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Transplants of olfactory mucosa in the rat brain I. A light microscopic study of transplant organization
Brain Research, 279 (1983) 241-245 Elsevier
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Transplants of olfactory mucosa in the rat brain I. A light microscopic study of transplant organization EDWARD E. MORRISON and PASQUALE P.C. GRAZIADEI* Department of Biological Sciences, Florida State University, Tallahasee, FL 32306 (U.S.A.)
(Accepted July 26th, 1983) Key words: transplantation - - olfactory neurons
Olfactory mucosa from neonatal rats has been transplanted into the fourth cerebral ventricle or into the parietal cortex of neonatal and adult rats. In these ectopic locations, olfactory neurons continue to differentiate from the neurogenetic matrix (basal cells) of the neuroepithelium. Sensory axon bundles from the newly formed olfactory neurons penetrate the host brain where they branch without forming the characteristic olfactory glomeruli. From the base of the neuroepithelium neural elements migrate into the host cerebral tissue losing their epithelial organization. The olfactory neuron is unique, within the vertebrate nervous sytem, in that it can replace itself normallyS,6,12 or when experimentally damagedl,2,n. New neurons develop from the basal cells of the neuroepithelium, however, the factors that govern the neuron replacement are not fully known. In an attempt to further understand the phenomena related to the neurogenesis and maturation of the olfactory neurons, and the p h e n o m e n a controlling their interaction with other neurons, we have utilized the method of transplantation. We have removed neonatal olfactory mucosa (P5-P10) from its normal locus, the nasal cavity, and transplanted it into the fourth ventricle or directly into the parietal cortex of adult and neonatal rats (P5-P10) (Table I). The fourth ventricle was accessed by the method described by Rosenstein and Brightman13, the parietal cortex was accessed through an opening in the parietal bone, incision of the meninges and introduction of the transplant with a glass needle in the underlying cortex. D a m a g e to the host brain was minimal in the fourth ventricle transplants. In the intracortical transplants the damage was limited to the barrel shaped lesion of the needle (0.5 m m in diameter) used to position the transplant. The post-operative period was 10-120 days. At the time of sacrifice, animals were anesthetized with pentobarbital and per* To whom all correspondence should be addressed. 0006-8993/83/$03.00 ~) 1983 Elsevier Science Publishers B .V.
fused intercardially with Ringer solution followed by Bouin's fixative. The brain was removed, embedded in paraffin and serially sectioned. Alternate sections were stained with iron hematoxylin and a silver method according to Loots et al. 9. Selected slides were processed for the determination of the olfactory marker protein (OMP) with the peroxidase-antiperoxidase methodl0A4. The various host sites did not affect the survival or morphological characteristics of the transplanted olfactory mucosa. The transplants survived and were rapidly vascularized by the choroid plexus or by blood vessels originating from the host brain. The transplants were in intimate contact with the host cerebrum, cerebellum, medulla or area postrema. The transplants form a series of interconnecting vesicles TABLE I Summary of olfactory mucosa transplants in adult and neonatal rats Transplantation site
Number of transplants*
Range of survival (days)
Adult cerebral cortex Neonatal cerebral cortex Adult cerebellum Neonatal cerebellum
10 20 20 15
10-100 days 10--120days 10-120 days 10-100 days
* 95% of transplants were successful.
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243 Fig. 1. Olfactory mucosa transplant within the host parietal cortex (PC). The transplant organizes into vesicles of varying sizes containing secretory material(s), a product of the epithelial supporting cells and the Bowman's glands (b) within the lamina propria. The olfactory neuroepithelium lining the vesicles is of varying thickness (see arrowheads) and at times resembles the typical pseudostratified columnar neuroepithelium (as indicated by the arrow in the large vesicle). Iron hematoxylin. (Host, neonate; transplant survival, 56 days; x63.) Fig. 2. Immunohistochemical technique for the demonstration of olfactory marker protein (OMP) confirms the morphological identification of olfactory axon fasicles (f) and olfactory sensory neurons (n). Some of the latter show a fine dendrite, at times unconventionally arranged in the neuropithelium (d). (Host, neonate; transplant survival, 56 days; x 100.) Fig. 3. The basal region of the olfactory neuroepithelium (at arrowheads) demonstrates partial disorganization. Olfactory neurons are observed forming cords (c) or nest-like clusters (cl) in the lamina propria. The process of migration of the olfactory neural elements leads to their penetration into the host brain (see Figs. 4 and 7). Secretion (s) partially fills the large vesicle formed by the transplanted neuroepithelium; lamina propria (lm) where numerous profiles of Bowman's glands (b) are obvious. Iron hematoxylin. (Host, neonate; transplant survival, 75 days; x 250.) Fig. 4. Olfactory mucosa transplant is shown in intimate contact with the host's medulla (m). The thickness of the neuroepithelium varies between the vesicles (v). Receptor's dendrites (d) can be seen on the free epithelial surface. Isolated clusters of olfactory neurons (n) are present in the host's tissue. Bundles of sensory fibres (sO originate from the sensory neuroepithelium and direct towards the host's medulla (arrowheads). Silver stain. (Host, adult; transplant survival, 35 days; x 150.)
