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Reinnervation of the olfactory bulb after section of the olfactory nerve in monkey (Saimiri sciureus)
Brain Research, 189 (1980) 343-354 © Elsevier/North-HollandBiomedicalPress
343
REINNERVATION OF THE OLFACTORY BULB AFTER SECTION OF THE OLFACTORY NERVE IN MONKEY (SAIMIRI SCIUREUS)
G. A. M O N T I G R A Z I A D E I , M. S. K A R L A N * , J. J. B E R N S T E I N * * a n d P. P. C. G R A Z I A D E I
Department of Biological Science, Florida State University, Tallahassee, Fla. 32306; (M.S.K.) Department of Otolaryngology and Center for Sensory Studies, University of Florida College of Medicine, and ( J.J.B.) Department of Neuroscience, Urdversity of Florida College of Medicine, Gainesville, Fla. 32610
(u.s.A.) (Accepted O c t o b e r 1 l t h , 1979)
Key words: olfactory bulb - - r e i n n e r v a t i o n - - m o n k e y olfactory nerve - - deafferentation
SUMMARY
Section of the ilia olfactoria in squirrel monkey, a non-human primate, induces rapid degeneration of the sensory axon terminals in the olfactory bulb glomeruli. A population of axons, from newly formed sensory neurons in the olfactory neuroepithelium, regrows, passes the lamina cribrosa and, upon reaching the olfactory bulb, reinnervates the glomeruli. A new set of synaptic contacts is reformed between the sensory terminals and the post-synaptic dendritic processes of the glomeruli. Our observations indicate that this portion of the CNS of a non-human primate can be reinnervated after deafferentation, and that active synaptogenesis occurs.
INTRODUCTION
The olfactory epithelium in lower vertebrates and rodents is unique because of its ability to generate new sensory neurons through the animal's entire life2,a,5,6. There is a cyclic neuronal renewal from the basal cells of this epithelium and the neurons appear to have a life span of approximately 30 40 days according to species1,4,6,11. The axons of these neurons join to form the ilia olfactoria and terminate in the bulb glomeruli, where they synapse upon the main dendrite of the mitral cells. Renewal of neurons perikarya in the neuroepithelium implies renewal of the synaptic contacts of their axon terminals in the olfactory bulb glomeruli. Following axotomy of the ilia olfactoria there is rapid degeneration of the olfactory sensory neurons in the nasal neuroepitheliuma,7,9,12. This process is followed
344
by basal cells division and differentiation into neurons, by regrowth of their axons into the olfactory bulb and synapses formation on the mitral cells dendrites. The process of olfactory neurons renewal has been recently shown to occur also in primates 10. In humans, sections and/or damage of the ilia olfactoria, which follows cribriform plate fracture, results in anosmia. This condition is in part determined by scar formation during dural repair and surgical protocol. Since it is known that the primate ilia regenerate 10, the present experiments were undertaken in a primate to determine if reinnervation of the olfactory bulb could occur following ilia olfactoria axotomy and regeneration. The regrowth of the newly formed axons and synaptogenesis have been followed over a 3 months postoperative period. MATERIAL AND METHODS Four adult, male squirrel monkeys (Sairniri sciureus), approximately 0.8 kg in weight, were used in this series of experiments. The animals were the same as used in a previous experiment TM. The animals were anesthetized (sodium pentobarbital, 25 mg/kg) and operated on under sterile conditions. Utilizing microsurgical techniques, the ilia olfactoria were approached by creating a window in the frontal bone, through a transorbital approach, and were sectioned above the lamina cribrosa. All animals were treated with Longicil, prior to and following surgery. Fila olfactoria section was performed as follows: animal no. 1 was operated on unilaterally 10 days prior to sacrifice; animal no. 2 was operated on 10 and 30 days (right and left, respectively) prior to sacrifice; animal no. 3 was operated on 60 and 90 days (right and left, respectively) prior to sacrifice; animal no. 4 was operated on 10 and 30 days (right and left, respectively) prior to sacrifice. Following the postoperative period the animals were anesthetized and perfused intracardially with 25 ml of Ringer solution with 0.I ~ xylocaine, followed by 2.0 ~ glutaraldehyde and 0.6 ~i paraformaldehyde, buffered at pH 7.3 with cacodylate. The animals were perfused for 1 h; the nasal epithelium and the olfactory bulb were then removed and washed in buffer before postfixation in 2 ~ Os04. Olfactory bulbs were sectioned in 4 segments along vertical and horizontal planes in the antero-posterior axis. The specimens, embedded in Araldite, were cut, mounted on slotted grids and stained with uranyl acetate and lead citrate. RESULTS There are no previous detailed ultrastructural reports on the synaptic organization of the olfactory bulb glomeruli in squirrel monkey. Normal olfactory glomeruli are, as observed in other mammals, rather discrete, globose structures surrounded by periglomerular cells (Fig. 1). The unmyelinated ilia enter the bulb from its external surface in small bundles. These fascicles of axons are arranged in large pockets of the sheath cells (Fig. 2). Contrary to rodents and amphibians, some of the periglomerular cells in primates have a complete or incomplete myelin sheath (Fig. 3). These cells have
Fig. 1. Normal animal. Glomerulus (G) contains dendrites (d) surrounded by dense, smaller profiles identified as sensory axon terminals (t). Periglomerular cells (pc) surround the giomerular neuropil. Scale bar 10 #m. Fig. 2. Normal animal. Sensory axons (ax) in the external fiber layer of the olfactory bulb are enclosed in sheath cells (S) channels. Scale bar 1 #m.
