EXPERIMENTAL
NEUROLOGY
115, 137-141 (19%)
Nerve Growth Factor Receptor and the Transplanted Rat Olfactory Bulb H.VICKLAND,*~t Departments
J.N. KOTT,* M. A. BOTHWELL,$ AND L.E. WEsTRuM*'?
of *Neurological University
Surgery, tBiologica1 Structure, and $Physiology of Washington, Seattle, Washington 98195
and Biophysics,
matrix (13). In any form, the NGF receptor is often expressed during regeneration after axonal injury (6, 15, 16). The expression of NGFR-immunoreactivity (IR) in the olfactory system is significant because of the following key features. The mammalian olfactory system is unique in that the primary sensory neurons are capable of regeneration at any time during the life of the animal (11). The olfactory nerve (ON) axons of the new neurons innervate the olfactory bulb (OB) directly, forming synaptic contact with dendrites of primary projection neurons of the OB in complex synaptic structures called glomeruli (OG). In the rat olfactory system, NGFR is developmentally regulated (2) and has been localized to the glial cells surrounding axons and synapses (18). In neonatal rats the low-affinity form of the receptor (as indicated by the monoclonal antibody 192 IgG) is immunolocalized to the ON, whereas in the adult it localizes to the OG (17, 18). Because of this differential age-related mode of expression of NGFR-IR, and its involvement in regeneration after axotomy (19), we investigated its expression during the recovery and reinnervation of the transplanted OB. Transplantation (TX) of OB into neonatal rats has been explored previously (20, 22, 23). The neural TX model we use involves the transection of the ON and will bring about the eventual regeneration of the ON (4, 11). The difference in the TX model is that, by removing the host OB, the host targets of the ON axons are lost, as are the connections of the OB with the cortex and limbic system. In place of the native tissue is the homotypic OB TX, and reinnervation of this tissue by host ON shows NGFR-IR in a changing pattern over a time course of recovery. The objective was to determine whether NGFR immunoreactivity in the TX paradigm parallels that of injured olfactory system observed in our laboratory (19).
Nerve growth factor receptor (NGFR) in the rat olfactory system is developmentally regulated, localizing to the olfactory nerve (ON) in the young rat, and to the olfactory bulb (OB) glomeruli in the adult (Vickland et al. 1991. Brain Res., in press). This pattern of immunoreactivity (IR) may indicate the state of axon growth in the young ON and synaptogenesis in the adult glomeruli. Additional experiments in our laboratory involving lesions to the ON in adult rats have shown a recapitulation of the developmental pattern of expression: NGFRIR is again found along the ON. Longer survival times after lesioning show the reexpression of the adult pattern of NGFR-IR. This phenomenon was further explored in a transplant (TX) model to determine the changes in NGFR-IR in both the host and TX tissue. A fetal OB labeled with [‘Hlthymidine is placed into the space created by the removal of the OB of a neonatal rat. After survival times of 1, 2, 8, and 13 weeks, the host animal is sacrificed and the OB TX is processed using monoclonal antibody 192 IgG for NGFR. The host ON shows strong NGFR-IR in TX of l- and Z-week survival times. In TX survival times of 8 weeks or more, NGFR-IR is observed in glomerulus-like structures. All of these glomerulus-like structures are near groups of neurons in the TX tissue, indicating that they may be functional, with appropriate synapses. Therefore, even though the adult pattern of NGFR-IR takes longer to become established than in normal rats, we have demonstrated that this pattern does so in the TX OB model. 0 1992 Academic Press, Inc.
