Improved Motor Function of Denervated Rat Hindlimb Muscles Induced by Embryonic Spinal Cord Grafts

Loss of motoneurons results in a decrease in force production by skeletal muscles and paralysis. Although it has been shown that missing motoneurons of rats can be replaced by embryonic homotopic neurons, attempts to guide their axons to their target muscles that have lost their innervation have been unsuccessful. In this study attempts were made to guide axons from grafted embryonic motoneurons to their target via a reimplanted ventral root. Adult hosts that received an embryonic graft prelabelled with 5-bromo-2′-deoxyuridine had their L4 ventral root avulsed and reimplanted into the spinal cord. Three to six months later, neurons that had their axons in the L4 ventral ramus were retrogradely labelled with fast blue and diamidino yellow. In five animals that had received an embryonic graft 116 ± 16 cells were retrogradely labelled, and of these at least 15% were of graft origin, since they were positive for 5-bromo-2′-deoxyuridine. In five animals that had their L4 ventral root reimplanted but did not receive a graft, only 12 ± 1.3 cells were retrogradely labelled. However, meaningful functional recovery could be achieved only if the regenerating axons of embryonic motoneurons found in the L4 ventral ramus were able to reverse the loss of force of muscles that had lost their innervation. This study shows that axons of embryonic motoneurons grafted into an adult rat spinal cord, as well as some axons of host origin, can be guided to denervated hindlimb muscles via reimplanted lumbar ventral roots. In normal rats ～30 motor axons innervated the extensor digitorurn longus and 60 innervated the tibialis anterior via the L4 ventral root. In rats that did not receive a graft only 3.7 ± 1.2 axons reached the extensor digitorum longus and 3.5 ± 0.4 reached the tibialis anterior muscle via the implanted L4 ventral root. In animals that had an embryonic graft, 7.6 ± 0.5 axons innervated the extensor digitorum longus and 8.5 ± 0.5 reached the tibialis anterior muscle via the implanted root. In rats without a transplant the maximum tetanic tension elicited by stimulating the implanted L4 root was 16 ± 7 g for the extensor digitorum longus and 53 ± 36 g for the tibialis anterior muscle, whereas the corresponding muscles in animals that had an embryonic graft developed 82 ± 16 and 281 ± 95 g respectively. Thus it appears that the grafted motoneurons contributed to the innervation and functional recovery of the denervated muscles.     

Electrical, mechanical, and physical properties of denervated latissimus dorsi muscles of the chicken

The anterior (ALD) and posterior (PLD) latissimus dorsi muscles of Cornish-cross chickens were examined after 1 wk of denervation to determine changes which might occur in their electrical and mechanical properties. Both muscles exhibited increased percentage of water and intracellular sodium concentrations, and decreased intracellular potassium concentrations. Although the denervated PLD had a relative increase in the volume of its extracellular space, the ALD experienced a relative decrease in extracellular space. The normal resting membrane potential dropped from ?70 mv (ALD) and ?74 mv (PLD) to ?57 mv after denervation. The mechanical threshold of the ALD, determined by potassium contracture studies, occurred at a lower [K]₀ in the denervated than in the control muscle; nevertheless, the resting potentials at threshold were the same. Similarly, the denervated ALD approached its maximum mechanical response at a lower [K]₀ than the control muscle, but at identical membrane potentials. This implies that the role of the membrane in excitation-contraction coupling was not eliminated by denervation. However, the denervated muscle developed only one-half the tension of the control muscle which suggests a deficiency in the excitation-contraction coupling process within the muscle fiber. The relationship between the resting potential and [K]₀ for the control ALD followed the Nernst equation, whereas potentials obtained from the denervated muscles were lower than predicted. This may be attributed to the effects of other ions on the resting potential, changes in the membrane permeability characteristics, or possible intracellular potassium binding.     

