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Spontaneous loss of axons in sympathetic unmyelinated nerve fibers of the rat during development
360
Brain Research, 54 (1973) 360-364 !~;, Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Spontaneous loss of axons in sympathetic unmyelinated nerve fibers of the rat during development
ALBERT J. AGUAYO, LEON C. TERRY AND GARTH M. BRAY
Division of Neurology, Department of Medicine, The Montreal General Hospital and McGill University, Montreal, Quebec (Canada) (Accepted February 1st, 1973)
Ultrastructural characteristics of neurogenesis have been documented for myelinated peripheral nerves3,4,6,8,13A4,20. The present study was undertaken to determine the ultrastructural features of axons and Schwann cells in unmyelinated nerve fibers during pre- and postnatal development. Rat cervical sympathetic trunks were studied because less than 1 ~ of axons in these nerves are myelinatedl, 5. Preliminary observations indicated that there were approximately twice as many axons in cervical sympathetic trunks from newborn rats as in the same nerves from adult animals 2. In the present report quantitative studies of axon and Schwann cell populations are described for rat cervical sympathetic trunks at various stages of development. One, 2, 3, 4, 5, 6, 10, 14 and 21 days after birth, and at 6 months of age, SpragueDawley rats were anesthetized with ether and systemically perfused with 3 ~ glutaraldehyde in 0.1 M phosphate buffer. For studies of prenatal animals, pregnant rats were anesthetized with ether on the nineteenth day after breeding (2 days pre-partum); the umbilical vessels of each fetus were perfused with fixative. Left carotid sheaths were dissected from the superior mediastinum to the base of the skull and processed for electron microscopy. Prior to polymerization in Epoxy resin, each carotid sheath was cut transversely at 2-3 mm intervals and embedded with an identifying label. Cervical sympathetic trunks were identified by phase microscopy and sectioned for electron microscopy. Montages of complete transverse sections at the midportion of each cervical sympathetic trunk were prepared from overlapping electron micrographs printed at a final magnification of × 10,000. For each nerve, diameters of 2000 unselected axons were determined with a Zeiss particle analyzer. Total numbers of axons, Schwann cell nuclei and Schwann cell units were counted from whole transverse-section montages. Schwann cell units were defined by the basal lamina which enclosed Schwann cell cytoplasm and axons, with or without Schwann cell nuclei at the same level. Cervical sympathetic trunks of newborn rats were composed of large bundles of minute axons enclosed by Schwann cells with elongated processes and a basal lamina.
361
SHORT COMMUNICATIONS 1200 SCHWANN CELL UNITS go0 600
~p,lllll
300
16000 12000 8000 4000 I I I l l l l l l
-2
~ 2
III
6
I I I I I I I I I I
10
14
II
.___J
21
MATURE
/
BIRTH
AGE(DAYS)
Fig. 1. Total numbers of Schwann cell units (top) and axons (bottom) in whole transverse sections of cervical sympathetic trunks from developing and mature rats.
The total number of axons per whole transverse section was approximately 16,000 two days before birth and decreased to 5470 six days after birth (Fig. 1). This striking reduction in the axonal population was associated with ultrastructural changes suggesting axonal breakdown. Surviving axons showed a steady increase in median axonal diameters (Fig. 2). The total number of Schwann cell units, as well as the number of Schwann cell nuclei per whole transverse section, progressively increased during the first 2 weeks after birth (Fig. 1). Schwann cell units with more than one nucleus were common prenatally and at birth, but declined during the first 6 days after birth
MEDIAN AXONAL DIAMETERS
.8 .7 ::t
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.5
N
,4 e...t
I l l , , l J , l , l l l J i J l l l l
-
~
2
6
10
14
,,l,
__.J
21
MATURE
BIRTH AGE (DAYS)
Fig. 2. Median axonal diameters in cervical sympathetic trunks of developing and mature rats. 2000 axons measured for each nerve.
362
SHORT COMMUNICATIONS
4000I. AXONS/SCHWANN CELL UNif 30g
100
• -2 ~, 2 BIRTH
6
10
14
21
MATURE
AGE
Fig. 3. Ratios of axons per Schwann cell in cervical sympathetic trunks of developing and mature rats.