INTRODUCTION
Cell signaling is important in neuron differentiation and axon outgrowth; in certain areas of the peripheral and central nervous systems, nerve growth factor receptor (NGFR) is involved in this process (14, 16). NGFR may have different functions depending on its molecular configuration. As the high-affinity form, it binds to neurotrophins such as nerve growth factor (NGF) (3,5), or brain-derived neurotrophic factor (BDNF) (12). The low-affinity form may have other functions as well, possibly involving interactions with the extracellular
METHODS
Procedures were carried out as described by Kott and colleagues (8). Rat fetal OBs at Embryonic Day 15 (E15) were placed into neonatal hosts at Postnatal Day 1 (PNl) after suction evacuation of host OB. The fetal 137 All
0014.4886192 $3.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
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bulbs had been labeled in utero by tritiated thymidine injection of the pregnant female so that transplant tissue could be identified in adjacent tissue sections processed for autoradiography. The neonatal hosts were perfused with 4% paraformaldehyde after survival times of 1,2,8, and greater than 13 weeks (adulthood). After a short (1 to 2 h) postfixation time in the perfusate solution, the tissue was cryoprotected in 30% sucrose, then embedded in egg yolk and serially sectioned at 24pm on a freezing microtome. Selected sections were immunoprocessed for NGF receptor using rat MAB 192 IgG and an avidin-biotin method. Free floating sections were blocked for nonspecific binding in a solution consisting of PBS, Triton X-100 (0.05%), 2.5% horse serum, and 2.5% rat serum. Monoclonal antibody 192 IgG for NGFR served as the primary antibody. This was added at a dilution of 3 pg/ml to a solution identical to the blocking solution above. After 6 PBS rinses, biotinylated horse anti-mouse IgG, rat absorbed, from Vector labs, served as the secondary antibody. Another series of PBS rinses were followed by streptavidin (from Zymed Labs). Diaminobenzidine served as the chromogen, and sections were rinsed in Tris buffer (0.5 M). Negative controls were processed using PBS instead of the primary antibody in the same blocking solution described above. For positive control, rat neonatal cerebellum was used. All controls were processed along with the experimental tissue. Sections were mounted on gelatin-coated slides, dehydrated, and coverslipped. Sections were studied and photographed using a Leitz Diaplan microscope. Adjacent sections were processed for autoradiography so that transplant boundaries were apparent. Other markers were investigated in a companion study (8). RESULTS
Rat OB TXs show progression of the pattern of NGFR immunoreactivity that in general parallels that of the pattern observed during development. However, in TXs, adult patterns take a longer time to become established. As discussed in our companion article (8), lamination typical of the normal OB fails to occur. Instead, cells with neuronal morphology remain in large areas, or “clones,” separated from other similar areas by groups of much smaller cells. Cells within the “clonal” area sometimes line up, as if to establish a laminar organization within the “clonal” area of cells, but no evidence is found of the normal laminar organization typical of OB. Sometimes the smaller cells surrounding the “clones” are immunoreactive for NGFR, and these may be glia. Some larger cells that were immunoreactive for NGFR were obviously neurons and will be discussed later. The most noteworthy result is the appearance of glomerulus-like regions in the older TX. Glomerulus-like
ET
AL.
regions are seen after 8 week survival (Figs. 3,5, and 6) and are also observed in the adult (13 week survival) TX (Fig. 4). These glomerulus-like regions are adjacent to cells with neuronal morphology, as observed in adjacent sections stained with a cell stain and processed for autoradiography. Figure 1 illustrates NGFR-IR in a TX fetal OB that survived for 1 week. Strong NGFR-IR is seen along the ON, which has ramified to surround the OB TX. There is little NGFR-IR found within the TX tissue itself. The NGFR-IR observed in the lower right of the TX tissue is located on the surface of the TX. (This figure shows a section which was cut near the surface of the TX.) The pattern of NGFR-IR observed along the host ON resembles that in normal neonatal rat ON. After a survival time of 2 weeks (Fig. 2), some significant differences can be seen. In the 2-week tissue, many fibers are seen ramifying throughout the TX OB. Some cell bodies are observed which appear to be neurons and many had the morphology of those neurons found in the septal nuclei (21). In some cases, neurons that have the appearance of those in the septal nuclei extend neurites that ramify among the axons of the ON. Some of the smaller cells may be glia or perhaps small interneurons. Glomerulus-like structures were not observed. Figure 3 shows OB TX tissue after an g-week survival time. Some of the tissue shows NGFR immunolabeled glomerulus-like structures which are near the periphery of the tissue and occur in groups rather than in laminar arrangement. Although NGFR-IR can be observed along the ON in the g-week TXs, there