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On the appearance of acetylcholine receptors in denervated rat diaphragm, and its dependence on nerve stump length
Brain Research, 153 (1978) 539-548
539
((~ Elsevier/North-HollandBiomedicalPress
ON THE APPEARANCE OF ACETYLCHOLINE RECEPTORS IN DENERVATED RAT DIAPHRAGM, AND 1TS DEPENDENCE ON NERVE STUMP LENGTH
O. UCHITEL and N. ROBBINS Department of Anatom.v, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 (U.S.A.) (Accepted January 18th, 1978)
SUMMARY
Acetylcholine (ACh) sensitivity and extrajunctional receptor distribution of the rat diaphragm were closely monitored during the early period following denervation. Both contracture in response to l0 #g/ml of ACh and extrajunctional binding of [leSl]alpha-bungarotoxin ([125I]a-BTX) were first detectable 30 h after cutting the phrenic nerve in the thorax. If the nerve were cut more proximally, leaving a 3.5 cm distal nerve stump, the same level o f A C h contracture and [125I]a-BTX binding did not appear until 40 h after operation. This 10-h delay was far longer than the 3-h delay in transmission failure reportedly dependent on stump length. The earliest detectable extrajunctional [125I]a-BTX binding appeared throughout the entire muscle fiber, and was not localized to the endplate region as would be expected if degeneration in the nerve terminal induced new receptors. However, later significant increases in [125I]a~ BTX binding at the endplate region could have resulted from such degeneration. All these results are consistent with neurotrophic regulation of muscle ACh receptors, working via a mechanism involving axonal transport.
INTRODUCTION
Acetylcholine receptors (AChR) in adult mammalian muscle fibers are mostly confined to the synaptic region, but after denervation they appear in the extrajunctional region as well2,t0, 21. Some workers assert that the lack of activity which follows denervation is the main f,~ctor in triggering the appearance of extra junctional AChR 17. However, silencing a normal muscle by blocking the motor nerve with tetrodotoxin does not produce extrajunctional receptors as rapidly as denervation15, 23. In addition, the time of development of denervation changes can be correlated with the length of the nerve stump. Acetylcholine (ACh) sensitivity~,V, 25, fibrillation as, membrane depo-
540 larization 6 and tetrodotoxin resistance 9 appear sooner when the nerve is cut close to the muscle than when it is cut more proximally. Since activity is presumably abolished in the distal and proximal denervated muscles at the same time, the delayed appearance of denervation phenomena in the long-stump dev.ervated muscle suggests that other factors in addition to activity control muscle properties. This important interpretation can be questioned in the light of two findings. First, nerve section but not short term tetrodotoxin exposure produces rapid degeneration of nerve terminals, and this degeneration is delayed by the presence of a long nerve stump 22. Second, the mere placement of a piece of degenerating nerve on an innervated muscle induces localized increase in ACh sensitivity in the underlying region 11. Indeed, it is claimed 5 (but contested 24) that innervated muscles lying adjacent to denervated muscles show denervation changes such as resistance to tetrodotoxin. Thus, the early appearance of AChR after short-nerve denervation, in comparison to long-nerve denervation or to tetrodotoxin paralysis, could result simply from nerve terminal degeneration, and not from the loss of atrophic factor. In view of the importance of the nerve stump experiments in understanding the neuronal control of AChR, we have subjected this contention to experimental test. If the initial appearance of extrajunctional AChR after denervation resulted only from nerve terminal degeneration 17, then the first extrajunctional AChR should appear only in the perisynaptic region, i.e., the initial findings should mimic those in which a degenerating nerve is placed on innervated muscle 1~. On the other hand, a metabolic response of the entire muscle to disuse or loss of trophic factors could occur initially either at the endplate or throughout the entire muscle surface. If the latter were true, the result would make it highly unlikely that nerve terminal degeneration products initiate the early extrajunctional AChR response after short-nerve denervation. Although results with iontophoretic techniques suggest that extrajunctional AChR after denervation can be several mm from the endplate, previous experiments were not specifically timed to catch and localize the earliest response. Furthermore, Hartzell and Fambrough 1° (see their Table II and Fig. 2) showed that the iontophoretic technique is not as sensitive as labelled bungarotoxin binding in detecting or quantitating tow levels of extrajunctional AChR. Indeed, standard deviations with contemporary iontophoretic techniques may be as high as 40 %oof the mean ~, so that an early increase in AChR might be missed. Therefore, we have used both the ACh contracture and in particular a-bungarotoxin binding, in order to characterize the locus and time of development of postdenervation extrajunctional AChR. In addition, by using these techniques in the early period after denervations with differing nerve stump lengths, we characterized the delay in onset of extrajunctional AChR as a function of time and nerve length. In this way, we could relate dependence on nerve stump to the literature on axonal transport. Again, the published data, using iontophoretic technique, did not always show a correlation with nerve length (cf. Table VI in Ref. 6), possibly due to difficulties in sampling and in the variability of the method. Therefore, a sensitive and quantitative averaging technique, such as abungarotoxin labeling, seemed appropriate.
