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Evidence for developmental plasticity of the rubrospinal tract. Studies using the North American opossum
DevelopmentalBrain Research, 39 (1988) 303-308 Elsevier
303
BRD 60255
Evidence for developmental plasticity of the rubrospinal tract. Studies using the North American opossum George F. Martin and Xiao Ming Xu Departmentof Anatomy, The Ohio State University, Collegeof Medicine, Columbus, OH 43210 (U.S.A.)
(Accepted 8 December 1987) Key words: Developmental plasticity; Opossum; Regeneration; Rubrospinal tract; Spinal cord
Using axonal tracing techniques we have shown that rubral axons are capable of growing around lesions of the rubrospinal tract during early stages of development in the North American opossum and that a critical period for such growth exists. The opossum was employed for study because it is born in a very immature state 12-13 days after conception and the entire development of its rubrospinal tract occurs postnatally.
It is generally assumed that the spinal cord is capable of greater plasticity during development than in the adult animal 1°~22'24. This principle is well illustrated by the plasticity of the pyramidal tract in newborn mammals demonstrated first by Kalil and Reh in the brainstem re, and subsequently by others in the spinal cord 2-4'21"23. When a lesion interrupts the developing pyramidal tract, cortical axons can grow around 3"4"12A3"23and possibly through 21 it to innervate generally appropriate areas more caudally. Such plasticity probably underlies the 'sparing' of tactile placing seen after spinal cord lesions which include the pyramidal tract in neonatal animals 3. In light of the results reported above, it seems paradoxical that rubral axons do not grow around or through lesions of the rubrospinal tract in neonatal rats 2° or cats 3'4. When the rubrospinal tract is transected at birth in those species, many axotomized neurons undergo retrograde degeneration (the socalled Gudden effect, see review by CowanS). This phenomenon also occurs in mature rubrospinal neurons ~'11'15'2°, but to a lesser degree. The rubrospinal tract of the rat and cat is relatively mature at birth, however, at least in comparison to the pyramidal tract which is still growing 3"4'21, and it may simply
have lost its potential for plasticity. If so, it should be possible to demonstrate rubrospinal plasticity at earlier stages of development. This hypothesis would be difficult to test in the rat or cat because rubrospinal development occurs prenatally 3,4,2°, but should be testable in the North American opossum because the entire development of its rubrospinal tract occurs after birth 5"7. Birth takes place 12-13 days after conception in the opossum 9'1s. In the initial experiments, the rubrospinal tract was lesioned at mid to caudal thoracic levels in 2 adult and 83 developing opossums. The developing animals were obtained from females captured in the wild or bred in captivity at The Ohio State University. The s n o u t - r u m p length (SRL) of each animal was measured by stretching it on a ruler and used to estimate its age, when unknown, from the growth curve of Cutts et al. 9. The adult animals were anesthetized by intraperitoneal injections of sodium pentobarbital (40 mg/kg) prior to surgical exposure of the appropriate level of the spinal cord. The lesion was made with a no. 11 surgical blade and, after closure of the surgical exposure, the animals were returned to the vivarium for recovery. Most of the developing animals, ranging in age from postnatal day (PD)12 to es-
Correspondence: G.F. Martin, Department of Anatomy, The Ohio State University, College of Medicine, 333 West 10th Avenue, Columbus, OH 43210, U.S,A.
