Effect of exercise on physiological age-related change at mouse neuromuscular junctions

To determine the effect of endurance exercise on physiological age-related change at the mouse neuromuscular junction (NMJ), synaptic function was studied for extensor digitorum longus (EDL) and soleus muscles of three C57BL/6J mouse groups, 1) young adult control (YC: 10 months), 2) old control (OC: 20 months), and 3) old mice which exercised (OE: 20 months) since young-adulthood. Electrophysiological properties were studied with intracellular recording techniques. Safety margin was studied by measuring indirect isometric twitch tension in different calcium concentrations. With sedentary aging, EDL and soleus quantal contents increased. Following aging combined with 10 months of exercise, the EDL quantal contents in OE and YC animals were similar. In contrast, soleus quantal content was greater in OE than in YC animals. Determined safety margins were OC greater than YC = OE for EDL, and OC = YC = OE for soleus. This is the first study to indicate that physiological age-related changes at NMJs of EDL and soleus muscles are affected differently by endurance exercise. Exercise prevented all physiological age-related changes in EDL NMJs but not in soleus NMJs, this suggests that EDL changes are associated with inactivity during aging, while soleus changes are "fundamental" age changes.     

The effects of heat stress on neuromuscular activity during endurance exercise

This study analysed the effect of hot (35 degrees C) and cold (15 degrees C) environments on electromyographic (EMG) signal characteristics, skin and rectal temperatures and heart rate during progressive endurance exercise. Eight healthy subjects performed three successive 15-min rides at 30%, 50% and 70% of their peak sustained power output and then cycled at increasing (15 W/min) work rates to exhaustion in both 35 degrees C and 15 degrees C environments. Skin and rectal temperatures, heart rate and EMG data were measured during the trials. The skin temperatures were higher and the subjects felt more uncomfortable in the hot conditions (Bedford scale) ( P<0.01). Rectal temperature was slightly, but not significantly, higher under hot conditions. Heart rate was significantly higher in the hot group (between condition P<0.05). Peak power output (267.4+/-67.7 W vs. 250.1+/-61.5 W) and time-to-exhaustion (55.7+/-16.7 min vs. 54.5+/-17.1 min) (COLD vs. HOT) were not different between conditions. There were no differences in integrated EMG (IEMG) or mean power frequency spectrum between conditions. Rating of perceived exertion increased similarly in both conditions over time. Although the hot conditions increased heart rate and skin temperature, there were no differences in muscle recruitment or maximal performance, which suggests that the thermal stress of 35 degrees C, in combination with exercise, did not impair maximal performance in this study.     

Effects of chronic corticosterone treatment on the morphology of rat neuromuscular junctions

This study examined the effects of chronic corticosterone (CORT) treatment on the morphology of two physiologically different muscles. Nerve terminals from the slow twitch soleus (SOL) and fast twitch extensor digitorum longus (EDL) of Fischer 344 rats were stained using the zinc iodide osmium (ZIO) technique. Nerve terminal area, perimeter, and longitudinal extent length were measured using computer-aided morphometry. Slow and fast muscle fibers from animals which received 5-10 mg CORT per day for 3 months were atrophied compared with controls. Treated animals failed to gain weight during the study, while controls gained 37%. Adrenal weights in treated animals were 30% less than controls, after correction for body weight. Morphological parameters for SOL nerve terminals were generally larger in the CORT group, while EDL nerve terminals from the CORT group did not differ significantly from controls. Soleus nerve terminal area was 43% greater, perimeter 14% longer, and longitudinal extent length 18% longer than the control nerve terminals. This study demonstrates a greater effect of CORT treatment on slow twitch muscle than has been demonstrated in previous studies. Changes in the nerve terminal morphology of the SOL were also greater than in previous studies and suggest that a functional adaptation or remodelling may occur following CORT treatment to maintain the neuromuscular interface during the enhanced catabolic effects of the steroid. These steroid-induced stress changes are similar in some respects to those observed in aging and disuse studies of the vertebrate neuromuscular junction. This suggests that glucocorticoid hormones may play an etiological role in the homeostasis of the neuromuscular junction in response to various stimuli.     