541 METHODS All experiments were done on diaphragm muscles of 90-120 g female SpragueDawley rats. Operations were performed under ether anesthesia. Rats were kept in a 12/12 h day-night cycle and the operations were usually performed around midnight. For a 'distal denervation', the left thorax was opened a few mm between the ribs, and the phrenic nerve was pulled with a glass hook and cut close to the muscle, leaving an extramuscular stump of 0.5 cm. The right thorax was opened in a similar manner as a sham operation. For a 'proximal denervation', the phrenic nerve was cut in the neck and the left and right thorax were opened as above for sham operations. The difference in the nerve stump length between proximal and distal denervation was approximately 3 cm. In all cases the right diaphragm was used as a normal control. In a few experiments, in order to have a distal and proximal denervated region in the same diaphragm, the left phrenic nerve was cut in the neck and in addition an intramuscular branch of the phrenic nerve was cut via an abdominal approach. Strips of muscle more than 2 m m from the intramuscular injury, supplied by either a long or a short nerve stump, were taken for analysis. One to 7 days after denervation, the rats were killed by concussion and the muscles were rapidly removed. In this series, muscles were tested for sensitivity to acetylcholine (ACh) by use of the isotonic contractile response. An 8 mm wide strip was dissected and pinned through the ribs to the bottom of a 4 cc vertical chamber filled with Krebs-Ringer solution and aerated with 95% 0 2 - 5 % COe at room temperature (22-24 °C). The tendon was connected to a Harvard isotonic transducer and the output voltage displayed on a Brush pen recorded system. ACh contractures were induced by rapidly changing the bath solution to Krebs-Ringer with ACh. Two contractures were elicited with a 10 min interval. The first contracture was expressed as per cent of the 0. I M K2SO4 contracture elicited at the end of the experiment. a-Bungarotoxin (Miami Serpentarium) was further purified by ion exchange column chromatography on Whatman CM-32. The toxin was labeled with 125I, using the chloramine T method 10, to a specific activity of 0.4 to 1.2 × 105 Ci/M. The toxicity was determined by studying the time to half or complete transmission block of a rat phrenic nerve-diaphragm preparation. Labeled toxin was 20 °/o less potent than cold toxin. Preliminary dose and exposure time studies showed that endplate receptors of normal diaphragms were saturated with toxin after a 3 h exposure at a concentration of 1.3 #g/ml. Specificity was demonstrated by the small per cent of non-endplate binding in normal muscles (see below). In all experiments, strips of diaphragm muscles were incubated in 1.5 #g/ml o f [1251]a-bungarotoxin in Krebs-Ringer solution for 3 h at 4 "C, then rinsed overnight in several changes of Krebs-Ringer, fixed in 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and stained for cholinesterase activity 1~. Under a dissection microscope, the stained muscles were cut into a 4 m m endplate strip and two 2 mm wide non-endplate strips. After being weighed, the strips were homogenized in 0.15 M N a C I , 0.4 m M sodium EDTA, 0.02 M Tris (pH 7.4) and 1% triton X-100 solution and the radioactivity counted by liquid scintillation spectrometry. Normal muscles
542 were always incubated in parallel with experimentals and the binding of the non-endplate region of these normal controls was considered 'non-specific' and subtracted on a weight basis from the binding of the experimental ones. The subtraction method has the problem that variation in 'non-specific' extrajunctional binding might obscure very minute increases in extrajunctional binding. Still, statistically significant increases in extrajunctional binding were detectable as early as 30 h after distal denervation (see Results). Binding at the 'endplate region', that is, in a segment of muscle containing endplates, was calculated by subtracting the amount of toxin bound to the nonendplate region, from the total amount of toxin bound to the endplate on a weight basis (endplate region binding/mg total endplate region binding/rag ...... nonendplate binding/mg). In some cases, a Sorvall tissue chopper was used to cut the muscle into 1 m m strips parallel to the end-plate. In these experiments, each strip was weighed, digested in concentrated nitric acid and assayed for radioactivity. All data are presented as mean i standard error of the mean. -
-
RESULTS
Isotonic contractile response to acetylcholine In order to find the earliest time of increased sensitivity to ACh, the isotonic response to ACh was tested in distal and proximal denervated muscles 24-40 h after denervation. Normal muscles showed no contraction in response to 10 #g/ml ACh. A significant response to this concentration first appeared in the 26 h distal denervated muscle and reached 35 ~ of the K contracture at 30 h after denervation (Fig. 1A). I n contrast, at 30 h after denervation, the proximal denervated muscle developed only a small response, and did not reach a comparable sensitivity until about 40 h after denervation (Fig. 1A). The dose-response curve also illustrates the profound differ-
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Fig. 1. A: isotonic contractile response to 10 t,g/ml ACh in proximal and distal (]enervated muscles as a function of time after denervation. Each point is the Mean --3-_-S.E. of 4 or more muscles (except 28 h, n ~ l ). B: ACh dose-response curve in 30 h proximal and distal denervated diaphragms.
543 ence in A C h sensitivity between muscles denervated 30 h with either a short or a long nerve stump (Fig. 1B). In two muscles studied after 55 h of denervation, a difference in sensitivity with lower doses o f A C h was also found. In order to eliminate any assymetries due to cholinesterase activity, neostigmine 2 • 10 -6 g/ml was added to the Ringer solution in a few experiments. A 6-8 fold decrease in ACh concentration was required to reproduce the same response as obtained in the absence of neostigmine, but the differential sensitivity of 30 h proximal and distal denervated muscle was still clearly present.