0165-3806/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
3(14
B
Fig. 1. A: photograph of pouch-young opossums at EPD20. B: Nissl-stained section showing the lesion (arrow) made 30 days earlier in one of the animals shown in A. The intact ventral funiculus (VF) and lateral funiculus (LF) are indicated. C: neurons in the red nucleus (RN) labeled by an injection of WGA-HRP placed two segments caudal to the lesion shown in B. The arrows point to labeled neurons: direction indicators are given. The bar in B indicates 240l¢m. that in C equals 60 f~m. timated P D (EPD)75, were o p e r a t e d while still in the m o t h e r ' s pouch. The m o t h e r was anesthetized first (see above) and placed on her back. During anesthesia, the pouch sphincter relaxes, exposing the litter. The pouch-young, still attached to the nipple, were then anesthetized individually by h y p o t h e r m i a or M e t o f a n e inhalation, so that the desired level of the spinal cord could be exposed and the rubrospinal tract cut with a no, 11 surgical blade and microscissors. A f t e r the incision was closed, the o p e r a t e d animals were r e t u r n e d with their m o t h e r to the vivarium. All animals were m a i n t a i n e d for at least 1 month (30-42 days) before being subjected to a second surgery and injections of Fast blue (FB) or wheat-germ agglutinin conjugated to horseradish peroxidase ( W G A - H R P ) , 2 segments caudal to the lesion and ipsilateral to it. The objective was to label rubral neu-
rons whose axons had grown caudal to the lesion. The adult animals were anesthetized as described above and after surgical exposure of the spinal cord, they were placed in a spinal frame. The injections (0.2-0.25 ~ul of 2% FB or 5% W G A - H R P ) were made stereotaxically using a glass micropipette attached to a 1.0 ~1 H a m i l t o n syringe. A f t e r the injections were c o m p l e t e d and the exposure closed, the animals were returned to the vivarium for a 2 - 3 day survival. The developing animals were r e m o v e d from the pouch for the second surgery and anesthetized either as described previously, or by intraperitoneal injections of sodium p e n t o b a r b i t o l (40-60 mg/kg). A f t e r exposure of the spinal cord and stabilization of the vertebral column, the injections (0.15-0.20 #1 of 10% W G A - H R P ) were m a d e as in the adult animals. The developing animals were maintained for 1 - 2 clays postoperatively.
305 All of the animals referred to above were sacrificed by an overdose of anesthetic and perfused intracardiaUy with phosphate-buffered saline, followed either by a citrate buffer-formaldehyde solution (FB experiments) or cold 1% paraformaldehyde-l.25% glutaraldehyde-1% sucrose in phosphate buffer followed by 10% sucrose in the same buffer (WGAH R P experiments). The spinal cords and brains were removed and placed either in the fixative (FB experiments) or 30% sucrose phosphate buffer for refrigeration at 4 °C. Frozen sections of the lesion site, the injection site and the brain were cut at 40/~m in all cases. The sections from the FB cases were mounted immediately and coverslipped with Entellan (Merck) for viewing and photography with a Leitz (Orthoplan) fluorescence photomicroscope using the A cube of the Ploem illumination system. The sections from the W G A - H R P cases were processed for H R P using tetramethyl benzidine as the chromagen 19 and coverslipped using piccolyte. Every third section was stained with Neutral red. Examination and photography of the H R P processed sections was carried out with a Leitz Dialux photomicroscope using bright and darkfield optics. The position of rubral neurons labeled by either FB or H R P was recorded on drawings of the sections using an X-Y plotter attached to the microscope stage by potentiometers. A total of 18 pouch-young ranging from PD18 to EPD54, were also used for orthograde transport experiments. They were subjected to lesions of the rubrospinal tract as already described, but after approximately 30 days, they were anesthetized (see above) for stereotaxic injections (0.12-0.15/tl) of 10% W G A - H R P into the red nucleus contralateral to the lesion. After about 48 h, the animals were anesthetized again and perfused so that the spinal cord and brain could be removed, sectioned on a freezing microtome at 40/zm, and processed for H R P as described above. Orthogradely labeled axons were photographed and their positions plotted using a Leitz Dialux photomicroscope equipped with light and darkfield condensors. When rubrospinal lesions were made in animals ranging in age from EPD54 to maturity, no evidence for retrograde labeling was present in the contralateral red nucleus one month later. The rubrospinal tract of the opossum 7'14, like that of other species, is almost entirely crossed. In all cases, neurons were
EPD18(SRL39MM)
Fig. 2. Plot of the rubrospinal tract labeling (arrows) at the first thoracic (T1) and lower thoracic (Low Thor) levels, 5 mm above and below the lesion and at lumbar (Lumb) levels produced by the rubral injection (Inj) of WGA-HRP shown at the top. The lesion (section indicated as Low Thor) was made at EPD18 (39 mm SRL); the injection was made 30 days later.
306
Fig. 3. Photomicrographs substantiating the results plotted in Fig. 2. A: darkfield photomicrograph of W G A - t t R P labeled rubrospinal axons (arrow) rostral to the lesion shown in B. B: darkficld photomicrograph of the lesion, Labeled rubrospinal axons are indicated (arrow). C: darkfield photomicrograph of rubrospinal labeling (arrow) caudal to the lesion. The bar in A equals 2l)[)llm and can also be used for B and C.
