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Neural regulation of glucose 6-phosphate dehydrogenase in rat muscle: effect of denervation and reinnervation
Brain Research, 220 (1981) 131-138
131
Elsevier/North-Holland Biomedical Press
N E U R A L R E G U L A T I O N OF G L U C O S E 6-PHOSPHATE D E H Y D R O G E N A S E I N RAT MUSCLE: E F F E C T OF D E N E R V A T I O N A N D R E I N N E R V A T I O N
STEPHEN R. MAX, JOSEPH L. YOUNG and RICHARD F. MAYER Department of Neurology, Universityof Maryland, School of Medicine, Baltimore, Md. 21201 (U.S.A.)
(Accepted January 29th, 1981) Key words: glucose 6-phosphate dehydrogenase - - muscle - - denervation - - reinnervation - -
choline acetyltransferase
SUMMARY We tested the hypothesis that glucose 6-phosphate dehydrogenase (G6PD) in rat extensor digitorum longus (EDL) muscle is under neural control by studying changes in G6PD activity in E D L muscles following nerve crush-induced denervation and reinnervation. Changes in G6PD were correlated with choline acetyltransferase activity, as well as with neurological function, muscle weights, and muscle isometric twitch tension. The data show a dramatic increase in G6PD following denervation. The gradual recovery of enzyme activity toward normal levels correlates with the return of functional synaptogenesis manifested by the return of neurological function, choline acetyltransferase, and muscle twitch tension. We conclude, therefore, that muscle G6PD is under neural control. G6PD activity provides a facile biochemical indicator of muscle reinnervation. INTRODUCTION The activity of glucose 6-phosphate dehydrogenase (G6PD) in rat skeletal muscles appears to be subject to neural regulation. G6PD activity is enhanced by denervationg,X°,la,14,19,2s, 2~, and the rate of the increase in enzyme activity is proportional to the length of the distal nerve stump2a,~L An important demonstration of neural control of muscle G6PD would be restoration of pre-denervation levels of activity upon reinnervation. We now report that reinnervation of the extensor digitorum longus muscle (EDL) following a crush lesion of the peroneal nerve restores G6PD activity to pre-denervation levels. The time-course of the return of G6PD to control values after reinnervation is correlated with temporal changes of enzymatic (i.e. choline acetyltransferase activity) and physiological (i.e. twitch tension) indicators of functional synaptogenesis. These data support the concept that this enzyme is controlled by neural influences.
132 MATERIALS AND METHODS
Animals. Female rats (CD-strain, Charles River Breeding Labs., Wilmington, Mass.) weighing 200-250 g were used in all experiments. They were fed Purina rat chow and water ad libitum. Eighty-four rats were used for the biochemical studies and 24 for electrophysiological measurements. Animals were anesthetized with ether. In reinnervation experiments, the right peroneal nerve was crushed with fine forceps approximately 1.0 cm from the entry of the nerve into the E D L muscle in the upper portion of the leg where the peroneal nerve winds around the head of the fibula. Care was taken to avoid damaging the blood supply to the muscle. For nerve section experiments, a 1.0 cm segment of the nerve was removed about 1 cm from its entrance into the E D L muscle. In both cases, the contralateral E D L muscle served as control. Biochemical studies. At 1, 5, 7, 9, 11, 14 and 16 days following nerve section or crush, rats were decapitated. The E D L muscles were dissected, weighed and homogenized in 50 m M Tris.HCl, p H 7.4, containing 0.5 mM dithiothreitol, in a Duall homogenizer (Kontes Glass, Vineland, N.J.) with a mechanically driven pestle (Sears Roebuck drill press). A portion of the homogenate (100 #1) was removed for assay of choline acetyltransferase (CAT) (vide infra). The remainder was centrifuged at 0-4 °C at 18,000 × g in a Sorvall RC-5B. Glucose 6-phosphate dehydrogenase was measured in supernatant fractions as described 26,27. Data were computed as nmoles NADPH/min/muscle and expressed as per cent of control (means :/_ S.E.M.). Choline acetyltransferase was assayed in homogenates by the method of Fonnum using acetyl CoA-[I-14C] (Amersham) 6. Assays were performed in the presence and absence of choline in order to assess the interference of carnitine acetyltransferase 1,17,21. Activity in the absence of choline was 10-20 ~ that in the presence of choline. Enzyme activity in the absence of choline (i.e. carnitine acetyltransferase) was subtracted from that in the presence of choline to yield choline acetyltransferase. The data were computed as cpm/h/muscle and presented as per cent of control (means _~- S.E.M.). Control values for G6PD and C A T were given in earlier reports 2°,2t. Statistical differences between experimental and control muscles were determined with a paired t-test.