Fig. 3. Normal animal. Periglomerular cell (pc) with a partial envelope o f myelin (my). Organelles in these small neurons are sparse. Scale bar 1 /tin. Fig. 4. Normal animal. Detail o f the glomerular neuropil. Sensory axon terminals (t) establish synaptic contacts with dendritic profiles (d). Serial synaptic arrangements are indicated at arrowheads. Scale bar I !;m.
Fig. 5. 10 days survival animal. A glomerulus (G) shows swollen dendritic profiles (d). On the left of the figure (L) corresponding to the external fiber layer, the sensory axons are absent. Astrocytes (a) show many dense bodies (arrowheads) with the characteristics of lysosomes. (bv) blood vessel. Scale bar 10/~m. Fig. 6. 10 days survival. Two periglomerular cells (pc), completely surrounded by a myelin sheath (my) show normal morphology, in contrast to the swollen appearance of several adjacent dendrites (d). Dendro-dendritic contacts are indicated at small arrowheads. Scale bar 1 /~m.
Fig. 7. 10 days survival. Detail of the glomerular neuropil. Only dendro-dendritic contacts remain, while sensory axons are absent at this time. Arrowheads indicate dendro-dendritic contacts. Scale bar I pro. Fig. 8. 10 days survival animal. Portion of the ilia olfactoria. Profiles of astrocytes (a) show abundant neuroglial fibrils. A few sensory axons (ax) and growth cones (gc) are observed even at this stage along the path of the olfactory ilia between the mucosa and the olfactory bulb. Scale bar I /~m.
349
Figs. 9 and 10. 30 days survival animal. Two areas of the fiber layer covering the olfactory bulb. New fibers (ax), enclosed in sheath cell channels, reappear with varying frequency in different portions of the bulb. Sheath cells (S) show fibrillar material (f) in their cytoplasm. Scale bar 1/~m.
Fig. 11.30 days survival animal. Detail of sensory fibers reaching the olfactory bulb. Small unmyelinated fibres and larger profiles with the characteristics of growth cones (gc) are invaginated in the cytoplasm of sheath cells (S). Scale bar l ,.m. Fig. 12. 30 days survival animal. A glomerulus (G) shows the reappearance of sensory terminals (t) and a normal component of dendrites (d). In the upper left corner of the figure a bundle of sensory axons (ax) is in a sheath cell channel. Scale bar l /~m.