Congruity of acetylcholine receptor,acetylcholinesterase, and Dolichos biflorus lectin binding glycoprotein in postsynaptic-like sarcolemmal specializations in noninnervated regenerating rat muscles

Noninnervated regenerating muscles are able to form focal postsynaptic-like sarcolemmal specializations either in places of the former motor endplates ( “junctional” specializations) or elsewhere along the muscle fibers (extrajunctional specializations). The triple labeling histochemical method was introduced to analyse the congruity of focalization in such specializations of 3 synaptic components: acetylcholinesterase (AChE), acetylcholine receptor (AChR), and a specific synaptic glycoprotein which binds Dolichos biflorus lectin (DBAR). Noninnervated regenerating soleus and extensor digitorum longus (EDL) muscles of the rat were examined and compared with denervated muscles of neonatal and adult rats. All junctional sarcolemmal specializations in noninnervated regenerating muscles accumulated AChE and AChR. Localization of the 2 components was identical within the limits of resolution of the method. DBAR could not be demonstrated in junctional specializations in 17-day-old regenerating muscles. It seems that an agrin-like inducing substance in the former junctional basal lamina invariably triggers the accumulation of both AChE and AChR in the underlying sarcolemma of the regenerating muscle fiber. However, accumulation of DBAR would probably require the presence of the motor nerve. In most of the extrajunctional sarcolemmal specializations in 8-day-old regenerating soleus and EDL muscles, both AChE and AChR accumulated. However, about 10 percent of AChE accumulations lacked AChR and about 35% of AChR accumulations lacked AChE. Even greater variability was observed in 17-day-old regenerating muscles. The presence of DBAR in the extrajunctional postsynaptic-like sarcolemmal specializations could not be demonstrated. Similar extrajunctional sarcolemmal specializations were observed in denervated postnatal rat muscles. About 70% contained both AChE and AChR, and 30% contained only AChR, but none contained DBAR. In denervated mature muscles, sparse extrajunctional AChR accumulations did not contain detectable amounts of AChE. The ability to form complex postsynaptic-like sarcolemmal specializations in the absence of nerve, which is probably inherent to noninnervated immature muscle fibers, may be reduced with muscle maturation. Variable accumulation of individual components in the postsynaptic-like specializations indicates that different triggering factors may be involved in their accumulation or, at least, the mechanisms of their accumulation can function relatively independently. © 1993 Wiley-Liss, Inc.     

Energy reserves and chemical changes in denervated anterior and posterior latissimus dorsi muscles of the chicken

Postdenervation changes in most muscles, whether experimentally produced in animals or resulting from disease or injury in man, are characterized by a largely irreversible atrophy. One notable exception is the prolonged hypertrophy exhibited by the denervated anterior latissimus dorsi (ALD) muscle of the chicken. We have determined the concentrations of several chemical compounds and nitrogen fractions in the innervated and denervated latissimus dorsi muscles of the young chicken at various intervals after denervation. The posterior latissimus dorsi (PLD), a fast muscle, has significantly decreased ATP, phosphocreatine (PC), and total creatine (TC) concentrations 1 week after denervation. It also has less noncollagen nitrogen (NCN), as well as sarcoplasmic and fibrillar nitrogen; however, collagen nitrogen is increased and the muscle weighs 15% less than its contralateral control. The slow ALD, on the other hand, has not yet decreased its level of energy stores (ATP or PC) or TC at 3 weeks after denervation; nor are there any changes in NCN or any individual nitrogen fraction at 1 week. The hypertrophy of the denervated ALD reaches its maximum by the end of the first week, when it is 43% heavier than the control muscle. The weight gain in both innervated and denervated ALD are similar after that with the denervated muscle still 41% heavier at 3 weeks.     