Physiological properties Isometric contractions of the extensor digitorum longus muscle were recorded in vivo 2-14 days after the peroneal nerve was crushed. The rats were initially anesthetized with ether and maintained in an anesthetized state by intraperitoneal injections of chloral hydrate (0.4 g/kg of body weight). The E D L muscle and peroneal nerve were dissected as free as possible from surrounding tissue without disturbing their blood supply. The leg was immobilized in a steel frame by pinning at the knee and tying at the ankle. The muscle surface temperature was maintained at 35-36 °C with a radiant heat lamp in a 28 °C laboratory. The exposed nerves and muscles were kept moist in a mineral oil bath. The distal E D L tendon was cut and attached to a strain gauge (Grass FT-03C) via a short length of 2-0 silk thread. A portion of the exposed peroneal nerve proximal
133 to the crush site was placed on a bipolar electrode for stimulation. The evoked muscle action potential (EMAP) was monitored by fine wires inserted into the EDL. The muscle was stretched to varing lengths to find the length that produced the maximum twitch. All measurements of the isometric contractions were made at this optimal length15,16, corresponding to a passive tension of 2-4 g. Supramaximal stimuli of 40-400 #sec duration were used. Twitch tension at optimal length was measured to peak 18. The conduction latency from onset of nerve stimulation to onset of the EMAP was monitored to demonstrate that muscular contraction was due to nerve stimulation and functional neuromuscular transmission. Contralateral limbs served as controls. RESULTS
Behavioral observations After crush injury or section of the peroneal nerve, the rats sustained a loss of the 'toe spreading' reflex, which was observed when they were picked up by the tail (for illustration, cf. ref. 8). This 'clubbing' persisted in the nerve sectioned animals. In the nerve crush rats, however, recovery of the 'toe spreading' reflex was apparent on the eigth-ninth day following the lesion; by 14 days, the neurological response appeared normal.
Muscle weights In both experimental groups (i.e. crush and nerve section), muscle fresh weights decreased after the first day following the procedure and declined to about 70 ~ of the weight of contralateral control muscles on the eighth day. At this point, muscle weights remained stable (through the sixteenth day) in the nerve crush animals, but continued to decline to about 5 0 ~ control in the nerve-sectioned rats (Fig. 1).
Glucose 6-phosphate dehydrogenase G6PD increased dramatically following denervation by nerve crush or section, as expected from our earlier studieslg,z0, z7 (Figs. 2, 3). In the nerve section rats, where
0 SECTION
Joo
90 ~_~SO ~
70 50
40
i
t
i
t
i
i12
i
I 6
2 4 6 8 I0 14 I DAYS AFTER NERVE CRUSH OR SECTION
Fig. 1. E D L muscle fresh weights after nerve c r u s h or nerve section. T h e data are expressed as per cent contralateral control muscle, m e a n s ± S.E.M. o f at least 6 muscles, Experimental procedures are described in the text.