351
Fig. 13.60 days survival animal. A fascicle of axons (ax) penetrates a glomerulus (G) which shows a pattern of profiles similar to normal. (a) astrocyte; (d) dendrites; arrowheads indicate synaptic contacts of the sensory axons. Scale bar 1/~m. an electron-lucent cytoplasm and an overall paucity of organelles. Profiles of endoplasmic reticulum (rER) are present but not arranged in the characteristic regular stacks which correspond to the Nissl bodies of the light microscope preparations. The sensory terminals in the glomerular neuropile can be recognized by their dense matrix, their agranular vesicles and the asymmetric contacts they establish with the dendrites. Dendro-dendritic contacts can also be seen in the glomeruli. Few degenerating axon terminals are commonly observed, together with others containing lysosomes. Glial profiles containing phagosomes are also present (Fig. 4). At 10 days survival the denervated olfactory bulb glomeruli show substantial alterations when compared with the normal olfactory bulb (Fig. 5). The sensory terminals have, with few exceptions, disappeared and the dendritic profiles have undergone considerable swelling. The alteration of the dendritic profiles is confined in the boundaries of the glomerular neuropil. The dendrites in the external plexiform layer are well preserved and so are the perikarya of periglomerular (Fig. 6), tufted and mitral cells. In the glomerular neuropil, the swelling seems to affect only portions of the dendrites, as we have been able to see in the same dendrites continuity between well preserved and swollen portions. In the swollen dendrites the matrix is electron-lucent, the organelles are sparse, but not appreciably altered. Dendro-dendritic contacts are preserved at this survival time and remain the only synaptic contacts that can be
Fig. 14. 60 days survival. Detail of the glomerulus with sensory terminals (t) establishing synaplic contacts with dendrites (d). Scale bar I um. Fig. 15. 90 days survival animal. Detail of the glomerulus with sensory terminals (t) establishing synaptic contacts with dendrites (d). Scale bar 1 tma.
353 observed in the glomerulus (Figs. 6 and 7). Neuroglial profiles are observed in the neuropil with higher frequency than in normal glomeruli, however, they do not have the extension described in mouse and rat at 2-4 days survival time4, 8. The sensory axons, which normally penetrate the glomeruli from the external fiber layer of the bulb, are lacking at this survival time. The neuroglia of this fiber layer is very hypertrophic and lacks the neuroglial channels containing the axons in normal preparations. However, along the course of the ilia olfactoria, a few axons are already growing toward the olfactory bulb and some growth cones can be observed (Fig. 8). At 30 days survival the external fiber layer of the olfactory bulb is replete with sensory axons. They are fasciculated in sheath cell channels. The sheath cells show the characteristic content of tonofilaments (Figs. 9 and 10). Some of the sheath cells have a characteristic dense cytoplasm (Fig. 9), which contrasts with the electron-lucent cytoplasm of other closely associated sheath cells. The axons are all unmyelinated and their diameter ranges from 0.2 to 0.4 #m. Among them, larger profiles can be recognized as growth cones (Fig. 11). At this survival time, the glomeruli have a rather clear appearance due to the predominance of dendritic profiles which are no longer swollen. There are a few sensory terminals present, showing the earliest stages of synaptogenesis (Fig. 12). At 60-90 days survival the glomeruli on the operated side are similar to normals. Bundles of sensory fibers penetrate the glomerular neuropil giving it the characteristic dense appearance (Fig. 13). The neuropil now has many dense patches of sensory terminals which establish synaptic contacts with the dendrites of mitral and periglomerular cells (Figs. 14, 15). CONCLUSIONS These data show that the axotomy of the olfactory sensory neurons at the level of the lamina cribrosa in a non-human primate does not necessarily mean permanent deafferentation of the olfactory bulb and, consequently, impairment of function. In spite of the surgical procedure and scar formation, the olfactory neurons have regenerated 1°, their axons have regrown by the 30th postoperative day and have reformed morphologically recognizable synapses on and before the 60th day. These results repeat previous observations in rodents4,7, s. However, their significance derives from being observed in a primate. The results seem to indicate that at least in the region of the olfactory bulb the central nervous system is not impermeable to the regrowth of new axons. Our data indicate that synaptogenesis and re-establishment of normal connections is not confined to the prenatal period but can be induced, after damage, in fully mature primates. ACKNOWLEDGEMENTS This work has been supported by Grants from the National Science Foundation (BNS 77/16737 to P.P.C.G.) and the National Institutes of Health (NS 08943 to P.P.C.G.; NS 06164 to J.J.B.; NS 14556 and NS 00281 to M.S.K.).