[rzsl]a-bungarotoxin binding in denervated muscles The extrajunctional binding of [125I]a-bungarotoxin (BTX) was studied in proximal and distal denervated muscles 30 h-7 days after denervation (Fig. 2). As early as 30 h after a distal denervation, a significant increase in extrajunctional toxin binding was detected. Similar results have been reported by Berg and Hall 3. Muscles treated with labeled toxin bound 104 -k 17 cpm/mg of non-endplate muscle (n=4). Normal and 30 h proximal denervated muscle bound 18.6 :k 4 (n=6) and 32.3 _-t:_5 (n--4) cpm/mg of non-endplate muscle, respectively. The increase in non-endplate binding at later times after denervation appeared to follow the same pattern in both types of denervated muscles but the proximal denervated muscles showed a delay of about 10 h compared to distal denervated muscles. Binding in the endplate region (after subtraction of extrajunctional binding, see Methods) of distal and proximal denervated muscles was not significantly different at any time. However, 40 h after denervation, the endplate region of both proximal and distal denervated muscles
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Fig. 3. Binding of [~zSI]a-BTXto end plate region of innervated (shaded) or of proximal and distal denervated muscles as a function of time after denervation. Each point is the Mean ~ S.Ei of 4 or more muscles (except 170 h proximal denervated muscles, n = 3). *, P < 0.02 for the null hypothesis between innervated and either proximal or distal denervated muscles. bound significantly more toxin than controls. This difference reached a maximum at 90 h and disappeared by 7 days after denervation (Fig. 3). In order to precisely determine the macroscopic distribution of extrajunctional receptors when they were first detectable, muscles denervated 30 h were treated with labeled toxin and then cut into 1 mm strips parallel to the line of endptates. The non-endplate segments of distal denervated muscle (segments 1--4 and 9-I 1 in Fig. 4) all showed significant increases in the amount of toxin binding compared to the same segments of normal or proximal denervated muscle, both of which showed similar and uniformly low levels of extrajunctional binding (Fig. 4). Further, the amount of toxin bound to the different non-endplate segments of distal denervated muscles was statistically the same, except for segment 1, where the lower number of binding sites may reflect an increased proportion of connective tissue. Localized binding in the endplate region could not be well characterized because of the variable numbers of endplates included per 1 mm segment in this region. Thus, small shifts in endplate location could produce considerable variation in the profiles of segments 5-8, as seen in Fig. 4. Nonetheless, as noted above, the summed binding in segments 5-8 (after subtraction of extrajunctional binding) was not increased 30 h after denervation (Fig. 3). Lomo and Westgaard TM found that simply exposing an innervated muscle during an operation could cause a transitory increase in ACh sensitivity. In the present study, it was necessary to pull the nerve in order to cut it close to the diaphragm. The sham operation of pulling without cutting would be ambiguous because of nerve injury due to pulling alone. Therefore, to eliminate the possibility of muscle injury as the basis of the early response to distal denervation, the left phrenic nerve of 3 rats was cut in the
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neck and at the same time the intramuscular ventral branch was cut via an intraabdominal approach. After this procedure, the same hemidiaphragm had a distal denervated ventral region and a proximal denervated dorsal one. At 40 h after this operation, extrajunctional binding of [125I]a-BTX in the distal denervated region was three times higher than in the proximal denervated muscle strip. DISCUSSION
Our results show a clear difference in the time course of appearance of extrajunctional acetylcholine sensitivity, depending on the length of nerve left attached to muscle after denervation. Comparable data have been reported by Deshpande and Albuquerque 6 using iontophoretic techniques and by Emmelin and Malm 7, who determined threshold doses of ACh for isotonic contracture. In the latter study, the maximum difference dependent on nerve stump length occurred at 4 days after denervation, while our results showed the greatest difference at 30-60 h. The apparent discrepancy may result from different measures of ACh sensitivity (threshold response
546 vs. response to a standard dose or [125I]a-BTX binding). Also, Emmelin and Maim 7 did not examine the early postdenervation period extensively. In our experiments, the isotonic ACh contracture was a sensitive method to detect the earliest postdenervation increase in ACh sensitivity, possibly because the measured response reflects the shortening of the most sensitive and superficial fibers to acetylcholine 19. Indeed, in preliminary experiments we found that the magnitude of the K-contracture was unaltered in diaphragm strips until more than 3,/4 of the muscle fibers were cut. In any event, both the ACh contracture and [12~I]a-BTX binding showed the same post-denervation time dependence on nerve length. It has been reported that merely exposing a muscle during an operation can produce a transitory increase in sensitivity to ACh 16. This explanation would not account for the results of experiments in which the same exposed diaphragm muscle was subjected to both distal and proximal denervation, in which the nerve stump dependent time delay was still present. Jones and VyskociP 1 have reported that nerve degeneration "per se" produces a localized increase in ACh sensitivity