307 labeled in those areas of the brainstem known to have axons in the spared white matter 16'17. In cases subjected to rubrospinal lesions between PD12 (30 m m SRL) and 43 (75 m m SRL), however, rubral neurons were labeled contralateral to the lesion (Fig. 1), suggesting that their axons had grown caudal to it. In general, the number of neurons labeled in the red nucleus contralateral to the lesion decreased with increasing age of the animal at the time of lesion. Some variation in the number of labeled neurons was present, however, even between cases subjected to lesions at the same age. Such variation is probably due to differences in the extent of the lesion and/or the size of the injection. When rubrospinal lesions were made between PD15 (36 m m SRL) and EPD33 (61 m m SRL), and the contralateral red nucleus was injected with W G A - H R P approximately a month later, rubrospinal axons were labeled caudal to the lesion. Fig. 2 shows a plot of the labeling obtained in a case with a particularly large lesion made at E P D 1 8 (39 mm SRL). Rostral to the lesion, rubrospinal axons were labeled in their normal position contralaterally (Figs. 2 and 3A), but at the lesion site (Figs. 2 and 3B), they were aggregated at the edge of the remaining cord. Caudal to the lesion, most of the labeled axons were again found contralateral to their origin, but they formed a tract in the dorsal funiculus (Figs. 2 and 3C), rather than within the lateral funiculus where they are found normally. In spite of their abnormal position, rubral axons appeared to innervate areas of the gray matter appropriate to them. Labeled axons could be traced to at least caudal lumbar segments of the cord. In other cases, the extent of the lesion was different, and the position of labeled axons caudal to the lesion varied accordingly. For example, when the lat-
eral funiculus was transected but the gray matter was spared, labeled axons formed a bundle at the lesion site which coursed through the lateral part of the gray matter. They maintained that position caudal to the lesion rather than returning to the lateral funiculus. As in the case described above, terminal labeling was present in areas appropriate for rubrospinal axons. There were not enough successful cases to make definitive statements concerning differences in the robustness of rubrospinal growth caudal to the lesion at different ages. The results of our study show that the rubrospinal tract is capable of plasticity at early stages of development and that a critical period for that plasticity exists. We know from the results of previous experiments 7 that some rubral axons reach thoracic levels of the cord prior to our earliest lesion and that rubral axons continue to grow into the thoracic cord, and beyond, well after that. It is impossible to determine, therefore, whether the rubrospinal plasticity described herein resulted from regeneration of axons cut by the lesion or the addition of new axons. It is possible, of course, that both occurred. It is our working hypothesis that all descending spinal pathways are capable of plasticity at some stage of development, but that the critical period for each is different. It is likely that such differences reflect differences in developmental history 5~6.
1 Barron, K.D., Dentinger, M.P., Nelson, L.R. and Scheibly, M.E., Incorporation of tritiated leucine by axotomized rubral neurons, Brain Res., 116 (1976) 251-266. 2 Bernstein, D.R. and Stelzner, D.I., Plasticity of the corticospinal tract following midthoracic spinal injury in the postnatal rat, J. Comp. Neurol., 221 (1983) 382-400. 3 Bregman, B.S. and Goldberger, M.E., Anatomical plasticity and sparing of function after spinal cord damage in neonatal cats, Science, 217 (1982) 553-555. 4 Bregman, B.S. and Goldberger, M.E., Infant lesion effect. III. Anatomical correlates of sparing and recovery of function after spinal cord damage in newborn and adult cats, Dev. Brain Res., 9 (1983) 137-154.
5 Cabana, T. and Martin, G.F., Developmental sequences in the origin of descending spinal pathways. Studies using retrograde transport techniques in the North American opossum (Didelphis virginiana), Dev. Brain Res., 15 (1984) 247-263. 6 Cabana, T. and Martin, G.F., Corticospinal development in the North American opossum: evidence for a sequence of growth of cortical axons in the spinal cord and for transient projections, Dev. Brain Res., 23 (1985) 69-80. 7 Cabana, T. and Martin, G.F., The development of the rubrospinal tract. An experimental study using the orthograde transport of WGA-HRP in the North American opossum, Dev. Brain Res., 30 (1986) 1-11.