is less of it than in the shorter survival times. The TX of an animal with a survival time of greater than 13 weeks is shown in Fig. 4. Small clumps of glomerulus-like structures that are strongly immunoreactive for NGFR can be seen along the periphery of the OB TX. In this and a few of the g-week animals, there is comparatively less NGFR-IR in the area of the ON; a finding also observed in the normal adult tissue. Figures 5 and 6 show higher magnification views of glomerulus-like areas from tissue of g-week animals. Sometimes these glomerulus-like structures are seen as “wispy” IR surrounded by NGFR-IR on the ON fascicles (Fig. 5). Figure 6 shows other tissue of g-week survival time that has a clear, unreactive space between the ON and the glomerulus-like areas, both of which show NGFR-IR. A clear space is also found in normal OB and is usually indicative of a normal nerve fiber layer. DISCUSSION
Our results show that the differential regulation of NGFR in the TX olfactory system generally parallels that seen in the olfactory system after injury to the ON (19); however, a longer time period is required for the changes to occur in the TX system. The NGFR immuno-
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FIG. 1. Parasaggital section of OB TX after 1 week survival. NGFR-IR is seen on ON fascicles surroun~ng the TX (arrows). Asterisk indicates NGFR-IR on ON fascicles that extend over the surface of the TX. 28~. FIG. 2. Parasaggital section of OB TX after 2 week survival. NGFR-IR neuron (arrow) is seen in lower right of TX; arrowheads indicate neurites of the neuron (left) and axons of ON (right). Small cell (asterisk) with NGFR-IR may be a glial cell or small neuron. Note fine NGFR-IR fibers throughout the TX. 28X. FIG. 3. Parasaggital section of OB TX after 8 week survival. Glomerulus-like structures (G) are seen in small groups. NGFR-IR ON fascicles are present (arrows) but are less dense than that of I- and 2-week TXs. 28~. FIG. 4. Parasaggital section of OR TX after survival of more than 13 weeks. NGFR-IR is seen in glomerulus-like structures (G). 28x. FIG. 5. GlomeNlus-like structures (G) in 8 week TX. Note the NGFR-IR along ON fascicles, which extend to the glomerulus-like structure. 50X. FIG. 6. Glomerulus-like structure in 8 week TX. Note the space (asterisk) between the structure and the ON fascicles. 50X.
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reactivity along the ON is observed even 8 weeks after TX, whereas in normal ON such expression would have disappeared by 2 to 3 weeks of age. In the ON of the 13-week TX, NGFR-IR is substantially reduced, possibly indicating that the normal adult pattern is being reestablished (17, 18). On the other hand, cell stains of the TX OBs show no reestablishment of the typical layering of cell types, and NGFR-labeled glomeruluslike structures are few in comparison to the number of glomeruli found in normal OB. Significantly, these results demonstrate that an apparently normal developmental pattern of NGFR-IR in the primary olfactory system can be established after OB TX. The lack of ON NGFR-IR in TX of 8 week and more than 13 week survival times may indicate that many of the axons no longer need local (glial) trophic support (10). This may be indicative of synapse formation of ON axons with target dendrites in the TX OB, just as in normal maturation. However, the precise role of the low affinity form of NGFR (recognized by the antibody 192 IgG) is not known. Because the gene for NGF is found in proliferating glia, and because axotomy induces the appearance of the NGF message in adult optic nerve (lo), it is thought that neurotrophic molecules are involved in the process of regrowth and recovery. However, NGFR-IR on glia may indicate other functions in addition to binding NGF and other neurotrophic factors, and the presence of NGFR-IR on glia is not adequate to implicate them as providers of trophic support. Although NGFR-IR in the TX may have other functions, its presence may indicate that neurotrophins are also present and may provide local support to growing axons in and around the TX. Recent evidence indicates that the trk protooncogene is related to the high-affinity form of NGFR (7). This and other new data have led to the hypothesis that the low-affinity form of NGFR (which the 192 IgG antibody recognizes; also known as ~75) and the trk protooncogene form a heterodimer which binds to neurotrophic factors (1). How this new hypothesis and the new data relate to axon regeneration and growth of the TX in the olfactory system remains to be determined. The lack of overall laminar arrangement in TXs was observed in all cases. It is noteworthy that glomeruluslike structures still form in areas observed to be populated by large neurons, presumed by their morphology, to be mitral cells or other projection neurons. This indicates that the ON axons do not require a laminar arrangement in order to make their synaptic contacts and attests to the aggressiveness of the regenerating ON neurons. However, because NGFR-IR is often found on glia, and the NGFR-IR along the ON in the TX may be localizing to glia surrounding the axons and not the axons themselves, the possible importance of glia in axon regeneration is emphasized.
ET
AL.
