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Axonal sprouting at the neuromuscular junction of adult and aged rats
EXPERIMENTAL
NEUROLOGY
74847-854
(1981)
Axonal Sprouting at the Neuromuscular Adult and Aged Rats GRAHAM
E. FAGG, STEPHEN
Junction
of
W. SCHEFF, AND CARL W. COTMAN'
Department of Psychobiology, University of California, Irvine, California 92717 Received June 9, 1981 We compared the extent of the repair response (terminal and nodal sprouting) that followed motor nerve damage in young (3 months old) and aged rats (27 months old). Two weeks after unilateral partial denervation of the soleus muscle (transection of the L4 spinal root), muscles were removed and stained using the zinc iodide-osmium tetroxide procedure. Sprouting was assessed by determining (a) the percentage of end-plates with terminal sprouts, (b) the percentage of preterminal axons with nodal sprouts, and the lengths of (c) the end-plate and (d) terminal sprouts. Relative to control (unoperated) animals, young adults showed significant increases in all four parameters after partial denervation. Aged animals exhibited evidence of considerable sprouting prior to any surgery: end-plates were long and complex, greater than 50% of preterminal axons had nodal sprouts, there was a high degree of terminal sprouting, and terminal sprouts were longer than in young animals. Partial denervation in the aged animals significantly increased only end-plate length and the degree of terminal sprouting. In both age groups, there were no differences between soleus muscles contralateral to the transection and those from control animals. The decreased response to partial denervation in aged animals may possibly be attributed to a longer latency or higher threshold for repair than in adults, or to the existence of an upper limit for axonal growth which has already been attained in the older age group.
INTRODUCTION Synapse replacement, whether in response to neural damage or as part of an ongoing process of natural turnover, occurs throughout the adult ’ We should like to thank Drs. S. F. Hoff, E. W. Harris, and M. Nieto-Sampedro for valuable discussions, and Ms. S. Bathgate for help in preparation of the manuscript. This work was supported by grant AGO0538 from the National Institute on Aging. Dr. Scheffs present address is Department of Anatomy, Albert B. Chandler Medical Center, University of Kentucky, Lexington, KY 40536. 847 0014-4886/81/120847-08SO2.00/0 Copyri&t All li&tc
8 1981 by Academic Pmm, Inc. of I-cpmductioll in my form TcltKd.
848
FAGG, SCHEFF, AND COTMAN
nervous system [for reviews, see (7, 9, 22)]. In the periphery, partial denervation of a skeletal muscle is followed within a few days by the growth of fine processes (sprouts) from the remaining intramuscular nerves, and these eventually reinnervate the denervated muscle fibers (2, 7, 10, 16). The sprouts may be derived either (a) from the end-plate or unmyelinated preterminal segment of the axon (terminal sprouting) or (b) from nodes of Ranvier (nodal or collateral sprouting). Recent studies suggest that different stimuli are required to elicit these two responses [see (7 and 9)]. One outstanding question is the relationship of the sprouting response to the age of the animal. Barker and Ip (2) suggested that motor neuron terminals may undergo cyclic renewal throughout life, and the observation that end-plates are morphologically more complex in aging animals than in adults (23) provided additional support for this proposal. More recently, Pestronk et al. (17) found that motor neuron terminal sprouting induced by botulinurn toxin decreased with the age of the animal, and a limited sprouting response was observed also in the central nervous system of aged rats [( 15, 20, 21); Hoff, Scheff, and Cotman, unpublished observations]. In the present study, we examined the sprouting response to partial denervation of the soleus muscle in young adult and aged rats. Our data indicate that aged animals exhibit a more limited capacity than adults to respond to nerve injury with compensatory axonal growth, although a high degree of sprouting is already present in the aged group prior to any surgical procedure. METHODS Surgical Procedures. Experiments were conducted using male SpragueDawley rats 3 months (young adult) or 27 months old (aged); care was taken, particularly in the case of the older age group, to ensure that all animals were of apparently normal limb function prior to any operative procedures. The animals were anesthetized with sodium pentobarbital (Nembutal; 45 mg/kg, i.p.), and a skin incision was made 1 cm to the left of the dorsal midline, extending over the lumbar vertebral segments. Spinal nerve L4 was exposed by blunt dissection and sectioned just distal to the intervertebral foramen. The wound was sutured and 45,000 Units of Bicillin (Wyeth) was administered subcutaneously. Histology. Two weeks postoperatively, the animals were deeply anesthetized and the soleus muscles both ipsilateral and contralateral to the nerve transection were removed and stained using the zinc iodide-osmium tetroxide procedure ( 1) exactly as described by Betz et al. (3). Bundles of teased muscle fibers were mounted in glycerol on microscope slides and examined under bright-field illumination with the aid of a camera lucida.