134 w
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2 4 6 DAYS A F T E R
8 I0 12 14 16 NERVE SECTION
Fig. 2. Glucose 6-phosphate dehydrogenase and choline acetyltransferase activities in rat EDL muscles following nerve section. Data are expressed as per cent contralateral control, means ± S.E.M. The number of muscles is in parentheses. G6PD activity at 1, 5, 7, 9, 14, and 16 days after nerve section was significantly greater than control (P < 0.05). Experimental procedures as described in the text. there w a s n o reinnervation, as evidenced by the persistence o f the neurological deficit a n d the fall in CAT activity, G6PD activity r e m a i n e d elevated t h r o u g h o u t the 16-day d u r a t i o n o f the e x p e r i m e n t (Fig. 2). In contrast, G 6 P D
initially rose to 180~ of control in the crush injury group, began to decline after 8 days and fell to within 20 of the c o n t r o l value by the f o u r t e e n t h day, r e m a i n i n g at this value until the sixteenth d a y (Fig. 3). The difference b e t w e e n the activities o f G 6 P D at day I following the nerve section (Fig. 2) or nerve crush (Fig. 3) w a s n o t significant. On the other hand, a significant difference w a s o b s e r v e d b e t w e e n these values on day 16 following nerve
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116
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2 4 6 8 I 0 12 14 DAYS AFTER NERVE CRUSH
Fig. 3. Glucose 6-phosphate dehydrogenase and choline acetyltransferase activities in denervated and reinnervated rat EDL muscles following crush lesion of the peroneal nerve. Data are expressed as per cent contralateral control, means ± S.E.M. The number of muscles is in parentheses. G6PD activity at t, 5, 7 and 9 days after nerve crush was significantly greater that control (P < 0.05). Experimental procedures as described in the text.
135 crush or section. That this recovery of G6PD corresponds to reinnervation and functional synaptogenesis after the crush lesion is demonstrated by the following results.
Choline acetyltransferase (CAT) Choline acetyltransferase activity, which was employed as a biochemical indicator of cholinergic synapses2, 4, declined after denervationl,a,19, ~4. In muscles, the nerves to which had been cut (Fig. 2), CAT activity declined steadily and reached 20 of control by day 16. In the nerve-crush animals, on the other hand, CAT activity declined to 20 ~o control by day 6, after which it began to recover and attained about 55 ~o control on day 16 (Fig. 3). The CAT data and the G6PD results are plotted on the same coordinates for ease of comparison. It can be seen that the recovery of CAT, a reflection of the reformation of neuromuscular synapses, correlates quite well with the phase of recovery of G6PD activity. In the nerve section animals where there was no opportunity for reinnervation, G6PD activity remained elevated and CAT activity showed no recovery (Fig. 2).
Physiological properties To ascertain whether the rise and fall of G6PD activity was associated with loss and recovery of functional innervation, the excitability of the E D L to electrical stimulation of its nerve was tested. The E D L became unexcitable to nerve stimulation immediately after the crush and this condition persisted through the eighth day. On day 9, muscular contraction following nerve stimulation was again detectable, a direct proof of motor reinnervation. This occurred on the same day that the activity of G6PD decreased toward normal values (Fig. 3). Twitch tension was measured at 2, 4, 6, 8, 9, 10, 12, and 14 days after nerve crush and was compared with controls (contralateral non-operated hind limbs) (Fig. 4). No muscle twitch was recorded immediately after nerve crush until day 9 at which time a small twitch with a prolonged twitch contraction time was recorded. The recovery of 40-
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DAYS AFTER NERVE CRUSH
Fig. 4. Isometric twitch tension at optimal length of rat EDL following a crush lesion of the peroneal nerve. Control values are plotted at day 0. No muscle response could be recorded immediately after nerve crush. Values are means and the range. The number of determinations is in parentheses. Experimental procedures as described in the text.