The NGFR-IR fibers often seen within the TX OBs of 2-week and longer survival may represent axons of neurons within the OB TX. At least some of these fibers belong to the NGFR-IR cells (possibly glia) that are often seen in TXs but not in normal OB. The fibers also could possibly belong to ON axons that have not received a signal to stop, because no glomerular layer has been established in the TX. Alternatively, some of the fibers may belong to large NGFR-IR neurons having a morphology similar to those of the septal nucleus (see Fig. 2). These cells were possibly “captured” in the excised TX tissue (because of the proximity of the septal cells and the OB in El5 rats) and grew within the TX to express their phenotype, which includes NGFR-IR (21). Even though these neurons are artifacts of the TX process, they survive and extend neurites which ramify near ingrowing ON axons. In any case, these NGFR-IR fibers, cells, and neurons are atypical of normal OB, and normally NGFR-IR is limited to the glomeruli and the glomerular layer. The olfactory nerve fiber layer in the TX sometimes shows NGFR-IR on nerve tracts coursing from the pial border to the putative glomerular layer (Fig. 5), whereas in the normal animal there is no immunolabeling in this area (see Fig. 6). In the normal OB, a signal may be present that brings about the disappearance of NGFRIR, this signal is perhaps missing in the OB TX. The possibility exists that the removal of the OB itself causes a release of trophic factors that support the TX and enhance its growth. It has been shown that neurons requiring NGF for survival will live, grow, and differentiate in medium to which soluble extracts of OB had been added (9). In normal medium without this extract, the neurons died within a few days. This evidence indicates the presence of trophic factors in the evacuated OB site that possibly enhance the growth and differentiation of the TX. In conclusion, NGFR and the neurotrophic factors may have an important role in the reinnervation of TX OB and may be essential for this reinnervation. NGFR may be part of the molecule which binds to neurotrophic factors, or it may have other functions important to axon outgrowth. Further work should establish the precise role of NGFR in the growth of TX tissue, just as its role is being established in development and recovery from injury.
ACKNOWLEDGMENTS This study was supported by NIH Grants NS09678, NS07144, and NS23343 and Training Grant 2T32HD07183-llA1. The authors thank Jeff Bedell for surgical assistance, Xiao-Ming Dong for technical assistance, and Janet Clardy and Paul Schwartz for photography. L.E.W. is a research affiliate of the CDMRC of the University of Washington.
NGFR IN OLFACTORY
REFERENCES 1. BOTHWELL, M. 1991. Keeping track of neurotrophin receptors. Cell 65: 915-918. 2. BUCK, C. R., H. J. I%~ARTINEZ,I. B. BLACK, AND M. V. CHAO. 1987. Developmentally regulated expression of the nerve growth factor receptor gene in the periphery and brain. Proc. Natl. Acud. Sci. USA
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3. CHANDLER, C. E., L. M. PARSONS, M. HOSANG, AND E. M. SHOOTER. 1984. A monocional antibody modulates the interaction of nerve growth factor with PC12 cells. J. Biol. Chem. 289: 6882-6889.
4. DOUCE~& J. R., J. A. KIERNAN, AND B. A. FLUMERPELT. 1983. The re-innervation of olfactory glomeruli following transection of primary olfactory axons in the central and peripheral nervous system. J. Anat. 137(l): 1-19. 5. GREEN, S. H., AND L. A. GREENE. 1986. A single M, 103,oO iz61beta-nerve growth factor-affinity-labeled species represents both the low and high affinity forms of the nerve growth factor receptor. J. Biol. Chem. 261: l&316-15,326. 6. HEUMANN, R., S. KORSCHING, C. BANDTLOW, AND H. THOENEN. 1987. Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection. J. Cell Biof. 104: 1623-1631. 7. KAPLAN, D. R., B. L. HEMPSTEAD, D. MARTIN-Z~CA, M. V. CHAO, AND L. F. PARADA. 1991. The trk proto-oncogene product: a signal transducing receptor for nerve growth factor. Science 252: 554-558. 8. Kolvr, J. N., H. VICKLAND, X-M. DONG, AND L. E. WESTRUM. 1992. Development of olfactory marker protein and tyrosine hydroxylase immunoreactivity in the transplanted rat olfactory bulb. Exp. Neural. 115: 000-000. 9. LAMBERT, M. P., MEGER~N, T., GARDEN, G., AND KLEIN, W. L. 1988. Soluble proteins form rat olfactory bulb promote the survival and differentiation of cultured basal forebrain neurons. Bruin
Res. 469:
263-276.
10. Lu, B., M. YOKOYAMA, C. F. DREYFUS, AND I. B. BLACK. 1991. NGF gene expression in actively growing brain glia. J. Neurosci. 11(2):318-326. 11. MONTI-GRAZIADEI, G. A., AND P. P. C. GRAZIADEI. 1979. Neurogenesis and neuron regeneration in the olfactory system of mammals. II. Degeneration and reconstitution of the olfactory sensory neurons after axotomy. J. Ne~~o~~o~. 8: 197-213.