MOTOR
NERVE
SPROUTING
AND
AGING
849
Soleus muscles from control (unoperated) animals were stained and examined in the same manner. “Sprouting” was quantitatively assessed by determining: (a) the length of the end-plate in the longitudinal direction of the muscle fiber (4, 17); (b) the percentage of nerve endings bearing sprouts (3, 4) [ultraterminal and preterminal outgrowths (2) were classified together as terminal sprouts (5)]; (c) the length of these terminal sprouts (3,4); and (d) the percentage of myelinated terminal axons bearing unmyelinated nodal sprouts. Nodal sprouting was assessed only in the terminal portions of axons (generally five to eight myelin nodes from the end-plate) where nerve fibers no longer traveled in bundles and could be clearly distinguished from each other. An average of 41 end-plates (20 to 102) was examined in each muscle. RESULTS Young Adult Rats. Soleus muscles from control adult animals had simple, ovoid end-plates with an average length of 43 pm (Fig. la and Table 1). End-plates were generally formed from one or two branches of the unmyelinated preterminal axon, with an occasional contribution from a sprout arising from the first node of Ranvier. Quantitatively, the number of terminal axons with either nodal sprouts or terminal sprouts was small (7 and 61, respectively), and the average length of those terminal sprouts present was 23 pm (Table 1). Partial denervation resulted in a significant sprouting response as judged by all indices used (Fig. lb and Table 1). The greatest increase from control values (3.8-fold, P < 0.01) was shown by the percentage of end-plates with terminal sprouts, with smaller changes in other parameters (end-plate length, 1.2-fold; terminal sprout length, 1.7-fold; nodal sprouting, 2.6fold-Table 1). The degree of terminal sprouting observed in these experiments was less than was reported previously for the mouse soleus muscle (5), probably reflecting different innervation patterns in the two species. [Weiss and Edds (24) reported that L4 contributes about 20% of fibers in the soleus nerve of the white rat.] Recently, Rotshenker and co-workers (18, 19) showed that unilateral denervation of the frog cutaneous pectoris muscle results in axonal sprouting in the contralateral muscle, and we therefore compared control and contralateral soleus muscles in the present studies. However, there were no significant differences (P > 0.05) in this regard for any parameter studied, although the mean end-plate length contralaterally did appear to be somewhat greater than in controls (Table 1). These data are in accord with the recent study by Brown et al. (6) in the mouse, and provide further evidence that transport of a “sprouting signal” from damaged to undam-
850
FAGG, SCHEFF, AND COTMAN
a
b
FIG. 1. Camera lucida drawing of zinc iodide-osmium tetroxide-stained end-plates and preterminal axons in the soleus muscles of (a), (b) young adult and (c), (d) aged rats. (a) and (c) illustrate end-plates from control animals; (c) shows an end-plate with two components, one arising from a nodal sprout. (b) and (d) show end-plates from animals with L4 transections; the end-plates are longer than in control animals and exhibit fairly extensive terminal sprouting. The scale bar (SO pm) is drawn in the longitudinal direction of the muscle fibers.