136 the twitch tension followed the rapid fall of G6PD during the 9-14 day post-crush period (Fig. 3). DISCUSSION The data described above add considerable support to our hypothesis that G6PD activity in rat skeletal muscle is neurally regulated. G6PD activity, which rises after denervation19,20, 22, returns to control values coincident with the recovery of functional reinnervation. This is seen in the time-course of behavioral changes, muscle wet weight (Fig. 1), choline acetyltransferase (Fig. 3), and evoked muscle response (twitch tension, Fig. 4). Indeed, all of these parameters recovered within essentially the same time-frame, viz. onset about 9 days after nerve crush and recovery near 14 days. This time-course correlates with reappearance of miniature endplate potentials (mepps), restoration of membrane potential, and return of ACh-sensitivity to control levels in a similar study by McArdle and Albuquerque2L Carlsson et al.~ also showed recovery of CAT during reinnervation of cat gastrocnemius muscles. That CAT showed no real increase at day 11 while G6PD decreased substantially at this time (Fig. 3), may indicate that CAT is insufficiently sensitive to detect very early reinnervation. We conclude that the recovery of G6PD is a concomitant of reinnervation, and that G6PD is under neural control. That axonal transport of putative trophic substances is involved in this control is suggested by the nerve stump lengthdependence of the denervation-mediated increase in G6PD23, 2v, which also tends to rule out 'usage', as a contributory factor since neuromuscular activity is terminated at any nerve stump length. The rapid increase in twitch tension (Fig. 4) and concomitant fall in G6PD (Fig. 3) found in this study correlate well with the increase in the number of fibers displaying mepps 22, suggesting that the twitch tension reflects the number of muscle fibers innervated. Our data indicate, therefore, that about 80-90 ~ of the muscle fibers in the EDL are reinnervated by day 14 following nerve crush approximately 1 cm from the muscle. That G6PD activity did not return completely to control levels (Fig. 3) argues for a correlation of G6PD activity with the degree of innervation of the muscle. Although the exact mechanisms involved remain unresolved, the present results provide compelling proof for neural control of G6PD. In summary, our results show changes in G6PD activity to correlate with the degree of innervation. While other proteins, such as myosin, are also neurally regulated 11, biochemical assays for these substances are cumbersome. Furthermore, recent research has cast doubt on the use of 16S AChE as a quantitative marker for the return of endplate AChE upon reinnervation 1'~,'z5. G6PD activity may prove to be a convenient and reliable biochemical indicator of functional reinnervation of mammalian skeletal muscle. ACKNOWLEDGEMENTS We thank Drs. B. H. Sohmer and G. Markelonis for helpful comments, Ms. B.
137 D y a s fo r expert assistance, a n d Ms. B. P a s k o for p r e p a r a t i o n o f the typescript. This research was s u p p o r t e d in p a r t by N I H G r a n t NS-15760.
REFERENCES 1 Butler, I. J., Drachman, D. B. and Goldberg, A. M., The effect of disuse on cholinergic enzymes, J. Physiol. (Lond.), 274 (1978) 593-600. 2 Carlson, B. M., Wagner, K. R. and Max, S. R., Reinnervation of rat extensor digitorum longus muscles after free grafting, Muscle and Nerve, 2 (1979) 304-307. 3 Carlsson, C.-A., Bolander, P. and Sjostrand, S., Biochemical and histochemical changes in the feline gastrocnemius muscle during regeneration of ventral roots, J. Neurol. Sci., 14 ( 1971) 95-105. 4 Chiapinelli, V., Giacobini, E., Pilar, G. and Uchimura, H., Induction of cholinergic enzymes in chick ciliary ganglion and iris muscle cells during synapse formation, J. Physiol. (Lond.), 257 (1975) 749-762. 5 Close, R., Dynamic properties of fast and slow skeletal muscles of rat during development, J. Physiol. (Lond.), 173 (1964) 74-95. 6 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. 7 Guth, L. and Samaha, F. J., Procedure for the histochemical demonstration of actomyosin ATPase, Exp. NeuroL, 28 (1970) 365-367. 8 Hasegawa, K., A new method of measuring functional recovery after crushing the peripheral nerves in unanesthetized and unrestrained rats, Experientia (Basel), 34 (1978) 272-273. 9 Hogan, E. L., Dawson, D. M. and Romanul, F. C. A., Enzymatic changes in denervated muscle. II. Biochemical studies, Arch. Neurol. (Chic.), 13 (1965) 274-282. 10 Ilyin, V. S.~ Razumovskaya, N. I. and Usatenko, M. S., Influence of nerve impulse on enzyme synthesis in skeletal muscle, Advanc. Enz. Regul., 13 (1975) 219-234. 11 Jean, D. H., Guth, L. and Albers, R. W., Neural regulation of the structure of myosin, Exp. Neurol., 38 (1973) 458-471. 