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12. RODRIGUEZ-TEBAR, A., DECHANT, G., AND BARDE, Y-A. 1990. Binding of brain-derived neurotrophic factor to the nerve growth factor receptor. Neuron 4: 487-492. 13. SEILHE~R, B., AND M. SCHACHNER. 1987. Regulation of neural cell adhesion molecule expression on cultured mouse Schwann cells by nerve growth factor. EMBO J. 6(6): 1611-1616. 14. SPRINGER, J. E. 1988. Nerve growth factor receptors in the eentral nervous system. Exp. Neurol. 102: 354-365. 15. TANIUCHI, M., H. B. CLARK, AND E. M. JOHNSON, JR. 1986. Induction of nerve growth factor receptor in Schwann cells after axotomy. Proc. Natl. Acad. Sci. USA 83: 4094-4098. 16. TANIUCHI, M., H. B. CLARK, J. B. SCHWEITZER, AND E. M. JOHNSON, JR. 1988. Expression of nerve growth factor receptors by Schwann cells of axotomized peripheral nerves: Ultrast~ctural location, suppression by axonal contact, and binding properties. J. Neurasci. S(2): 664-681. 17. TAYRIEN, M. W., S. KOH, J. E. SPRINGER, AND R. Lou. 1986. Immunocytochemical localization of nerve growth factor (NGF) and NGF receptor in the rat olfactory bulb. Anat. Rec. 214: 133A. 18. VICKLAND, II., L. E. WESTRUM, J. N. KOTT, S. L. PATTERSON, AND M. A. BOTHWELL. 1991. Nerve growth factor receptor expression in young and adult rat olfactory system. Brain Res., in press. 19. VICKLAND, H., L. E. WESTRUM, AND M. A. BOTHWELL. 1991. Recapitulation of the developmental pattern of NGF receptor immunoreactivity in regenerating rat olfactory nerve. Neurosci. 17: 1315. [Abstr] 20. WESTRUM, L. E., J. N. KOTT, H. VICKLAND, M. H. HANKIN, AND R. D. LUND. 1990. Fetal olfactory bulb transplants send projeetions to host olfactory cortex in the rat. Neurosci. Lett. 119: 265-268. 21, YAN, Q., AND E. M. JOHNSON, JR. 1989. Immunohistochemical localization end biochemical characterization of nerve growth factor receptor in adult rat brain. J. Comp. Neural. 290: 586598.
22. ZIGOVA, T., P. P. C. GRAZIADEI, AND A. G. MONTI-GRAZIADEI. 1990. Olfactory bulb transplantation into the olfactory bulb of neonatal rats. Brain Res. 513: 315-319. 23. ZIGOVA, T., P. P. C. GRAZIADEI, AND A. G. MONTI-GEZA~IADEI. 1991. Olfactory bulb transplantation into olfactory bulbs of neonatal rats: An autoradiographic study. Brain Res. 539: 51-58.
NEUROLOGY
115, 137-141 (19%)
Nerve Growth Factor Receptor and the Transplanted Rat Olfactory Bulb H.VICKLAND,*~t Departments
J.N. KOTT,* M. A. BOTHWELL,$ AND L.E. WEsTRuM*'?
of *Neurological University
Surgery, tBiologica1 Structure, and $Physiology of Washington, Seattle, Washington 98195
and Biophysics,
matrix (13). In any form, the NGF receptor is often expressed during regeneration after axonal injury (6, 15, 16). The expression of NGFR-immunoreactivity (IR) in the olfactory system is significant because of the following key features. The mammalian olfactory system is unique in that the primary sensory neurons are capable of regeneration at any time during the life of the animal (11). The olfactory nerve (ON) axons of the new neurons innervate the olfactory bulb (OB) directly, forming synaptic contact with dendrites of primary projection neurons of the OB in complex synaptic structures called glomeruli (OG). In the rat olfactory system, NGFR is developmentally regulated (2) and has been localized to the glial cells surrounding axons and synapses (18). In neonatal rats the low-affinity form of the receptor (as indicated by the monoclonal antibody 192 IgG) is immunolocalized to the ON, whereas in the adult it localizes to the OG (17, 18). Because of this differential age-related mode of expression of NGFR-IR, and its involvement in regeneration after axotomy (19), we investigated its expression during the recovery and reinnervation of the transplanted OB. Transplantation (TX) of OB into neonatal rats has been explored previously (20, 22, 23). The neural TX model we use involves the transection of the ON and will bring about the eventual regeneration of the ON (4, 11). The difference in the TX model is that, by removing the host OB, the host targets of the ON axons are lost, as are the connections of the OB with the cortex and limbic system. In place of the native tissue is the homotypic OB TX, and reinnervation of this tissue by host ON shows NGFR-IR in a changing pattern over a time course of recovery. The objective was to determine whether NGFR immunoreactivity in the TX paradigm parallels that of injured olfactory system observed in our laboratory (19).