The authors wish to thank Ms. Mary Ann Jarrell for excellent technical assistance and for typing the manuscript. We are also grateful to Mr. Karl Rubin for photographic help and to Drs. Michael Beattie and Albert Humbertson, Jr. for helpful suggestions. This investigation was supported by USPHS Grants BNS-8309245 and NS-25095.
308 8 Cowan, W.M., Anterograde and retrograde transneuronal degeneration in the central and peripheral nervous system. In W.J.H. Nauta and S.O.E. Ebbeson (Eds.). Contemporary Research Methods in Neuroanatorny, Springer, New York, 1970, pp. 217-251. 9 Cutts, J.W., Krause, W.J. and Leeson, C.R., General observations on the growth and development of the pouchyoung opossum, Biol. Neonate, 33 (1978) 264-272. 10 Goldberger, M.E. and Murray, M., Recovery of function and anatomical plasticity after damage to the adult and neonatal spinal cord. In C. Cotman (Ed.), Synaptic Plasticity, Guilford, New York, 1985, pp. 77-110. 11 Goshgarian, H.G., Koistinen, J.M. and Schmidt, E.R., Cell death and changes in the retrograde transport of horseradish peroxidase in rubrospinal neurons following spinal cord hemisection in the adult rat, J. Comp. Neurol., 214 (1983) 251-257. 12 Kalil, K. and Reh, T., Regrowth of severed axons in the neonatal CNS, Science, 205 (1979) 1158-1161. 13 Kalil, K. and Reh, T,, A light and electron microscopic study of regrowing pyramidal tract fibers, J. Cornp. Neurol., 211 (1982) 265-275. 14 Martin, G.F. and Dora, R., The rubrospinal tract of the opossum, Didelphis virginiana, J. Comp. Neurol., 138 (1970) 19-30. 15 Martin, G.F., Dora, R., Katz, S. and King, J.S., The organization of projection neurons in the opossum red nucleus, Brain Res., 78 (1974) 17-34. 16 Martin, G.F., Humbertson, A.O., Laxson, L.C., Panneton, W.M. and Tschismadia, I., Spinal projections from the mesencephalic and pontine reticular formation in the North American opossum. A study using axonal transport tech-
niques, J. Cornp. Neurol., 187 (1979) 373-400. 17 Martin, G.F., Cabana, T., ttumbertson, AA)., I,axson, L.C. and Panneton, W.M., Spinal projections from the medullary reticular formation of the North American opossum: evidence for connectional heterogeneity..l ('ornp. Neurol., 196 ( 1981 ) 663-682. 18 McCrady, E., The embryology of the opossum, Am. Anat. Memoirs, No. 16, The Wistar Institute of Embryology and Biology, Philadelphia, 1938. 19 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. C)'tochern., 26 (1978) 1(/6-117. 20 Prendergast, J. and Stelzner, D.J., Changes in the magnocellular portion of the red nucleus following thoracic hemisection in the neonatal and adult rat, J. Comp. Neurol., 166 (1976) 163-172. 21 Schreyer, D.J. and Jones, E.G., Growing corticospinal axons bypass lesions of neonatal rat spinal cord, Neuroscience, 9 (1983) 31-40. 22 Stelzner, D.J., Weber, E.P. and Prendergast, J., A comparison of the effect of mid-thoracic spinal hemisection in the neonatal and weanling rat on the distribution of dorsal root axons in the lumbosacral spinal cord of the adult, Brain Res., 172 (1979) 407-426. 23 Tolbert, D.L. and ti Der, T., Redirected growth ol7pyramidal tract axons tollowing neonatal pyramidotomy in cats, J. Comp. Neurol., 260 (1987) 299-311. 24 Weber, E.D. and Stelzner, D.J., Behavioral effects of spinal cord transection in the developing rat, Brain Res., 125 (1977) 241-255.
303
BRD 60255
Evidence for developmental plasticity of the rubrospinal tract. Studies using the North American opossum George F. Martin and Xiao Ming Xu Departmentof Anatomy, The Ohio State University, Collegeof Medicine, Columbus, OH 43210 (U.S.A.)