aged motor neurons is probably not a major factor influencing axonal sprouting in rodents. Aged Ruts. End-plates in the soleus muscles of aged rats were significantly larger ( 1.5fold, P < 0.01 -Fig. lc and Table 1) than those in young adults. Moreover, the end-plates were of a more complex nature than those in the younger age group, frequently consisting of two or three components arising from the terminal axon and from one or two collateral sprouts (Fig. lc); quantitatively, 55% of terminal axons possessed nodal sprouts (compared with 7% in young adults,P < O.Ol-Table 1). Similar observations
MOTOR
NERVE SPROUTING
851
AND AGING
were made both in aged cats (23) and in 18-month-old rats (17) although Pestronk et al. (17) found that 28-month-old Wistar rats had relatively small and noncomplex end-plates. The present data also revealed significant (P < 0.01) increases in both the degree of terminal sprouting (3.2-fold) and terminal sprout length (2.2-fold) in aged compared with young rats. Hence, by all parameters examined, the motor nerve terminals in the soleus muscles of aged rats appear to have undergone a substantial amount of sprouting prior to any operative procedures. In contrast to the generalized response seen in young adult rats, partial denervation of the soleus muscles in aged rats induced changes in only the end-plate length (P < 0.01) and the degree of terminal sprouting (P TABLE
1
Motor Nerve Sprouting in Control and Partially Denervated Soleus Muscles from Young Adult and Aged Rats Experimental group” Control
Partially denervated
Contralateral
Paramete@
Young adult
Aged
EPL (pm) WTS TSL Gm) %NS
43 f 1 (6) 6+2 23 + 2 7+2
65 k 4* (6) 19k 2* 51+ 7’ 55 + 10*
EPL (rm) WTS TSL (t.4 %NS
50 23 38 18
82 f 34 f 42+ 48+
EPL (pm) ITS TSL bm) INS
51 + 4 (5) 5+2 25 f 9 5+2
+ k f +
l**(5) 2** 2** 3**
3** (4) 5*** 6 6
76 + 1 14+ 5 49 + 10 48k 4
(4)
“Three groups of muscles were examined: controls-from unoperated animals; partially denervated-ipsilateral to a transection of the L4 spinal nerve root; contralateral-contralateral to an L4 transection. ’ Sprouting was assessed by determining: end-plate length (EPL), the percentage of endplates with terminal sprouts (%TS), terminal sprout length (TSL), and the percentage of preterminal axons with nodal sprouts (%NS). Values are means + SE. The number of muscles in each group is indicated in parentheses. Statistical significance was assessed using Student’s t test. * P < 0.01 for comparisons between young adult controls and aged controls. ** P < 0.01 and *** P < 0.05 for comparisons between controls of each age and the corresponding partially denervated or contralateral group. Unmarked comparisons were not significantly different (P > 0.05).
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FAGG, SCHEFF, AND COTMAN
< 0.05-see Fig. Id and Table 1). There was no effect on either the average length of terminal sprouts nor on the degree of nodal sprouting. Moreover, the percentage of end-plates with terminal sprouts was only 1.8 times the control value, compared with a 3.8-fold increase in the young adult age group. In agreement with the observations in the younger animals, partial denervation did not appear to affect the degree of axonal sprouting in the contralateral soleus muscle (Table 1). DISCUSSION The present study was undertaken to determine the extent of the repair response that followed motor nerve damage in young adult and aged animals. Our data indicate that aged rats exhibit a more limited ability than their young adult counterparts to respond to nerve injury with compensatory axonal growth. This reduced capacity is apparent as (a) an absence of any change in nodal sprouting, (b) a smaller increase in terminal sprouting than in adult animals, and (c) no alteration in the average length of terminal sprouts. However, both age groups respond to partial denervation with a similar increase in end-plate length. Although the present study indicates that aged animals have a reduced capacity for repair, one should be cautious at this stage of concluding that this is due to an inability to sustain neuronal growth. In fact, the total extent of sprouting (74%-terminal plus nodal) in control aged rats was almost 6-fold higher than in control young adults and nearly twice as great as in the young adult experimental group. However, this value increased only slightly (to 82%) after partial denervation. Hence, one explanation for the reduced response in aged animals is that there may be a maximum limit to the amount of sprout