of varying sizes lined by a respiratory and olfactory epithelium, with B o w m a n glands in the lamina propria (Fig. 1). At early survival times (10 days) the mature neurons of the transplant degenerate and subsequently are replaced by new neurons developed from the neurogenetic matrix (basal cells), where mitotic figures are frequently observed. At later survival times ( > 10 days) morphologically identifiable neurons with dendrites and axons characterized the neuroepithelium (Figs. 2-4). N u m e r o u s argentophilic olfactory axonal fascicles were seen within the lamina propria and penetrate the host brain directly (Fig. 4-8). Host myelinated fibers were also observed entering the transplant. Slides prepared to determine the presence of the olfactory marker protein (OMP) demonstrated that morphologically identifiable olfactory neurons and their fibers O M P positive (Fig. 2). The behavior of O M P in the transplanted olfactory neuroepithelium will be addressed more completely in a future report. The olfactory neuroepithelium was of varying thickness and at times displayed the typical pseudostratified organization with the sensory neuron dendrites extending to the luminal surface and axon fibers below the basal lamina (Fig. 4). However, the transplants did demonstrate some peculiar characteristics at all transplantation sites. Portions of the basal neuroepithelium appeared to lose their epithelial character. Olfactory neurons formed cords or nestlike structures below the basal lamina, streaming away from the epithelium (Figs. 3, 6 and 7). At times the neuroepithelium appeared nearly devoid of neurons except for isolated aggregations of neurons near
or below the basal lamina (Figs. 6 and 7). It appears that these olfactory neurons migrate away from the epithelium and penetrate the host brain. Olfactory fiber bundles originating from the epithelium were also observed penetrating the host brain (Figs. 4, 6 and 8). Examination of serial sections did demonstrate that the bundles diverge into the host brain in smaller units but never organize in glomerular structures, even at late survival times (120 days). Our results are similar with those reported in olfactory mucosa transplants in the eye 8 and demonstrate the continued neurogenetic property of the olfactory neuroepithelial matrix, and the maturation of olfactory neurons even when removed from their natural environmental locus. A surprising observation, however, was the occurrence of epithelial breakdown and olfactory neuron migration, and the penetration of olfactory sensory fibers into the host's brain with their apparent failure to form glomeruli in any of our transplantation sites. Although neuronal migration during development is a well established phenomenon, it has never been observed in the post-natal olfactory neuroepithelium. In previous published experiments where total bulbectomy was performed or the olfactory bulb was replaced by means of transplanting occipital or cerebellar cortexes, the in situ reconstituted olfactory neurons were able to grow their axons into the unconventional targets and consistently formed glomeruli. The newly developed axons established synaptic contacts with the unconventional target neurons and modified the organization of their dendrites 3,4,7. However, in the present experiment, epithelial disorganization with extraepithelial
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245 Fig. 5. The olfactory mucosa transplant has developed into several large vesicles containing secretory material and cellular debris (s). The vesicles are attached to the host cerebellum (c) and olfactory axon fasicles (fa, between arrowheads) can be observed penetrating the host cerebellum. No distinct glomerular structures were observed. A cluster of migrating neural cells (mn) is illustrated in more detail in Fig. 6. Silver stain. (Host, neonate; transplant survival, 75 days; x 100.) Fig. 6. Detail of Fig. 5 showing a thin layer of neuroepithelium (ne) where neuronal dendrites (d) can be recognized. Numerous olfactory neural elements (n') are found below the basal lamina, migrating towards the host cerebellum (c). Large olfactory axon fasicles (fa) penetrate the host tissue x250. Fig. 7. Transplanted olfactory neuroepithelium (ep) demonstrates a sparse population of sensory receptor neurons identified by their dendrites (d). Olfactory neural elements (ne) are found below the epithelial layer and directly within the host's cerebral cortex (cc). Silver stain. (Host, adult; transplant survival, 44 days; x250.) Fig. 8. Low power view of an olfactory mucosa transplant located in the parietal cortex. Several olfactory axon fasicles (arrowheads) can be seen originating from the transplant and directed to the host tissue where they penetrate without forming the characteristic glomeruli. The silver method used in this preparation stains these fasicles in a characteristic pink and black color (not obvious in the B/W illustration) and allows their tracing through the serial sections of the entire preparation. Several vesicles (v) are lined by sensory epithelium and some neural elements (ne) can be seen dispersing into the host's parenchyma. Silver stain. (Host, adult; transplant survival, 50 days; × 100).