(Accepted 8 December 1987) Key words: Developmental plasticity; Opossum; Regeneration; Rubrospinal tract; Spinal cord
Using axonal tracing techniques we have shown that rubral axons are capable of growing around lesions of the rubrospinal tract during early stages of development in the North American opossum and that a critical period for such growth exists. The opossum was employed for study because it is born in a very immature state 12-13 days after conception and the entire development of its rubrospinal tract occurs postnatally.
It is generally assumed that the spinal cord is capable of greater plasticity during development than in the adult animal 1°~22'24. This principle is well illustrated by the plasticity of the pyramidal tract in newborn mammals demonstrated first by Kalil and Reh in the brainstem re, and subsequently by others in the spinal cord 2-4'21"23. When a lesion interrupts the developing pyramidal tract, cortical axons can grow around 3"4"12A3"23and possibly through 21 it to innervate generally appropriate areas more caudally. Such plasticity probably underlies the 'sparing' of tactile placing seen after spinal cord lesions which include the pyramidal tract in neonatal animals 3. In light of the results reported above, it seems paradoxical that rubral axons do not grow around or through lesions of the rubrospinal tract in neonatal rats 2° or cats 3'4. When the rubrospinal tract is transected at birth in those species, many axotomized neurons undergo retrograde degeneration (the socalled Gudden effect, see review by CowanS). This phenomenon also occurs in mature rubrospinal neurons ~'11'15'2°, but to a lesser degree. The rubrospinal tract of the rat and cat is relatively mature at birth, however, at least in comparison to the pyramidal tract which is still growing 3"4'21, and it may simply
have lost its potential for plasticity. If so, it should be possible to demonstrate rubrospinal plasticity at earlier stages of development. This hypothesis would be difficult to test in the rat or cat because rubrospinal development occurs prenatally 3,4,2°, but should be testable in the North American opossum because the entire development of its rubrospinal tract occurs after birth 5"7. Birth takes place 12-13 days after conception in the opossum 9'1s. In the initial experiments, the rubrospinal tract was lesioned at mid to caudal thoracic levels in 2 adult and 83 developing opossums. The developing animals were obtained from females captured in the wild or bred in captivity at The Ohio State University. The s n o u t - r u m p length (SRL) of each animal was measured by stretching it on a ruler and used to estimate its age, when unknown, from the growth curve of Cutts et al. 9. The adult animals were anesthetized by intraperitoneal injections of sodium pentobarbital (40 mg/kg) prior to surgical exposure of the appropriate level of the spinal cord. The lesion was made with a no. 11 surgical blade and, after closure of the surgical exposure, the animals were returned to the vivarium for recovery. Most of the developing animals, ranging in age from postnatal day (PD)12 to es-
Correspondence: G.F. Martin, Department of Anatomy, The Ohio State University, College of Medicine, 333 West 10th Avenue, Columbus, OH 43210, U.S,A.
0165-3806/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
3(14
B
Fig. 1. A: photograph of pouch-young opossums at EPD20. B: Nissl-stained section showing the lesion (arrow) made 30 days earlier in one of the animals shown in A. The intact ventral funiculus (VF) and lateral funiculus (LF) are indicated. C: neurons in the red nucleus (RN) labeled by an injection of WGA-HRP placed two segments caudal to the lesion shown in B. The arrows point to labeled neurons: direction indicators are given. The bar in B indicates 240l¢m. that in C equals 60 f~m. timated P D (EPD)75, were o p e r a t e d while still in the m o t h e r ' s pouch. The m o t h e r was anesthetized first (see above) and placed on her back. During anesthesia, the pouch sphincter relaxes, exposing the litter. The pouch-young, still attached to the nipple, were then anesthetized individually by h y p o t h e r m i a or M e t o f a n e inhalation, so that the desired level of the spinal cord could be exposed and the rubrospinal tract cut with a no, 11 surgical blade and microscissors. A f t e r the incision was closed, the o p e r a t e d animals were r e t u r n e d with their m o t h e r to the vivarium. All animals were m a i n t a i n e d for at least 1 month (30-42 days) before being subjected to a second surgery and injections of Fast blue (FB) or wheat-germ agglutinin conjugated to horseradish peroxidase ( W G A - H R P ) , 2 segments caudal to the lesion and ipsilateral to it. The objective was to label rubral neu-
rons whose axons had grown caudal to the lesion. The adult animals were anesthetized as described above and after surgical exposure of the spinal cord, they were placed in a spinal frame. The injections (0.2-0.25 ~ul of 2% FB or 5% W G A - H R P ) were made stereotaxically using a glass micropipette attached to a 1.0 ~1 H a m i l t o n syringe. A f t e r the injections were c o m p l e t e d and the exposure closed, the animals were returned to the vivarium for a 2 - 3 day survival. The developing animals were r e m o v e d from the pouch for the second surgery and anesthetized either as described previously, or by intraperitoneal injections of sodium p e n t o b a r b i t o l (40-60 mg/kg). A f t e r exposure of the spinal cord and stabilization of the vertebral column, the injections (0.15-0.20 #1 of 10% W G A - H R P ) were m a d e as in the adult animals. The developing animals were maintained for 1 - 2 clays postoperatively.