growth for each motor neuron (perhaps determined by the synthetic capacity of the cell) and a response is possible only if that limit has not been attained; in aged animals, natural processes of degeneration ( 1 I- 13) have already served to induce a high degree of intramuscular nerve sprouting, and additional neuronal growth cannot be supported. Alternatively, the latency of axonal sprouting may be greater in aged than in younger animals. This may account for the larger increase in end-plate length observed in the present study (14 days postlesion) compared with that seen by Pestronk et al. [7 days after botulinurn treatment (17)]. Moreover, recent results from this laboratory indicate that, after a unilateral lesion of the entorhinal cortex, reinnervation of the molecular layer in the dentate gyrus proceeds at essentially the same rate in both adult and aged rats, but that there is a delayed onset in the older animals (25). In this system, the growth response appears to depend on the rate
MOTOR
NERVE SPROUTING
AND AGING
853
at which degeneration products are cleared from the area (26), although decreased rates of protein synthesis and axonal transport in old animals may also play a role in the motoneuron-skeletal muscle system (14). Interestingly, the denervation-induced sprouting observed in the aged rats was only of the terminal type. Current evidence indicates that terminal sprouting is produced by treatments which simply render the target muscle inactive (such as botulinum toxin and tetrodotoxin), whereas nodal sprouting appears either to require an additional stimulus or to have a different threshold (7, 9). Selective synapse stabilization is thought to be involved in the development and maturation of the nervous system (8) and this type of mechanism has been invoked recently to explain the low lesion-induced synaptic turnover in the central nervous system of aged rats (15). Thus, another possibility to account for thi present data would be the existence of a higher threshold for sprouting (or greater degree of stabilization) in aged than in adult animals. In summary, partial denervation of a skeletal muscle in aged rats was shown to induce a smaller repair response (axonal and terminal sprouting) than in young adults. This does not appear to be due to an inability of aged motor neurons to sustain neuronal growth, since there was a high degree of sprouting in aged control animals. The diminished response in aged animals may be due to a raised threshold or longer latency for sprouting, or to the existence of an upper limit to axonal growth for each motor neuron. REFERENCES 1. AKERT, K., AND C. SANDRI. 1968. An elcctronmicroscopic study of zinc iodide-osmium impregnation of neurons. I. Staining of synaptic vesicles at cholinergic junctions. Brain Res. 7: 286-295. 2. BARKER, D., AND M. C. IP. 1966. Sprouting and degeneration of mammalian motor axons in normal and dc-affercntated skeletal muscle. Proc. R Sot. (BioZ) 153: 538-554. 3. BETZ. W. J., J. H. CALDWELL, AND R. R. RIBCHESTTJR.1980. Sprouting of active nerve terminals in partially inactive muscles of the rat. J. Physiol. (London) 303 281-297. 4. BROWN, M. C., AND R. IRONTON. 1977. Motor neurone sprouting induced by prolonged tetrodotoxin block of nerve action potentials. Nature (London) 265: 459-461. 5. BROWN, M. C., AND R. L. HOLLAND. 1979. A central role for denervated tissues in causing nerve sprouting. Nature (London) 282: 724-726. 6. BROWN, M. C., R. L. HOLLAND, AND R. IRONTON. 1980. Nodal and terminal sprouting from motor nerves in fast and slow muscles of the mouse. J. Physiol. (London) 306: 493-510. 7. BROWN. M. C., R. L. HOLLAND, AND W. G. HOPKINS. 1981. Motor nerve sprouting. Annu. Rev. Neurosci. 4: 17-42. 8. CHANGEUX. J.-P., AND A. DANCHIN. 1976. Selective stabilisation of developing synapses as a mechanism for the specification of neuronal networks. Nature (London) 264: 705712.