restricted to 1 mm on either side of the site of the nerve degeneration. Since degeneration of the terminals occurs more rapidly if the nerve is cut close to the muscle than if it is cut far away '~z, it has been argued that the nerve stump dependence of the development of denervation effects is due to the degeneration of the terminals 17. This explanation now seems unlikely in the case of extrajunctional AChR of rat diaphragm. We found that the difference in the amount of toxin binding sites between short and long stump denervated muscles is present in extrajunctional segments as far as 4 mm from the endplate region, i.e.. at sites considerably far away from regions reportedly affected by nerve degeneration 11. On the other hand, the increase in the number of toxin binding sites at the endplate region, which first occurred 10 h after such sites had appeared throughout the whole muscle fiber, would be consistent with a localized 'inflammatory' response to degenerating nerve. Nonetheless, without additional data (e.g. autoradiography with labeled BTX), we cannot distinguish between a regional increase in receptors around the endplate and an increase in junctional endplate receptors per se. No significant differences were found between distal and proximal denervated muscles at the endplate region at any time after denervation. Biochemical assay of a-bungarotoxin binding sites yields an average density of receptors in a given sample, and obscures the microheterogeneity of patches of high density of AChR known to occur in denervated muscle 13. However, at 4 days after denervation, these AChR patches are extremely small and not invariably present ~3. Since much of our data was obtained after shorter periods of denervation, an autoradiographic approach would not be as useful as the biochemical averaging technique we employed. In any event, regardless of any microheterogeneity underlying the averaged data, the point remains that the early effect of denervation of AChR is not localized to the endplate region, whereas nerve terminal degeneration, as a cause of extrajunctional AChR increase shortly after denervation, would exert an effect localized to the endplate region. Our finding may not apply to all denervated muscles, since there is electrophysiological evidence that in rat limb muscles, increased extra-
547 junctional ACh sensitivity is not uniform throughout the length of the muscle fiberL Our results also give quantitative information on the time delay in the development of extrajunctional receptors as a function of nerve stump length. Miledi and Slater 22 reported that after phrenic nerve section, failure of neuromuscular transmission, disappearance of miniature endplate potentials, and nerve terminal degeneration were delayed about 45 min/cm of nerve stump length. In our case, the delaying effect of a long nerve stump length was considerably longer (180 min/cm). Therefore, it is unlikely that the lack of miniature endplate potentials, the failure of transmission, or, most important, nerve terminal degeneration is directly related to the nerve length dependent increase in sensitivity to ACh. In addition, the electrical activity of muscles denervated with either a short or long nerve stump is assumed to be either nill or identical. Thus, the experiments reported here are consistent with the hypothesis that extrajunctional ACh receptors are at least under partial control of some trophic factor which depends on axonal transport. This axonal transport could either maintain the terminal neurotrophic function or, more directly, convey the trophic substance itself. Since 3 cm of nerve stump delays the appearance of extrajunctional A C h R for at least 10 h, the trophic factor(s) could be transported at a rate no less than 72 mm/day. Indeed, axonal transport in this range, which is intermediate between slow and fast transport, has been reported4,t4, z0. However, our computation assumes a single rate of transport, whereas multiple rates, retrograde transport, or recycling of transported material within the nerve stump could be involved. ACKNOWLEDGEMENT This grant was supported by a grant from the Muscular Dystrophy Associations of America to N.R., and an International Postdoctoral Fellowship to O.U.
REFERENCES I Albuquerque, E. X. and McIsaac, R. J., Early development of acetylcholinereceptors on fast and slow mammalian skeletal muscle, Life Sci., 8 (1969) 409-416. 2 Axelsson,J. and Thesleff, S., A study of supersensitivity in denervated mammalian skeletal muscle, J. Physiol. (Lond.), 147 (1959) 178-193. 3 Berg, D. K. and Hall, Z. W., Increased extrajunctional acetylcholinesensitivity produced by chronic post-synaptic neuromuscular blockade, J. Physiol. (Lond.), 244 (1975) 659-676. 4 Bradley, W. G., Murchison, D. and Day, M. J., The range of velocities of axoplasmic flow. A new approach, and its application to mice with genetically inherited spinal muscular atrophy, Brain Research, 35 (1973) 185-197. 5 Cangiano, A. and Lutzemberger, L., Partial denervation affects both denervated and innervated fibers in the mammalian skeletal muscle, Science, 196 (1977) 542-545. 6 Desbpande, S. S., Albuquerque, E. X. and Guth, L., Neurotrophic regulation of prejunctional and postjunctional membrane at the mammalian motor endplate. Exp. Neurol., 53 (1976) 151-165. 7 Emmelin,N. and Malm,L.,Development ofsupersensitivity as dependent on thelength ofdegenerating nerve fibres, Quart. J. exp. Physiol., 50 (1965) 142-145. 8 Fambrough, D. M.,Acetylcholinesensitivityofmusclefibermembranes:mechanismofregulation by motoneurons, Science, 168 (1970) 372-373. 9 Harris, J, B. and Thesleff, S., Nerve stump length and membrane changes in denervated skeletal muscle, Nature New Biol., 236 (1972) 60-61.
