Reinnervation and recovery of mouse soleus muscle after long-term denervation

Reinnervation and recovery of the mouse soleus muscle were studied 2-10 months after denervation periods of about 7 months. To maintain denervation the right sciatic nerve was frozen 14 times at 2-week intervals. Though initially intermittent muscle reinnervation occurred, contractile force of denervated muscles was reduced to less than 10% of the contralateral muscles by the fifth nerve freezing and further declined thereafter. Following reinnervation, recovery of soleus muscle force proceeded slowly to reach plateau values after 5-6 months. Tetanic muscle force reached on average 72% (range 58-86%, n = 12) of contralateral muscles after 5-10 months, (P less than 0.01, t-test for absolute values) and 87% of unoperated animals after 10 months (P less than 0.05, n = 5). Muscle fibre diameters were significantly reduced in reinnervated muscles, but frequency distributions were normal and similarly shaped in reinnervated and control muscles, suggesting complete muscle reinnervation and the absence of denervated fibres even at 2 months of reinnervation. Total numbers of muscle fibres were similar in reinnervated (842 +/- 73 S.D., n = 15), contralateral (854 +/- 104 S.D., n = 15) and control soleus muscles (853 +/- 77 S.D., n = 5). The number of myelinated axons in regenerating soleus nerves reached control values by 3 months after the last freezing, continued to increase till 6 months (150% of control), and declined thereafter (125% at 9-10 months). In the contralateral soleus nerves the number of myelinated axons remained constant during this period. Nerve fibre diameters remained abnormally small; even after 10 months of reinnervation fibre diameters were unimodally distributed with a mean diameter of 3.3 microns in contrast to the bimodal distribution in intact nerves (mean values 3.9 and 9.0 microns, respectively). Total fibre cross-section area per nerve increased with time but reached only 54% +/- 6 S.D., (n = 3) of contralateral nerves by 10 months. The relative thickness of the myelin sheath (g-ratio) returned to normal after 9-10 months. Anatomically, muscle reinnervation appeared to be complete by 7-8 weeks since unusually small muscle fibre profiles were absent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increase of elastic fibres in muscle spindles of rats following single or repeated denervation with or without reinnervation

Summary Muscle spindles in the lower lumbrical muscles of rats were studied by transmission electron microscopy following denervation with or without reinnervation. The number and total area of elastic fibres per muscle spindle increased at 3–12 months following various experimental procedures: (1) denervation and reinnervation after a single crush lesion to the sciatic nerve; (2) reinnervation after four-fold repeated crush injuries; and (3) transection and suture of the nerve. The increased number of oxytalan and elaunin fibres, the precursors of mature elastic fibres, within these muscle spindles provided further evidence for their numerical and dimensional increase. An attachment site of elastic fibres at the spindle pole was identified at the inner cells of the outer spindle capsule. The processes of these cells embraced terminating elastic fibres tightly. Attachment of elastic fibres to intrafusal muscle fibres was less conspicuous since they were not similarly embraced but were rather indistinctly, though closely, associated with the basal lamina along longitudinal surface indentations of intrafusal muscle fibres. It is concluded from this series of experiments that muscle spindles, as dynamic mechanoreceptors, maintain their elastic properties even under pathological conditions. The increase of elastic fibres following denervation and reinnervation represents an obviously meaningful reaction that may compensate for loss of tonic properties of muscle spindles without causing stiffness.     

Altered patterns of innervation in frog muscle after denervation

Summary The pattern of reinnervation of muscle fibres after a nerve crush was examined in the cutaneous pectoris muscle of the frog by microscopy and electrophysiology. Normally, about 16% of the muscle fibres are innervated by more than one motor neuron. Two months after reinnervation, about 50% of the fibres are polyneuronally innervated and this high incidence persists for at least seven months. The total number of neurons reinnervating the muscle, as well as the number of muscle fibres comprising the muscle, are normal. However, nerve fibres sprout branches at the site of the crush, and, therefore, the number of axons entering the muscle is greater than normal. Regenerating axons contact muscle fibres precisely at the original synaptic sites and the terminal branches from different axons that end on the same muscle fibre often run side by side occupying stretches of original postsynaptic membrane normally covered by one terminal. Our findings indicate that the amount of synaptic contact during regeneration is limited by the amount of original postsynaptic membrane and that any number of axons that reach vacant portions of original postsynaptic membrane can make synaptic contact with it.Surprisingly, control cutaneous pectoris muscles, situated contralaterally to those that were denervated, also exhibited an abnormal pattern of innervation. Although neither nerve, nor muscle was disturbed by the operation, there was a higher incidence of polyneuronal innervation (27% vs 16%) than in muscles of normal animals.This study was supported by USPHS Grants NS 02253, NS 12922, NS 70606 and a grant from the Lady Davis Fellowship Trust.     

