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A maturational increase in rat neuromuscular junctional acetylcholine receptors despite disuse or denervation
Brain Research, 266 (1983) 155-158
155
ElsevierBiomedicalPress
A maturaUonal Increase in rat neuromuscular junctional acetylcholine receptors despite disuse or denervaUon S. S. LABOVITZand N. ROBBINS Department of Anatomy, Case WesternReserve University, School of Medicine, Cleveland, OH 44106 (U.S.A.)
(Accepted December 21st, 1982) Key words: denervation - disuse- acetylcholinereceptor - rat neuromuscularjunction - receptor development
Junctional acetylcholine receptors (AChR) of rat skeletal muscles were microassayed4-7 days after denervation or total disuse. The normal growth-related increase in AChR number occurred despite denervation or muscleatrophy, but in disused muscles, this increase was less.Thus, at least for short periods, the developmentaladdition ofjunctional AChR is independent of muscle fiber size or innervation and partly independent &usage. Extensive studies have revealed that acetylcholine receptors (AChR), normally restricted to the junctional region of mature skeletal muscle appear in extrajunctional membrane after denervation 1.7,9 or disuse 8,1°. The response of junctional A C h R under similar conditions is considerably less clear; the acute effects of denervation are variable 4 and the changes, if any, after disuse alone are entirely unexplored. These issues are the subject of this communication. In addition, since junctional A C h R increases during maturation 2.~3, the experiments reported here also test the effect of denervation or disuse on this developmental increase in junctional AChR. Maturational increase of junctional A C h R should abruptly cease if it were dependent on increase in muscle fiber size, the presence of a nerve terminal (absent after denervation), or nerve-muscle activity (in the case of induced disuse). The lack of consistent data on junctional A C h R arises in part from technical difficulties in estimating the number of junctional A C h R in the presence of substantial extrajunctional AChR. In this study, we have employed a recently described method for more precise measurement of junctional AChW ~. In addition, we have obtained disuse without denervation by use of an implanted minipump continuously de-
livering tetrodotoxin (TTX) to the nerve. Ether-anesthetized 90-110 g male SpragueDawley rats were subjected to either unilateral sciatic nerve section at the mid-thigh or to paralysis of one leg by a subcutaneous implant of a minipump (Alzet, model 2001) which delivered TTX (1 #l/h of 0.6 M TTX) via a Silastic catheter into a Silastic cuff fitted loosely around the nerve. Contralateral sham operations or control implantations were routinely performed. The efficacy of the nerve block was assessed by observing the twitch response to sciatic nerve stimulation before and after the initial application of TTX and at the end of the treatment period. Paralysis was also checked on a daily basis by inspection of the toe-spreading reflex 3. The possibility of cuff-related nerve injury was assessed at the end of the treatment by electrical stimulation of the nerve distal to the block, by measurement of extrajunctional A C h R (see below), and by examination of osmium-stained cross-sections of nerve distal to the chronic block. TTX-treated animals showing evidence of denervation or partial failure of nerve block by any of these criteria were excluded. The extensor digitorum longus (EDL) and soleus (SOL) muscles were removed 4-7 days after the initial operation, exposed to [125I]a-bungarotoxin (NEN, 10-20 /tCi//~g), and assayed for
0006-8993/83/0000-0000/$03.00 © 1983ElsevierSciencePublishers
156 TABLE I
Effects of denervation and TTX-disuse on extrajunctional A ChR density Results are expressed as A C h R (number of[125I]a-bungarotoxin binding sites) per #m 2 muscle fiber surface area (mean _ S.E.); 5-6 muscle pairs were used for each comparison.