548 10 Hartzell, H. C. and Fambrough, D. M., Acetylcholine receptors: distribution anti extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity, J. eeo. Physiol.. 60 (1972) 248-262. 11 Jones, R. and Vyskocil, F., An electrophysiological examination of the changes in skeletal muscle fibres in response to degenerating nerve tissue, Brain Research, 88 (1975) 309 317. 12 Karnovsky, J. and Roots, L., A 'direct-coloring' thiocholine method for cholinesterase, .I. Hi.~tochem. Cytochem., 12 (t964) 219-221. 13 Ko, P. K., Anderson, M. J. and Cohen, M. W., Denervated skeletal muscle fibers develop discrete patches of high acetylcholine receptor density, Science, 196 (1977) 540-542. 14 Lasek, R. J., Protein transport in neurons, Int. Rev. Neurobiol., 13 (1970) 289 32t. 15 Lavoie, P. A., Collier, B. and Tenenhouse, A., Comparison of a-bungarotoxin binding to skeletal muscle after inactivity or denervation, Nature (Lond.), 260 (1976) 349-350. 16 Lomo, T. and Westgaard, R. H., Further studies on the control of ACh sensitivity by muscle activity in the rat, J. Physiol. (Lond.), 252 (1975) 603-626. 17 Lomo, T. and Westgaard, R. H., Control of ACh sensitivity in rat muscle fibers, Cold Spr. Harb. Syrnp. quant. Biol., 40 (1976) 263-274. 18 Luco, J. V. and Eyzaguirre, C., Fibrillation and hypersensitivity to ACh in denervated muscle: effect of length of degenerating nerve fibers, J. Neurophysiol., 18 (1955) 65 73. 19 Lullmann, H., Preuner, J. and Schaube, H., A kinetic approach for an interpretation of the acetylcholine-D-tubocurarine interaction on chronically denervated skeletal muscle, Namo,n-Schmiedeberg's Arch. exp. Path. Pharrnaeol., 281 (1974) 415-426. 20 Lux, H. D., Schubert, P., Kreutzberg, G. W. and Globus, A., Excitation and axonal flow: autoradiographic study on motoneurons intracellularly injected with a 3H-amino acid, Exp. Brain Res., 10 (1970) 197-204. 21 Miledi, R., The acetylcholine sensitivity of frog muscle fibers after complete or partial denervation, J. Physiol. (Lond.), 151 (1960) 1 23. 22 Miledi, R. and Slater, C. R., On the degeneration of rat neuromuscular junctions after nerve section, J. Physiol. (Lond.), 207 (1970) 507-528. 23 Pestronk, A., Drachman, D. B. and Griffin, J. W., Effect of muscle disuse on acetylchotine receptors, Nature (Lond.), 260 (1976) 352- 354. 24 Tiedt, T. N., Albuquerque, E. X. and Guth, L., Degenerating nerve fiber products do not alter physiological properties of adjacent innervated skeletal muscle fibers, Science, 198 (t977) 839 841. 25 Uchitel, O. D.,Development of supersensitivity in denervated slow muscle of the frog. ltsdependence on the nerve stump length, Acta physiol, lat.-amer., 25 (1975) 34-38.
539
((~ Elsevier/North-HollandBiomedicalPress
ON THE APPEARANCE OF ACETYLCHOLINE RECEPTORS IN DENERVATED RAT DIAPHRAGM, AND 1TS DEPENDENCE ON NERVE STUMP LENGTH
O. UCHITEL and N. ROBBINS Department of Anatom.v, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 (U.S.A.) (Accepted January 18th, 1978)
SUMMARY
Acetylcholine (ACh) sensitivity and extrajunctional receptor distribution of the rat diaphragm were closely monitored during the early period following denervation. Both contracture in response to l0 #g/ml of ACh and extrajunctional binding of [leSl]alpha-bungarotoxin ([125I]a-BTX) were first detectable 30 h after cutting the phrenic nerve in the thorax. If the nerve were cut more proximally, leaving a 3.5 cm distal nerve stump, the same level o f A C h contracture and [125I]a-BTX binding did not appear until 40 h after operation. This 10-h delay was far longer than the 3-h delay in transmission failure reportedly dependent on stump length. The earliest detectable extrajunctional [125I]a-BTX binding appeared throughout the entire muscle fiber, and was not localized to the endplate region as would be expected if degeneration in the nerve terminal induced new receptors. However, later significant increases in [125I]a~ BTX binding at the endplate region could have resulted from such degeneration. All these results are consistent with neurotrophic regulation of muscle ACh receptors, working via a mechanism involving axonal transport.
INTRODUCTION
Acetylcholine receptors (AChR) in adult mammalian muscle fibers are mostly confined to the synaptic region, but after denervation they appear in the extrajunctional region as well2,t0, 21. Some workers assert that the lack of activity which follows denervation is the main f,~ctor in triggering the appearance of extra junctional AChR 17. However, silencing a normal muscle by blocking the motor nerve with tetrodotoxin does not produce extrajunctional receptors as rapidly as denervation15, 23. In addition, the time of development of denervation changes can be correlated with the length of the nerve stump. Acetylcholine (ACh) sensitivity~,V, 25, fibrillation as, membrane depo-
540 larization 6 and tetrodotoxin resistance 9 appear sooner when the nerve is cut close to the muscle than when it is cut more proximally. Since activity is presumably abolished in the distal and proximal denervated muscles at the same time, the delayed appearance of denervation phenomena in the long-stump dev.ervated muscle suggests that other factors in addition to activity control muscle properties. This important interpretation can be questioned in the light of two findings. First, nerve section but not short term tetrodotoxin exposure produces rapid degeneration of nerve terminals, and this degeneration is delayed by the presence of a long nerve stump 22. Second, the mere placement of a piece of degenerating nerve on an innervated muscle induces localized increase in ACh sensitivity in the underlying region 11. Indeed, it is claimed 5 (but contested 24) that innervated muscles lying adjacent to denervated muscles show denervation changes such as resistance to tetrodotoxin. Thus, the early appearance of AChR after short-nerve denervation, in comparison to long-nerve denervation or to tetrodotoxin paralysis, could result simply from nerve terminal degeneration, and not from the loss of atrophic factor. In view of the importance of the nerve stump experiments in understanding the neuronal control of AChR, we have subjected this contention to experimental test. If the initial appearance of extrajunctional AChR after denervation resulted only from nerve terminal degeneration 17, then the first extrajunctional AChR should appear only in the perisynaptic region, i.e., the initial findings should mimic those in which a degenerating nerve is placed on innervated muscle 1~. On the other hand, a metabolic response of the entire muscle to disuse or loss of trophic factors could occur initially either at the endplate or throughout the entire muscle surface. If the latter were true, the result would make it highly unlikely that nerve terminal degeneration products initiate the early extrajunctional AChR response after short-nerve denervation. Although results with iontophoretic techniques suggest that extrajunctional AChR after denervation can be several mm from the endplate, previous experiments were not specifically timed to catch and localize the earliest response. Furthermore, Hartzell and Fambrough 1° (see their Table II and Fig. 2) showed that the iontophoretic technique is not as sensitive as labelled bungarotoxin binding in detecting or quantitating tow levels of extrajunctional AChR. Indeed, standard deviations with contemporary iontophoretic techniques may be as high as 40 %oof the mean ~, so that an early increase in AChR might be missed. Therefore, we have used both the ACh contracture and in particular a-bungarotoxin binding, in order to characterize the locus and time of development of postdenervation extrajunctional AChR. In addition, by using these techniques in the early period after denervations with differing nerve stump lengths, we characterized the delay in onset of extrajunctional AChR as a function of time and nerve length. In this way, we could relate dependence on nerve stump to the literature on axonal transport. Again, the published data, using iontophoretic technique, did not always show a correlation with nerve length (cf. Table VI in Ref. 6), possibly due to difficulties in sampling and in the variability of the method. Therefore, a sensitive and quantitative averaging technique, such as abungarotoxin labeling, seemed appropriate.