Precision of reinnervation of original postsynaptic sites in frog muscle after a nerve crush

Summary Regenerating neuromuscular junctions in the cutaneous pectoris muscle of the frog were examined by light and electron microscopy up to three months after crushing the motor nerve. The aim was to determine the precision of reinnervation of the original synaptic sites. More than 95% of the original postsynaptic membrane is recovered by nerve terminals and little, if any, synaptic contact is made on other portions of the muscle fibre surface. Even after prolonged denervation when the Schwann cells have retracted from 70–80% of the postsynaptic membrane, regenerating terminals return to and cover a large fraction of it. Although synapses are confined to the original synaptic sites, the pattern of innervation of muscle fibres is altered in several ways: (a) regenerating axon terminals can fail to branch leaving small stretches of postsynaptic membrane uncovered; (b) two terminal branches can lie side by side over a stretch of postsynaptic membrane normally occupied by one terminal; and (c) after growing along a stretch of postsynaptic membrane on one muscle fibre, terminals can leave it to end either in extracellular space or on the postsynaptic membrane of another fibre. Altogether the results demonstrate a strong and specific affinity between the original synaptic sites and regenerating nerve terminals.     

Axonal sprouting and changes in fibre types after running-induced muscle damage

Summary We have recently observed increase in Type I fibres in mouse soleus — but not extensor digitorum longus — muscles as a result of repeated muscle damage induced by voluntary wheel running. The most likely mechanism underlying the changes in fibre type composition is a redistribution of motor units with axonal sprouting and formation of new synapses. To test this hypothesis we exercised mice on a motor-driven treadmill once (3 × 3 h with 30 min rest periods in between, 14 m min^–1, slope 6 °) or repeatedly (8–10 times at intervals of 3–5 days) and quantified axonal sprouting after staining with zinc iodide-osmium. In the contralateral solei, muscle damage and fibre type changes were evaluated with standard histochemical techniques.Significant numbers of damaged muscle fibres were found 0–15 days after a single exercise as compared to unexercised control animals (range 0.0–0.3% of the fibres in sedentary,n=5,vs 2.1–14.8% in exercised muscles,n=10) and repeated damage occurred in repeatedly exercised animals. In muscles of sedentary animals 3.8 ± 1.4% SD of the examined endplates (n=880, 5 muscles) had nodal or terminal sprouts. The incidence of sprouting was significantly elevated 3–21 days after a single exercise (7.5 ± 1.8%,n=2855, 12 muscles,P < 0.01 signed-rank test), and more so after repeated running (12.0 ± 2.5%,n=1505, 6 muscles,P < 0.01). Fibre type distributions were not different from controls 3 weeks after a single running episode, but after the 6–7 weeks of repeated running a significant increase in undifferentiated fibres at the cost of Type II fibres was found (9.7 ± 3.4% versus 1.0 ± 0.5% in sedentary controls,P < 0.05,t-test); undifferentiated fibres express both Type I and Type II myofibrillar ATPase and are considered as fibres in the process of changing their types. These observations strongly support the assumption that sprouting and formation of new synapses — followed by motor unit enlargement and redistribution — occur as a result of muscle damage.     

