELO,C., LLIN.~S, R., AND BAKER, R., Structural study of inferior olivary nucleus of the cat: morphological correlates of electronic coupling, J. NeurophysioL, 37 (1974) 541-559. 24 SOUSA-PINTO,A., AND BRODAL, A., Demonstration of a somatotopical pattern in the corticoolivary projection in the cat, Exp. Brain Res., 8 (1969) 364-386. 25 VENABLE,J. H., AND COGGESHALL,R., A simplified lead citrate stain for use in electron microscopy, J. Cell Biol., 25 (1965) 407408. 26 WALBERG,F., Descending connections to the inferior olive. An experimental study in the cat, J. comp. Neurol., 104 (1956) 77-173. 27 WALBERG,F., An electron microscopic study of the inferior olive of the cat, J. comp. Neurol., 120 (1963) 1-18. 28 WALBERG, F., Further electron microscopical investigations of the inferior olive of the cat. In W. BARGMANNAND J. P. SCHAD~ (Eds.), Topics in Basic Neurology, Progr. Brain Res., Vol. 6, Elsevier, Amsterdam, 1964, pp. 59-75. 29 WALBERG, F., Descending connections from the mesencephalon to the inferior olive: an experimental study in the cat, Exp. Brain Res., 21 (1974) 145-156.
303
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Short Communications
An electron microscope study of synaptic terminals of the spino-olivary fibers in the cat
NOBORU MIZUNO, AKIRA KONISHI AND YASUHISA NAKAMURA Department of Anatomy, Faculty of Medicine, Kyoto University, Kyoto 606 (Japan.)
(Accepted November 26th, 1975)
The inferior olivary nucleus (IO) has been well known as one of the important precerebeUar nuclei and reported to receive afferent fibers from various sources in the central nervous systemL The distribution pattern of terminals of spino-olivary fibers has been described in the cat on the basis of light microscopical findings6,S, 17. On the other hand, electron microscope studies on the mode of termination of spinal afferent fibers to the IO in the cat are rather sporadic 1, although the ultrastructures of the IO of the cat are relatively well known23, 27,2s. In view of these circumstances the present study was undertaken in an attempt to analyze quantitatively the synaptic terminals of spinal fibers terminating within the IO areas. Of 12 cats used in the present study, 9 were allowed to survive for 2-7 days following hemicordotomy at the level of the second or third cervical cord segment; the remaining 3 served as controls. Surgical procedures were carried out under deep general anesthesia (sodium pentobarbital, 35-40 mg/kg i.p.), and all animals were treated postoperatively with penicillin. After a survival period the cats were deeply anesthetized with an overdose of pentobarbital and perfused intravitally through the ascending aorta with 1.5-2 1 of a mixture composed of 4 70 paraformaldehyde, 0.5 70 glutaraldehyde and 0.002 70 calcium chloride in Millonig's phosphate buffer adjusted to pH 7.2-7.4. Perfusion was extended over a period of 15 min from a height of about 1 m. After perfusion, thin tissue slices containing the caudal portions of the IO ipsilateral to the lesion were postfixed for 90-120 min in a chilled 2 ~ solution of osmium tetroxide in the same buffer used for preparing the perfusion fixative. Embedding was performed in an Epoxy resin after dehydration in a graded series of ethanol. The location of the IO areas was verified in semithin sections which were cut from each block and stained with 0.5 70 toluidine blue in 1 700borax. Of the various subdivisions of the IO, the caudolateral portions of the medial (MO) and dorsal (DO) accessory olivary nuclei were selected for ultrastructural study of thin sections, because the spino-olivary fibers were reported to terminate most numerously in these portions of the 10 6,8,17. Ultrathin sections were mounted on uncoated or collodion-coated copper grid and
304 stained with lead acetatO 6 or lead citrate zS, and viewed with a Hitachi HU-12 electron microscope. The location and extent of the lesion in each of the operated cats were verified histologically as described elsewhere is. In the central parts of each grid hole (120-130 Hm diameter), 2l rectangular fields (8 #m x 9 #m) were photographed serially without reference to their contents at an initial magnification of x 10,000, and subsequently enlarged on paper to a final magnification of x 25,000. On these prints, both axosomatic (AS) and axodendritic (AD) synaptic knobs were categorized into knobs with round vesicles and those with pleomorphic ones (mixture of varying ratios of round and elongated vesicles), and counts were made of the knobs of each category. The density of AS synapses (the number of AS knobs with synaptic active zones per 100/~m length of the cell membrane) was also calculated on each of the somatic profiles cut through the nucleolar plane. Details pertaining to the procedures for these quantitative analyses have been described elsewhere 18,19. In the 10 areas examined in the normal cats, about 20')0 of the AS