Regulation of putative muscle-derived neurotrophic factors by muscle activity and innervation: in vivo and in vitro studies

The normal embryonic development of spinal cord motoneurons (MNs) involves the proliferation of precursor cells followed by the degeneration of approximately 50% of postmitotic MNs during the period when nerve-muscle connections are being established. The death of MNs in vivo can be ameliorated by activity blockade and by treatment with muscle extracts. Muscle activity and innervation have been suggested to regulate the availability of putative muscle-derived neurotrophic agent(s), and MNs are thought to compete for limited amounts of these trophic agents during normal development. Thus, activity and innervation are thought to regulate MN survival by modulating trophic factor availability. We have tested this notion by examining MN survival in vivo and ChAT development in spinal cord neurons in vitro following treatments with partially purified muscle extracts from normally active, paralyzed (genetically or pharmacologically), aneural, denervated, slow tonic, and fast-twitch muscles from embryonic and postnatal animals. Extracts from active and chronically inactive embryonic avian and mouse muscles were found to be equally effective in promoting the in vivo survival of MNs in the chick embryo. Similarly, extracts from fast-twitch and slow tonic postnatal avian muscles did not differ in their ability to promote both MN survival in vivo and ChAT activity in vitro. Although aneural and control embryonic muscle extract had similar effects on ChAT development in vitro, aneural muscle extract contained somewhat less MN survival-promoting activity when tested in vivo. By contrast, denervated postnatal muscle extract was more effective in promoting both MN survival in vivo and ChAT activity in vitro than age-matched control muscle extract.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of nerve and tension in maturation of posthatching slow-tonic muscle in chicken

The role of motor innervation and muscle tension in the posthatching maturation of the slow-tonic anterior latissimus dorsi (ALD) muscle of the chicken has been investigated. Modification of the muscle tension was obtained either by maintaining ALD in a shortened state or by stretching, after or without denervation. In denervated as well as in innervated ALD, shortening resulted in atrophy and inhibition of developmental change in muscle fiber population. In contrast, stretch causes hypertrophy, transformation of all 3B fibers, increase in SM2 isomyosin expression, and decrease in Ca2+-activated myosin ATPase in innervated or denervated ALD. On the other hand oxidative activity in ALD fibers was strikingly reduced after denervation even in presence of stretch-induced hypertrophy. This study suggests that a passive stretch can be involved in some, but not all, changes in ALD characteristics occurring after denervation and may be also involved in normal posthatching development of the slow-tonic muscle. Possible clinical implications of these results in relation to treatments for preventing muscle atrophy resulting from immobilization or disuse are suggested.     

Accelerated degradation of junctional acetylcholine receptor-α-bungarotoxin complexes in denervated rat diaphragm

[¹²⁵I]α-bungarotoxin was administered to rats in vivo to label acetylcholine receptors in innervated diaphragm, 5-day denervated diaphragm, or diaphragm which had been denervated immediately before labeling. The rate of degradation of junctional toxin-receptor complexes was followed by sacrificing animals at various times after labeling. The rate of degradation of junctional toxin-receptor complexes was significantly faster in 5-day denervated left hemidiaphragm (t 1/2 = 2.0 days) than in innervated left hemidiaphragm (t 1/2 = 10.7 days). The rate of degradation of junctional toxin-receptor complexes in left hemidiaphragm denervated at the time of labeling was essentially identical to that in innervated muscle for 3 days but then increased to a significantly more rapid rate (t 1/2 = 3.7 days in the period 3–13 days after denervation and labelling). These findings support the concert that continous innervation is needed to maintain the metabolic stability of junctional acetylcholine receptors.     

Myosin light and heavy chains in muscle regenerating in absence of the nerve: Transient appearance of the embryonic light chain

We examined myosin of fast and slow skeletal rat muscles regenerating after ischemia and bupivacaine injection in denervated limbs. Four days after injury two-dimensional gel electrophoresis revealed the presence of the embryonic light chain in the myosin isolated from the portion of muscle showing a homogeneous population of new small fibers by histological examination. Two weeks after injury this subunit was absent, whereas the two light chains, LC1F and LC2F, became prominent. One month after injury the still denervated soleus muscle maintained this light chain pattern. Gel electrophoresis in native condition of the myosin and peptide mapping of electrophoretically purified heavy chains confirmed that the muscle regenerating in absence of the nerve accumulated a myosin that had the general features of a fast, not slow, myosin but contained definite differences from the former.     