Motor unit numbers,motor unit sizes and innervation of single muscle fibres in hyperinnervated adult mouse soleus muscle

The sural and the lateral plantar nerves were implanted simultaneously into the denervated soleus muscle of adult mice. Each of these nerves contained approximately the normal number of soleus motor axons. This procedure therefore allowed a study of how an initial excessive number of motor axons provided by two different, foreign nerves and terminating into the soleus muscle affected the final pattern of muscle innervation. In muscles examined two months or more after the implantation of the foreign nerves all muscle fibres were innervated. The fraction of the muscle innervated by either nerve varied widely from one preparation to another. However, all the motor axons which were implanted into the muscle appeared to make permanent synapses. Moreover, the distribution of motor unit sizes of each foreign nerve relative to the total number of muscle fibres innervated by that nerve was similar to the distribution of motor unit sizes in muscles cross-innervated by that nerve alone, although the absolute motor unit sizes were reduced. Estimated by intracellular recording, 20–30% of the muscle fibres were polyneuronally innervated. A similar fraction of teased muscle fibres stained for acetylcholinesterase had more than one endplate.     

Formation of new muscle fibres and tumours after injection of cultured myogenic cells

Summary We examined the effects of implantation of cultured myogenic cells from a permanent cell line into soleus muscles of histocompatible adult mice. Myogenic cells (10⁶ or 10⁴) were implanted into intact muscles, muscles frozen with liquid nitrogen, paralysed with botulinum toxin or reinnervated after long-term (seven months) denervation. Formation of numerous muscle fibres in myogenic cell-injected muscles raised the total number of fibres up to ten times above control by four weeks. Larger effects were found in freeze-damaged than in paralysed muscles. The new fibres had small calibers, considerable length (> 1.3mm, maximum distance over which serial sections were made), were multinucleated and were oriented parallel to the large-diameter fibres of the host muscles. In some experiments -galactosidase, introduced into myogenic cells via retroviral transfection, was detected in small and large muscle fibres 4–20 weeks after implantation, indicating survival of the grafted cells and formation of mosaic (host-donor) and new fibres of donor origin. Muscle weight increased significantly and, rather surprisingly, a parallel increase was found in isometric tetanic tension of isolated nerve-muscle preparations; thus tension per mg muscle tissue was not different from normal. By eight weeks reduction of acetylcholine sensitivity and down-regulation of neural cell adhesion molecule to normal were observed, indicating that synaptic transmission at the new fibres was mature. After different periods of time (5–20 weeks, depending on the subclone used) tumours developed in most but not all injected limbs (37 out of 39). The tumours were destructive to the muscles and were classified asrhabdomyosarcomas. Prior to tumour formation, neural cell adhesion molecule positive cells reappeared in the muscles; since the myogenic cells initially produced differentiated muscle fibres, it appears that malignant growth is induced by factorsin vivo. Thus, at present the outcome of such implantation is unpredictable.     

Reinnervation of the mammalian spinal cord after neonatal dorsal root crush

Summary In the adult mammal, nerve fibres do not regrow into the spinal cord after a dorsal root lesion. The elongation of dorsal root nerve fibres into the spinal cord of neonatal rats was examined: L4 and L5 dorsal roots were crushed in rat pups. After 3–6 months, the dorsal root-spinal cord junction was investigated morphologically in several long series of ultrathin cross-sections. In rats which had been operated on at birth (0–2 days old), axons from the lesioned roots could be followed into the CNS tissue of the spinal cord. In contrast to normal development, the usual short segment of CNS glia did not grow into the neonatally lesioned roots. Instead, the CNS-PNS border was located within the spinal cord. The nerve fibres, which were of normal diameter, had regrown across the PNS-CNS border and elongated further into the CNS environment of the spinal cord. In rats operated on at the end of the first postnatal week or later, the largest dorsal root nerve fibres were only half the size of those in unoperated animals and reinnervation of the spinal cord had not occurred. An astrocyte-dominated CNS segment had developed in these roots. The impact of an early neuronal lesion on the development of certain glia cells and their importance in the outcome of spinal cord reinnervation are discussed.     

Differential expression of tenascin after denervation, damage or paralysis of mouse soleus muscle