354 REFERENCES 1 Graziadei, P. P. C., Cell dynamics in the olfactory mucosa, Tissue and Cell, 5 (1973) 113-131. 2 Graziadei, P. P. C. and Metcalf, J. F., Autoradiographic and ultrastructural observations oil the frogs olfactory mucosa, Z. Zellforsch., 116 (1971) 305-318. 3 Graziadei, P. P. C. and Delian, R. S., Neuronal regeneration in frog olfactory system, J. Cell BioL, 59 (1973) 525-530. 4 Graziadei, P. P. C. and Monti Graziadei, G. A., The olfactory system: A model for the study of neurogenesis and axon regeneration in mammals. In C. W. Cotman fEd.), Neuronal Plasticity, Raven Press, New York, 1978, pp. 131-153. 5 Graziadei, P. P. C. and Monti Graziadei, G. A., Continuous nerve cell renewal in the olfactory system. In M. Jacobson (Ed.), Handbook of Sensory Physiology, Vol. IX, Springer Verlag, New York, 1978, pp. 55-83. 6 Graziadei, P. P. C. and Monti Graziadei, G. A., Neurogenesis and neuron regeneration in the olfactory system of mammals. I. Morphological aspects of differentiation and structural organization of the olfactory sensory neurons, J. Neurocytol., 8 (1979) 1-18. 7 Monti Graziadei, G. A. and Graziadei, P. P. C., Neurogenesis and neuron regeneration in the olfactory system of mammals. II. Degeneration and reconstitution of the olfactory sensory neurons after axotomy, J. Neurocytol., 8 (1979) 197-213. 8 Graziadei, P. P. C. and Mooti Graziadei, G. A., Neurogenesis and neuron regeneration in the olfactory system of mammals. III. Deafferentation and reinnervation of the olfactory bulb following section of the ilia olfactoria in rat, J. NeuroeytoL, 8 (1979) (in press). 9 Graziadei, P. P. C. and Okano, M., Neuronal degeneration and regeneration in the olfactory epithelium of pigeon following transection of the first cranial nerve, Acta anat. (Basel), 104 (t 979) 220-236. 10 Graziadei, P. P. C., Karlan, M. G., Monti Graziadei, G. A. and Bernstein, J. J., Neurogenesis of sensory neurons in the primate olfactory system after section of the fila olfactoria, Brain Research, in press. 11 Moulton, D. G., Dynamics of cell populations in the olfactory epithelium, Ann. N. Y. Acad. Sci., 237 (1974) 52-61. 12 Oley, N., Delian, R. S., Tucker, D., Smith, J. C. and Graziadei, P. P. C., Recovery of structure and function following transection of the primary olfactory nerves in pigeons, J. comp. physioL PsychoL, 88 (1975) 477~195.
343
REINNERVATION OF THE OLFACTORY BULB AFTER SECTION OF THE OLFACTORY NERVE IN MONKEY (SAIMIRI SCIUREUS)
G. A. M O N T I G R A Z I A D E I , M. S. K A R L A N * , J. J. B E R N S T E I N * * a n d P. P. C. G R A Z I A D E I
Department of Biological Science, Florida State University, Tallahassee, Fla. 32306; (M.S.K.) Department of Otolaryngology and Center for Sensory Studies, University of Florida College of Medicine, and ( J.J.B.) Department of Neuroscience, Urdversity of Florida College of Medicine, Gainesville, Fla. 32610
(u.s.A.) (Accepted O c t o b e r 1 l t h , 1979)
Key words: olfactory bulb - - r e i n n e r v a t i o n - - m o n k e y olfactory nerve - - deafferentation
SUMMARY
Section of the ilia olfactoria in squirrel monkey, a non-human primate, induces rapid degeneration of the sensory axon terminals in the olfactory bulb glomeruli. A population of axons, from newly formed sensory neurons in the olfactory neuroepithelium, regrows, passes the lamina cribrosa and, upon reaching the olfactory bulb, reinnervates the glomeruli. A new set of synaptic contacts is reformed between the sensory terminals and the post-synaptic dendritic processes of the glomeruli. Our observations indicate that this portion of the CNS of a non-human primate can be reinnervated after deafferentation, and that active synaptogenesis occurs.