EDL SOL
Innervated
Contralateral innervated
Denervated
Contralateral control
TTX
Contralateral control
3 _ 0.82 9 _ 2.5
6 _ 1.5 5 _ 1.2
96 _ 8.6* 130 +__ 11.0"
6 +_ 0.41 6 + 0.82
28 _ 6.3*§ 54 _ 19.0"§
4 _ 1.8 7 _ 0.89
* Treated greater than control, P <( 0.05. § TTX less than denervated, P ~ 0.01.
junctional and extrajunctional AChR (number of a-toxin binding sites) according to a mass dissociation, single fiber method previously described 1. In this method, preincubation of muscle in unlabeled toxin blocked over 90% of the binding of labeled toxin. The lengths and widths of muscle fibers were measured by use of a graticule under light microscopy, and the fiber surface area was computed from these data. Muscles from unoperated animals of ages comparable to those at the start and finish of the denervation and TTX experiments were similarly processed. Extrajunctional AChR density was increased over control levels in denervated and, to a lesser degree, in TTX-disused muscle fibers (Table I) in agreement with previous reports8,'°. Values of AChR density obtained from right and left sides of unoperated animals were statistically identical. A far different result emerged at the junction. At 4-7 days after treatment, junctional AChR numbers in the EDL were preserved in denervated muscles compared to their contralateral controls. On the other hand, the method of paired comparisons revealed that the number of AChR per junction in TTX-disused muscles was reduced compared to the contralateral controls (Fig. 1). However, regardless of treatment, junctional AChR was generally greater in the more mature animals ('final' age) than in younger ('initial' age) innervated controls (Fig. 1). That is, both experimental and the respective contralateral control muscles developed greater junctional toxin-binding than was present in innervated muscles from unoperated animals one week
~
7 . 0
i
30-o
°
2o- ~ • ,0
O.
~ 5.0
Inltllll
FInJl
Den. Control
Don.
TTX Control
TTX
Fig. 1. Fiber diameter and junctional A C h R n u m b e r ([]25I]abungarotoxin binding sites) in the EDL as a function of age (weight) and treatment of nerve. N u m b e r of muscles in each group is indicated at base of bars and standard errors by vertical lines at the top of bars. In initial and final groups, two muscles per animal were used. In all other groups one muscle per animal was used. Animals were studied under a number of different conditions: (1) initial, unoperated innervated muscle at the same initial age and weight (4 weeks and 108 --- 0.6 g) as experimental animals at the time of operation: (2) final, unoperated animals at the same age and weisht (5 weeks and 135 --+ 15 g) as experimental animals at the Iermination of the experiments; (3) Den and Den control, 4 to 7-day denervated muscle and contralateral controls. respectively; and (4) TTX and TTX control, 4 to 7-day TTX paralyzed muscle and contralateral controls, respectively. Each diameter bar represents the mean diameter of 40-170 fibers, with an equal n u m b e r of fibers drawn from each muscle in a given muscle group. Each A C h R bar represents counts of 20-50 endplates per muscle. Statistical comparisons were made using Student's t-test: * greater than initial innervated. P < 0.05; and o less than contralaterat control P 0.02 by standard t-test or by the method of paired comparisons.