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9. COTMAN, C. W., M. NIETO-SAMPEDRO, AND E. W. HARRIS. 198 1. Synapse replacement in the adult nervous system of vertebrates. Physiol. Rev. 61: 684-784. 10. EDDS, M. V. 1950. Collateral regeneration of residual motor axons in partially denervated muscles. J. Exp. Zool. 113: 517-552. 11. FUJISAWA, K. 1976. Some observations on the skeletal musculature of aged rats. III. Abnormalities of terminal axons found in motor end-plates. Exp. Geront. 11: 43-47. 12. GUTMANN, E., AND V. HANZLIKOVA. 1965. Age changes of motor endplates in muscle fibres of the rat. Gerontologia 1 I: 12-24. 13. GUTMANN, E., AND V. HANZLIKOVA. 1966. Motor unit in old age. Nature (London) 209: 921-922. 14. GUTMANN, E., V. HANZLIKOVA, AND B. JAKOUBEK. 1968. Changes in the neuromuscular system during old age. Exp. Geronr. 3: 141-146. 15. HOFF, S. F., S. W. SCHEFF, A. Y. KWAN, AND C. W. COTMAN. 1981. A new type of lesion-induced synaptogenesis. II. The effects of aging on synaptic turnover in nondenervated zones. Brain Res., 222: 15-29. 16. HOFFMAN, H. 1950. Local reinnervation in partially denervated muscle: a histophysiological study. Avst. J. Exp. Biol. Med. Sci. 28: 383-397. 17. PESTRONK, A., D. B. DRACHMAN, AND J. W. GRIFFIN. 1980. Effects of aging on nerve sprouting and regeneration. Exp. Neural. 70: 65-82. 18. REICHART, F., AND S. ROTSHENKER. 1979. Motor axon terminal sprouting in intact muscles. Brain Rex 170: 187-189. 19. ROTSHENKER, S., AND U. J. MCMAHAN. 1976. Altered patterns of innervation in frog muscle after denervation. J. Neurocyfol. 5: 719-730. 20. SCHEFF, S. W., L. S. BENARDO, AND C. W. COTMAN. 1978. Decrease in adrenergic axon sprouting in the senescent rat. Science 202: 775-778. 21. SCHEFF, S. W., L. S. BENARDO, AND C. W. COTMAN. 1980. Decline in reactive fiber growth in the dentate gyrus of aged rats compared to young adult rats following entorhinal cortex removal. Brain Res. 199: 21-38. 22. TSUKAHARA, N. 198 1. Synaptic plasticity in the mammalian central nervous system. Annu. Rev. Neurosci. 4: 351-379. 23. TUFFERY, A. R. 1971. Growth and degeneration of motor end-plates in normal cat hindlimb muscles. J. Anat. 110: 221-247. 24. WEISS, P., AND M. V. EDDS. 1946. Spontaneous recovery of muscle following partial denervation. Am. J. Physiol. 145: 587-607. 25. HOFF, S. F., S. W. SCHEFF, L. S. BENARDO, AND C. W. COTMAN. 1981. Lesion-induced synaptogenesis in the dentate gyrus of aged rats. I. Loss and reacquisition of normal synaptic density. J. Comp. Neural.. in press. 26. HOFF, S. F., S. W. SCHEFF, AND C. W. COTMAN. 1981. Lesion-induced synaptogenesis in the dentate gyrus of aged rats. II. Demonstration of an impaired degeneration clearing response. J. Comp. Neurol.. in press.
NEUROLOGY
74847-854
(1981)
Axonal Sprouting at the Neuromuscular Adult and Aged Rats GRAHAM
E. FAGG, STEPHEN
Junction
of
W. SCHEFF, AND CARL W. COTMAN'
Department of Psychobiology, University of California, Irvine, California 92717 Received June 9, 1981 We compared the extent of the repair response (terminal and nodal sprouting) that followed motor nerve damage in young (3 months old) and aged rats (27 months old). Two weeks after unilateral partial denervation of the soleus muscle (transection of the L4 spinal root), muscles were removed and stained using the zinc iodide-osmium tetroxide procedure. Sprouting was assessed by determining (a) the percentage of end-plates with terminal sprouts, (b) the percentage of preterminal axons with nodal sprouts, and the lengths of (c) the end-plate and (d) terminal sprouts. Relative to control (unoperated) animals, young adults showed significant increases in all four parameters after partial denervation. Aged animals exhibited evidence of considerable sprouting prior to any surgery: end-plates were long and complex, greater than 50% of preterminal axons had nodal sprouts, there was a high degree of terminal sprouting, and terminal sprouts were longer than in young animals. Partial denervation in the aged animals significantly increased only end-plate length and the degree of terminal sprouting. In both age groups, there were no differences between soleus muscles contralateral to the transection and those from control animals. The decreased response to partial denervation in aged animals may possibly be attributed to a longer latency or higher threshold for repair than in adults, or to the existence of an upper limit for axonal growth which has already been attained in the older age group.
INTRODUCTION Synapse replacement, whether in response to neural damage or as part of an ongoing process of natural turnover, occurs throughout the adult ’ We should like to thank Drs. S. F. Hoff, E. W. Harris, and M. Nieto-Sampedro for valuable discussions, and Ms. S. Bathgate for help in preparation of the manuscript. This work was supported by grant AGO0538 from the National Institute on Aging. Dr. Scheffs present address is Department of Anatomy, Albert B. Chandler Medical Center, University of Kentucky, Lexington, KY 40536. 847 0014-4886/81/120847-08SO2.00/0 Copyri&t All li&tc
8 1981 by Academic Pmm, Inc. of I-cpmductioll in my form TcltKd.