migration of olfactory n e u r o n s and failure of the olfactory axon terminals to form distinct glomerular structures have been consistently observed. Apparently, the presence of the lamina propria mucosae and even the occasional presence of bony fragments in the transplants is not sufficient to preserve the epithelial integrity nor the ability of the sensory n e u r o n s to form glomeruli. However, we cannot exclude that the olfactory axons may form glomeruli when different parameters of age and location of the donor-host
by ultrastructural examination and intracerebral transplants of isolated n e u r o n a l staminal cells (basal cells), provide further insight into the behavior of this neuronal population when removed from its natural environment. It may also contribute to our understanding of the potential of a matrix which persists even in adult mammals.
relationship are examined. The present studies, which will be c o m p l e m e n t e d
This work was supported by a grant from the National Institutes of Health, R01-NS16421. The authors wish to thank Dr. F. L. Margolis, Roche Institutes of Molecular Biology for the O M P antibody.
1 Graziadei, P. P. C., Cell dynamics in the olfactory mucosa, Tissue Cell, 5 (1973) 113-131. 2 Graziadei, P. P. C. and Delian, R. S., Neuronal regeneration in frog olfactory system, J. Cell Biol., 59 (1973) 525-530. 3 Graziadei, P. P. C. and Kaplan, M. S., Regrowth of the olfactory sensory axons into transplanted neural tissue. I. Development of connections with the occipital cortex, Brain Research, 201 (1980) 39-44. 4 Graziadei, P. P. C., Levine, R. R. and Monti Graziadei, G. A., Plasticity of connections of the olfactory sensory neuron: regeneration into the forebrain following bulbectomy in the neonatal mouse, Neuroscience, 4 (1979) 713-727. 5 Graziadei, P. P. C. and Metcalf, J. F., Autoradiographic and ultrastructural observations on the frogs olfactory mucosa, Z. Zellforsch, 116 (1971) 305-318. 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 Oraziadei, P. P. C. and Monti Graziadei, G. A., Plasticity of connections in the olfactory pathway; transplantation studies. In Olfaction and Taste VII, IRL Press, Ltd., London, 1980, pp. 1980. 8 Heckroth, J. A., Monti Graziadei, G. A. and Graziadei, P.
P. C., Intraocular transplants of olfactory neuroepithelium in rat, Int. J. Develop. Neurosci., in Press, 1983. Loots, G. P., Loots, J. M., Brown, J. M. M. and Schoeman, J. L., A rapid silver impregnation method for nervous tissue: a modified protargolperoxide technique, Stain Technol., 54 (1979) 97-100. Monti Graziadei, G. A., Margolis, F. L., Harding, J. W. and Graziadei, P. P. C., Immunocytochemistry of the olfactory marker protein, J. Histochem. Cytochern., 25 (1977) 1311-1316. Monti Graziadei, G. A. and Graziadei, P. P. C., Neurogenesis and neuron regeneration in the olfactory system of mammals. (2) Degeneration and reconstitution of the olfactory sensory neurons after axotomy, J. Neurocytol., 8 (1979) 197-213. Moulton, D. G., Dynamics of cell population i the olfactory epithelium, Ann. N. Y. Acad. Sci. 237 (1974) 52-61. Rosenstein, J. M. and Brightman, M. W., Intact cerebral ventricle as a site for tissue transplantation, Nature (Lond.), 276 (1978) 83--85. Sternberger, L. A., Hardy, P. H., Jr., Cuculis, J. J. and Meyer, H. G., The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (HorseradishperoxidaseAntihorseradish Peroxidase) and its use in identification of Spirochetes, J. Histochern. Cytochem., 18 (1970)315-333.