Summary The expression of the extracellular matrix molecule tenascin was studied by immunocytochemistry and Western blotting in soleus muscles of adult mice after nerve damage (denervation), muscle injury (induced by enforced running or freezing) and functional block of synaptic transmission (botulinum toxin). Enhanced expression of tenascin in the extracellular spaces around focally damaged muscle fibres was found already 10 h after onset of running on a motor-driven treadmill which causes muscle injury in soleus muscle. Tenascin expression reached a peak at 2–3 days post-exercise, after which it declined gradually and became undetectable by two weeks after injury. Similarly, cryo-damage of soleus musclesin situ led to upregulation of tenascin. Chronic muscle denervation after sciatic nerve transection caused a persistent (studied up to 31 days) expression of tenascin at denervated endplates and in intramuscular nerve branches but not in other tissue compartments. Local application of botulinum toxin Type A, which results in muscle inactivity but not in tissue degeneration, however, did not induce tenascin expression 12 h to 12 days post-injection. Expression of tenascin after denervation and muscle damage, but its absence after paralysis, were verified by SDS-PAGE and Western blot analysis. Independent of the type of injury (muscle, nerve or both) the known major isoforms of mouse tenascin, as judged by M_r comparison, were re-expressed, with no preponderance of individual M_r forms. These results show that tenascin expression in adult muscles is induced by both axon and muscle fibre damage but not by muscle inactivity. In contrast, NCAM, in accordance with previous observations, showed enhanced expression both as a result of inactivity and in association with tissue repair.     

Polyclonal antibodies against NCAM and tenascin delay endplate reinnervation

Summary Experiments were performed to block molecules with antibodies which are upregulated in nerve and muscle following denervation. The delay in endplate reinnervation was taken as a measure for their involvement in regeneration. Gluteus maximus muscles of 86 male CBA/J mice were hemidenervated by freezing the caudal gluteal nerve at a defined position. The degree of reinnervation was evaluated in identified endplates by repeated vital staining of ACh receptors with rhodaminated -bungarotoxin and of axons with 4D1-2ASP. Normally, endplates were completely reinnervated by 13–14 days (108 endplates in seven muscles). After daily application of polyclonal antibodies against NCAM or tenascin, reinnervation was significantly delayed. Preimmune serum, rabbit immunoglobulins or saline did not show this effect. Several monoclonal antibodies against NCAM (H-28) and tenascin (576, 578, 630, 633) showed a tendency but no significant effect. It is concluded that both NCAM and tenascin, upregulated after denervation, are involved in axon guidance and/or endplate reinnervation.     

Differentiation of fast and slow muscles in the rat after neonatal denervation: A physiological study

Summary Whether an intact innervation is essential for postnatal muscle differentiation was examined in the rat by recording physiological contraction parameters. Muscles in one leg were denervated neonatally (within 24 h of birth) and, between 3–28 days after the operation, their contractions were compared with those of the contralateral control muscles. Experiments were performed on the extensor digitorum longus (edl, a fast muscle) and the soleus (a slow muscle) muscles and contractions were recordedin vitro, at 35 C and with direct stimulation. When compared with the control muscles, 3–4-day-old neonatally denervated fast and slow muscles had longer twitch contractions, higher twitch/tetanus ratios and certain other specific differences in their contraction parameters. These denervation-induced changes in neonatal muscles were essentially similar to those produced 3–7 days after denervation in the differentiated (4-week-old) fast muscle. Despite differences in their absolute values, the contraction parameters of neonatally denervated and control edl muscles changed similarly during development, indicating that postnatal differentiation of fast muscle fibres is independent of a neuronal influence. In the case of the neonatally denervated soleus muscle, the developmental changes in contraction parameters, i.e. shortening of the twitch duration, increase of rate of rise and rate of relaxation in the tetanus and increase of the maximum shortening velocity, were more pronounced than in the control slow muscle; also, there were similarities with the pattern of fast muscle differentiation. Thus, muscle fibre differentiation in soleus becomes altered towards that of a fast muscle after neonatal denervation.     

The effect of long-term administration of α1-blocker prazosin on capillary density in cardiac and skeletal muscle