848
FAGG, SCHEFF, AND COTMAN
nervous system [for reviews, see (7, 9, 22)]. In the periphery, partial denervation of a skeletal muscle is followed within a few days by the growth of fine processes (sprouts) from the remaining intramuscular nerves, and these eventually reinnervate the denervated muscle fibers (2, 7, 10, 16). The sprouts may be derived either (a) from the end-plate or unmyelinated preterminal segment of the axon (terminal sprouting) or (b) from nodes of Ranvier (nodal or collateral sprouting). Recent studies suggest that different stimuli are required to elicit these two responses [see (7 and 9)]. One outstanding question is the relationship of the sprouting response to the age of the animal. Barker and Ip (2) suggested that motor neuron terminals may undergo cyclic renewal throughout life, and the observation that end-plates are morphologically more complex in aging animals than in adults (23) provided additional support for this proposal. More recently, Pestronk et al. (17) found that motor neuron terminal sprouting induced by botulinurn toxin decreased with the age of the animal, and a limited sprouting response was observed also in the central nervous system of aged rats [( 15, 20, 21); Hoff, Scheff, and Cotman, unpublished observations]. In the present study, we examined the sprouting response to partial denervation of the soleus muscle in young adult and aged rats. Our data indicate that aged animals exhibit a more limited capacity than adults to respond to nerve injury with compensatory axonal growth, although a high degree of sprouting is already present in the aged group prior to any surgical procedure. METHODS Surgical Procedures. Experiments were conducted using male SpragueDawley rats 3 months (young adult) or 27 months old (aged); care was taken, particularly in the case of the older age group, to ensure that all animals were of apparently normal limb function prior to any operative procedures. The animals were anesthetized with sodium pentobarbital (Nembutal; 45 mg/kg, i.p.), and a skin incision was made 1 cm to the left of the dorsal midline, extending over the lumbar vertebral segments. Spinal nerve L4 was exposed by blunt dissection and sectioned just distal to the intervertebral foramen. The wound was sutured and 45,000 Units of Bicillin (Wyeth) was administered subcutaneously. Histology. Two weeks postoperatively, the animals were deeply anesthetized and the soleus muscles both ipsilateral and contralateral to the nerve transection were removed and stained using the zinc iodide-osmium tetroxide procedure ( 1) exactly as described by Betz et al. (3). Bundles of teased muscle fibers were mounted in glycerol on microscope slides and examined under bright-field illumination with the aid of a camera lucida.
MOTOR
NERVE
SPROUTING
AND
AGING
849
Soleus muscles from control (unoperated) animals were stained and examined in the same manner. “Sprouting” was quantitatively assessed by determining: (a) the length of the end-plate in the longitudinal direction of the muscle fiber (4, 17); (b) the percentage of nerve endings bearing sprouts (3, 4) [ultraterminal and preterminal outgrowths (2) were classified together as terminal sprouts (5)]; (c) the length of these terminal sprouts (3,4); and (d) the percentage of myelinated terminal axons bearing unmyelinated nodal sprouts. Nodal sprouting was assessed only in the terminal portions of axons (generally five to eight myelin nodes from the end-plate) where nerve fibers no longer traveled in bundles and could be clearly distinguished from each other. An average of 41 end-plates (20 to 102) was examined in each muscle. RESULTS Young Adult Rats. Soleus muscles from control adult animals had simple, ovoid end-plates with an average length of 43 pm (Fig. la and Table 1). End-plates were generally formed from one or two branches of the unmyelinated preterminal axon, with an occasional contribution from a sprout arising from the first node of Ranvier. Quantitatively, the number of terminal axons with either nodal sprouts or terminal sprouts was small (7 and 61, respectively), and the average length of those terminal sprouts present was 23 pm (Table 1). Partial denervation resulted in a significant sprouting response as judged by all indices used (Fig. lb and Table 1). The greatest increase from control values (3.8-fold, P < 0.01) was shown by the percentage of end-plates with terminal sprouts, with smaller changes in other parameters (end-plate length, 1.2-fold; terminal sprout length, 1.7-fold; nodal sprouting, 2.6fold-Table 1). The degree of terminal sprouting observed in these experiments was less than was reported previously for the mouse soleus muscle (5), probably reflecting different innervation patterns in the two species. [Weiss and Edds (24) reported that L4 contributes about 20% of fibers in the soleus nerve of the white rat.] Recently, Rotshenker and co-workers (18, 19) showed that unilateral denervation of the frog cutaneous pectoris muscle results in axonal sprouting in the contralateral muscle, and we therefore compared control and contralateral soleus muscles in the present studies. However, there were no significant differences (P > 0.05) in this regard for any parameter studied, although the mean end-plate length contralaterally did appear to be somewhat greater than in controls (Table 1). These data are in accord with the recent study by Brown et al. (6) in the mouse, and provide further evidence that transport of a “sprouting signal” from damaged to undam-
850
FAGG, SCHEFF, AND COTMAN
a
b
FIG. 1. Camera lucida drawing of zinc iodide-osmium tetroxide-stained end-plates and preterminal axons in the soleus muscles of (a), (b) young adult and (c), (d) aged rats. (a) and (c) illustrate end-plates from control animals; (c) shows an end-plate with two components, one arising from a nodal sprout. (b) and (d) show end-plates from animals with L4 transections; the end-plates are longer than in control animals and exhibit fairly extensive terminal sprouting. The scale bar (SO pm) is drawn in the longitudinal direction of the muscle fibers.