541 METHODS All experiments were done on diaphragm muscles of 90-120 g female SpragueDawley rats. Operations were performed under ether anesthesia. Rats were kept in a 12/12 h day-night cycle and the operations were usually performed around midnight. For a 'distal denervation', the left thorax was opened a few mm between the ribs, and the phrenic nerve was pulled with a glass hook and cut close to the muscle, leaving an extramuscular stump of 0.5 cm. The right thorax was opened in a similar manner as a sham operation. For a 'proximal denervation', the phrenic nerve was cut in the neck and the left and right thorax were opened as above for sham operations. The difference in the nerve stump length between proximal and distal denervation was approximately 3 cm. In all cases the right diaphragm was used as a normal control. In a few experiments, in order to have a distal and proximal denervated region in the same diaphragm, the left phrenic nerve was cut in the neck and in addition an intramuscular branch of the phrenic nerve was cut via an abdominal approach. Strips of muscle more than 2 m m from the intramuscular injury, supplied by either a long or a short nerve stump, were taken for analysis. One to 7 days after denervation, the rats were killed by concussion and the muscles were rapidly removed. In this series, muscles were tested for sensitivity to acetylcholine (ACh) by use of the isotonic contractile response. An 8 mm wide strip was dissected and pinned through the ribs to the bottom of a 4 cc vertical chamber filled with Krebs-Ringer solution and aerated with 95% 0 2 - 5 % COe at room temperature (22-24 °C). The tendon was connected to a Harvard isotonic transducer and the output voltage displayed on a Brush pen recorded system. ACh contractures were induced by rapidly changing the bath solution to Krebs-Ringer with ACh. Two contractures were elicited with a 10 min interval. The first contracture was expressed as per cent of the 0. I M K2SO4 contracture elicited at the end of the experiment. a-Bungarotoxin (Miami Serpentarium) was further purified by ion exchange column chromatography on Whatman CM-32. The toxin was labeled with 125I, using the chloramine T method 10, to a specific activity of 0.4 to 1.2 × 105 Ci/M. The toxicity was determined by studying the time to half or complete transmission block of a rat phrenic nerve-diaphragm preparation. Labeled toxin was 20 °/o less potent than cold toxin. Preliminary dose and exposure time studies showed that endplate receptors of normal diaphragms were saturated with toxin after a 3 h exposure at a concentration of 1.3 #g/ml. Specificity was demonstrated by the small per cent of non-endplate binding in normal muscles (see below). In all experiments, strips of diaphragm muscles were incubated in 1.5 #g/ml o f [1251]a-bungarotoxin in Krebs-Ringer solution for 3 h at 4 "C, then rinsed overnight in several changes of Krebs-Ringer, fixed in 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and stained for cholinesterase activity 1~. Under a dissection microscope, the stained muscles were cut into a 4 m m endplate strip and two 2 mm wide non-endplate strips. After being weighed, the strips were homogenized in 0.15 M N a C I , 0.4 m M sodium EDTA, 0.02 M Tris (pH 7.4) and 1% triton X-100 solution and the radioactivity counted by liquid scintillation spectrometry. Normal muscles
542 were always incubated in parallel with experimentals and the binding of the non-endplate region of these normal controls was considered 'non-specific' and subtracted on a weight basis from the binding of the experimental ones. The subtraction method has the problem that variation in 'non-specific' extrajunctional binding might obscure very minute increases in extrajunctional binding. Still, statistically significant increases in extrajunctional binding were detectable as early as 30 h after distal denervation (see Results). Binding at the 'endplate region', that is, in a segment of muscle containing endplates, was calculated by subtracting the amount of toxin bound to the nonendplate region, from the total amount of toxin bound to the endplate on a weight basis (endplate region binding/mg total endplate region binding/rag ...... nonendplate binding/mg). In some cases, a Sorvall tissue chopper was used to cut the muscle into 1 m m strips parallel to the end-plate. In these experiments, each strip was weighed, digested in concentrated nitric acid and assayed for radioactivity. All data are presented as mean i standard error of the mean. -
-
RESULTS
Isotonic contractile response to acetylcholine In order to find the earliest time of increased sensitivity to ACh, the isotonic response to ACh was tested in distal and proximal denervated muscles 24-40 h after denervation. Normal muscles showed no contraction in response to 10 #g/ml ACh. A significant response to this concentration first appeared in the 26 h distal denervated muscle and reached 35 ~ of the K contracture at 30 h after denervation (Fig. 1A). I n contrast, at 30 h after denervation, the proximal denervated muscle developed only a small response, and did not reach a comparable sensitivity until about 40 h after denervation (Fig. 1A). The dose-response curve also illustrates the profound differ-
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543 ence in A C h sensitivity between muscles denervated 30 h with either a short or a long nerve stump (Fig. 1B). In two muscles studied after 55 h of denervation, a difference in sensitivity with lower doses o f A C h was also found. In order to eliminate any assymetries due to cholinesterase activity, neostigmine 2 • 10 -6 g/ml was added to the Ringer solution in a few experiments. A 6-8 fold decrease in ACh concentration was required to reproduce the same response as obtained in the absence of neostigmine, but the differential sensitivity of 30 h proximal and distal denervated muscle was still clearly present.