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Transplants of olfactory mucosa in the rat brain I. A light microscopic study of transplant organization EDWARD E. MORRISON and PASQUALE P.C. GRAZIADEI* Department of Biological Sciences, Florida State University, Tallahasee, FL 32306 (U.S.A.)
(Accepted July 26th, 1983) Key words: transplantation - - olfactory neurons
Olfactory mucosa from neonatal rats has been transplanted into the fourth cerebral ventricle or into the parietal cortex of neonatal and adult rats. In these ectopic locations, olfactory neurons continue to differentiate from the neurogenetic matrix (basal cells) of the neuroepithelium. Sensory axon bundles from the newly formed olfactory neurons penetrate the host brain where they branch without forming the characteristic olfactory glomeruli. From the base of the neuroepithelium neural elements migrate into the host cerebral tissue losing their epithelial organization. The olfactory neuron is unique, within the vertebrate nervous sytem, in that it can replace itself normallyS,6,12 or when experimentally damagedl,2,n. New neurons develop from the basal cells of the neuroepithelium, however, the factors that govern the neuron replacement are not fully known. In an attempt to further understand the phenomena related to the neurogenesis and maturation of the olfactory neurons, and the p h e n o m e n a controlling their interaction with other neurons, we have utilized the method of transplantation. We have removed neonatal olfactory mucosa (P5-P10) from its normal locus, the nasal cavity, and transplanted it into the fourth ventricle or directly into the parietal cortex of adult and neonatal rats (P5-P10) (Table I). The fourth ventricle was accessed by the method described by Rosenstein and Brightman13, the parietal cortex was accessed through an opening in the parietal bone, incision of the meninges and introduction of the transplant with a glass needle in the underlying cortex. D a m a g e to the host brain was minimal in the fourth ventricle transplants. In the intracortical transplants the damage was limited to the barrel shaped lesion of the needle (0.5 m m in diameter) used to position the transplant. The post-operative period was 10-120 days. At the time of sacrifice, animals were anesthetized with pentobarbital and per* To whom all correspondence should be addressed. 0006-8993/83/$03.00 ~) 1983 Elsevier Science Publishers B .V.
fused intercardially with Ringer solution followed by Bouin's fixative. The brain was removed, embedded in paraffin and serially sectioned. Alternate sections were stained with iron hematoxylin and a silver method according to Loots et al. 9. Selected slides were processed for the determination of the olfactory marker protein (OMP) with the peroxidase-antiperoxidase methodl0A4. The various host sites did not affect the survival or morphological characteristics of the transplanted olfactory mucosa. The transplants survived and were rapidly vascularized by the choroid plexus or by blood vessels originating from the host brain. The transplants were in intimate contact with the host cerebrum, cerebellum, medulla or area postrema. The transplants form a series of interconnecting vesicles TABLE I Summary of olfactory mucosa transplants in adult and neonatal rats Transplantation site
Number of transplants*
Range of survival (days)
Adult cerebral cortex Neonatal cerebral cortex Adult cerebellum Neonatal cerebellum
10 20 20 15
10-100 days 10--120days 10-120 days 10-100 days
* 95% of transplants were successful.
6
LP
t
243 Fig. 1. Olfactory mucosa transplant within the host parietal cortex (PC). The transplant organizes into vesicles of varying sizes containing secretory material(s), a product of the epithelial supporting cells and the Bowman's glands (b) within the lamina propria. The olfactory neuroepithelium lining the vesicles is of varying thickness (see arrowheads) and at times resembles the typical pseudostratified columnar neuroepithelium (as indicated by the arrow in the large vesicle). Iron hematoxylin. (Host, neonate; transplant survival, 56 days; x63.) Fig. 2. Immunohistochemical technique for the demonstration of olfactory marker protein (OMP) confirms the morphological identification of olfactory axon fasicles (f) and olfactory sensory neurons (n). Some of the latter show a fine dendrite, at times unconventionally arranged in the neuropithelium (d). (Host, neonate; transplant survival, 56 days; x 100.) Fig. 3. The basal region of the olfactory neuroepithelium (at arrowheads) demonstrates partial disorganization. Olfactory neurons are observed forming cords (c) or nest-like clusters (cl) in the lamina propria. The process of migration of the olfactory neural elements leads to their penetration into the host brain (see Figs. 4 and 7). Secretion (s) partially fills the large vesicle formed by the transplanted neuroepithelium; lamina propria (lm) where numerous profiles of Bowman's glands (b) are obvious. Iron hematoxylin. (Host, neonate; transplant survival, 75 days; x 250.) Fig. 4. Olfactory mucosa transplant is shown in intimate contact with the host's medulla (m). The thickness of the neuroepithelium varies between the vesicles (v). Receptor's dendrites (d) can be seen on the free epithelial surface. Isolated clusters of olfactory neurons (n) are present in the host's tissue. Bundles of sensory fibres (sO originate from the sensory neuroepithelium and direct towards the host's medulla (arrowheads). Silver stain. (Host, adult; transplant survival, 35 days; x 150.)