Myosin expression in hypertrophied fast twitch and slow tonic muscles of normal and dystrophic chickens.

Disruption of the development program of myosin gene expression has been reported in chicken muscular dystrophy. In the present report, the relationship between muscular dystrophy and the ability of muscle to respond to an increased work load with a transition in the myosin phenotype has been investigated. Hypertrophy of slow tonic anterior latissimus dorsi (ALD) and fast twitch patagialis (PAT) muscles was induced by overloading for 35 days and myosin expression was analyzed by electrophoresis and immunocytochemistry. Normal and dystrophic chicken ALD muscles have nearly identical proportions of SM-1 and SM-2 isomyosins and both exhibit an age-related repression of the SM-1 isomyosin which is enhanced and accelerated by overloading. Immunocytochemistry with anti-myosin heavy chain (MHC) antibodies demonstrates the appearance of nascent myofibers in overloaded ALD muscles from both normal and dystrophic chickens. A minor fast twitch fiber population is also identified which doubles in number with overloading in normal ALD muscles. There are only half as many fast twitch fibers in control dystrophic ALD muscles and this number does not increase with overloading. In contrast to ALD muscles, the isomyosin profile of normal and dystrophic PAT muscles is quite different. There is significantly more FM-3 and significantly less FM-1 isomyosin in the dystrophic PAT muscle. However, both normal and dystrophic PAT muscles exhibit an overload-induced accumulation of the FM-3 isomyosin. Immunocytochemistry reveals that, unlike the normal PAT muscle, the dystrophic PAT muscle contains a population of myofibers which express slow MHCs. As in the ALD muscle, overload-induced hypertrophy is associated with a repression of the SM-1 MHC in these fibers. Nascent myofiber formation does not occur in either normal or dystrophic overloaded PAT muscles.     

Expression of superfast myosin in aneural regenerates of cat jaw muscle

We investigated whether innervation is necessary for the expression of superfast myosin in regenerating cat jaw-closing muscle. Strips of jaw muscle were permitted to regenerate bilaterally in the beds of a fast limb muscle with innervation on one side being prevented surgically. Immunocytochemical analyses using anti-myosin heavy chain antibodies were done at various times postoperatively, the latest being after 78 days. We found little difference between innervated and uninnervated regenerates up to 27 days postoperatively. All regenerating myotubes expressed fetal myosin. In addition, some myotubes stained for superfast or slow myosin, while others stained for both superfast and slow myosins. Subsequently, uninnervated myotubes became atrophic but continued to express fetal, slow, and superfast myosins while innervated myofibers suppressed fetal and slow myosin expression. These results are consistent with the notion that satellite cells of jaw-closing muscles are committed to express superfast myosin during myogenesis, and that the expression of this program is independent of innervation.     

Increase of Cardiotrophin‐1 immunoreactivity in regenerating and overloaded but not denervated muscles of rats