157 younger ('initial' data, Fig. 1), and the number of such binding sites was the same as that observed in innervated muscles from unoperated animals of the same (older) age ('final' data, Fig. 1). Similar results were obtained in SOL muscles. As in the EDL, the junctional AChR content of SOL muscles 4-7 days after denervation was not different from that of contralateral controls (5.9 x 106 binding sites per control junction, 6.1 × 106 sites denervated), but was significantly greater than junctional AChR of innervated muscles of animals one week younger (P < 0.05). Data obtained from the TTX-disused SOL were qualitatively similar to those of the TTX-disused EDL, but the differences were only at the P ~ 0.1 level. The finding of increased junctional bungarotoxin binding with growth of innervated muscles is consistent with other studies2,~3. From the present results it appears that, in the short-term, neither denervation nor disuse interferes with this developmental program. Also, although fiber diameter and junctional size (and AChR in turn 2) are normally correlated6, the reduced muscle fiber growth in the denervated or disused EDL was without influence on AChR development (Fig. 1). This finding was also observed in the denervated or disused SOL where both cessation of growth and atrophy were even more pronounced. In sum, in muscles denervated for one week or less, junctional toxin-binding was not different from that in contralateral controls. In contrast, EDL muscles subjected to TTX-disuse for the
same time developed somewhat less junctional AChR than contralateral controls. These results may be due to different rates of junctional AChR turnover following denervation and disuse. Despite this difference, denervated and TTX-disused muscles both accumulated junctional AChR in the developmental period studied here. The significant variable underlying these findings may be a pre-existent store of internal AChR ready for insertion into the membrane or the continued growth of the specialized junctional area, despite disuse or atrophy. Indeed, scanning electron micrography of 4 to 7day TTX-paralyzed EDL and SOL muscles revealed no change in primary cleft area compared to contralateral controls, depite atrophy (Labovitz, Robbins and Fahim, unpublished observation). On the other hand, junctional AChR content for periods greater than those studied here might be altered after denervation. For example, a slow decline in junctional AChR several weeks after denervation has been reported5,t2. The main conclusion is that in contrast to early nerve-muscle junction formation, where innervation is required for the formation of densely clustered AChR and postsynaptic specialization (e.g. ref. 14), later maturation is to some extent independent of neuronal regulation.
1 Axelsson, D. and Thesleff, S., A study of supersensitivity in denervated mammalian skeletal muscle, J. Physiol. (Lond.), 147 (1959) 178-193. 2 Bevan, S. and Steinbach, J. H., The disiribution of abungarotoxin binding sites on mammali~/n skeletal muscle developing in vivo, J. Physiol. (Lond.), 267 (1977) 195-213. 3 Blunt, R. J. and Vrbova, G., Pfli~gers Arch. Europ. J. Physiol., 357 (1975) 187-199. 4 Edwards, C., The effects ofinnervation on the properties of acetylcholine receptors in muscle, Neuroscience, 4 (1979) 565- 584. 5 Frank, E., Gautrik, K. and Sommerschild, H., Cholinergic receptors at denervated mammalian motor endplate, A ctaphysiol, scand., 95 (1975) 66-76.
6 Grinnell, A. D. and Herrera, A. A., Specificity and plasticity of neuromuscular connections: long-term regulation of motoneuron function, Progr. Neurobiol., 17 (1981) 203-282. 7 Hartzell, H. C. and Fambrough, D. M., Acetylcholine receptors: distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity,J. gen. Physiol., 60 (! 972) 248-262. 8 Lavoie, P. A., Collier, B. and Tenenhouse, A., Comparison of a-bungarotoxin binding to skeletal muscles after inactivity or denervation, Nature (Lond.), 260 (1976) 349-350. 9 Miledi, R., The acetylcholine sensitivity of frog muscle fibers after complete or partial denervation, J. Physiol. (Lond.), 151 (1960) 1-23.
This work was supported by NIH Grants AG 00795 and NS 18694 as well as a Muscular Dystrophy Association Summer Scholarship awarded to S.S.L.
158 10 Pestronk, A., Drachman, D. D. and GrilTm, J. W., Effect of muscle disuse on acetylcholine receptors, Nature (Lond.), 260 (1976) 352-353. 11 Robbins, N., Olek, A., Kelly, S. S., Takach, P. and Christopher, M., Quantitative study of motor endplates in muscle fibers dissociated by a simple procedure, Proc. roy. Soc. B., 209 (1980) 555-563. 12 Steinbach, J. H., Neuromuscular junctions and a-bungarotoxin binding sites in denervated and contralateral
cat skeletal muscles, J. PhysioL (Lond.), 313 (1981) 513528. 13 Steinbach, J. H., Developmental changes in acetylcholine receptor aggregates at rat skeletal neuromuscular junctions, Develop. Biol., 84 (198 I) 267-276. 14 Frank, E. and Fischbach, G. D., Early events in neuromuscular junction formation in vitro, J. Cell Biol., 83 (1979) 143-158.