knobs were filled with round synaptic vesicles, whereas the AD knobs with round vesicles constituted approximately 60 ~/o of the total A D synaptic population. Although the association between the shape of synaptic vesicle and the type of synaptic active zone was not consistent, the A D knobs filled with round vesicles were associated usually with the asymmetrical active zone of the synapses; the vast majority of the A D knobs containing pleomorphic vesicles and the AS knobs were associated with the symmetrical active zones. In all of 9 cats subjected to hemicordotomy, degenerated changes of synaptic knobs were observed in the spinal areas of the IO: the caudolateral portions of the MO and DO ipsilateral to the lesion. Electron-dense degenerated synaptic knobs with discernible active zones were encountered most numerously in the cats with survival period of 3-4 days. Electron-dense knobs and dense bodies engulfed by glial profiles were seen even in a cat with survival period of 2 days, but these were observed most numerously in the cats with survival period of 5-7 days. In a total of 274 electrondense knobs encountered in the MO and DO, 269 were found upon dendritic profiles (Fig. 1) and 5 upon somatic membranes (Fig. 2); the synaptic active zones could be identified in 43 of the former and in 2 of the latter. In addition to these electron-dense knobs, other features of possible degeneration of synaptic knobs, such as the increase of neurofilaments, the paleness of the matrix, the accumulation of glycogen granules and the enlargement of synaptic vesicles were observed. Similar features, however, were also seen in normal cats, although only infrequently and in lower grades. In the present study, therefore, these features of the synaptic knobs were not considered as definitely reliable criteria of degeneration of axon terminals. in the grid holes where electron-dense degenerated synaptic knobs were encountered most frequently, the ratio between degenerated and normal synaptic knobs was calculated. Within a DO area of about 1200 sq. ~m in a cat with survival period of 4 days, of 355 synaptic knobs upon dendritic profiles, 24 (6.8 ~o) were electrondense; one electron-dense knob was also found in contact with a somatic profile as well as 30 dense bodies enveloped by glial profiles. Within a MO area of the same cat,
305
Fig. 1. An electron-dense degenerated axodendritic synaptic knob encountered in the MO of a cat with a spinal lesion in the second cervical cord segment. An arrow indicates the synaptic active zone. 3 days survival, x 76,000. Fig. 2. A degenerated axosomatic synaptic knob found in the DO following hemisection of the third cervical cord segment. An arrow points to the synaptic active zone. 4 days survival, x 63,000.
306 TABLE I AXODENDRITIC SYNAPTIC KNOBS
Knobs counted
Controls Medial accessory olive Dorsal accessory olive
Knobs with round vesicles ( d: S.E.M.)
Knobs with pleomorphic vesicles ( ± S.E.M.)
1476 1266
859 (58.2 ± 1.28%) 775 (61.2 ± 1.37%)
617 (41.8 :c 1.28%) 491 (38.8 ± 1.37%)
Experimentals (7 days survival) Medial accessory olive* 1513 Dorsal accessory olive** 1671
974 (64.4 ± 1.23%) 1069 (64.0 ± 1.17%)
539 (35.6 ± 1.23 %) 602 (36.0 ± 1.17%)
* Medial accessory olive: Z~ 12.02,n -- 1, 0.01 3> P. ** Dorsal accessory olive: Z2 -- 2.34, n -- 1, 0.13 ~ P ~-- 0.12. out of 273 synaptic k n o b s u p o n dendritic profiles 13 (4.8 %) were electron-dense; 18 dense bodies were also observed in this M O area. Thus, degenerated knobs constituted only a small fraction of the total synaptic k n o b s in the areas examined; they never exceeded more t h a n 7 % even in the area where degenerated k n o b s were observed most numerously. Such unexpected sparseness of degenerated k n o b s at a particular survival time, however, could be a t t r i b u t a b l e to variable time courses of degeneration process. The ratio of the two types of intact A D k n o b s a n d / o r the density of intact AS k n o b s could then be expected to be changed accordingly in the animals with survival period of 7 days; in these cats almost all of the degenerated k n o b s were observed to be enveloped by glial profiles a n d the vast majority of the synaptic knobs of severed axons were considered to be in a detectable stage of degeneration or to have disappeared. The data thus o b t a i n e d are summarized in Tables I a n d II: the ratio of A D k n o b s with r o u n d vesicles to those with pleomorphic vesicles was somewhat increased in the spinal areas of the M O of the operated cats; no other significant differences were n o t e d at this level o f accuracy between the data o b t a i n e d from the operated cats a n d those from the controls. Even in the spinal TABLE lI THE DENSITY OF AXOSOMATIC SYNAPSES (THE NUMBER OF AXOSOMATIC SYNAPTIC KNOBS PER
100 //na
LENGTH OF THE SOMATIC MEMBRANE)
Number of cells
Range o] density
Mean of density ( ± S.E.M.)