The original report by Pennica et al. on Cardiotrophin‐1 (CT‐1) states that it markedly stimulates hypertrophy in cardiac myocytes both in vitro and in vivo and is predominantly expressed in the early mouse embryonic heart tube. CT‐1 is a member of the interleukin‐6 superfamily and past studies have shown that it exerts trophic effects on neurons, glial cells and their precursors, and is expressed during myogenesis. Thus CT‐1 is associated with physical and pathological changes in skeletal muscle. In this study, we examined whether CT‐1 is expressed in mechanically overloaded, regenerating, and denervated muscles of rats using immunohistochemistry. In the overloaded plantaris muscles at 1 and 3 days postsurgery, CT‐1 immunoreactivity was detected in the mononuclear cells that had infiltrated the extracellular space. CT‐1 immunoreactivity was also observed in the mononuclear cells invading the extracellular space at 2, 4, and 6 days after a bupivacaine injection and in degenerative and necrotic muscle fibers at 2 days postinjection. In the denervated muscles, the CT‐1 immunoreactivity did not change in intensity during the entire period of the denervation (2, 7, and 14 days postsurgery). The cells invading extracellular space and in necrotic muscle fibers possessing CT‐1 immunoreactivity might be muscle precursor cells (satellite cells) or migrating macrophages undergoing phagocytosis. Using double‐immunostainings for anti‐CT‐1/antic‐met, anti‐CT‐1/ anti‐M‐cadherin, and anti‐CT‐1/anti‐ED1, we found that satellite cells and macrophages exhibited CT‐1 immunoreactivity in the damaged muscles after bupivacaine injection. We therefore believe that CT‐1 plays a key role in regeneration and hypertrophy in the skeletal muscle of rats.     

Conversion of muscle fiber types in regenerating chicken muscles following cross-reinnervation

Summary Slow-tonic anterior latissimus dorsi (ALD) and fast-twitch posterior latissimus dorsi (PLD) muscles of 7 to 10-day-old White Leghorn chickens were (a) crushed and allowed to be reinnervated by their own nerve, or (b) crushed and transplanted to the other side and allowed to be reinnervated by the nerve of the side to which they were transplanted. Following transplantation, changes in the weight of the muscle, fiber-type composition and innervation pattern during regeneration were investigated. Normal growth rate of PLD was about twice that of ALD. Regenerating PLD, however, atrophied rapidly after crushing and denervation whether innervated by its own nerve or the other nerve type, whereas ALD reinnervated by its own nerve showed marked hypertrophy. PLD fibers transformed rapidly to fast-twitch or slow-tonic (ST) fibers when they were reinnervated by PLD or ALD nerve, respectively. When ALD fibers were reinnervated by their own nerve, they differentiated into ST fibers that were surrounded by smaller immature fibers. ALD fibers were, however, resistant to complete control by fast-twitch PLD nerve and contained a large number of slow fibers (ST and ) long after transplantation. Slow fibers in regenerates were initially multiply innervated, but later transformed into fast-twitch fibers that were focally innervated. The mode of differentiation and innervation pattern of different muscle fiber types in regenerating muscles are discussed.Supported by the Muscular Dystrophy Association of America (Dr. T. Kikuchi) and partly by the National Center for Nervous, Mental and Muscular Disorder of Ministry of Health and Welfare, Japan (grant No. 84-04-05)     

Motoneurone survival factor produced by muscle increases in parallel with messenger RNA sequences homologous to beta NGF-cDNA

Motoneurone survival in culture is enhanced by growth factors present in embryonic rat muscles and denervated adult muscles: extracts from mature muscle and 2-day denervated mature muscle do not produce survival effects whereas extracts from embryonic muscle and 4-day denervated mature muscle produce maximum survival effects. Here we show, using dot hybridization of mRNA with a 32P-labelled cDNA probe for beta-nerve growth factor (beta NGF), that mRNA sequences homologous to beta NGF appear in muscle in parallel with increased motoneurone survival activity. RNA gel blot hybridizations show that embryonic mRNA contains a sequence in the same molecular weight range as the mRNA for beta NGF.     

Contractile and histochemical properties of sliced muscle grafts regenerating in normal and denervated rat limbs.