155
ElsevierBiomedicalPress
A maturaUonal Increase in rat neuromuscular junctional acetylcholine receptors despite disuse or denervaUon S. S. LABOVITZand N. ROBBINS Department of Anatomy, Case WesternReserve University, School of Medicine, Cleveland, OH 44106 (U.S.A.)
(Accepted December 21st, 1982) Key words: denervation - disuse- acetylcholinereceptor - rat neuromuscularjunction - receptor development
Junctional acetylcholine receptors (AChR) of rat skeletal muscles were microassayed4-7 days after denervation or total disuse. The normal growth-related increase in AChR number occurred despite denervation or muscleatrophy, but in disused muscles, this increase was less.Thus, at least for short periods, the developmentaladdition ofjunctional AChR is independent of muscle fiber size or innervation and partly independent &usage. Extensive studies have revealed that acetylcholine receptors (AChR), normally restricted to the junctional region of mature skeletal muscle appear in extrajunctional membrane after denervation 1.7,9 or disuse 8,1°. The response of junctional A C h R under similar conditions is considerably less clear; the acute effects of denervation are variable 4 and the changes, if any, after disuse alone are entirely unexplored. These issues are the subject of this communication. In addition, since junctional A C h R increases during maturation 2.~3, the experiments reported here also test the effect of denervation or disuse on this developmental increase in junctional AChR. Maturational increase of junctional A C h R should abruptly cease if it were dependent on increase in muscle fiber size, the presence of a nerve terminal (absent after denervation), or nerve-muscle activity (in the case of induced disuse). The lack of consistent data on junctional A C h R arises in part from technical difficulties in estimating the number of junctional A C h R in the presence of substantial extrajunctional AChR. In this study, we have employed a recently described method for more precise measurement of junctional AChW ~. In addition, we have obtained disuse without denervation by use of an implanted minipump continuously de-
livering tetrodotoxin (TTX) to the nerve. Ether-anesthetized 90-110 g male SpragueDawley rats were subjected to either unilateral sciatic nerve section at the mid-thigh or to paralysis of one leg by a subcutaneous implant of a minipump (Alzet, model 2001) which delivered TTX (1 #l/h of 0.6 M TTX) via a Silastic catheter into a Silastic cuff fitted loosely around the nerve. Contralateral sham operations or control implantations were routinely performed. The efficacy of the nerve block was assessed by observing the twitch response to sciatic nerve stimulation before and after the initial application of TTX and at the end of the treatment period. Paralysis was also checked on a daily basis by inspection of the toe-spreading reflex 3. The possibility of cuff-related nerve injury was assessed at the end of the treatment by electrical stimulation of the nerve distal to the block, by measurement of extrajunctional A C h R (see below), and by examination of osmium-stained cross-sections of nerve distal to the chronic block. TTX-treated animals showing evidence of denervation or partial failure of nerve block by any of these criteria were excluded. The extensor digitorum longus (EDL) and soleus (SOL) muscles were removed 4-7 days after the initial operation, exposed to [125I]a-bungarotoxin (NEN, 10-20 /tCi//~g), and assayed for
0006-8993/83/0000-0000/$03.00 © 1983ElsevierSciencePublishers
156 TABLE I
Effects of denervation and TTX-disuse on extrajunctional A ChR density Results are expressed as A C h R (number of[125I]a-bungarotoxin binding sites) per #m 2 muscle fiber surface area (mean _ S.E.); 5-6 muscle pairs were used for each comparison.