Nerve growth factor receptor (NGFR) in the rat olfactory system is developmentally regulated, localizing to the olfactory nerve (ON) in the young rat, and to the olfactory bulb (OB) glomeruli in the adult (Vickland et al. 1991. Brain Res., in press). This pattern of immunoreactivity (IR) may indicate the state of axon growth in the young ON and synaptogenesis in the adult glomeruli. Additional experiments in our laboratory involving lesions to the ON in adult rats have shown a recapitulation of the developmental pattern of expression: NGFRIR is again found along the ON. Longer survival times after lesioning show the reexpression of the adult pattern of NGFR-IR. This phenomenon was further explored in a transplant (TX) model to determine the changes in NGFR-IR in both the host and TX tissue. A fetal OB labeled with [‘Hlthymidine is placed into the space created by the removal of the OB of a neonatal rat. After survival times of 1, 2, 8, and 13 weeks, the host animal is sacrificed and the OB TX is processed using monoclonal antibody 192 IgG for NGFR. The host ON shows strong NGFR-IR in TX of l- and Z-week survival times. In TX survival times of 8 weeks or more, NGFR-IR is observed in glomerulus-like structures. All of these glomerulus-like structures are near groups of neurons in the TX tissue, indicating that they may be functional, with appropriate synapses. Therefore, even though the adult pattern of NGFR-IR takes longer to become established than in normal rats, we have demonstrated that this pattern does so in the TX OB model. 0 1992 Academic Press, Inc.
INTRODUCTION
Cell signaling is important in neuron differentiation and axon outgrowth; in certain areas of the peripheral and central nervous systems, nerve growth factor receptor (NGFR) is involved in this process (14, 16). NGFR may have different functions depending on its molecular configuration. As the high-affinity form, it binds to neurotrophins such as nerve growth factor (NGF) (3,5), or brain-derived neurotrophic factor (BDNF) (12). The low-affinity form may have other functions as well, possibly involving interactions with the extracellular
METHODS
Procedures were carried out as described by Kott and colleagues (8). Rat fetal OBs at Embryonic Day 15 (E15) were placed into neonatal hosts at Postnatal Day 1 (PNl) after suction evacuation of host OB. The fetal 137 All
0014.4886192 $3.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
138
VICKLAND
bulbs had been labeled in utero by tritiated thymidine injection of the pregnant female so that transplant tissue could be identified in adjacent tissue sections processed for autoradiography. The neonatal hosts were perfused with 4% paraformaldehyde after survival times of 1,2,8, and greater than 13 weeks (adulthood). After a short (1 to 2 h) postfixation time in the perfusate solution, the tissue was cryoprotected in 30% sucrose, then embedded in egg yolk and serially sectioned at 24pm on a freezing microtome. Selected sections were immunoprocessed for NGF receptor using rat MAB 192 IgG and an avidin-biotin method. Free floating sections were blocked for nonspecific binding in a solution consisting of PBS, Triton X-100 (0.05%), 2.5% horse serum, and 2.5% rat serum. Monoclonal antibody 192 IgG for NGFR served as the primary antibody. This was added at a dilution of 3 pg/ml to a solution identical to the blocking solution above. After 6 PBS rinses, biotinylated horse anti-mouse IgG, rat absorbed, from Vector labs, served as the secondary antibody. Another series of PBS rinses were followed by streptavidin (from Zymed Labs). Diaminobenzidine served as the chromogen, and sections were rinsed in Tris buffer (0.5 M). Negative controls were processed using PBS instead of the primary antibody in the same blocking solution described above. For positive control, rat neonatal cerebellum was used. All controls were processed along with the experimental tissue. Sections were mounted on gelatin-coated slides, dehydrated, and coverslipped. Sections were studied and photographed using a Leitz Diaplan microscope. Adjacent sections were processed for autoradiography so that transplant boundaries were apparent. Other markers were investigated in a companion study (8). RESULTS
Rat OB TXs show progression of the pattern of NGFR immunoreactivity that in general parallels that of the pattern observed during development. However, in TXs, adult patterns take a longer time to become established. As discussed in our companion article (8), lamination typical of the normal OB fails to occur. Instead, cells with neuronal morphology remain in large areas, or “clones,” separated from other similar areas by groups of much smaller cells. Cells within the “clonal” area sometimes line up, as if to establish a laminar organization within the “clonal” area of cells, but no evidence is found of the normal laminar organization typical of OB. Sometimes the smaller cells surrounding the “clones” are immunoreactive for NGFR, and these may be glia. Some larger cells that were immunoreactive for NGFR were obviously neurons and will be discussed later. The most noteworthy result is the appearance of glomerulus-like regions in the older TX. Glomerulus-like
ET
AL.