of varying sizes lined by a respiratory and olfactory epithelium, with B o w m a n glands in the lamina propria (Fig. 1). At early survival times (10 days) the mature neurons of the transplant degenerate and subsequently are replaced by new neurons developed from the neurogenetic matrix (basal cells), where mitotic figures are frequently observed. At later survival times ( > 10 days) morphologically identifiable neurons with dendrites and axons characterized the neuroepithelium (Figs. 2-4). N u m e r o u s argentophilic olfactory axonal fascicles were seen within the lamina propria and penetrate the host brain directly (Fig. 4-8). Host myelinated fibers were also observed entering the transplant. Slides prepared to determine the presence of the olfactory marker protein (OMP) demonstrated that morphologically identifiable olfactory neurons and their fibers O M P positive (Fig. 2). The behavior of O M P in the transplanted olfactory neuroepithelium will be addressed more completely in a future report. The olfactory neuroepithelium was of varying thickness and at times displayed the typical pseudostratified organization with the sensory neuron dendrites extending to the luminal surface and axon fibers below the basal lamina (Fig. 4). However, the transplants did demonstrate some peculiar characteristics at all transplantation sites. Portions of the basal neuroepithelium appeared to lose their epithelial character. Olfactory neurons formed cords or nestlike structures below the basal lamina, streaming away from the epithelium (Figs. 3, 6 and 7). At times the neuroepithelium appeared nearly devoid of neurons except for isolated aggregations of neurons near
or below the basal lamina (Figs. 6 and 7). It appears that these olfactory neurons migrate away from the epithelium and penetrate the host brain. Olfactory fiber bundles originating from the epithelium were also observed penetrating the host brain (Figs. 4, 6 and 8). Examination of serial sections did demonstrate that the bundles diverge into the host brain in smaller units but never organize in glomerular structures, even at late survival times (120 days). Our results are similar with those reported in olfactory mucosa transplants in the eye 8 and demonstrate the continued neurogenetic property of the olfactory neuroepithelial matrix, and the maturation of olfactory neurons even when removed from their natural environmental locus. A surprising observation, however, was the occurrence of epithelial breakdown and olfactory neuron migration, and the penetration of olfactory sensory fibers into the host's brain with their apparent failure to form glomeruli in any of our transplantation sites. Although neuronal migration during development is a well established phenomenon, it has never been observed in the post-natal olfactory neuroepithelium. In previous published experiments where total bulbectomy was performed or the olfactory bulb was replaced by means of transplanting occipital or cerebellar cortexes, the in situ reconstituted olfactory neurons were able to grow their axons into the unconventional targets and consistently formed glomeruli. The newly developed axons established synaptic contacts with the unconventional target neurons and modified the organization of their dendrites 3,4,7. However, in the present experiment, epithelial disorganization with extraepithelial
4~
245 Fig. 5. The olfactory mucosa transplant has developed into several large vesicles containing secretory material and cellular debris (s). The vesicles are attached to the host cerebellum (c) and olfactory axon fasicles (fa, between arrowheads) can be observed penetrating the host cerebellum. No distinct glomerular structures were observed. A cluster of migrating neural cells (mn) is illustrated in more detail in Fig. 6. Silver stain. (Host, neonate; transplant survival, 75 days; x 100.) Fig. 6. Detail of Fig. 5 showing a thin layer of neuroepithelium (ne) where neuronal dendrites (d) can be recognized. Numerous olfactory neural elements (n') are found below the basal lamina, migrating towards the host cerebellum (c). Large olfactory axon fasicles (fa) penetrate the host tissue x250. Fig. 7. Transplanted olfactory neuroepithelium (ep) demonstrates a sparse population of sensory receptor neurons identified by their dendrites (d). Olfactory neural elements (ne) are found below the epithelial layer and directly within the host's cerebral cortex (cc). Silver stain. (Host, adult; transplant survival, 44 days; x250.) Fig. 8. Low power view of an olfactory mucosa transplant located in the parietal cortex. Several olfactory axon fasicles (arrowheads) can be seen originating from the transplant and directed to the host tissue where they penetrate without forming the characteristic glomeruli. The silver method used in this preparation stains these fasicles in a characteristic pink and black color (not obvious in the B/W illustration) and allows their tracing through the serial sections of the entire preparation. Several vesicles (v) are lined by sensory epithelium and some neural elements (ne) can be seen dispersing into the host's parenchyma. Silver stain. (Host, adult; transplant survival, 50 days; × 100).