12 Kato, A. C., Vrachliotis, A., Fulpius, B. and Durant, Y., Molecular forms of acetylcholinesterase in chick muscle and ciliary ganglion: embryonic tissues and cultured cells, Develop. Biol., 76 (1980) 222-228. 13 Kauffman, F. C., Albuquerque, E. X., Warnick, J. E. and Max, S. R., Effect of vinblastine on neural regulation in rat muscle, Exp. Neurol., 50 (1976) 60--66. 14 Koski, C. L. and Max, S. R., Substrate utilization by the denervated rat hemidiaphragm, Exp. Neurol., 48 (1974) 547-554. 15 Lewis, D. M., The effect of denervation on the mechanical and electrical properties of fast and slow mammalian twitch muscle, J. Physiol. (Lond.), 222 (1972) 51-75. 16 Lewis, D. M., Kean, C. J. C. and McGarrick, J. D., Dynamic properties of slow and fast muscle and their trophic regulation, Ann. N. Y. Acad. Sci., 228 (1974) 105-120. 17 Luine, V., Nottebohm, F., Harding, C. and McEwen, B. S., Androgen affects cholinergic enzymes in syringeal motor neurons and muscle, Brain Research, 192 (1980) 89-107. 18 Mano, Y., Mano, K., Mayer, R. F., Deshpande, S. S. and Albuquerque, E. X., Effects of paraplegia produced by intrathecal 6-aminonicotinamide on motor units in the rat, Exp. Neurol., 65 (1979) 435-456. 19 Max, S. R., Effect of estrogen on denervated muscle, J. Neurochem., 36 (1981) 1077-1082. 20 Max, S. R. and Knudsen, J. F., Neural and endocrine interaction in muscle, Brain Research, 190 (1980) 54%553. 21 Max, S. R. and Rifenberick, D. H., Choline acetyltransferase and cholinesterase in skeletal muscle regeneration, J. Neurochem., 24 (1975) 771-773. 22 McArdle, J. J. and Albuquerque, E. X., A study of the reinnervation of fast and slow muscles, J. gen. Physiol., 61 (1973) 1-23. 23 Robbins, N. and Carlson, D., Early changes in muscle glucose 6-phosphate dehydrogenase activity after denervation: locus and dependence on nerve stump length, Brain Research, 177 (1979) 145-156. 24 Tucek, S., Choline acetyltransferase activity in skeletal muscles after denervation, Exp. Neurol., 40 (1973) 23-25.
138 25 Vigny, M., Koenig, J. and Rieger, F., The motor endplate specific form of ACHE: appearance during embryogenesis and reinnervation of rat muscle, J. Neurochem., 27 (1976) 1347-1353. 26 Wagner, K. R., Kauffman, F.C. and Max, S. R., The pentose phosphate pathway in regenerating skeletal muscle, Biochem. J., 170 (1978) 17-22. 27 Wagner, K. R. and Max, S. R., Neurotrophic regulation of glucose 6-phosphate dehydrogenase in rat skeletal muscle, Brain Research, 170 (1979) 572-576.
131
Elsevier/North-Holland Biomedical Press
N E U R A L R E G U L A T I O N OF G L U C O S E 6-PHOSPHATE D E H Y D R O G E N A S E I N RAT MUSCLE: E F F E C T OF D E N E R V A T I O N A N D R E I N N E R V A T I O N
STEPHEN R. MAX, JOSEPH L. YOUNG and RICHARD F. MAYER Department of Neurology, Universityof Maryland, School of Medicine, Baltimore, Md. 21201 (U.S.A.)
(Accepted January 29th, 1981) Key words: glucose 6-phosphate dehydrogenase - - muscle - - denervation - - reinnervation - -
choline acetyltransferase
SUMMARY We tested the hypothesis that glucose 6-phosphate dehydrogenase (G6PD) in rat extensor digitorum longus (EDL) muscle is under neural control by studying changes in G6PD activity in E D L muscles following nerve crush-induced denervation and reinnervation. Changes in G6PD were correlated with choline acetyltransferase activity, as well as with neurological function, muscle weights, and muscle isometric twitch tension. The data show a dramatic increase in G6PD following denervation. The gradual recovery of enzyme activity toward normal levels correlates with the return of functional synaptogenesis manifested by the return of neurological function, choline acetyltransferase, and muscle twitch tension. We conclude, therefore, that muscle G6PD is under neural control. G6PD activity provides a facile biochemical indicator of muscle reinnervation. INTRODUCTION The activity of glucose 6-phosphate dehydrogenase (G6PD) in rat skeletal muscles appears to be subject to neural regulation. G6PD activity is enhanced by denervationg,X°,la,14,19,2s, 2~, and the rate of the increase in enzyme activity is proportional to the length of the distal nerve stump2a,~L An important demonstration of neural control of muscle G6PD would be restoration of pre-denervation levels of activity upon reinnervation. We now report that reinnervation of the extensor digitorum longus muscle (EDL) following a crush lesion of the peroneal nerve restores G6PD activity to pre-denervation levels. The time-course of the return of G6PD to control values after reinnervation is correlated with temporal changes of enzymatic (i.e. choline acetyltransferase activity) and physiological (i.e. twitch tension) indicators of functional synaptogenesis. These data support the concept that this enzyme is controlled by neural influences.