regions are seen after 8 week survival (Figs. 3,5, and 6) and are also observed in the adult (13 week survival) TX (Fig. 4). These glomerulus-like regions are adjacent to cells with neuronal morphology, as observed in adjacent sections stained with a cell stain and processed for autoradiography. Figure 1 illustrates NGFR-IR in a TX fetal OB that survived for 1 week. Strong NGFR-IR is seen along the ON, which has ramified to surround the OB TX. There is little NGFR-IR found within the TX tissue itself. The NGFR-IR observed in the lower right of the TX tissue is located on the surface of the TX. (This figure shows a section which was cut near the surface of the TX.) The pattern of NGFR-IR observed along the host ON resembles that in normal neonatal rat ON. After a survival time of 2 weeks (Fig. 2), some significant differences can be seen. In the 2-week tissue, many fibers are seen ramifying throughout the TX OB. Some cell bodies are observed which appear to be neurons and many had the morphology of those neurons found in the septal nuclei (21). In some cases, neurons that have the appearance of those in the septal nuclei extend neurites that ramify among the axons of the ON. Some of the smaller cells may be glia or perhaps small interneurons. Glomerulus-like structures were not observed. Figure 3 shows OB TX tissue after an g-week survival time. Some of the tissue shows NGFR immunolabeled glomerulus-like structures which are near the periphery of the tissue and occur in groups rather than in laminar arrangement. Although NGFR-IR can be observed along the ON in the g-week TXs, there is less of it than in the shorter survival times. The TX of an animal with a survival time of greater than 13 weeks is shown in Fig. 4. Small clumps of glomerulus-like structures that are strongly immunoreactive for NGFR can be seen along the periphery of the OB TX. In this and a few of the g-week animals, there is comparatively less NGFR-IR in the area of the ON; a finding also observed in the normal adult tissue. Figures 5 and 6 show higher magnification views of glomerulus-like areas from tissue of g-week animals. Sometimes these glomerulus-like structures are seen as “wispy” IR surrounded by NGFR-IR on the ON fascicles (Fig. 5). Figure 6 shows other tissue of g-week survival time that has a clear, unreactive space between the ON and the glomerulus-like areas, both of which show NGFR-IR. A clear space is also found in normal OB and is usually indicative of a normal nerve fiber layer. DISCUSSION
Our results show that the differential regulation of NGFR in the TX olfactory system generally parallels that seen in the olfactory system after injury to the ON (19); however, a longer time period is required for the changes to occur in the TX system. The NGFR immuno-
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FIG. 1. Parasaggital section of OB TX after 1 week survival. NGFR-IR is seen on ON fascicles surroun~ng the TX (arrows). Asterisk indicates NGFR-IR on ON fascicles that extend over the surface of the TX. 28~. FIG. 2. Parasaggital section of OB TX after 2 week survival. NGFR-IR neuron (arrow) is seen in lower right of TX; arrowheads indicate neurites of the neuron (left) and axons of ON (right). Small cell (asterisk) with NGFR-IR may be a glial cell or small neuron. Note fine NGFR-IR fibers throughout the TX. 28X. FIG. 3. Parasaggital section of OB TX after 8 week survival. Glomerulus-like structures (G) are seen in small groups. NGFR-IR ON fascicles are present (arrows) but are less dense than that of I- and 2-week TXs. 28~. FIG. 4. Parasaggital section of OR TX after survival of more than 13 weeks. NGFR-IR is seen in glomerulus-like structures (G). 28x. FIG. 5. GlomeNlus-like structures (G) in 8 week TX. Note the NGFR-IR along ON fascicles, which extend to the glomerulus-like structure. 50X. FIG. 6. Glomerulus-like structure in 8 week TX. Note the space (asterisk) between the structure and the ON fascicles. 50X.