migration of olfactory n e u r o n s and failure of the olfactory axon terminals to form distinct glomerular structures have been consistently observed. Apparently, the presence of the lamina propria mucosae and even the occasional presence of bony fragments in the transplants is not sufficient to preserve the epithelial integrity nor the ability of the sensory n e u r o n s to form glomeruli. However, we cannot exclude that the olfactory axons may form glomeruli when different parameters of age and location of the donor-host
by ultrastructural examination and intracerebral transplants of isolated n e u r o n a l staminal cells (basal cells), provide further insight into the behavior of this neuronal population when removed from its natural environment. It may also contribute to our understanding of the potential of a matrix which persists even in adult mammals.
relationship are examined. The present studies, which will be c o m p l e m e n t e d
This work was supported by a grant from the National Institutes of Health, R01-NS16421. The authors wish to thank Dr. F. L. Margolis, Roche Institutes of Molecular Biology for the O M P antibody.
1 Graziadei, P. P. C., Cell dynamics in the olfactory mucosa, Tissue Cell, 5 (1973) 113-131. 2 Graziadei, P. P. C. and Delian, R. S., Neuronal regeneration in frog olfactory system, J. Cell Biol., 59 (1973) 525-530. 3 Graziadei, P. P. C. and Kaplan, M. S., Regrowth of the olfactory sensory axons into transplanted neural tissue. I. Development of connections with the occipital cortex, Brain Research, 201 (1980) 39-44. 4 Graziadei, P. P. C., Levine, R. R. and Monti Graziadei, G. A., Plasticity of connections of the olfactory sensory neuron: regeneration into the forebrain following bulbectomy in the neonatal mouse, Neuroscience, 4 (1979) 713-727. 5 Graziadei, P. P. C. and Metcalf, J. F., Autoradiographic and ultrastructural observations on the frogs olfactory mucosa, Z. Zellforsch, 116 (1971) 305-318. 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 Oraziadei, P. P. C. and Monti Graziadei, G. A., Plasticity of connections in the olfactory pathway; transplantation studies. In Olfaction and Taste VII, IRL Press, Ltd., London, 1980, pp. 1980. 8 Heckroth, J. A., Monti Graziadei, G. A. and Graziadei, P.
P. C., Intraocular transplants of olfactory neuroepithelium in rat, Int. J. Develop. Neurosci., in Press, 1983. Loots, G. P., Loots, J. M., Brown, J. M. M. and Schoeman, J. L., A rapid silver impregnation method for nervous tissue: a modified protargolperoxide technique, Stain Technol., 54 (1979) 97-100. Monti Graziadei, G. A., Margolis, F. L., Harding, J. W. and Graziadei, P. P. C., Immunocytochemistry of the olfactory marker protein, J. Histochem. Cytochern., 25 (1977) 1311-1316. Monti Graziadei, G. A. and Graziadei, P. P. C., Neurogenesis and neuron regeneration in the olfactory system of mammals. (2) Degeneration and reconstitution of the olfactory sensory neurons after axotomy, J. Neurocytol., 8 (1979) 197-213. Moulton, D. G., Dynamics of cell population i the olfactory epithelium, Ann. N. Y. Acad. Sci. 237 (1974) 52-61. Rosenstein, J. M. and Brightman, M. W., Intact cerebral ventricle as a site for tissue transplantation, Nature (Lond.), 276 (1978) 83--85. Sternberger, L. A., Hardy, P. H., Jr., Cuculis, J. J. and Meyer, H. G., The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (HorseradishperoxidaseAntihorseradish Peroxidase) and its use in identification of Spirochetes, J. Histochern. Cytochem., 18 (1970)315-333.
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