and were not seen thereafter. Axon-Schwann cell ratios (Fig. 3), which were initially high, rapidly approached normal values as a result of axonal loss and the increase in the number of Schwann cells; concomitantly there was a rapid decrease in the crosssectional diameters of individual axon-Schwann cell complexes. Individual axons were not surrounded by Schwann cell processes at birth. After the first week, however, axons were usually totally enclosed by Schwann cell processes. Serial transverse sections of developing rat cervical sympathetic trunks demonstrated that cytoplasmic connections between consecutive Schwann cell complexes during the first 6 days of life were loose. Schwann cell processes were often transversely directed and lacked the longitudinal orientation and close interdigitation which characterizes the alignment of Schwann cell columns in adult animals. The results of this study indicate that normal development of unmyelinated nerve fibers in rat cervical sympathetic trunks involves a complex reorganization of axon-Schwann cell relationships. Beginning prenatally and continuing during the first 6 days after birth, there is a 4-fold reduction in the total number of axons, a gradual increase in the Schwann celt population, a progressive decline in the number of axons per axon-Schwann cell complex and a rapid increase in axonal diameters. Thus, it can no longer be assumed that axonal populations in the peripheral nervous system are static after birth or that the reduction in axon-Schwann cell ratios observed during development 4,14 is solely due to segregation of axons by increasing numbers of Schwann cells. However, it remains to be determined whether the spontaneous reduction in axons is due to the loss of entire neurons or just axonal branches. Cell death occurring during normal development was first described as part of a process called 'histogenetic differentiation'7. A loss of approximately 75 ~ of neurons occurs in the ventral horns of developing spinal cords in anuran tadpoles15; this is associated with a loss of ventral root axons 16. Spontaneous postnatal axonal degeneration has been demonstrated in the sciatic nerves of newborn ratslS,2L These observations suggest a balanced mechanism between factors responsible for axonal loss and those responsible for axonal preservation at this particular stage of neurogenesis.
SHORT COMMUNICATIONS
363
Peripheral factors influence the number of surviving neurons in the spinal cord; the effects of limb amputation and supernumerary limbs at a critical period of development have been documented9,15. Histogenetic differentiation is also influenced by thyroxine administration, which produces precocious cytodifferentiation17; axonal maturation is delayed by neonatal thyroidectomyag. After X-irradiation, there is an initial delay in cell differentiation which, however, is accelerated several weeks later 1°. Nerve growth factor (NGF) influences the development of sensory and sympathetic ganglia in newborn animals 12 and neonatal administration of NGF antiserum causes a marked reduction in axons and Schwann cells of rat cervical sympathetic trunks 1. Since the effect of NGF-antiserum is maximal during the first week of life H, this period of relative vulnerability may be related to the changes occurring in axons and Schwann cells at this time~. The technical assistance of Margaret Attiwell, Wendy Downey, Adrienne Liberman, Susan Pierer and Jane Trecarten is acknowledged with gratitude. This project was supported by grants from the Medical Research Council and Muscular Dystrophy Association of Canada. 1 AGUAYO, A., MARTIN, J. B., AND BRAY, G. M., Effects of nerve growth factor antiserum on peripheral unmyelinated nerve fibers, Acta neuropath. (BerL), 20 (1972) 288-298. 2 AGUAYO, A., TERRY, L. C., BRAY, G. M., AND MARTIN, J. B., Axonal loss in rat unmyelinated nerve fibers (UNF) during normal development and following administration of nerve growth factor antiserum (AS-NGF), Clin. Res., 20 (1972) 948. 3 ASBURY, A. K., Schwann cell proliferation in developing mouse sciatic nerve, J. Cell Biol., 34 (1967) 735-743. 4 CRAVIOTO, H., The role of Schwann cells on the development of human peripheral nerves. An electron microscope study, J. U#rastruct. Res., 12 (1965) 634-651. 5 DYCK, P. J., AND HOPKtNS, A. P., Electron microscopic observations on degeneration and regeneration of unmyelinated fibres, Brain, 95 (1972) 223-234. 6 FRIEDE,R. L., AND SAMORAJSKI,T., Myelin formation in the sciatic nerve of the rat, J. Neuropath. exp. Neurol., 27 (1968) 546-570. 7 GLUCKSMANN,A., Cell deaths in normal vertebrate ontogeny, Biol. Rev., 26 (1951) 59-86. 8 GUTRECHT,J. A., AND DYCK, P. J., Quantitative teased-fiber and histologic studies of human sural nerve during postnatal development, J. comp. Neurol., 138 (1970) 117-130. 9 HUGHES, A., An experimental study on the relationships between limb and spinal cord in the embryo of Eleutherodactylus martinicensis, J. Embryol. exp. Morph., 10 (1962) 575-601. 10 HUGHES,A. F., AND FOZZARD,J. A., The effect of irradiation on cell degeneration among developing neurones in Xenopus laevis, Brit. J. RadioL, 34 (1961) 302-307. 11 LEvI-MONTALCINI,R., The morphological effects of immunosympathetectomy. In G. STEINERAND E. SCH6NBAUM (Eds.), Immunosympathectomy, Elsevier Publishing Company, Amsterdam, 1972, pp. 55-78. 12 LEvI-MONTALCINI,R., ANGELETTI, R. H., AND ANGELETTI,P. U., The nerve growth factor. In G. H. BOURNE (Ed.), The Structure and Function of Nervous Tissue, VoL 5, Academic Press, New York, 1972, pp. 1-38. 13 OCHOA,J., The sural nerve of the human foetus: Electron microscope observations and counts of axons, J. Anat. (Lond.), 108 (1971) 231-245. 14 PETERS,A., AND MUIR, A. R., The relationship between axons and Schwann cells during development of peripheral nerves in the rat, Quart. J. exp. Physiol., 44 (1959) 117-130. 15 PRESTIGE, M. C., Differentiation, degeneration and the role of the periphery: Quantitative considerations. In F. O. SCHMITT(Ed.), The Neurosciences, Rockefeller Univ. Press, New York, 1970. pp. 73-82. 16 PRESTIGE, M. C., AND WILSON, M. A., Loss of axons from ventral roots during development, Brain Research, 41 (1972) 467-470.