Rat extensor digitorum longus muscles were transversely sliced into 7–8 segments. The muscle slices were autografted back into their original beds. In one series the recipient limbs were normal and in the other, limbs were denervated. At postoperative intervals of 7, 14, 30, and 60 days, the contractile properties (Latency period, contraction and half relaxation times, time parameters of contraction of twitch and tetanus, and twitch and tetanic tension) and histochemical properties (succinic dehydrogenase and myofibrillar ATPase) were analyzed. Sliced grafts regenerating in normally innervated legs followed a typical conversion from slow to fast contraction times, whereas regenerates in denervated limbs remained slow. Histochemically, innervated regenerates developed a heterogeneous pattern of muscle fiber type staining during the second month, whereas different histochemical types of muscle fibers did not appear in noninnervated regenerates. As in ontogeny, denervation retards or prevents the full structural and functional differentiation of regenerating muscle fibers.     

The effect of denervation on the morphology of regenerating rat soleus muscles

Summary This study examines the level to which muscle regeneration proceeds in the absence of innervation. Regeneration was monitored in rat soleus muscles following localised injection of a snake toxin, notexin. Muscles which had been concomittantly denervated were compared with those that were normally innervated. Until 3–4 days following toxin administration regeneration is identical in both groups. The muscles contain new myotubes in place of the degenerated parent fibres. Thereafter, the non-denervated muscles grow rapidly and by 28 days their myofibres attain the size of those from the contralateral controls. Growth of denervated regenerating muscles, however, is retarded and is superseded by a gradual atrophy. In such muscles we further identify ultrastructural abnormalities from 7 days post-injection. These a re loss of individual myosin filaments and the presence of immature and abnormal configurations of the transverse system and triads. We, thus, conclude that innervation is an obligatory requirement for the restoration of normal myofibrillar and sarcotubular morphology, as well as growth, but is not necessary for the neo-formation of myofibres.     

Lectin receptors in the cockroach neuromuscular system. II. Effects of denervation on muscle lectin receptors

The binding of fluorescent derivatives of plant lectins to the 6 coxal depressor muscles in the leg of the cockroach was examined at 1, 2, 3 and 5 weeks after denervation. Histochemical analysis of the binding of the lectins to frozen sections of the muscles demonstrated that all detectable binding was to the surfaces of the muscle fibers. In addition, within one week of denervation those muscles which in the innervated state have different amounts of D-galactose and/or alpha-N-acetylgalactosamine on their surfaces, now all have identical amounts of these carbohydrates. Biochemical analysis of concanavalin A and wheat germ agglutinin glycoprotein receptors by sodium dodecylsulphate polyacrylamide gel electrophoresis indicated that approximately 17% of all the receptors detected altered their relative levels in denervated muscles. These changes were observed in those receptors that were present in equal amounts in each of the muscles as well as in those receptors that were distributed among the 6 muscles in a manner that correlated with innervation by an identified motor neuron. However, in spite of these changes that reduced the biochemical differences in the denervated CDMs it was still possible to distinguish among the 6 muscles by the nature of the Con A and WGA glycoprotein receptors. Denervated muscles still present biochemically different cell surfaces to regenerating motor neurons.     

Regeneration following denervation of minced gastrocnemius muscles in mice.

Gastrocnemius muscles in mice were minced and orthotopically implanted. At the same time all nervous supply to this muscle was completely removed. It was observed that initially the pattern of muscle regeneration was similar to what was observed in a normally innervated implant. But by day 6, distinct degeneration of regenerated muscle fibres sets in, which continues unabated so that by about 3 weeks there usually remains only a thin band of connective tissue in place of the implant. Histochemically, there is a gradual loss of SDH, myofibrillar ATPase and cholinesterase activities within the degenerating muscle fibres and a corresponding appearance of these enzymes in the regenerating fibres. In the denervated implants, with the onset of degeneration of the regenerating fibres, the enzymatic activities were also lost. Histochemical fibre typing was not achieved within the regenerating fibres. The regeneration and degeneration pattern of the denervated muscle observed in the present study is compared with the one observed in other animals.     