The effect of prazosin on heart and muscle blood flow and capillary density was studied in rats. In acute experiments, ₁-blocker prazosin almost trebled blood flow in fast skeletal muscles [tibialis anterior (TA) and extensor digitorum longus (EDL)], but did not affect coronary flow when infused i.v. at a dose of 0.5 g · ml^–1 · min^–1 for 30 min. Prazosin in an equivalent dose was then given orally over a period of 5 weeks to investigate its effect on capillarisation in heart and skeletal muscle. Capillary density (CD, capillaries · mm^–2), estimated in frozen sections stained for alkaline phosphatase, was similar in the hearts of prazosin-treated and control rats. Capillary/fibre ratio in skeletal muscles increased from 1.52±0.019 in control EDL to 1.69±0.01 (P<0.001) and from 1.56±0.04 in control TA to 2.16±0.04 (P<0.001). In TA, the increase was greater than in EDL both in the glycolytic periphery (from 1.30±0.13 to 1.75±0.11, P<0.025) and the oxidative core of the muscle (from 1.837±0.14 to 2.51±0.12, P<0.005). Unilateral crush of the lateral peroneal nerve and subsequent reinnervation over the next 7 weeks resulted in redistribution of fibre types from a typical mosaic pattern into groups composed of fibres of similar oxidative capacity. Capillary density as well as capillary/fibre ratio in purely glycolytic areas was lower when compared to supply of glycolytic fibres in normal muscles. Oral administration of prazosin over the whole period of reinnervation not only maintained the original level of capillarity associated with fast glycolytic fibres in control muscles, but considerably increased it. Thus long-term prazosin administration not only causes an increase in capillary supply in normal muscles but also prevents loss of capillaries during reinnervation. The fact that it only increases capillarisation in tissues where it increases flow further supports the hypothesis that capillary growth can be initiated by mechanical factors connected with high blood flow.     

Muscle differentiation after sciatic nerve transection and reinnervation in adult rats

Reinnervation after peripheral nerve transections generally leads to poor functional recovery. In order to study whether changes in muscles might be a contributing factor in this phenomenon we studied muscle morphology and fibre type distributions after sciatic nerve transection in the rat hind limb. Proximally, before the bifurcation in the tibial and common peroneal nerve, a 12 mm segment of the sciatic nerve was resected, reversed and re-implanted as an autologous nerve graft. After survival periods of 7, 15 and 21 weeks the lateral gastrocnemius, tibialis anterior and soleus muscles were dissected, stained with mATP-ase, and fibre type distributions were studied. In addition, numbers of muscle fibres were counted, and cross sectional areas were calculated. After 7 weeks, cross sectional areas were decreased in all muscles. In the gastrocnemius and tibialis anterior muscles the fibre number remained unaltered but the hypotrophy had been reversed at later ages. The number of muscle fibres in the soleus muscle remained decreased over the entire period of observation. The percentages of type II fibres in the gastrocnemius and tibialis anterior muscles were decreased at 7 and 15 weeks but these again approached normal values at 21 weeks. The type I fibres, however, remained arranged in groups. In the soleus muscle a large increase in the percentage of type II muscle fibres was observed and this remained until 21 weeks. We conclude that a non-selective reinnervation and later readjustments by regression of polyneural innervation may in part explain the changes in distributions of various fibre types.     

Effects of growth hormone on skeletal muscle. II. Studies on regeneration and denervation in adult rats

The effects of human recombinant growth hormone (rhGH) on regenerating skeletal muscle after ischaemic necrosis and on denervated skeletal muscle were studied in normal adult rats. One group of rats was treated with 4 IE rhGH daily by subcutaneous injections, while control rats were injected with saline. The treatment with rhGH resulted in increased levels of insulin-like growth factor-I (IGF-I) in serum. Ischaemic necrosis was achieved in the extensor digitorum longus (EDL) muscle by cutting the supplying vessels and nerve fascicles at the entrance into the muscle. The wet weight and DNA: protein ration in the regenerating muscle were determined 2 and 4 weeks after the operation. The weight of the regenerating muscles in the rats treated with rhGH during the period of study was larger than in the control rats, while the DNA:protein ratio did not differ significantly between the groups. Denervation of the EDL and soleus muscles followed by subsequent reinnervation was obtained by freezing the sciatic nerve with a forceps chilled in liquid nitrogen. Rats treated with rhGH during the period of denervation and reinnervation, i.e. during the 4 weeks after the freezing of the sciatic nerve, revealed increased weight of both the reinnervated and normal muscles compared to corresponding muscles of control rats. Denervation of the EDL and soleus muscles without subsequent reinnervation was achieved by cutting the sciatic nerve at the level of the thigh. Four weeks after denervation the muscles showed atrophy, mainly affecting type 2 fibres in the EDL muscle and both type 1 and type 2 fibres in the soleus muscle.     

