Endplate postsynaptic structure dependent upon muscle activity

Neuroscience Letters, 43 (1983) 277-283

277

Elsevier Scientific Publishers Ireland Ltd.

ENDPLATE POSTSYNAPTIC STRUCTURE DEPENDENT UPON MUSCLE ACTIVITY

BRUCE R. PACHTER and ARTHUR EBERSTEIN

Department o f Rehabilitation Medicine, New York University Medical Center, 400 East 34th Street, New York, N Y 10016 (U.S.A.) (Received October 6th, 1983; Revised version received October 19th, 1983; Accepted October 20th, 1983)

Key words: denervation - endplate - contractile activity - electrical stimulation - muscle fibers morphometry The influence of contractile activity on the preservation of the denervated postsynaptic region of the endplate was quantitatively assessed by electron microscopy. The extensor digitorum Iongus muscle of rats were denervated for 21 days. Denerva~ed animals were divided into two groups, those receiving electrical stimulation treatment (I h/day for 21 days) and those left untreated. The postsynaptic area of clefts and folds in endplates of type i and Ii muscle fibers from controls and denervated-stimulated animals were found to be comparable in size whereas the postsynaplic areas in the denervated-non-stimulated muscles were significantly reduced. The results show that electrically-induced contractile activity plays a significant role in the maintenance of the postsynaptic region of the endplate.

Nerve transection produces two effects in muscle, loss of activity and the loss of neurotrophic influences [7]. Studies over many years have defined some of the changes which accompany denervation: the muscle membrane becomes sensitive to acetylcholine (ACh) over its entire length instead of simply at the endplate [1], the resting membrane potential falls, spontaneous fibrillation occurs [6], the muscle fibers become atrophic [4], there is a reduction in the activity of metabolic enzymes [12], and the neuromuscular junction begins to degenerate [18]. There appears to be growing evidence to favor the concept that the loss of contractile activity by the denervated muscle might be responsible for many of the above changes [9]. For example, electrical stimulation of denervated muscle has been shown by several investigators to be effective in preventing the development of ACh sensitivity and fibrillation activity, restoring the normal electrical characteristics of the membrane [9, 20], retarding muscle atrophy [14], and preventing the loss of oxidative enzymes [12]. Several studies have shown that the level of junctional acetylcholinesterase appears to be dependent upon muscle activity as well [10, 171. The neuromuscular junctional apparatus following denervation also undergoes a series of degenerative changes which eventually leads to removal of the nerve terminal and its replacement by the Schwann cell. The postsynaptic area of the endplate flattens out and the postjunctional folds decrease in amount, size, and become 0304-3940/83/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.

278

irregular. There appears to be a controversy in the literature as to what controls the maintenance of the postsynaptic region of the muscle. Some investigators are of the opinion that it is dependent upon neurotrophic factors [11, 16], while other studies seem to indicate that activity is necessary [2]. The present study was designed to test the role of muscular contractions on the preservation of the endplate using the experimental model Lomo et al. [81 employed for examining contractile properties. Muscle contractions are produced by means of direct electrical stimulation of denervated muscles. Nine male Wistar rats (average body weight, 300 g) were chosen for this study. The right hind limbs of 6 rats were surgically denervated under ether anesthesia by excision of a 0.5 cm segment of the sciatic nerve just proximal to the bifurcation of the tibial and peroneal nerves. The cut end of the proximal portion of the nerve was capped with Silastic tubing to prevent reinnervation. The procedure used by Gilliatt et al. [5] was used in stimulating the denervated muscles. Sterile electrodes (EMG monopolar) were i~,troduced subcutaneously at each end of the extensor digitorum Iongus (EDL) muscle at the start of stimulation and removed at the end of the stimulation period. One group of 3 denervated rats, while in a restrainer cage, received electrical stimulation treatment at 10 Hz (1 h/day for 21 days) beginning 4 h after surgery. The current intensity was adjusted to evoke a visible toe extension (approximately 4 mA). Another group of 3 denervated rats was treated identically to those receiving stimulation, except they were not stimulated electrically. Three rats served as normal controls. At 21 days postdenervation, the EDL muscles were exposed and the proximal end of the sciatic nerve was stimulated to ascertain if any reinnervation had occurred. No response was observed in any of the experimental animals. The EDL muscles were fixed in situ by 4°7o glutaraldehyde in phosphate buffer (0.1 M, pH 7.2) for I0-15 rain and then removed in total. The muscles were then transferred to cold 4°/o glutaraldehyde solution overnight, postfixed in 1~0 osmium tetroxide for 2 h, dehydrated in graded alcohols, and embedded in Epon 812. The muscles were embedded flat and serially sectioned transversely at 15 #m by a steel knife on a sliding microtome. These thick Epon sections were cleared for light microscopy by curing a layer of Epon onto them within a sandwich of polystyrene film [14], the muscles could thus be initially surveyed by phase contrast to locate the endplate zone of the muscles. Sections containing endplates were remounted on a Beem capsule and ultrathin sections were cut, stained with lead citrate and uranyl acetate, and examined with a Zeiss EM-10 electron microscope at an accelerating voltage of 60 kV. All endplates observed on type I and type I1 muscle fibers in the electron microscope were photographed and analyzed at a final magnification of 15,750x. The uitrastructural identification of type 1 and type 11 muscle fibers was based on mitochondrial content and configuration of the sarcoplasmic reticulum [15, 19]. For