aged motor neurons is probably not a major factor influencing axonal sprouting in rodents. Aged Ruts. End-plates in the soleus muscles of aged rats were significantly larger ( 1.5fold, P < 0.01 -Fig. lc and Table 1) than those in young adults. Moreover, the end-plates were of a more complex nature than those in the younger age group, frequently consisting of two or three components arising from the terminal axon and from one or two collateral sprouts (Fig. lc); quantitatively, 55% of terminal axons possessed nodal sprouts (compared with 7% in young adults,P < O.Ol-Table 1). Similar observations
MOTOR
NERVE SPROUTING
851
AND AGING
were made both in aged cats (23) and in 18-month-old rats (17) although Pestronk et al. (17) found that 28-month-old Wistar rats had relatively small and noncomplex end-plates. The present data also revealed significant (P < 0.01) increases in both the degree of terminal sprouting (3.2-fold) and terminal sprout length (2.2-fold) in aged compared with young rats. Hence, by all parameters examined, the motor nerve terminals in the soleus muscles of aged rats appear to have undergone a substantial amount of sprouting prior to any operative procedures. In contrast to the generalized response seen in young adult rats, partial denervation of the soleus muscles in aged rats induced changes in only the end-plate length (P < 0.01) and the degree of terminal sprouting (P TABLE
1
Motor Nerve Sprouting in Control and Partially Denervated Soleus Muscles from Young Adult and Aged Rats Experimental group” Control
Partially denervated
Contralateral
Paramete@
Young adult
Aged
EPL (pm) WTS TSL Gm) %NS
43 f 1 (6) 6+2 23 + 2 7+2
65 k 4* (6) 19k 2* 51+ 7’ 55 + 10*
EPL (rm) WTS TSL (t.4 %NS
50 23 38 18
82 f 34 f 42+ 48+
EPL (pm) ITS TSL bm) INS
51 + 4 (5) 5+2 25 f 9 5+2
+ k f +
l**(5) 2** 2** 3**
3** (4) 5*** 6 6
76 + 1 14+ 5 49 + 10 48k 4
(4)
“Three groups of muscles were examined: controls-from unoperated animals; partially denervated-ipsilateral to a transection of the L4 spinal nerve root; contralateral-contralateral to an L4 transection. ’ Sprouting was assessed by determining: end-plate length (EPL), the percentage of endplates with terminal sprouts (%TS), terminal sprout length (TSL), and the percentage of preterminal axons with nodal sprouts (%NS). Values are means + SE. The number of muscles in each group is indicated in parentheses. Statistical significance was assessed using Student’s t test. * P < 0.01 for comparisons between young adult controls and aged controls. ** P < 0.01 and *** P < 0.05 for comparisons between controls of each age and the corresponding partially denervated or contralateral group. Unmarked comparisons were not significantly different (P > 0.05).