INTRODUCTION
The olfactory epithelium in lower vertebrates and rodents is unique because of its ability to generate new sensory neurons through the animal's entire life2,a,5,6. There is a cyclic neuronal renewal from the basal cells of this epithelium and the neurons appear to have a life span of approximately 30 40 days according to species1,4,6,11. The axons of these neurons join to form the ilia olfactoria and terminate in the bulb glomeruli, where they synapse upon the main dendrite of the mitral cells. Renewal of neurons perikarya in the neuroepithelium implies renewal of the synaptic contacts of their axon terminals in the olfactory bulb glomeruli. Following axotomy of the ilia olfactoria there is rapid degeneration of the olfactory sensory neurons in the nasal neuroepitheliuma,7,9,12. This process is followed
344
by basal cells division and differentiation into neurons, by regrowth of their axons into the olfactory bulb and synapses formation on the mitral cells dendrites. The process of olfactory neurons renewal has been recently shown to occur also in primates 10. In humans, sections and/or damage of the ilia olfactoria, which follows cribriform plate fracture, results in anosmia. This condition is in part determined by scar formation during dural repair and surgical protocol. Since it is known that the primate ilia regenerate 10, the present experiments were undertaken in a primate to determine if reinnervation of the olfactory bulb could occur following ilia olfactoria axotomy and regeneration. The regrowth of the newly formed axons and synaptogenesis have been followed over a 3 months postoperative period. MATERIAL AND METHODS Four adult, male squirrel monkeys (Sairniri sciureus), approximately 0.8 kg in weight, were used in this series of experiments. The animals were the same as used in a previous experiment TM. The animals were anesthetized (sodium pentobarbital, 25 mg/kg) and operated on under sterile conditions. Utilizing microsurgical techniques, the ilia olfactoria were approached by creating a window in the frontal bone, through a transorbital approach, and were sectioned above the lamina cribrosa. All animals were treated with Longicil, prior to and following surgery. Fila olfactoria section was performed as follows: animal no. 1 was operated on unilaterally 10 days prior to sacrifice; animal no. 2 was operated on 10 and 30 days (right and left, respectively) prior to sacrifice; animal no. 3 was operated on 60 and 90 days (right and left, respectively) prior to sacrifice; animal no. 4 was operated on 10 and 30 days (right and left, respectively) prior to sacrifice. Following the postoperative period the animals were anesthetized and perfused intracardially with 25 ml of Ringer solution with 0.I ~ xylocaine, followed by 2.0 ~ glutaraldehyde and 0.6 ~i paraformaldehyde, buffered at pH 7.3 with cacodylate. The animals were perfused for 1 h; the nasal epithelium and the olfactory bulb were then removed and washed in buffer before postfixation in 2 ~ Os04. Olfactory bulbs were sectioned in 4 segments along vertical and horizontal planes in the antero-posterior axis. The specimens, embedded in Araldite, were cut, mounted on slotted grids and stained with uranyl acetate and lead citrate. RESULTS There are no previous detailed ultrastructural reports on the synaptic organization of the olfactory bulb glomeruli in squirrel monkey. Normal olfactory glomeruli are, as observed in other mammals, rather discrete, globose structures surrounded by periglomerular cells (Fig. 1). The unmyelinated ilia enter the bulb from its external surface in small bundles. These fascicles of axons are arranged in large pockets of the sheath cells (Fig. 2). Contrary to rodents and amphibians, some of the periglomerular cells in primates have a complete or incomplete myelin sheath (Fig. 3). These cells have
Fig. 1. Normal animal. Glomerulus (G) contains dendrites (d) surrounded by dense, smaller profiles identified as sensory axon terminals (t). Periglomerular cells (pc) surround the giomerular neuropil. Scale bar 10 #m. Fig. 2. Normal animal. Sensory axons (ax) in the external fiber layer of the olfactory bulb are enclosed in sheath cells (S) channels. Scale bar 1 #m.
Fig. 3. Normal animal. Periglomerular cell (pc) with a partial envelope o f myelin (my). Organelles in these small neurons are sparse. Scale bar 1 /tin. Fig. 4. Normal animal. Detail o f the glomerular neuropil. Sensory axon terminals (t) establish synaptic contacts with dendritic profiles (d). Serial synaptic arrangements are indicated at arrowheads. Scale bar I !;m.
Fig. 5. 10 days survival animal. A glomerulus (G) shows swollen dendritic profiles (d). On the left of the figure (L) corresponding to the external fiber layer, the sensory axons are absent. Astrocytes (a) show many dense bodies (arrowheads) with the characteristics of lysosomes. (bv) blood vessel. Scale bar 10/~m. Fig. 6. 10 days survival. Two periglomerular cells (pc), completely surrounded by a myelin sheath (my) show normal morphology, in contrast to the swollen appearance of several adjacent dendrites (d). Dendro-dendritic contacts are indicated at small arrowheads. Scale bar 1 /~m.