132 MATERIALS AND METHODS
Animals. Female rats (CD-strain, Charles River Breeding Labs., Wilmington, Mass.) weighing 200-250 g were used in all experiments. They were fed Purina rat chow and water ad libitum. Eighty-four rats were used for the biochemical studies and 24 for electrophysiological measurements. Animals were anesthetized with ether. In reinnervation experiments, the right peroneal nerve was crushed with fine forceps approximately 1.0 cm from the entry of the nerve into the E D L muscle in the upper portion of the leg where the peroneal nerve winds around the head of the fibula. Care was taken to avoid damaging the blood supply to the muscle. For nerve section experiments, a 1.0 cm segment of the nerve was removed about 1 cm from its entrance into the E D L muscle. In both cases, the contralateral E D L muscle served as control. Biochemical studies. At 1, 5, 7, 9, 11, 14 and 16 days following nerve section or crush, rats were decapitated. The E D L muscles were dissected, weighed and homogenized in 50 m M Tris.HCl, p H 7.4, containing 0.5 mM dithiothreitol, in a Duall homogenizer (Kontes Glass, Vineland, N.J.) with a mechanically driven pestle (Sears Roebuck drill press). A portion of the homogenate (100 #1) was removed for assay of choline acetyltransferase (CAT) (vide infra). The remainder was centrifuged at 0-4 °C at 18,000 × g in a Sorvall RC-5B. Glucose 6-phosphate dehydrogenase was measured in supernatant fractions as described 26,27. Data were computed as nmoles NADPH/min/muscle and expressed as per cent of control (means :/_ S.E.M.). Choline acetyltransferase was assayed in homogenates by the method of Fonnum using acetyl CoA-[I-14C] (Amersham) 6. Assays were performed in the presence and absence of choline in order to assess the interference of carnitine acetyltransferase 1,17,21. Activity in the absence of choline was 10-20 ~ that in the presence of choline. Enzyme activity in the absence of choline (i.e. carnitine acetyltransferase) was subtracted from that in the presence of choline to yield choline acetyltransferase. The data were computed as cpm/h/muscle and presented as per cent of control (means _~- S.E.M.). Control values for G6PD and C A T were given in earlier reports 2°,2t. Statistical differences between experimental and control muscles were determined with a paired t-test.
Physiological properties Isometric contractions of the extensor digitorum longus muscle were recorded in vivo 2-14 days after the peroneal nerve was crushed. The rats were initially anesthetized with ether and maintained in an anesthetized state by intraperitoneal injections of chloral hydrate (0.4 g/kg of body weight). The E D L muscle and peroneal nerve were dissected as free as possible from surrounding tissue without disturbing their blood supply. The leg was immobilized in a steel frame by pinning at the knee and tying at the ankle. The muscle surface temperature was maintained at 35-36 °C with a radiant heat lamp in a 28 °C laboratory. The exposed nerves and muscles were kept moist in a mineral oil bath. The distal E D L tendon was cut and attached to a strain gauge (Grass FT-03C) via a short length of 2-0 silk thread. A portion of the exposed peroneal nerve proximal
133 to the crush site was placed on a bipolar electrode for stimulation. The evoked muscle action potential (EMAP) was monitored by fine wires inserted into the EDL. The muscle was stretched to varing lengths to find the length that produced the maximum twitch. All measurements of the isometric contractions were made at this optimal length15,16, corresponding to a passive tension of 2-4 g. Supramaximal stimuli of 40-400 #sec duration were used. Twitch tension at optimal length was measured to peak 18. The conduction latency from onset of nerve stimulation to onset of the EMAP was monitored to demonstrate that muscular contraction was due to nerve stimulation and functional neuromuscular transmission. Contralateral limbs served as controls. RESULTS
Behavioral observations After crush injury or section of the peroneal nerve, the rats sustained a loss of the 'toe spreading' reflex, which was observed when they were picked up by the tail (for illustration, cf. ref. 8). This 'clubbing' persisted in the nerve sectioned animals. In the nerve crush rats, however, recovery of the 'toe spreading' reflex was apparent on the eigth-ninth day following the lesion; by 14 days, the neurological response appeared normal.