279 TABLE 1 TOTAL POSTSYNAPTIC AREA (~rn2) OF ENDPLATES FROM TYPE I AND TYPE !i MUSCLE FIBERS Values are means ± S.D. from control, denervated-non-stimulated, and denervated-stimulated rat extensor digitorum iongus muscles. *Significantly different (P<0.001) from control and denervated-stimulated muscle fibers. A tninimum of 20 endplates/muscle fiber type/muscle were analyzed for each experimental situation.

Control Denervated-non-stimulated Denervated-stimulated

Type 1

Type 11

21.07 ± 7.22 13.74 ± 6.74* 23.13 ± 10.49

20.84 ± 6.71 13.69 ± 5.54* 20.11 ± 8.33

each group, the postsynaptic area of junctional folds and clefts associated with a given muscle fiber was measured by morphometry using the line sampling method [19] and expressed in square microns. The significance of the differences between the groups was determined by Student's t-test. All P values below 0.05 were considered significant. In control animals, the mean postsynaptic area of endplates from type I and type II muscle fibers were found not to be significantly different from each other, being 21.07 + 7.22 and 20.84 +_ 6.71 ~m 2, respectively (see Table I). Both type 1 (Fig. IA) and type II (Fig. IB) endplates from control animals exhibited abundant primary and secondary postjunctional folding and overlying nerve terminals. in the denervated-non-stimulated muscles, the postsynaptic area of type 1 (Fig. 2A) and type II (Fig. 2B) fibers were found to be significantly reduced as compared to controls. In the denervated-stimulated muscles, the type I (Fig. 2C) and type II (Fig. 2D) fibers postsynaptic area was observed to be similar in size to controls (Table I). Ultrastructurally, the junctional folding in both the type I and type 11 endplates tended to be more regular in the denervated-stimulated group as compared to that seen in the denervated-non-stimulated group. The synaptic clefts of type I and type II endplates in the denervated-stimulated muscles were better delineated, and tended to have a cup-shaped appearance, than corresponding endplates found in the denervated-non-stimulated muscles. Additionally, the postsynaptic regions of type l and type II fibers in the denervated-non-stimulated muscles exhibited greater amounts of junctional fold breakdown as well as globular residues in their synaptic spaces. In the present study, the postsynaptic area of clefts and folds of normal control type 1 and type II muscle fibers were found to be similar to each other. This is in agreement with the findings of Nystrom'[13] and Santa and Engel [19] that endplates on fast (type II) and slow (type I) muscle fibers are comparable in size. Following denervation, endplates from type I and type II muscle fibers were observed to undergo comparable degenerative changes, the most prominent alteration be-