Effects of chronic corticostorone treatment on the morphology of rat neuromuscular junctions

This study examined the effects of chronic corticosterone (CORT) treatment on the morphology of two physiologically different muscles. Nerve terminals from the slow twitch soleus (SOL) and fast twitch extensor digitorum longus (EDL) of Fischer 344 rats were stained using the zinc iodide osmium (ZIO) technique. Nerve terminal area, perimeter, and logitudinal extent legth were measured using computer-aided morphometry. Slow and fast muscle fibers from animals which received 5–10 mg CORT per day for 3 months were atrophied compared with controls. Treated animals failed to gain weight during the study, while controls gained 37%. Adrenal weights in treated animals were 30% less than controls, after correction for body weight. Morphological parameters for SOL nerve terminals were generally larger in the CORT group, while EDL nerve terminals from the CORT group did not differ significantly from controls. Soleus nerve terminal area was 43% greater, perimeter 14% longer, and logitudinal extent length 18% longer than the control nerve terminals. This study demonstrates a greater effect of CORT treatment on slow twitch muscle than has been demonstrated in previous studies. Changes in the nerve terminal morphology of the SOL were also greater than in previous studies and suggest that a functional adaptation or remodelling may occur following CORT treatment to maintain the neuromuscular interface during the enhanced catabolic effects of the steroid. These steroid-induced stress changes are similar in some respects to those observed in aging and disuse studies of the vertebrate neuromuscular junction. This suggests that glucocorticoid hormones may play an etiological role in the homeostasis of the neuromuscular junction in response to various stimuli.     

Effects of ageing and endurance exercise training on alpha-actinin isoforms in rat plantaris muscle

Aim: We recently reported that α‐actinin adaptation occurs at the isoform level. This study was undertaken to clarify the effects of: (1) ageing‐induced shift of myosin heavy chain (MyHC) composition and (2) endurance exercise training on α‐actinin isoforms in rat plantaris muscle. Methods: Adult (18 mo) and old (28 mo) male Fischer 344 rats were assigned to either sedentary control or endurance exercise training groups. Animals in the training groups ran on a treadmill for 8 week with training intensity adjusted to be equal for adult and old groups. After the training was completed, the plantaris muscles were taken for analyses of α‐actinin‐2, α‐actinin‐3, and MyHC composition and metabolic enzyme activities. Results: The proportion of type IIb MyHC was lower, and that of type I MyHC was higher in old animals than in adult animals. α‐actinin‐3 was significantly lower in old animals than in adult animals. No significant difference was found in α‐actinin‐2 and citrate synthase (CS) activity between adult and old animals. Citrate synthase activity was higher in trained animals than in sedentary animals. Endurance training produced a fast‐to‐slow shift within type II MyHC isoforms in both adult and old animals. α‐actinin‐2 was significantly higher in trained animals than in sedentary animals. No significant difference was found in α‐actinin‐3 between trained and sedentary animals. Conclusion: These results support the α‐actinin adaptation at the isoform level and show that the α‐actinin‐3 expression depends on the amount of type II MyHC, whereas α‐actinin‐2 expression is associated with improvement of muscular aerobic capacity.     

Fluorescent staining of living mouse neuromuscular junctions

A technique for staining living neuromuscular junctions is described and used for electrophysiological recording with both intra- and extra-cellular micro-electrodes. This technique may be used on thicker muscles (eg. mouse soleus), has no effect on any of several physiological parameters tested, and should have several useful applications.     

Ultrastructural studies of young and old mouse neuromuscular junctions

Summary The ultrastructure of the neuromuscular junction of young and old male CBF-1 mice was analysed both qualitatively and quantitatively. The age-related findings were similar in both the phasic extensor digitorum longus muscle and the tonic soleus muscle but more pronounced in the latter. Presynaptic terminals of old mice compared to young showed decreases in nerve terminal area, mitochondria and synaptic vesicles, but increases in smooth endoplasmic reticulum, coated vesicles, cisternae, microtubules and probably neurofilaments. On the postsynaptic side there were increases in complexity of junctional folds and subsarcolemmal vesicles, and the appearance of lipofuscin deposits. Occasional denervated postsynaptic regions were encountered in old neuromuscular junctions, but the predominant characteristics of aging changes were not those of denervation. Rather, a unique and uniform process involving most of the population of nerve terminals, possibly of physiologically adaptive significance, appears to occur with age in both phasic and tonic limb muscles.     