[rzsl]a-bungarotoxin binding in denervated muscles The extrajunctional binding of [125I]a-bungarotoxin (BTX) was studied in proximal and distal denervated muscles 30 h-7 days after denervation (Fig. 2). As early as 30 h after a distal denervation, a significant increase in extrajunctional toxin binding was detected. Similar results have been reported by Berg and Hall 3. Muscles treated with labeled toxin bound 104 -k 17 cpm/mg of non-endplate muscle (n=4). Normal and 30 h proximal denervated muscle bound 18.6 :k 4 (n=6) and 32.3 _-t:_5 (n--4) cpm/mg of non-endplate muscle, respectively. The increase in non-endplate binding at later times after denervation appeared to follow the same pattern in both types of denervated muscles but the proximal denervated muscles showed a delay of about 10 h compared to distal denervated muscles. Binding in the endplate region (after subtraction of extrajunctional binding, see Methods) of distal and proximal denervated muscles was not significantly different at any time. However, 40 h after denervation, the endplate region of both proximal and distal denervated muscles
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neck and at the same time the intramuscular ventral branch was cut via an intraabdominal approach. After this procedure, the same hemidiaphragm had a distal denervated ventral region and a proximal denervated dorsal one. At 40 h after this operation, extrajunctional binding of [125I]a-BTX in the distal denervated region was three times higher than in the proximal denervated muscle strip. DISCUSSION
Our results show a clear difference in the time course of appearance of extrajunctional acetylcholine sensitivity, depending on the length of nerve left attached to muscle after denervation. Comparable data have been reported by Deshpande and Albuquerque 6 using iontophoretic techniques and by Emmelin and Malm 7, who determined threshold doses of ACh for isotonic contracture. In the latter study, the maximum difference dependent on nerve stump length occurred at 4 days after denervation, while our results showed the greatest difference at 30-60 h. The apparent discrepancy may result from different measures of ACh sensitivity (threshold response
546 vs. response to a standard dose or [125I]a-BTX binding). Also, Emmelin and Maim 7 did not examine the early postdenervation period extensively. In our experiments, the isotonic ACh contracture was a sensitive method to detect the earliest postdenervation increase in ACh sensitivity, possibly because the measured response reflects the shortening of the most sensitive and superficial fibers to acetylcholine 19. Indeed, in preliminary experiments we found that the magnitude of the K-contracture was unaltered in diaphragm strips until more than 3,/4 of the muscle fibers were cut. In any event, both the ACh contracture and [12~I]a-BTX binding showed the same post-denervation time dependence on nerve length. It has been reported that merely exposing a muscle during an operation can produce a transitory increase in sensitivity to ACh 16. This explanation would not account for the results of experiments in which the same exposed diaphragm muscle was subjected to both distal and proximal denervation, in which the nerve stump dependent time delay was still present. Jones and VyskociP 1 have reported that nerve degeneration "per se" produces a localized increase in ACh sensitivity restricted to 1 mm on either side of the site of the nerve degeneration. Since degeneration of the terminals occurs more rapidly if the nerve is cut close to the muscle than if it is cut far away '~z, it has been argued that the nerve stump dependence of the development of denervation effects is due to the degeneration of the terminals 17. This explanation now seems unlikely in the case of extrajunctional AChR of rat diaphragm. We found that the difference in the amount of toxin binding sites between short and long stump denervated muscles is present in extrajunctional segments as far as 4 mm from the endplate region, i.e.. at sites considerably far away from regions reportedly affected by nerve degeneration 11. On the other hand, the increase in the number of toxin binding sites at the endplate region, which first occurred 10 h after such sites had appeared throughout the whole muscle fiber, would be consistent with a localized 'inflammatory' response to degenerating nerve. Nonetheless, without additional data (e.g. autoradiography with labeled BTX), we cannot distinguish between a regional increase in receptors around the endplate and an increase in junctional endplate receptors per se. No significant differences were found between distal and proximal denervated muscles at the endplate region at any time after denervation. Biochemical assay of a-bungarotoxin binding sites yields an average density of receptors in a given sample, and obscures the microheterogeneity of patches of high density of AChR known to occur in denervated muscle 13. However, at 4 days after denervation, these AChR patches are extremely small and not invariably present ~3. Since much of our data was obtained after shorter periods of denervation, an autoradiographic approach would not be as useful as the biochemical averaging technique we employed. In any event, regardless of any microheterogeneity underlying the averaged data, the point remains that the early effect of denervation of AChR is not localized to the endplate region, whereas nerve terminal degeneration, as a cause of extrajunctional AChR increase shortly after denervation, would exert an effect localized to the endplate region. Our finding may not apply to all denervated muscles, since there is electrophysiological evidence that in rat limb muscles, increased extra-