364
SHORT COMMUNICATIONS
17 RACE, J., Thyroid hormone control of development of lateral motor column cells in the lumbosacral cord in hypophysectomized Rana pipiens, Gen. comp. Endocr., 1 (1961) 322-331. t8 REIER, P. J., AND HUGHES, A., Evidence for spontaneous axon degeneration during peripheral nerve maturation, Amer. J. Anat., 135 (1972) 147 152. 19 REIER, P. J., AND HUGHES, A., An effect of neonatal radio-thyroidectomy upon nonmyelinated axons and association Schwann cells during maturation of the mouse sciatic nerve, Brain Research, 41 (1972) 263-282. 20 WEBSTER, H. DEF., The geometry of peripheral myelin sheaths during their formation and growth in rat sciatic nerves, J. Cell Biol., 48 (1971) 348-367. 21 WEBSTER, H. DEF., AND O'CONNELL, M. F., Myelin formation in peripheral nerves. A morphological reappraisal and its neuropathological significance. In Proc. V1 Congrds International de Neuropathologie, Masson, Paris, 1970, pp. 579-588.
Brain Research, 54 (1973) 360-364 !~;, Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Spontaneous loss of axons in sympathetic unmyelinated nerve fibers of the rat during development
ALBERT J. AGUAYO, LEON C. TERRY AND GARTH M. BRAY
Division of Neurology, Department of Medicine, The Montreal General Hospital and McGill University, Montreal, Quebec (Canada) (Accepted February 1st, 1973)
Ultrastructural characteristics of neurogenesis have been documented for myelinated peripheral nerves3,4,6,8,13A4,20. The present study was undertaken to determine the ultrastructural features of axons and Schwann cells in unmyelinated nerve fibers during pre- and postnatal development. Rat cervical sympathetic trunks were studied because less than 1 ~ of axons in these nerves are myelinatedl, 5. Preliminary observations indicated that there were approximately twice as many axons in cervical sympathetic trunks from newborn rats as in the same nerves from adult animals 2. In the present report quantitative studies of axon and Schwann cell populations are described for rat cervical sympathetic trunks at various stages of development. One, 2, 3, 4, 5, 6, 10, 14 and 21 days after birth, and at 6 months of age, SpragueDawley rats were anesthetized with ether and systemically perfused with 3 ~ glutaraldehyde in 0.1 M phosphate buffer. For studies of prenatal animals, pregnant rats were anesthetized with ether on the nineteenth day after breeding (2 days pre-partum); the umbilical vessels of each fetus were perfused with fixative. Left carotid sheaths were dissected from the superior mediastinum to the base of the skull and processed for electron microscopy. Prior to polymerization in Epoxy resin, each carotid sheath was cut transversely at 2-3 mm intervals and embedded with an identifying label. Cervical sympathetic trunks were identified by phase microscopy and sectioned for electron microscopy. Montages of complete transverse sections at the midportion of each cervical sympathetic trunk were prepared from overlapping electron micrographs printed at a final magnification of × 10,000. For each nerve, diameters of 2000 unselected axons were determined with a Zeiss particle analyzer. Total numbers of axons, Schwann cell nuclei and Schwann cell units were counted from whole transverse-section montages. Schwann cell units were defined by the basal lamina which enclosed Schwann cell cytoplasm and axons, with or without Schwann cell nuclei at the same level. Cervical sympathetic trunks of newborn rats were composed of large bundles of minute axons enclosed by Schwann cells with elongated processes and a basal lamina.