Muscle weights In both experimental groups (i.e. crush and nerve section), muscle fresh weights decreased after the first day following the procedure and declined to about 70 ~ of the weight of contralateral control muscles on the eighth day. At this point, muscle weights remained stable (through the sixteenth day) in the nerve crush animals, but continued to decline to about 5 0 ~ control in the nerve-sectioned rats (Fig. 1).
Glucose 6-phosphate dehydrogenase G6PD increased dramatically following denervation by nerve crush or section, as expected from our earlier studieslg,z0, z7 (Figs. 2, 3). In the nerve section rats, where
0 SECTION
Joo
90 ~_~SO ~
70 50
40
i
t
i
t
i
i12
i
I 6
2 4 6 8 I0 14 I DAYS AFTER NERVE CRUSH OR SECTION
Fig. 1. E D L muscle fresh weights after nerve c r u s h or nerve section. T h e data are expressed as per cent contralateral control muscle, m e a n s ± S.E.M. o f at least 6 muscles, Experimental procedures are described in the text.
134 w
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0 z
140
60 ~, 40
~- u
(6)
20
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2 4 6 DAYS A F T E R
8 I0 12 14 16 NERVE SECTION
Fig. 2. Glucose 6-phosphate dehydrogenase and choline acetyltransferase activities in rat EDL muscles following nerve section. Data are expressed as per cent contralateral control, means ± S.E.M. The number of muscles is in parentheses. G6PD activity at 1, 5, 7, 9, 14, and 16 days after nerve section was significantly greater than control (P < 0.05). Experimental procedures as described in the text. there w a s n o reinnervation, as evidenced by the persistence o f the neurological deficit a n d the fall in CAT activity, G6PD activity r e m a i n e d elevated t h r o u g h o u t the 16-day d u r a t i o n o f the e x p e r i m e n t (Fig. 2). In contrast, G 6 P D
initially rose to 180~ of control in the crush injury group, began to decline after 8 days and fell to within 20 of the c o n t r o l value by the f o u r t e e n t h day, r e m a i n i n g at this value until the sixteenth d a y (Fig. 3). The difference b e t w e e n the activities o f G 6 P D at day I following the nerve section (Fig. 2) or nerve crush (Fig. 3) w a s n o t significant. On the other hand, a significant difference w a s o b s e r v e d b e t w e e n these values on day 16 following nerve
z
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120
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2 4 6 8 I 0 12 14 DAYS AFTER NERVE CRUSH
Fig. 3. Glucose 6-phosphate dehydrogenase and choline acetyltransferase activities in denervated and reinnervated rat EDL muscles following crush lesion of the peroneal nerve. Data are expressed as per cent contralateral control, means ± S.E.M. The number of muscles is in parentheses. G6PD activity at t, 5, 7 and 9 days after nerve crush was significantly greater that control (P < 0.05). Experimental procedures as described in the text.
135 crush or section. That this recovery of G6PD corresponds to reinnervation and functional synaptogenesis after the crush lesion is demonstrated by the following results.
Choline acetyltransferase (CAT) Choline acetyltransferase activity, which was employed as a biochemical indicator of cholinergic synapses2, 4, declined after denervationl,a,19, ~4. In muscles, the nerves to which had been cut (Fig. 2), CAT activity declined steadily and reached 20 of control by day 16. In the nerve-crush animals, on the other hand, CAT activity declined to 20 ~o control by day 6, after which it began to recover and attained about 55 ~o control on day 16 (Fig. 3). The CAT data and the G6PD results are plotted on the same coordinates for ease of comparison. It can be seen that the recovery of CAT, a reflection of the reformation of neuromuscular synapses, correlates quite well with the phase of recovery of G6PD activity. In the nerve section animals where there was no opportunity for reinnervation, G6PD activity remained elevated and CAT activity showed no recovery (Fig. 2).