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< 0.05-see Fig. Id and Table 1). There was no effect on either the average length of terminal sprouts nor on the degree of nodal sprouting. Moreover, the percentage of end-plates with terminal sprouts was only 1.8 times the control value, compared with a 3.8-fold increase in the young adult age group. In agreement with the observations in the younger animals, partial denervation did not appear to affect the degree of axonal sprouting in the contralateral soleus muscle (Table 1). DISCUSSION The present study was undertaken to determine the extent of the repair response that followed motor nerve damage in young adult and aged animals. Our data indicate that aged rats exhibit a more limited ability than their young adult counterparts to respond to nerve injury with compensatory axonal growth. This reduced capacity is apparent as (a) an absence of any change in nodal sprouting, (b) a smaller increase in terminal sprouting than in adult animals, and (c) no alteration in the average length of terminal sprouts. However, both age groups respond to partial denervation with a similar increase in end-plate length. Although the present study indicates that aged animals have a reduced capacity for repair, one should be cautious at this stage of concluding that this is due to an inability to sustain neuronal growth. In fact, the total extent of sprouting (74%-terminal plus nodal) in control aged rats was almost 6-fold higher than in control young adults and nearly twice as great as in the young adult experimental group. However, this value increased only slightly (to 82%) after partial denervation. Hence, one explanation for the reduced response in aged animals is that there may be a maximum limit to the amount of sprout growth for each motor neuron (perhaps determined by the synthetic capacity of the cell) and a response is possible only if that limit has not been attained; in aged animals, natural processes of degeneration ( 1 I- 13) have already served to induce a high degree of intramuscular nerve sprouting, and additional neuronal growth cannot be supported. Alternatively, the latency of axonal sprouting may be greater in aged than in younger animals. This may account for the larger increase in end-plate length observed in the present study (14 days postlesion) compared with that seen by Pestronk et al. [7 days after botulinurn treatment (17)]. Moreover, recent results from this laboratory indicate that, after a unilateral lesion of the entorhinal cortex, reinnervation of the molecular layer in the dentate gyrus proceeds at essentially the same rate in both adult and aged rats, but that there is a delayed onset in the older animals (25). In this system, the growth response appears to depend on the rate
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NERVE SPROUTING
AND AGING
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at which degeneration products are cleared from the area (26), although decreased rates of protein synthesis and axonal transport in old animals may also play a role in the motoneuron-skeletal muscle system (14). Interestingly, the denervation-induced sprouting observed in the aged rats was only of the terminal type. Current evidence indicates that terminal sprouting is produced by treatments which simply render the target muscle inactive (such as botulinum toxin and tetrodotoxin), whereas nodal sprouting appears either to require an additional stimulus or to have a different threshold (7, 9). Selective synapse stabilization is thought to be involved in the development and maturation of the nervous system (8) and this type of mechanism has been invoked recently to explain the low lesion-induced synaptic turnover in the central nervous system of aged rats (15). Thus, another possibility to account for thi present data would be the existence of a higher threshold for sprouting (or greater degree of stabilization) in aged than in adult animals. In summary, partial denervation of a skeletal muscle in aged rats was shown to induce a smaller repair response (axonal and terminal sprouting) than in young adults. This does not appear to be due to an inability of aged motor neurons to sustain neuronal growth, since there was a high degree of sprouting in aged control animals. The diminished response in aged animals may be due to a raised threshold or longer latency for sprouting, or to the existence of an upper limit to axonal growth for each motor neuron. REFERENCES 1. AKERT, K., AND C. SANDRI. 1968. An elcctronmicroscopic study of zinc iodide-osmium impregnation of neurons. I. Staining of synaptic vesicles at cholinergic junctions. Brain Res. 7: 286-295. 2. BARKER, D., AND M. C. IP. 1966. Sprouting and degeneration of mammalian motor axons in normal and dc-affercntated skeletal muscle. Proc. R Sot. (BioZ) 153: 538-554. 3. BETZ. W. J., J. H. CALDWELL, AND R. R. RIBCHESTTJR.1980. Sprouting of active nerve terminals in partially inactive muscles of the rat. J. Physiol. (London) 303 281-297. 4. BROWN, M. C., AND R. IRONTON. 1977. Motor neurone sprouting induced by prolonged tetrodotoxin block of nerve action potentials. Nature (London) 265: 459-461. 5. BROWN, M. C., AND R. L. HOLLAND. 1979. A central role for denervated tissues in causing nerve sprouting. Nature (London) 282: 724-726. 6. BROWN, M. C., R. L. HOLLAND, AND R. IRONTON. 1980. Nodal and terminal sprouting from motor nerves in fast and slow muscles of the mouse. J. Physiol. (London) 306: 493-510. 7. BROWN. M. C., R. L. HOLLAND, AND W. G. HOPKINS. 1981. Motor nerve sprouting. Annu. Rev. Neurosci. 4: 17-42. 8. CHANGEUX. J.-P., AND A. DANCHIN. 1976. Selective stabilisation of developing synapses as a mechanism for the specification of neuronal networks. Nature (London) 264: 705712.
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