305 All of the animals referred to above were sacrificed by an overdose of anesthetic and perfused intracardiaUy with phosphate-buffered saline, followed either by a citrate buffer-formaldehyde solution (FB experiments) or cold 1% paraformaldehyde-l.25% glutaraldehyde-1% sucrose in phosphate buffer followed by 10% sucrose in the same buffer (WGAH R P experiments). The spinal cords and brains were removed and placed either in the fixative (FB experiments) or 30% sucrose phosphate buffer for refrigeration at 4 °C. Frozen sections of the lesion site, the injection site and the brain were cut at 40/~m in all cases. The sections from the FB cases were mounted immediately and coverslipped with Entellan (Merck) for viewing and photography with a Leitz (Orthoplan) fluorescence photomicroscope using the A cube of the Ploem illumination system. The sections from the W G A - H R P cases were processed for H R P using tetramethyl benzidine as the chromagen 19 and coverslipped using piccolyte. Every third section was stained with Neutral red. Examination and photography of the H R P processed sections was carried out with a Leitz Dialux photomicroscope using bright and darkfield optics. The position of rubral neurons labeled by either FB or H R P was recorded on drawings of the sections using an X-Y plotter attached to the microscope stage by potentiometers. A total of 18 pouch-young ranging from PD18 to EPD54, were also used for orthograde transport experiments. They were subjected to lesions of the rubrospinal tract as already described, but after approximately 30 days, they were anesthetized (see above) for stereotaxic injections (0.12-0.15/tl) of 10% W G A - H R P into the red nucleus contralateral to the lesion. After about 48 h, the animals were anesthetized again and perfused so that the spinal cord and brain could be removed, sectioned on a freezing microtome at 40/zm, and processed for H R P as described above. Orthogradely labeled axons were photographed and their positions plotted using a Leitz Dialux photomicroscope equipped with light and darkfield condensors. When rubrospinal lesions were made in animals ranging in age from EPD54 to maturity, no evidence for retrograde labeling was present in the contralateral red nucleus one month later. The rubrospinal tract of the opossum 7'14, like that of other species, is almost entirely crossed. In all cases, neurons were
EPD18(SRL39MM)
Fig. 2. Plot of the rubrospinal tract labeling (arrows) at the first thoracic (T1) and lower thoracic (Low Thor) levels, 5 mm above and below the lesion and at lumbar (Lumb) levels produced by the rubral injection (Inj) of WGA-HRP shown at the top. The lesion (section indicated as Low Thor) was made at EPD18 (39 mm SRL); the injection was made 30 days later.
306
Fig. 3. Photomicrographs substantiating the results plotted in Fig. 2. A: darkfield photomicrograph of W G A - t t R P labeled rubrospinal axons (arrow) rostral to the lesion shown in B. B: darkficld photomicrograph of the lesion, Labeled rubrospinal axons are indicated (arrow). C: darkfield photomicrograph of rubrospinal labeling (arrow) caudal to the lesion. The bar in A equals 2l)[)llm and can also be used for B and C.