547 junctional ACh sensitivity is not uniform throughout the length of the muscle fiberL Our results also give quantitative information on the time delay in the development of extrajunctional receptors as a function of nerve stump length. Miledi and Slater 22 reported that after phrenic nerve section, failure of neuromuscular transmission, disappearance of miniature endplate potentials, and nerve terminal degeneration were delayed about 45 min/cm of nerve stump length. In our case, the delaying effect of a long nerve stump length was considerably longer (180 min/cm). Therefore, it is unlikely that the lack of miniature endplate potentials, the failure of transmission, or, most important, nerve terminal degeneration is directly related to the nerve length dependent increase in sensitivity to ACh. In addition, the electrical activity of muscles denervated with either a short or long nerve stump is assumed to be either nill or identical. Thus, the experiments reported here are consistent with the hypothesis that extrajunctional ACh receptors are at least under partial control of some trophic factor which depends on axonal transport. This axonal transport could either maintain the terminal neurotrophic function or, more directly, convey the trophic substance itself. Since 3 cm of nerve stump delays the appearance of extrajunctional A C h R for at least 10 h, the trophic factor(s) could be transported at a rate no less than 72 mm/day. Indeed, axonal transport in this range, which is intermediate between slow and fast transport, has been reported4,t4, z0. However, our computation assumes a single rate of transport, whereas multiple rates, retrograde transport, or recycling of transported material within the nerve stump could be involved. ACKNOWLEDGEMENT This grant was supported by a grant from the Muscular Dystrophy Associations of America to N.R., and an International Postdoctoral Fellowship to O.U.
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548 10 Hartzell, H. C. and Fambrough, D. M., Acetylcholine receptors: distribution anti extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity, J. eeo. Physiol.. 60 (1972) 248-262. 11 Jones, R. and Vyskocil, F., An electrophysiological examination of the changes in skeletal muscle fibres in response to degenerating nerve tissue, Brain Research, 88 (1975) 309 317. 12 Karnovsky, J. and Roots, L., A 'direct-coloring' thiocholine method for cholinesterase, .I. Hi.~tochem. Cytochem., 12 (t964) 219-221. 13 Ko, P. K., Anderson, M. J. and Cohen, M. W., Denervated skeletal muscle fibers develop discrete patches of high acetylcholine receptor density, Science, 196 (1977) 540-542. 14 Lasek, R. J., Protein transport in neurons, Int. Rev. Neurobiol., 13 (1970) 289 32t. 15 Lavoie, P. A., Collier, B. and Tenenhouse, A., Comparison of a-bungarotoxin binding to skeletal muscle after inactivity or denervation, Nature (Lond.), 260 (1976) 349-350. 16 Lomo, T. and Westgaard, R. H., Further studies on the control of ACh sensitivity by muscle activity in the rat, J. Physiol. (Lond.), 252 (1975) 603-626. 17 Lomo, T. and Westgaard, R. H., Control of ACh sensitivity in rat muscle fibers, Cold Spr. Harb. Syrnp. quant. Biol., 40 (1976) 263-274. 18 Luco, J. V. and Eyzaguirre, C., Fibrillation and hypersensitivity to ACh in denervated muscle: effect of length of degenerating nerve fibers, J. Neurophysiol., 18 (1955) 65 73. 19 Lullmann, H., Preuner, J. and Schaube, H., A kinetic approach for an interpretation of the acetylcholine-D-tubocurarine interaction on chronically denervated skeletal muscle, Namo,n-Schmiedeberg's Arch. exp. Path. Pharrnaeol., 281 (1974) 415-426. 20 Lux, H. D., Schubert, P., Kreutzberg, G. W. and Globus, A., Excitation and axonal flow: autoradiographic study on motoneurons intracellularly injected with a 3H-amino acid, Exp. Brain Res., 10 (1970) 197-204. 21 Miledi, R., The acetylcholine sensitivity of frog muscle fibers after complete or partial denervation, J. Physiol. (Lond.), 151 (1960) 1 23. 22 Miledi, R. and Slater, C. R., On the degeneration of rat neuromuscular junctions after nerve section, J. Physiol. (Lond.), 207 (1970) 507-528. 23 Pestronk, A., Drachman, D. B. and Griffin, J. W., Effect of muscle disuse on acetylchotine receptors, Nature (Lond.), 260 (1976) 352- 354. 24 Tiedt, T. N., Albuquerque, E. X. and Guth, L., Degenerating nerve fiber products do not alter physiological properties of adjacent innervated skeletal muscle fibers, Science, 198 (t977) 839 841. 25 Uchitel, O. D.,Development of supersensitivity in denervated slow muscle of the frog. ltsdependence on the nerve stump length, Acta physiol, lat.-amer., 25 (1975) 34-38.