Fig. 7. 10 days survival. Detail of the glomerular neuropil. Only dendro-dendritic contacts remain, while sensory axons are absent at this time. Arrowheads indicate dendro-dendritic contacts. Scale bar I pro. Fig. 8. 10 days survival animal. Portion of the ilia olfactoria. Profiles of astrocytes (a) show abundant neuroglial fibrils. A few sensory axons (ax) and growth cones (gc) are observed even at this stage along the path of the olfactory ilia between the mucosa and the olfactory bulb. Scale bar I /~m.
349
Figs. 9 and 10. 30 days survival animal. Two areas of the fiber layer covering the olfactory bulb. New fibers (ax), enclosed in sheath cell channels, reappear with varying frequency in different portions of the bulb. Sheath cells (S) show fibrillar material (f) in their cytoplasm. Scale bar 1/~m.
Fig. 11.30 days survival animal. Detail of sensory fibers reaching the olfactory bulb. Small unmyelinated fibres and larger profiles with the characteristics of growth cones (gc) are invaginated in the cytoplasm of sheath cells (S). Scale bar l ,.m. Fig. 12. 30 days survival animal. A glomerulus (G) shows the reappearance of sensory terminals (t) and a normal component of dendrites (d). In the upper left corner of the figure a bundle of sensory axons (ax) is in a sheath cell channel. Scale bar l /~m.
351
Fig. 13.60 days survival animal. A fascicle of axons (ax) penetrates a glomerulus (G) which shows a pattern of profiles similar to normal. (a) astrocyte; (d) dendrites; arrowheads indicate synaptic contacts of the sensory axons. Scale bar 1/~m. an electron-lucent cytoplasm and an overall paucity of organelles. Profiles of endoplasmic reticulum (rER) are present but not arranged in the characteristic regular stacks which correspond to the Nissl bodies of the light microscope preparations. The sensory terminals in the glomerular neuropile can be recognized by their dense matrix, their agranular vesicles and the asymmetric contacts they establish with the dendrites. Dendro-dendritic contacts can also be seen in the glomeruli. Few degenerating axon terminals are commonly observed, together with others containing lysosomes. Glial profiles containing phagosomes are also present (Fig. 4). At 10 days survival the denervated olfactory bulb glomeruli show substantial alterations when compared with the normal olfactory bulb (Fig. 5). The sensory terminals have, with few exceptions, disappeared and the dendritic profiles have undergone considerable swelling. The alteration of the dendritic profiles is confined in the boundaries of the glomerular neuropil. The dendrites in the external plexiform layer are well preserved and so are the perikarya of periglomerular (Fig. 6), tufted and mitral cells. In the glomerular neuropil, the swelling seems to affect only portions of the dendrites, as we have been able to see in the same dendrites continuity between well preserved and swollen portions. In the swollen dendrites the matrix is electron-lucent, the organelles are sparse, but not appreciably altered. Dendro-dendritic contacts are preserved at this survival time and remain the only synaptic contacts that can be
Fig. 14. 60 days survival. Detail of the glomerulus with sensory terminals (t) establishing synaplic contacts with dendrites (d). Scale bar I um. Fig. 15. 90 days survival animal. Detail of the glomerulus with sensory terminals (t) establishing synaptic contacts with dendrites (d). Scale bar 1 tma.