EDL SOL
Innervated
Contralateral innervated
Denervated
Contralateral control
TTX
Contralateral control
3 _ 0.82 9 _ 2.5
6 _ 1.5 5 _ 1.2
96 _ 8.6* 130 +__ 11.0"
6 +_ 0.41 6 + 0.82
28 _ 6.3*§ 54 _ 19.0"§
4 _ 1.8 7 _ 0.89
* Treated greater than control, P <( 0.05. § TTX less than denervated, P ~ 0.01.
junctional and extrajunctional AChR (number of a-toxin binding sites) according to a mass dissociation, single fiber method previously described 1. In this method, preincubation of muscle in unlabeled toxin blocked over 90% of the binding of labeled toxin. The lengths and widths of muscle fibers were measured by use of a graticule under light microscopy, and the fiber surface area was computed from these data. Muscles from unoperated animals of ages comparable to those at the start and finish of the denervation and TTX experiments were similarly processed. Extrajunctional AChR density was increased over control levels in denervated and, to a lesser degree, in TTX-disused muscle fibers (Table I) in agreement with previous reports8,'°. Values of AChR density obtained from right and left sides of unoperated animals were statistically identical. A far different result emerged at the junction. At 4-7 days after treatment, junctional AChR numbers in the EDL were preserved in denervated muscles compared to their contralateral controls. On the other hand, the method of paired comparisons revealed that the number of AChR per junction in TTX-disused muscles was reduced compared to the contralateral controls (Fig. 1). However, regardless of treatment, junctional AChR was generally greater in the more mature animals ('final' age) than in younger ('initial' age) innervated controls (Fig. 1). That is, both experimental and the respective contralateral control muscles developed greater junctional toxin-binding than was present in innervated muscles from unoperated animals one week
~
7 . 0
i
30-o
°
2o- ~ • ,0
O.
~ 5.0
Inltllll
FInJl
Den. Control
Don.
TTX Control
TTX
Fig. 1. Fiber diameter and junctional A C h R n u m b e r ([]25I]abungarotoxin binding sites) in the EDL as a function of age (weight) and treatment of nerve. N u m b e r of muscles in each group is indicated at base of bars and standard errors by vertical lines at the top of bars. In initial and final groups, two muscles per animal were used. In all other groups one muscle per animal was used. Animals were studied under a number of different conditions: (1) initial, unoperated innervated muscle at the same initial age and weight (4 weeks and 108 --- 0.6 g) as experimental animals at the time of operation: (2) final, unoperated animals at the same age and weisht (5 weeks and 135 --+ 15 g) as experimental animals at the Iermination of the experiments; (3) Den and Den control, 4 to 7-day denervated muscle and contralateral controls. respectively; and (4) TTX and TTX control, 4 to 7-day TTX paralyzed muscle and contralateral controls, respectively. Each diameter bar represents the mean diameter of 40-170 fibers, with an equal n u m b e r of fibers drawn from each muscle in a given muscle group. Each A C h R bar represents counts of 20-50 endplates per muscle. Statistical comparisons were made using Student's t-test: * greater than initial innervated. P < 0.05; and o less than contralaterat control P 0.02 by standard t-test or by the method of paired comparisons.