361
SHORT COMMUNICATIONS 1200 SCHWANN CELL UNITS go0 600
~p,lllll
300
16000 12000 8000 4000 I I I l l l l l l
-2
~ 2
III
6
I I I I I I I I I I
10
14
II
.___J
21
MATURE
/
BIRTH
AGE(DAYS)
Fig. 1. Total numbers of Schwann cell units (top) and axons (bottom) in whole transverse sections of cervical sympathetic trunks from developing and mature rats.
The total number of axons per whole transverse section was approximately 16,000 two days before birth and decreased to 5470 six days after birth (Fig. 1). This striking reduction in the axonal population was associated with ultrastructural changes suggesting axonal breakdown. Surviving axons showed a steady increase in median axonal diameters (Fig. 2). The total number of Schwann cell units, as well as the number of Schwann cell nuclei per whole transverse section, progressively increased during the first 2 weeks after birth (Fig. 1). Schwann cell units with more than one nucleus were common prenatally and at birth, but declined during the first 6 days after birth
MEDIAN AXONAL DIAMETERS
.8 .7 ::t
t.tJ t---
.5
N
,4 e...t
I l l , , l J , l , l l l J i J l l l l
-
~
2
6
10
14
,,l,
__.J
21
MATURE
BIRTH AGE (DAYS)
Fig. 2. Median axonal diameters in cervical sympathetic trunks of developing and mature rats. 2000 axons measured for each nerve.
362
SHORT COMMUNICATIONS
4000I. AXONS/SCHWANN CELL UNif 30g
100
• -2 ~, 2 BIRTH
6
10
14
21
MATURE
AGE
Fig. 3. Ratios of axons per Schwann cell in cervical sympathetic trunks of developing and mature rats.
and were not seen thereafter. Axon-Schwann cell ratios (Fig. 3), which were initially high, rapidly approached normal values as a result of axonal loss and the increase in the number of Schwann cells; concomitantly there was a rapid decrease in the crosssectional diameters of individual axon-Schwann cell complexes. Individual axons were not surrounded by Schwann cell processes at birth. After the first week, however, axons were usually totally enclosed by Schwann cell processes. Serial transverse sections of developing rat cervical sympathetic trunks demonstrated that cytoplasmic connections between consecutive Schwann cell complexes during the first 6 days of life were loose. Schwann cell processes were often transversely directed and lacked the longitudinal orientation and close interdigitation which characterizes the alignment of Schwann cell columns in adult animals. The results of this study indicate that normal development of unmyelinated nerve fibers in rat cervical sympathetic trunks involves a complex reorganization of axon-Schwann cell relationships. Beginning prenatally and continuing during the first 6 days after birth, there is a 4-fold reduction in the total number of axons, a gradual increase in the Schwann celt population, a progressive decline in the number of axons per axon-Schwann cell complex and a rapid increase in axonal diameters. Thus, it can no longer be assumed that axonal populations in the peripheral nervous system are static after birth or that the reduction in axon-Schwann cell ratios observed during development 4,14 is solely due to segregation of axons by increasing numbers of Schwann cells. However, it remains to be determined whether the spontaneous reduction in axons is due to the loss of entire neurons or just axonal branches. Cell death occurring during normal development was first described as part of a process called 'histogenetic differentiation'7. A loss of approximately 75 ~ of neurons occurs in the ventral horns of developing spinal cords in anuran tadpoles15; this is associated with a loss of ventral root axons 16. Spontaneous postnatal axonal degeneration has been demonstrated in the sciatic nerves of newborn ratslS,2L These observations suggest a balanced mechanism between factors responsible for axonal loss and those responsible for axonal preservation at this particular stage of neurogenesis.