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282

ing a flattening of the primary and secondary synaptic clefts associated with an irregularity of the postjunctional folds. Such morphological f'mdings are consistent with the results of several other investigators [2, 18]. In contrast to these observatior, s, PuUiam and April [16] have reported that endplates on type II muscle fibers were more sensitive to the degenerative changes following denervation. Their results indicated that the primary cleft rather than the secondary deft structure exhibited greater sensitivity to degeneration. They concluded that the maintenance of the cupshaped primary cleft is dependent upon neurotrophic factors while secondary cleft structures were not. Our results, however, provide quantitative evidence to show that activity, as seen in the denervated-stimulated muscles, is also an important fac.tot in maintaining both primary and secondary cleft structure. In support of our findings, Brown et al. [2] have demonstrated in mouse muscle that activity is needed for the maintenance of endplate structure. They used botulinum toxin to produce paralysis in mouse soleus and EDL muscles and compared them to muscles which were denervated. They found that endplates from both experimental groups exhibited comparable changes and concluded that activity was therefore necessary for endplate maintenance. Additionally, Duxson [3] has shown that inactivation of the A Ch receptors by e~-bungarotoxin in developing rat soleus muscle retards the maturation of the neuromuscular junction. He concluded that activity of the postsynaptic structures, rather than just the presence of the nerve is necessary for the normal development of the junctional folding of the muscle membrane. In conclusion, our results demonstrate that electrically-induced contractile activitiy is important in maintaining endplate structure; however, they do not rule out the possible parallel role(s) of axonally-transported neurotrophic influences. The authors wish to acknowledge the technical assistance of Ruth Johnston, Kevin Phelan, and Barbara Zimmer. This study was supported by Grant G008300071 from the National Institute of Handicapped Research, U.S. Department of Education, Washington, DC. ! A×elsson, J. and Thesleff, S., Study of supersensitivity in denervated mammalian skeletal muscle, J. Physiol. (Lond.), 147 tl959) 178-193. 2 Brown, M.C., Hopkins, W.G., Keynes, R.J. and White, !., A comparison of early morphological changes at denervated and paralyzed endplates ia fast and slow muscles of the mouse, Brain Res., 248 (1982) 382-386. 3 Duxson, M.J., The effect of postsynaptic block on development on the neuromuscular junction in postnatal rats, J. Neurocytol., il (1982) 395-408. 4 Engel, A.G. and Stonnington, H.H., Morphological effects of denervation of muscle: quantitative ultrastructural study, Ann. N.Y. Acad. Sci., 228 (1974) 68-88. 5 Gilliatt, R.W., Westgaard, R.H. and Williams, i.R., Extrajunctional acetylcholine sensitivity of inactive muscle fibers in baboon during prolonged nerve pressure block, J. Physiol. (Lond.), 280 (1978) 499-514. 6 Guth, L. and Albuquerque, E.X., Neurotrophic regulation of resting membrane potential and extrajunctional acetylcholine sensitivity in mammaliai~ skeletal muscle. In A. Mauro (Ed.), Muscle Regeneration, Raven Press, 1979, pp. 405-415.

283 7 Guth, L., Kemerer, V.F., Samaras, T.A., Warnick, J.E. and Albuquerque, E.X., The roles of disuse and loss of neurotrophic function in denervation atrophy of skeletal muscles, Exp. Neurol., 73 (1981) 20-36. 8 Lomo, T., Westgaard, R.H. and Dahl, H.A., Contractile properties of muscle control by pattern of muscle activity in the rat, Proc. roy. Soc. B, 187 (1974) 99-103. 9 Lomo, T., Role of activity in control of membrane and contractile properties of skeletal muscle. In S. Thesleff (Ed.), Motor Innervation of Muscle, Academic Press, 1976, pp. 289-321. 10 Lomo, T. and Slater, C.R., Control of junctional acetylcholinesterase by neural and muscular "~,~fluences in the rat, J. Physiol. (Lond.), 303 (1980) 191-202. I 1 Miledi, R. and Slater, C.R., On the degeneration of rat neuromuscular junction after nerve ~ction, J. ?hysiol. (Lond.), 207 (1970) 507-528. 12 Nemeth, P.M., Electrical stimulation of denervated muscle prevents decreases in oxidative enzymes, Muscle and Nerve, 5 (1982) 134-139. 13 Nystrom, B., Postnatal development of motor nerve terminals in 'slow-red' and 'fast-white' cat muscles, Acta neurol, scand., 44 (1968) 363-383. 14 Pachter, B.R., Eberstein, A. and Goodgold, J., Electrical stimulation effect on denel ,~ated skeletal myofibers in rats: a light and electron microscopic study, Arch. Phys. Med. Rehabi1., 63 (1982) 427-430. 15 Padykula, H.A. and Gauthier, G.F., Morphological and cytochemical characteristics o~" fiber types in normal mammalian skeletal muscle, Excerpta Med. Int. Congr. S¢~r. No. 147, 1967, I'P- 117-128. 16 Pulliam, D.L. and April, E.W., Degenerative changes at neuromuscular !unctions of red, white and intermediate muscle fibers, J. Ne,jrol., 43 (1979) 205-222. 17 Rubin, L., Schuetze, S., Weill, C. and Fischbach, G., Regulation of acetylcholines-:erase appearance at neuromuscular junctions in vitro, Nature (Lond.), 283 (1980) 264-267. 18 Saito, A. and Zacks, S.I., Fine structure observations of degeaeration and reinn-.rvation of neuromuscular junctions in mouse foot muscle, J. Bone Jt. Surg., 51 (1967) 1163-1178. 19 Santa, T. and Engel, A.G., Histometric analysis of neuromuscular junction ultrastructure in rat red, white and intermediate muscle fibers. In J.E. Desmedt (Ed.), New Developments in Elcctromyography and Clinical Nettrophysiology, Vol. I, Karger, Basel, 1973, pp. 41-54. 20 Westgaard, R.H., Influence of activity on the passive electrical properties of denervated soleus muscle fibers in the rat, J. Physiol. (Lond.), 215 (1975) 683-697.