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reactivity along the ON is observed even 8 weeks after TX, whereas in normal ON such expression would have disappeared by 2 to 3 weeks of age. In the ON of the 13-week TX, NGFR-IR is substantially reduced, possibly indicating that the normal adult pattern is being reestablished (17, 18). On the other hand, cell stains of the TX OBs show no reestablishment of the typical layering of cell types, and NGFR-labeled glomeruluslike structures are few in comparison to the number of glomeruli found in normal OB. Significantly, these results demonstrate that an apparently normal developmental pattern of NGFR-IR in the primary olfactory system can be established after OB TX. The lack of ON NGFR-IR in TX of 8 week and more than 13 week survival times may indicate that many of the axons no longer need local (glial) trophic support (10). This may be indicative of synapse formation of ON axons with target dendrites in the TX OB, just as in normal maturation. However, the precise role of the low affinity form of NGFR (recognized by the antibody 192 IgG) is not known. Because the gene for NGF is found in proliferating glia, and because axotomy induces the appearance of the NGF message in adult optic nerve (lo), it is thought that neurotrophic molecules are involved in the process of regrowth and recovery. However, NGFR-IR on glia may indicate other functions in addition to binding NGF and other neurotrophic factors, and the presence of NGFR-IR on glia is not adequate to implicate them as providers of trophic support. Although NGFR-IR in the TX may have other functions, its presence may indicate that neurotrophins are also present and may provide local support to growing axons in and around the TX. Recent evidence indicates that the trk protooncogene is related to the high-affinity form of NGFR (7). This and other new data have led to the hypothesis that the low-affinity form of NGFR (which the 192 IgG antibody recognizes; also known as ~75) and the trk protooncogene form a heterodimer which binds to neurotrophic factors (1). How this new hypothesis and the new data relate to axon regeneration and growth of the TX in the olfactory system remains to be determined. The lack of overall laminar arrangement in TXs was observed in all cases. It is noteworthy that glomeruluslike structures still form in areas observed to be populated by large neurons, presumed by their morphology, to be mitral cells or other projection neurons. This indicates that the ON axons do not require a laminar arrangement in order to make their synaptic contacts and attests to the aggressiveness of the regenerating ON neurons. However, because NGFR-IR is often found on glia, and the NGFR-IR along the ON in the TX may be localizing to glia surrounding the axons and not the axons themselves, the possible importance of glia in axon regeneration is emphasized.
ET
AL.
The NGFR-IR fibers often seen within the TX OBs of 2-week and longer survival may represent axons of neurons within the OB TX. At least some of these fibers belong to the NGFR-IR cells (possibly glia) that are often seen in TXs but not in normal OB. The fibers also could possibly belong to ON axons that have not received a signal to stop, because no glomerular layer has been established in the TX. Alternatively, some of the fibers may belong to large NGFR-IR neurons having a morphology similar to those of the septal nucleus (see Fig. 2). These cells were possibly “captured” in the excised TX tissue (because of the proximity of the septal cells and the OB in El5 rats) and grew within the TX to express their phenotype, which includes NGFR-IR (21). Even though these neurons are artifacts of the TX process, they survive and extend neurites which ramify near ingrowing ON axons. In any case, these NGFR-IR fibers, cells, and neurons are atypical of normal OB, and normally NGFR-IR is limited to the glomeruli and the glomerular layer. The olfactory nerve fiber layer in the TX sometimes shows NGFR-IR on nerve tracts coursing from the pial border to the putative glomerular layer (Fig. 5), whereas in the normal animal there is no immunolabeling in this area (see Fig. 6). In the normal OB, a signal may be present that brings about the disappearance of NGFRIR, this signal is perhaps missing in the OB TX. The possibility exists that the removal of the OB itself causes a release of trophic factors that support the TX and enhance its growth. It has been shown that neurons requiring NGF for survival will live, grow, and differentiate in medium to which soluble extracts of OB had been added (9). In normal medium without this extract, the neurons died within a few days. This evidence indicates the presence of trophic factors in the evacuated OB site that possibly enhance the growth and differentiation of the TX. In conclusion, NGFR and the neurotrophic factors may have an important role in the reinnervation of TX OB and may be essential for this reinnervation. NGFR may be part of the molecule which binds to neurotrophic factors, or it may have other functions important to axon outgrowth. Further work should establish the precise role of NGFR in the growth of TX tissue, just as its role is being established in development and recovery from injury.
ACKNOWLEDGMENTS This study was supported by NIH Grants NS09678, NS07144, and NS23343 and Training Grant 2T32HD07183-llA1. The authors thank Jeff Bedell for surgical assistance, Xiao-Ming Dong for technical assistance, and Janet Clardy and Paul Schwartz for photography. L.E.W. is a research affiliate of the CDMRC of the University of Washington.
NGFR IN OLFACTORY
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