The formation of synapses in mammalian striated muscle reinnervated with autonomic preganglionic nerves

1. A study has been made of the formation of synapses during reinnervation of the hemidiaphragm of adult rabbits with preganglionic fibres of the thoracic vagus, using histological, ultrastructural and electrophysiological techniques.2. Following reinnervation with preganglionic axons, silver-stained nerve terminals were found in association with cholinesterase-stained end-plates only in the region of the muscle corresponding to the original innervation band.3. The fine preganglionic axons retained their normal structure in striated muscle, but were found to synapse over discrete areas of dimensions not larger than those of the original end-plates.4. The regenerated varicose preganglionic nerve terminals were observed with the electronmicroscope in positions either overlying or in the vicinity of the old synaptic folds.5. Spontaneous potentials and evoked synaptic potentials were recorded only in the middle of the muscle fibres after vagus reinnervation.6. In a few cases, multiple synaptic potentials with similar time courses were recorded, suggesting that several axons had formed synapses at a single site on a muscle fibre.7. It has been shown that, during reinnervation of adult mammalian striated muscle fibres with nerves other than those of the somatic system, synapses are formed preferentially in the region of the old end-plates.     

Regeneration and retrograde degeneration of axons in the rat optic nerve

Summary The retinal stump of the rat optic nerve was examined histologically 1–64 weeks after intracranial section of the nerve with or without grafting of autologous peripheral nerve segments. Single unmyelinated axons and bundles of unmyelinated axons appeared in cut optic nerves and were most abundant 2–4 weeks after section. With light and electron microscope radioautography after injection of tritiated amino acids into the globe, it was confirmed that many unmyelinated fibres arose from the optic nerve rather than from nearby peripheral nerves and it was estimated that some axons regenerated as far as 0.5 mm. At or near the end of retinofugal axons, structures resembling growth cones were seen at 2 weeks and vesicle-containing swellings similar to synapses were found at 1–2 months. Outgrowth from optic nerve axons was not obviously enhanced by peripheral nerve grafts although a few retinofugal axons became ensheathed by Schwann cells. Retrograde axonal degeneration was rapid in both cut and grafted optic nerves, the number of nerve fibres near the globe falling to less than 10% of normal after 4 weeks. A few myelinated and unmyelinated fibres were still present 64 weeks after nerve transection. In conclusion, some cut axons in the rat optic nerve display a transient regenerative response before undergoing retrograde degeneration.     

Lysosomal and energy enzyme activities in hypertrophied rat soleus muscle after denervation

Muscle hypertrophy was induced in the soleus muscle of young rats by tenotomy of the gastrocnemius and plantaris muscles. Three and 7 days afterwards the sciatic nerve was sectioned. The loss of weight of muscles subjected to this combined procedure three days after denervation was 30–40%. Lysosomal enzyme activities (acid phosphatase, -glucosidase, -galactosidase and N-acetyl--d-glucosaminidase) and energy enzyme activities (lactate dehydrogenase, LDH, triose-3-phosphate dehydrogenase, TPDH,d-hexokinase, HK and citrate synthase, CS) were determined 3 days after denervation, 3, 7 and 10 days after hypertrophy had been induced and 3 days after denervation of hypertrophying muscles on day 3 and 7. Normal non-operated rats of corresponding body wieght served as controls and their enzyme activities were estimated on the same day. In the course of muscle hypertrophy, the 4 lysosomal enzyme activities increased progressively. Although 3 days' denervation of control muscles did not alter lysomal enzyme activities, denervation of hypertrophying muscles greatly enhanced the activity of these enzymes. Enzymes of energy metabolism were affected to a lesser degree. The results suggest that denervation of hypertrophying muscles causes more extreme changes in muscle weight and lysosomal enzyme activities than denervation alone. The possible implications of this finding are discussed in relation to the rapid atrophy.     