Physiological properties To ascertain whether the rise and fall of G6PD activity was associated with loss and recovery of functional innervation, the excitability of the E D L to electrical stimulation of its nerve was tested. The E D L became unexcitable to nerve stimulation immediately after the crush and this condition persisted through the eighth day. On day 9, muscular contraction following nerve stimulation was again detectable, a direct proof of motor reinnervation. This occurred on the same day that the activity of G6PD decreased toward normal values (Fig. 3). Twitch tension was measured at 2, 4, 6, 8, 9, 10, 12, and 14 days after nerve crush and was compared with controls (contralateral non-operated hind limbs) (Fig. 4). No muscle twitch was recorded immediately after nerve crush until day 9 at which time a small twitch with a prolonged twitch contraction time was recorded. The recovery of 40-
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Fig. 4. Isometric twitch tension at optimal length of rat EDL following a crush lesion of the peroneal nerve. Control values are plotted at day 0. No muscle response could be recorded immediately after nerve crush. Values are means and the range. The number of determinations is in parentheses. Experimental procedures as described in the text.
136 the twitch tension followed the rapid fall of G6PD during the 9-14 day post-crush period (Fig. 3). DISCUSSION The data described above add considerable support to our hypothesis that G6PD activity in rat skeletal muscle is neurally regulated. G6PD activity, which rises after denervation19,20, 22, returns to control values coincident with the recovery of functional reinnervation. This is seen in the time-course of behavioral changes, muscle wet weight (Fig. 1), choline acetyltransferase (Fig. 3), and evoked muscle response (twitch tension, Fig. 4). Indeed, all of these parameters recovered within essentially the same time-frame, viz. onset about 9 days after nerve crush and recovery near 14 days. This time-course correlates with reappearance of miniature endplate potentials (mepps), restoration of membrane potential, and return of ACh-sensitivity to control levels in a similar study by McArdle and Albuquerque2L Carlsson et al.~ also showed recovery of CAT during reinnervation of cat gastrocnemius muscles. That CAT showed no real increase at day 11 while G6PD decreased substantially at this time (Fig. 3), may indicate that CAT is insufficiently sensitive to detect very early reinnervation. We conclude that the recovery of G6PD is a concomitant of reinnervation, and that G6PD is under neural control. That axonal transport of putative trophic substances is involved in this control is suggested by the nerve stump lengthdependence of the denervation-mediated increase in G6PD23, 2v, which also tends to rule out 'usage', as a contributory factor since neuromuscular activity is terminated at any nerve stump length. The rapid increase in twitch tension (Fig. 4) and concomitant fall in G6PD (Fig. 3) found in this study correlate well with the increase in the number of fibers displaying mepps 22, suggesting that the twitch tension reflects the number of muscle fibers innervated. Our data indicate, therefore, that about 80-90 ~ of the muscle fibers in the EDL are reinnervated by day 14 following nerve crush approximately 1 cm from the muscle. That G6PD activity did not return completely to control levels (Fig. 3) argues for a correlation of G6PD activity with the degree of innervation of the muscle. Although the exact mechanisms involved remain unresolved, the present results provide compelling proof for neural control of G6PD. In summary, our results show changes in G6PD activity to correlate with the degree of innervation. While other proteins, such as myosin, are also neurally regulated 11, biochemical assays for these substances are cumbersome. Furthermore, recent research has cast doubt on the use of 16S AChE as a quantitative marker for the return of endplate AChE upon reinnervation 1'~,'z5. G6PD activity may prove to be a convenient and reliable biochemical indicator of functional reinnervation of mammalian skeletal muscle. ACKNOWLEDGEMENTS We thank Drs. B. H. Sohmer and G. Markelonis for helpful comments, Ms. B.
137 D y a s fo r expert assistance, a n d Ms. B. P a s k o for p r e p a r a t i o n o f the typescript. This research was s u p p o r t e d in p a r t by N I H G r a n t NS-15760.
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