SHORT COMMUNICATIONS
363
Peripheral factors influence the number of surviving neurons in the spinal cord; the effects of limb amputation and supernumerary limbs at a critical period of development have been documented9,15. Histogenetic differentiation is also influenced by thyroxine administration, which produces precocious cytodifferentiation17; axonal maturation is delayed by neonatal thyroidectomyag. After X-irradiation, there is an initial delay in cell differentiation which, however, is accelerated several weeks later 1°. Nerve growth factor (NGF) influences the development of sensory and sympathetic ganglia in newborn animals 12 and neonatal administration of NGF antiserum causes a marked reduction in axons and Schwann cells of rat cervical sympathetic trunks 1. Since the effect of NGF-antiserum is maximal during the first week of life H, this period of relative vulnerability may be related to the changes occurring in axons and Schwann cells at this time~. The technical assistance of Margaret Attiwell, Wendy Downey, Adrienne Liberman, Susan Pierer and Jane Trecarten is acknowledged with gratitude. This project was supported by grants from the Medical Research Council and Muscular Dystrophy Association of Canada. 1 AGUAYO, A., MARTIN, J. B., AND BRAY, G. M., Effects of nerve growth factor antiserum on peripheral unmyelinated nerve fibers, Acta neuropath. (BerL), 20 (1972) 288-298. 2 AGUAYO, A., TERRY, L. C., BRAY, G. M., AND MARTIN, J. B., Axonal loss in rat unmyelinated nerve fibers (UNF) during normal development and following administration of nerve growth factor antiserum (AS-NGF), Clin. Res., 20 (1972) 948. 3 ASBURY, A. K., Schwann cell proliferation in developing mouse sciatic nerve, J. Cell Biol., 34 (1967) 735-743. 4 CRAVIOTO, H., The role of Schwann cells on the development of human peripheral nerves. An electron microscope study, J. U#rastruct. Res., 12 (1965) 634-651. 5 DYCK, P. J., AND HOPKtNS, A. P., Electron microscopic observations on degeneration and regeneration of unmyelinated fibres, Brain, 95 (1972) 223-234. 6 FRIEDE,R. L., AND SAMORAJSKI,T., Myelin formation in the sciatic nerve of the rat, J. Neuropath. exp. Neurol., 27 (1968) 546-570. 7 GLUCKSMANN,A., Cell deaths in normal vertebrate ontogeny, Biol. Rev., 26 (1951) 59-86. 8 GUTRECHT,J. A., AND DYCK, P. J., Quantitative teased-fiber and histologic studies of human sural nerve during postnatal development, J. comp. Neurol., 138 (1970) 117-130. 9 HUGHES, A., An experimental study on the relationships between limb and spinal cord in the embryo of Eleutherodactylus martinicensis, J. Embryol. exp. Morph., 10 (1962) 575-601. 10 HUGHES,A. F., AND FOZZARD,J. A., The effect of irradiation on cell degeneration among developing neurones in Xenopus laevis, Brit. J. RadioL, 34 (1961) 302-307. 11 LEvI-MONTALCINI,R., The morphological effects of immunosympathetectomy. In G. STEINERAND E. SCH6NBAUM (Eds.), Immunosympathectomy, Elsevier Publishing Company, Amsterdam, 1972, pp. 55-78. 12 LEvI-MONTALCINI,R., ANGELETTI, R. H., AND ANGELETTI,P. U., The nerve growth factor. In G. H. BOURNE (Ed.), The Structure and Function of Nervous Tissue, VoL 5, Academic Press, New York, 1972, pp. 1-38. 13 OCHOA,J., The sural nerve of the human foetus: Electron microscope observations and counts of axons, J. Anat. (Lond.), 108 (1971) 231-245. 14 PETERS,A., AND MUIR, A. R., The relationship between axons and Schwann cells during development of peripheral nerves in the rat, Quart. J. exp. Physiol., 44 (1959) 117-130. 15 PRESTIGE, M. C., Differentiation, degeneration and the role of the periphery: Quantitative considerations. In F. O. SCHMITT(Ed.), The Neurosciences, Rockefeller Univ. Press, New York, 1970. pp. 73-82. 16 PRESTIGE, M. C., AND WILSON, M. A., Loss of axons from ventral roots during development, Brain Research, 41 (1972) 467-470.
364
SHORT COMMUNICATIONS
17 RACE, J., Thyroid hormone control of development of lateral motor column cells in the lumbosacral cord in hypophysectomized Rana pipiens, Gen. comp. Endocr., 1 (1961) 322-331. t8 REIER, P. J., AND HUGHES, A., Evidence for spontaneous axon degeneration during peripheral nerve maturation, Amer. J. Anat., 135 (1972) 147 152. 19 REIER, P. J., AND HUGHES, A., An effect of neonatal radio-thyroidectomy upon nonmyelinated axons and association Schwann cells during maturation of the mouse sciatic nerve, Brain Research, 41 (1972) 263-282. 20 WEBSTER, H. DEF., The geometry of peripheral myelin sheaths during their formation and growth in rat sciatic nerves, J. Cell Biol., 48 (1971) 348-367. 21 WEBSTER, H. DEF., AND O'CONNELL, M. F., Myelin formation in peripheral nerves. A morphological reappraisal and its neuropathological significance. In Proc. V1 Congrds International de Neuropathologie, Masson, Paris, 1970, pp. 579-588.