Ultrastructural studies of normal and degenerating mouse neuromuscular junctions

Summary A quantitative study of the ultrastructural changes occurring at degenerating motor axon terminals of the mouse hemidiaphragm in the first 26 h following unilateral phrenicotomy has been made. Several ultrastructural characteristics of axon terminals from normal diaphragms and phrenicotomized and control hemidiaphragms of phrenicotomized preparations were analyzed. Considerable swelling and disruption of mitochondria occurred in terminals of control hemidiaphragms at 3, 6 and 9 h post-phrenicotomy after which no more damage than that seen in normal terminals after fixation for electron microscopy was observed. At terminals of the phrenicotomized hemidiaphragm, much mitochondrial damage was observed from 3 h post-phrenicotomy onwards. In phrenicotomized hemidiaphragms all terminals had completely degenerated by 26 h post-phrenicotomy. Reduced synaptic vesicle population densities occurred during degeneration. At many axon terminals on phrenicotomized hemidiaphragms the population densities of synaptic vesicles were reduced compared with controls and vesicle aggregates were noted in many engulfed or partially engulfed nerve terminals.These results are discussed with respect to the vesicle hypothesis for nerve-muscle transmission. The mechanisms underlying Schwann cell hyperactivity are also considered.     

Effects of endurance training on lactate removal by oxidation and gluconeogenesis during exercise

This report describes the effects of 9 weeks of endurance-training on the relative rates of lactate removal via oxidation and gluconeogenesis in humans. Before and after training, eight subjects performed incremental (60 W plus 40 W every 6 min) exercise tests, while¹⁴C-lactate was infused into one forearm vein and arterialized venous blood was sampled from the other forearm. During the trial, the volume of expired¹⁴CO₂ and circulating¹⁴C-lactate and¹⁴C-glucose specific radioactivities were measured. Such measurements revealed that training increased the estimated oxidation of equivalent venous blood lactate concentrations [VLa] of greater than 1.6 mmol/l. These increases in lactate oxidation were more than would be predicted from the approximately 40% higher O₂ uptake values at any [VLa] after training. At a [VLa] of 6 mmol/l, rates of lactate oxidation were increased by some 100% following training, from 105±12 to 208±33 mol/min/kg (P<0.01). Improvements in lactate oxidation after training reduced the estimated rates of lactate-to-glucose conversion from 40±3 to 9±2 mol/min/kg at a [VLa] of 2.5 mmol/l (P<0.01). Thus, unlike in rats, human endurance-training does not increase gluconeogenesis. In the final stages of progressive exercise after training, more than 80% of lactate was oxidised and accounted for approximately 45% of overall carbohydrate oxidation.     

Effects of endurance training on hyperammonaemia during a 45-min constant exercise intensity

Summary Eleven laboratory-pretrained subjects (initial =54 ml·kg⁻¹·min⁻¹) took part in a study to evaluate the effect of a short endurance training programme [8–12 sessions, 1 h per session, with an intensity varying from 60% to 90% maximal oxygen consumption ] on the responses of blood ammonia (b[NH ₄ ⁺ ]) and lactate (b[la]) concentrations during progressive and constant exercise intensities. After training, during which did not increase, significant decreases in b[NH ₄ ⁺ ], b[la] and muscle proton concentration were observed at the end of the 80% constant exercise intensity, although b[NH ₄ ⁺ ] and b[la] during progressive exercise were unchanged. On the other hand, no correlations were found between muscle fibre composition and b[NH ₄ ⁺ ] in any of the exercise procedures. This study demonstrated that a constant exercise intensity was necessary to reveal the effect of training on muscle metabolic changes inducing the decrease in b[NH ₄ ⁺ ] and b[la]. At a relative power of exercise of 80% , there was no effect of muscle fibre composition on b[NH ₄ ⁺ ] accumulation.     