Controls Medial accessory olive Dorsal accessory olive
61 45
0-12.0 0- 8. l
2.4 ± 0.34 3.0 ± 0.33
Experimentals (7 days survival) Medial accessory olive* Dorsal accessory olive**
30 32
0- 6.5 0- 8.5
2.2 ± 0.37 3.0 :L 0.42
* Medial accessory olive: F296° 1.698,t 0.173,n 89, 0.9 :-~ P ** Dorsal accessory olive: F3144 1.141,t - 0.025, n := 75, P ~ 0.9.
0.8.
307
areas of the MO of the operated animals, however, the subjective impression was that the number of the intact synaptic knobs did not seem to be decreased, although no further quantitative measurements were undertaken. As regards the numerous knobs remaining intact in the spinal areas of the IO even after deafferentation of spinal afferents, several possible sources for these could be assumed. A considerable number of fibers arising from the gracile nucleus are reported to terminate contralaterally in the spinal areas of the IO (especially of the DO), but the amount of these afferent fibers does not seem to exceed that of the spinal fibersS,6,10, la. The MO and DO areas were also reported to receive afferent fibers from the cerebral cortex24, 26, mesodiencephalic areas15, 29, cerebellar nuclei°, 12 and cuneate nucleus6,1L In the spinal areas of the IO examined in the present study, however, these extra-spinal fibers did not seem to terminate so numerously as the spino-olivary fibers. Other possible sources for the remaining normal synaptic knobs could be the medullary reticular formation4,14, zz and/or fibers of intrinsic origin. The presence of axon collaterals of IO neurons terminating within the confines of the IO was indicated electrophysiologicallyS, 11 as well as histologically 2°. There is no clear evidence suggesting the existence of internuncial neurons within the 10 7,21. In summary, the majority of synaptic knobs in the spinal areas of the IO are tentatively considered to be axon terminals of afferent fibers of extra-spinal origin, and/or terminals of axon collaterals of IO neurons, although precise knowledge concerning the sites of origin of these extra-spinal fibers is still lacking. It is inferred that the spinal areas of the IO may function as more than simple relay areas for spinal inputs.
1 ALKSNE,J. F., BLACKSTAD,TH. W., WALBERG,F., AND WHITE, L. E., JR., Electron microscopy of axon degeneration: a valuable tool in experimental neuroanatomy, Ergebn. Anat. Entwickl.Gesch., 39 (1966) 1-32. 2 ARMSTRONG,D. M., Functional significance of connections of the inferior olive, PhysioL Rev., 54 (1974) 358--417. 3 ARMSTRONG,D. M., AND HARVEY, R. J., Responses in the inferior olive to stimulation of the cerebellar and cerebral cortices in the cat, J. PhysioL (Lond.), 187 (1966) 553-574. 4 BAUMGARTEN,H. G., BJORKLUND,A., LACKENMAYER,t . , RENSCH, A., AND ROSENGREN,E., Deand regeneration of the bulbospinal serotonin neurons in the rat following 5,6- or 5,7-dihydroxytryptamine treatment, Cell Tiss. Res., 152 (1974) 271-281. 5 BERKLEY, K. J., Different targets of different neurons in nucleus gracilis of the cat, J. comp. Neurol., 163 (1975) 285-304. 6 BOESTEN,A. J. P., AND VOOCD, J., Projections of the dorsal column nuclei and the spinal cord on the inferior olive in the cat, J. comp. Neurol., 161 (1975) 215-238. 7 BRODAL, A., Experimentelle Untersuchungen iiber die olivocerebellare Lokalisation, Z. ges. Neurol. Psychiat., 169 (1940) 1-153. 8 BRODAL, A., WALBERG, F., AND BLACKSTAD,TH. W., Termination of spinal afferents to inferior olive in cats, J. NeurophysioL, 13 (1950) 431454. 9 DOM, R., KING, J. S., AND MARTIN, G. F., Evidence for two direct cerebello-olivary connections, Brain Research, 57 (1973) 498-501. 10 EBBESSON,S. O. E., A connection between the dorsal column nuclei and the dorsal accessory olive, Brain Research, 8 (1968) 393-397. 11 ECCLES,J. C., LLIN.~S, R., AND SASAKI,K., The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum, J. Physiol. (Lond.), 182 (1966) 268-296. 12 GRAYBIEL,A. M., NAUTA, H. J. W., LASEK,R. J., AND NAUTA, W. J. H., A cerebello-olivary path-
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