307 labeled in those areas of the brainstem known to have axons in the spared white matter 16'17. In cases subjected to rubrospinal lesions between PD12 (30 m m SRL) and 43 (75 m m SRL), however, rubral neurons were labeled contralateral to the lesion (Fig. 1), suggesting that their axons had grown caudal to it. In general, the number of neurons labeled in the red nucleus contralateral to the lesion decreased with increasing age of the animal at the time of lesion. Some variation in the number of labeled neurons was present, however, even between cases subjected to lesions at the same age. Such variation is probably due to differences in the extent of the lesion and/or the size of the injection. When rubrospinal lesions were made between PD15 (36 m m SRL) and EPD33 (61 m m SRL), and the contralateral red nucleus was injected with W G A - H R P approximately a month later, rubrospinal axons were labeled caudal to the lesion. Fig. 2 shows a plot of the labeling obtained in a case with a particularly large lesion made at E P D 1 8 (39 mm SRL). Rostral to the lesion, rubrospinal axons were labeled in their normal position contralaterally (Figs. 2 and 3A), but at the lesion site (Figs. 2 and 3B), they were aggregated at the edge of the remaining cord. Caudal to the lesion, most of the labeled axons were again found contralateral to their origin, but they formed a tract in the dorsal funiculus (Figs. 2 and 3C), rather than within the lateral funiculus where they are found normally. In spite of their abnormal position, rubral axons appeared to innervate areas of the gray matter appropriate to them. Labeled axons could be traced to at least caudal lumbar segments of the cord. In other cases, the extent of the lesion was different, and the position of labeled axons caudal to the lesion varied accordingly. For example, when the lat-
eral funiculus was transected but the gray matter was spared, labeled axons formed a bundle at the lesion site which coursed through the lateral part of the gray matter. They maintained that position caudal to the lesion rather than returning to the lateral funiculus. As in the case described above, terminal labeling was present in areas appropriate for rubrospinal axons. There were not enough successful cases to make definitive statements concerning differences in the robustness of rubrospinal growth caudal to the lesion at different ages. The results of our study show that the rubrospinal tract is capable of plasticity at early stages of development and that a critical period for that plasticity exists. We know from the results of previous experiments 7 that some rubral axons reach thoracic levels of the cord prior to our earliest lesion and that rubral axons continue to grow into the thoracic cord, and beyond, well after that. It is impossible to determine, therefore, whether the rubrospinal plasticity described herein resulted from regeneration of axons cut by the lesion or the addition of new axons. It is possible, of course, that both occurred. It is our working hypothesis that all descending spinal pathways are capable of plasticity at some stage of development, but that the critical period for each is different. It is likely that such differences reflect differences in developmental history 5~6.
1 Barron, K.D., Dentinger, M.P., Nelson, L.R. and Scheibly, M.E., Incorporation of tritiated leucine by axotomized rubral neurons, Brain Res., 116 (1976) 251-266. 2 Bernstein, D.R. and Stelzner, D.I., Plasticity of the corticospinal tract following midthoracic spinal injury in the postnatal rat, J. Comp. Neurol., 221 (1983) 382-400. 3 Bregman, B.S. and Goldberger, M.E., Anatomical plasticity and sparing of function after spinal cord damage in neonatal cats, Science, 217 (1982) 553-555. 4 Bregman, B.S. and Goldberger, M.E., Infant lesion effect. III. Anatomical correlates of sparing and recovery of function after spinal cord damage in newborn and adult cats, Dev. Brain Res., 9 (1983) 137-154.
5 Cabana, T. and Martin, G.F., Developmental sequences in the origin of descending spinal pathways. Studies using retrograde transport techniques in the North American opossum (Didelphis virginiana), Dev. Brain Res., 15 (1984) 247-263. 6 Cabana, T. and Martin, G.F., Corticospinal development in the North American opossum: evidence for a sequence of growth of cortical axons in the spinal cord and for transient projections, Dev. Brain Res., 23 (1985) 69-80. 7 Cabana, T. and Martin, G.F., The development of the rubrospinal tract. An experimental study using the orthograde transport of WGA-HRP in the North American opossum, Dev. Brain Res., 30 (1986) 1-11.
The authors wish to thank Ms. Mary Ann Jarrell for excellent technical assistance and for typing the manuscript. We are also grateful to Mr. Karl Rubin for photographic help and to Drs. Michael Beattie and Albert Humbertson, Jr. for helpful suggestions. This investigation was supported by USPHS Grants BNS-8309245 and NS-25095.
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