Critical periods in the development of motoneurons

We define "critical periods" in motoneuron development as periods when neurons can express a particular aspect of their sequence of differentiation only within a limited time interval. Embryonic rat motoneurons were axotomized and their survival and capacity to regenerate their axons studied at later times. A critical period of absolute target dependence extended from embryonic day 14 (E14) until E17. All motoneurons axotomized during this interval died, while cells axotomized earlier or later could survive. During the period E18- postnatal day 14 (PN14) regenerating motoneurons accurately reinnervated motor unit territories of normal size, and intact motoneurons did not exhibit collateral sprouting in response to denervation of adjacent muscle fibres. After PN21, muscle fibres were reinnervated in a spatially indiscriminate manner, and collateral sprouting occurred in response to partial denervation of muscles. We conclude that adult motoneurons have a powerful capacity to reinnervate denervated muscles, even in an inappropriate manner, in contrast to motoneurons in neonates which can recapitulate embryogenesis and accurately reinnervate their proper motor unit territories.     

The multifaceted character of lymphotoxin β in inflammatory myopathies and muscular dystrophies

Lymphotoxin beta (LTβ) regulates some inflammatory mechanisms that could be operative in idiopathic inflammatory myopathies (IM). We studied LTβ and LTβR in inflammatory myopathies, normal and disease controls with immunohistochemistry, Western blotting and in situ hybridisation. LTβ occurs in myonuclei of normal controls, implying its role in normal muscle physiology. LTβ is strongly upregulated in regenerating muscle fibres in all myopathies, but not in denervated myofibres. Normal-appearing myofibres in inflammatory myopathies and muscular dystrophies express LTβ possibly reflecting early myofibre damage, representing a hitherto undescribed pathologic hallmark. Furthermore, we visualised LTβ in several inflammatory cell types in inflammatory myopathies, suggesting its involvement in the different inflammatory mechanisms underlying inflammatory myopathy subgroups.     

Sodium influx during action potential in innervated and denervated rat skeletal muscles.

Resting Na(+) influx (J(i)(Na)) was measured in innervated and denervated (1-6 days) rat extensor digitorum longus muscle in the absence and presence of 2 micromol/L tetrodotoxin (TTX).The mean value of Na(+) permeability (P(Na)) in innervated muscles was 49.6 +/- 2.6 pm.s(-1). At the second day postdenervation, it decreased by about 45%. This was followed, between the second and fourth days, by a sharp rise, which by the sixth day reached a steady value approximately 2.5 times greater than that of innervated muscles. This, most likely, generated the 30% increase in internal [Na(+)] concentration ([Na(+)](I)) observed at this time. Tetrodotoxin reduced P(Na) of both innervated and denervated muscles by about 25%. In 6-day denervated muscles, virtually all the TTX effect on P(Na) represents the blockage of TTX-resistant Na(+) channels. Denervation produced a depolarization of about 20 mV by the sixth day. The extra J(i)(Na) per action potential (AP) decreased monotonically with time after denervation from 20.0 +/- 3.8 in innervated to 11.1 +/- 1.0 nmol.g(-1).AP(-1) in 6-day denervated muscles. The overshoot of the AP decreased from 15 +/- 1 in innervated to 7 +/- 1 mV in 6-day denervated muscles. Likewise, the maximum rate of rise (+dV/dt), an expression of the inward Na(+) current, fell from 305 +/- 14 in innervated to 188 +/- 18 V.s(-1) in 6-day denervated muscles. The estimated 6-day denervated/innervated ratio of peak Na(+) conductance (g(Na)) was 0.67. The changes in AP parameters promoted by denervation were substantially reduced when both innervated and denervated fibers were hyperpolarized to -90 mV. These results suggest that the depolarization, mainly due to the increase in P(Na) /P(K) ratio, increases Na(+) inactivation and consequently reduces peak g(Na), in spite of the absolute increment in resting TTX-sensitive P(Na). This, in addition to the moderate reduction in the inward driving force on Na(+), decreases the inward Na(+) current and the extra J(i)(Na) per AP.