157 younger ('initial' data, Fig. 1), and the number of such binding sites was the same as that observed in innervated muscles from unoperated animals of the same (older) age ('final' data, Fig. 1). Similar results were obtained in SOL muscles. As in the EDL, the junctional AChR content of SOL muscles 4-7 days after denervation was not different from that of contralateral controls (5.9 x 106 binding sites per control junction, 6.1 × 106 sites denervated), but was significantly greater than junctional AChR of innervated muscles of animals one week younger (P < 0.05). Data obtained from the TTX-disused SOL were qualitatively similar to those of the TTX-disused EDL, but the differences were only at the P ~ 0.1 level. The finding of increased junctional bungarotoxin binding with growth of innervated muscles is consistent with other studies2,~3. From the present results it appears that, in the short-term, neither denervation nor disuse interferes with this developmental program. Also, although fiber diameter and junctional size (and AChR in turn 2) are normally correlated6, the reduced muscle fiber growth in the denervated or disused EDL was without influence on AChR development (Fig. 1). This finding was also observed in the denervated or disused SOL where both cessation of growth and atrophy were even more pronounced. In sum, in muscles denervated for one week or less, junctional toxin-binding was not different from that in contralateral controls. In contrast, EDL muscles subjected to TTX-disuse for the
same time developed somewhat less junctional AChR than contralateral controls. These results may be due to different rates of junctional AChR turnover following denervation and disuse. Despite this difference, denervated and TTX-disused muscles both accumulated junctional AChR in the developmental period studied here. The significant variable underlying these findings may be a pre-existent store of internal AChR ready for insertion into the membrane or the continued growth of the specialized junctional area, despite disuse or atrophy. Indeed, scanning electron micrography of 4 to 7day TTX-paralyzed EDL and SOL muscles revealed no change in primary cleft area compared to contralateral controls, depite atrophy (Labovitz, Robbins and Fahim, unpublished observation). On the other hand, junctional AChR content for periods greater than those studied here might be altered after denervation. For example, a slow decline in junctional AChR several weeks after denervation has been reported5,t2. The main conclusion is that in contrast to early nerve-muscle junction formation, where innervation is required for the formation of densely clustered AChR and postsynaptic specialization (e.g. ref. 14), later maturation is to some extent independent of neuronal regulation.
1 Axelsson, D. and Thesleff, S., A study of supersensitivity in denervated mammalian skeletal muscle, J. Physiol. (Lond.), 147 (1959) 178-193. 2 Bevan, S. and Steinbach, J. H., The disiribution of abungarotoxin binding sites on mammali~/n skeletal muscle developing in vivo, J. Physiol. (Lond.), 267 (1977) 195-213. 3 Blunt, R. J. and Vrbova, G., Pfli~gers Arch. Europ. J. Physiol., 357 (1975) 187-199. 4 Edwards, C., The effects ofinnervation on the properties of acetylcholine receptors in muscle, Neuroscience, 4 (1979) 565- 584. 5 Frank, E., Gautrik, K. and Sommerschild, H., Cholinergic receptors at denervated mammalian motor endplate, A ctaphysiol, scand., 95 (1975) 66-76.
6 Grinnell, A. D. and Herrera, A. A., Specificity and plasticity of neuromuscular connections: long-term regulation of motoneuron function, Progr. Neurobiol., 17 (1981) 203-282. 7 Hartzell, H. C. and Fambrough, D. M., Acetylcholine receptors: distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity,J. gen. Physiol., 60 (! 972) 248-262. 8 Lavoie, P. A., Collier, B. and Tenenhouse, A., Comparison of a-bungarotoxin binding to skeletal muscles after inactivity or denervation, Nature (Lond.), 260 (1976) 349-350. 9 Miledi, R., The acetylcholine sensitivity of frog muscle fibers after complete or partial denervation, J. Physiol. (Lond.), 151 (1960) 1-23.
This work was supported by NIH Grants AG 00795 and NS 18694 as well as a Muscular Dystrophy Association Summer Scholarship awarded to S.S.L.
158 10 Pestronk, A., Drachman, D. D. and GrilTm, J. W., Effect of muscle disuse on acetylcholine receptors, Nature (Lond.), 260 (1976) 352-353. 11 Robbins, N., Olek, A., Kelly, S. S., Takach, P. and Christopher, M., Quantitative study of motor endplates in muscle fibers dissociated by a simple procedure, Proc. roy. Soc. B., 209 (1980) 555-563. 12 Steinbach, J. H., Neuromuscular junctions and a-bungarotoxin binding sites in denervated and contralateral
cat skeletal muscles, J. PhysioL (Lond.), 313 (1981) 513528. 13 Steinbach, J. H., Developmental changes in acetylcholine receptor aggregates at rat skeletal neuromuscular junctions, Develop. Biol., 84 (198 I) 267-276. 14 Frank, E. and Fischbach, G. D., Early events in neuromuscular junction formation in vitro, J. Cell Biol., 83 (1979) 143-158.