Postnatal growth of motor nerve terminals in muscles of the mouse

Summary A reduced silver stain was used to examine the development of complexity of motor nerve terminals in the postnatal period. Terminals in three histochemically different muscles were examined in mice aged 12 days to 3 years. The total number and total length of intraterminal axon branches increases with age, but only until animals are 3 months old. Terminals become longest and most branched in the histochemically glycolytic tensor fascia latae (TFL) muscle, shortest and least branched in the oxidative diaphragm, and intermediate in the histochemically mixed gluteus muscle. In addition, myelinated terminal branches develop in TFL and to a lesser extent in the gluteus between 1 and 3 months of age. These myelinated branches appear to be produced by nodal sprouting from the penultimate node of Ranvier of the terminal axon, and also by myelination of pre-existing terminal branches. The diameter of muscle fibres also increases until animals are 3 months old, and there is a good correlation between mean fibre diameter and either mean terminal length or mean number of terminal branches when all muscles at all ages are compared. This suggests that terminal growth could be determined by muscle fibre growth; however, within any given muscle there is little or no correlation between the diameter of a muscle fibre and either the length or number of branches of its nerve terminal, suggesting that terminal morphology is not controlled solely by muscle fibre growth. The presence of a myelinated branch in a nerve terminal is also unrelated to fibre diameter within a given muscle, but again when means are compared there are good, but significantly different correlations for the three different muscles. Thus some kind of muscle or nerve type-specific property additional to a general effect of muscle fibre size influences the development of myelinated terminal branches.Between 3 and 12 months of age terminal complexity remains constant or may decrease slightly. At 19 months or older, when mice are becoming senile, a large proportion of synapses have terminal sprouts and muscle fibres become innervated by two or more distinct axons. These changes can be attributed to the death of some motor neurons and sprouting of the remaining axons.     

The influence of secretin on the secretion of pepsin in response to acid stimulants in the anaesthetized cat.

1. The problem of selectivity during reinnervation of skeletal muscle fibres was investigated in the rat using the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus muscles and their nerves. 2. After an operation on these nerves permitting them to compete for reinnervation of one or the other muscle (hereafter called Y-union), virtually the total isometric tetanic tension of EDL muscle could be elicited by stimulating the EDL nerve, while stimulating the soleus nerve yielded little or no tension. In the case of the soleus muscle, stimulation of either nerve elicited about half of the total isometric tetanic tension. 3. During the course of reinnervation of these muscles in non-competitive situations, the time course of increase in the ratio of tension elicited by nerve stimulation to that by direct stimulation was slower in the case of soleus nerve reinnervating EDL muscle, compared with cross-reinnervation in the reverse direction or reinnervation of each muscle by its own nerve. 4. Crushing the common peroneal nerve 12 days after a Y-union in an attempt to retard the EDL nerve did not favour reinnervation of the EDL by soleus nerve, but crushing the nerve again or just once at 1 month after the original operation produced substantial partial reinnervation of the EDL by the soleus nerve. 5. It is concluded that soleus nerve fibres form functioning neuromuscular synapses on EDL muscle fibres only with difficulty. The pattern of reinnervation reveals characteristic differences between fast-twitch and slow-twitch muscles on the one hand and between their respective nerves on the other.     

Late reinnervation of the rat soleus muscle is differentially suppressed by chronic stimulation and by ectopic innervation

Soleus (SOL) and extensor digitorum longus (EDL) muscles in adult rats were kept denervated for 2 months by four repeated freezes at 2-week intervals of the sciatic nerve. Reinnervation was studied in the absence or presence of chronic muscle stimulation, starting 1 month before reinnervation began. In addition, reinnervation was studied in SOL muscles where a previously transplanted fibular (FIB) nerve had formed ectopic neuromuscular junctions outside the original endplate area. After repeated freezes only, reinnervation was complete judged by tension measurements and histochemical examinations in SOL (n = 7) and EDL (n = 8) muscles. In directly stimulated muscles reinnervation was incomplete, and the force tensions evoked from indirect stimulation was on average 87 (n = 5) and 82% (n = 5) of direct muscle stimulation in SOL and EDL muscles, respectively. Of ectopically innervated SOL muscle fibres, only 26% became reinnervated in 12 muscles. Denervation and reinnervation increased the number of muscle fibres in stimulated (n = 4) and unstimulated (n = 5) EDL muscles by 18 and 15%, respectively. In stimulated (n = 4) and unstimulated (n = 7) SOL muscles, on the other hand, the number of muscle fibres remained normal. The stronger suppression of reinnervation in ectopically reinnervated compared to chronically stimulated SOL fibres indicates that reinnervation can also be suppressed by activity independent influences from the foreign nerve.