353 observed in the glomerulus (Figs. 6 and 7). Neuroglial profiles are observed in the neuropil with higher frequency than in normal glomeruli, however, they do not have the extension described in mouse and rat at 2-4 days survival time4, 8. The sensory axons, which normally penetrate the glomeruli from the external fiber layer of the bulb, are lacking at this survival time. The neuroglia of this fiber layer is very hypertrophic and lacks the neuroglial channels containing the axons in normal preparations. However, along the course of the ilia olfactoria, a few axons are already growing toward the olfactory bulb and some growth cones can be observed (Fig. 8). At 30 days survival the external fiber layer of the olfactory bulb is replete with sensory axons. They are fasciculated in sheath cell channels. The sheath cells show the characteristic content of tonofilaments (Figs. 9 and 10). Some of the sheath cells have a characteristic dense cytoplasm (Fig. 9), which contrasts with the electron-lucent cytoplasm of other closely associated sheath cells. The axons are all unmyelinated and their diameter ranges from 0.2 to 0.4 #m. Among them, larger profiles can be recognized as growth cones (Fig. 11). At this survival time, the glomeruli have a rather clear appearance due to the predominance of dendritic profiles which are no longer swollen. There are a few sensory terminals present, showing the earliest stages of synaptogenesis (Fig. 12). At 60-90 days survival the glomeruli on the operated side are similar to normals. Bundles of sensory fibers penetrate the glomerular neuropil giving it the characteristic dense appearance (Fig. 13). The neuropil now has many dense patches of sensory terminals which establish synaptic contacts with the dendrites of mitral and periglomerular cells (Figs. 14, 15). CONCLUSIONS These data show that the axotomy of the olfactory sensory neurons at the level of the lamina cribrosa in a non-human primate does not necessarily mean permanent deafferentation of the olfactory bulb and, consequently, impairment of function. In spite of the surgical procedure and scar formation, the olfactory neurons have regenerated 1°, their axons have regrown by the 30th postoperative day and have reformed morphologically recognizable synapses on and before the 60th day. These results repeat previous observations in rodents4,7, s. However, their significance derives from being observed in a primate. The results seem to indicate that at least in the region of the olfactory bulb the central nervous system is not impermeable to the regrowth of new axons. Our data indicate that synaptogenesis and re-establishment of normal connections is not confined to the prenatal period but can be induced, after damage, in fully mature primates. ACKNOWLEDGEMENTS This work has been supported by Grants from the National Science Foundation (BNS 77/16737 to P.P.C.G.) and the National Institutes of Health (NS 08943 to P.P.C.G.; NS 06164 to J.J.B.; NS 14556 and NS 00281 to M.S.K.).
354 REFERENCES 1 Graziadei, P. P. C., Cell dynamics in the olfactory mucosa, Tissue and Cell, 5 (1973) 113-131. 2 Graziadei, P. P. C. and Metcalf, J. F., Autoradiographic and ultrastructural observations oil the frogs olfactory mucosa, Z. Zellforsch., 116 (1971) 305-318. 3 Graziadei, P. P. C. and Delian, R. S., Neuronal regeneration in frog olfactory system, J. Cell BioL, 59 (1973) 525-530. 4 Graziadei, P. P. C. and Monti Graziadei, G. A., The olfactory system: A model for the study of neurogenesis and axon regeneration in mammals. In C. W. Cotman fEd.), Neuronal Plasticity, Raven Press, New York, 1978, pp. 131-153. 5 Graziadei, P. P. C. and Monti Graziadei, G. A., Continuous nerve cell renewal in the olfactory system. In M. Jacobson (Ed.), Handbook of Sensory Physiology, Vol. IX, Springer Verlag, New York, 1978, pp. 55-83. 6 Graziadei, P. P. C. and Monti Graziadei, G. A., Neurogenesis and neuron regeneration in the olfactory system of mammals. I. Morphological aspects of differentiation and structural organization of the olfactory sensory neurons, J. Neurocytol., 8 (1979) 1-18. 7 Monti Graziadei, G. A. and Graziadei, P. P. C., Neurogenesis and neuron regeneration in the olfactory system of mammals. II. Degeneration and reconstitution of the olfactory sensory neurons after axotomy, J. Neurocytol., 8 (1979) 197-213. 8 Graziadei, P. P. C. and Mooti Graziadei, G. A., Neurogenesis and neuron regeneration in the olfactory system of mammals. III. Deafferentation and reinnervation of the olfactory bulb following section of the ilia olfactoria in rat, J. NeuroeytoL, 8 (1979) (in press). 9 Graziadei, P. P. C. and Okano, M., Neuronal degeneration and regeneration in the olfactory epithelium of pigeon following transection of the first cranial nerve, Acta anat. (Basel), 104 (t 979) 220-236. 10 Graziadei, P. P. C., Karlan, M. G., Monti Graziadei, G. A. and Bernstein, J. J., Neurogenesis of sensory neurons in the primate olfactory system after section of the fila olfactoria, Brain Research, in press. 11 Moulton, D. G., Dynamics of cell populations in the olfactory epithelium, Ann. N. Y. Acad. Sci., 237 (1974) 52-61. 12 Oley, N., Delian, R. S., Tucker, D., Smith, J. C. and Graziadei, P. P. C., Recovery of structure and function following transection of the primary olfactory nerves in pigeons, J. comp. physioL PsychoL, 88 (1975) 477~195.