Acetylcholine receptors at mature and aged mouse neuromuscular junctions

The density and distribution of junctional and perijunctional ACh receptors (AChR) were studied in young (8-12 months) and old (24-25 months) C57 mice to determine: (1) if increased amplitude of spontaneous postsynaptic potentials previously reported in old C57 muscle was due to increased junctional AChR; (2) if increased extrajunctional AChR would be found in association with previously reported nerve terminal complexity; and (3) if extrajunctional AChR was present as in disused or denervated muscle. Microdissection of individual muscle fibers combined with I125-alpha-bungarotoxin labeling, gamma counting, measurement of surface area, cholinesterase stains, and autoradiography were used to obtain the results. In both young and old mice there was a sharp gradient in AChR between the end-plate and the perijunctional region. End-plate AChR densities and total AChR per end-plate were the same at old and young end-plates, as were perijunctional values. Thus, neither end-plate nor extrajunctional AChR density changes with age. An increased mepp amplitude reported previously in old CB57 animals must be due to other factors. The perijunctional AChR in old mice show no changes characteristic of disuse or denervation, or those which might give rise to the observed nerve terminal complexity.     

Filopodia, lamellipodia and retractions at mouse neuromuscular junctions

Summary In order to determine if mature motor nerve terminals retain structures associated with development such as filopodia and lamellipodia, we studied whole mounts of mature mouse neuromuscular junctions stained with both fluorescent-labelled tetanus toxin C-fragment and alpha-bungarotoxin, and employed electron microscopy in parallel. The rapid fluorescent stain may be of general usefulness. Both filopodia and lamellipodia were found, extending beyond the border of the established postsynaptic receptors. Filopodia often appeared in clusters, were devoid of a synaptic vesicle antigen, and many withdrew in response to cytochalasin D. Control experiments demonstrated that filopodia were not induced by the toxin treatment. The mean number of filopodia per endplate varied from about one in phasic muscle to three in tonic muscle, and was twice as great in immature mouse muscle. Postsynaptic receptor-rich regions without overlying terminals were less numerous than filopodial and lamellipodial projections without underlying receptors. Electron microscopy showed that lamellipodia contained actin-like filaments and immunoreactivity to actin, but no neurofilaments, microtubules, mitochondria or vesicles. Therefore, these structures would not be visualized byin vivo mitochondrial stains. The lamellipodia protruded into the gap between muscle and a closely overlying Schwann cell process. Lamellipodia occupied about 5% of the linear extent of the terminal arbor in whole mounts, but appeared in 16% of random electron micrographic fields. Thus, the lamellipodia and filopodia typical of developing terminals are present in adulthood and represent a distinctive specialization of the nerve terminal, which may interact with the adjacent Schwann and muscle cell. The frequency of filopodia is a function of age and of muscle or motoneuron type. We suggest that some of the factors known to regulate growth of filopodia and lamellipodiain vitro or in development may continue to act at adult presynaptic nerve terminals.     

Effects ofAnemonia sulcata toxin II on presynaptic currents and evoked transmitter release at neuromuscular junctions of the mouse

The effect ofAnemonia sulcata toxin II (ATX-II) on the amount of transmitter released by nerve impulses was investigated in motor end-plates of the mouse. ATX-II (80 nM) caused repetitive end-plate potentials in response to a single nerve stimulus and a 3- to 4-fold increase in the quantal content of the phasic end-plate potential. This increase is less than what would be expected if ATX-II induced plateau action potentials at the motor endings. To solve this discrepancy presynaptic currents were recorded by focal extracellular electrodes. It was found that the K current present at the endings is strong enough to prevent the development of presynaptic plateau action potentials, in contrast to what has been observed in other excitable membranes (unmyelinated axons, nodes of Ranvier and skeletal muscle fibres). By using tetraethylammonium and 3,4-diaminopyridine to block K channels and Co²⁺ to block Ca channels, ATX-II allowed the development of prolonged plateau responses at the endings upon motor nerve stimulation. These results suggest that the mouse motor endings are endowed with a relatively powerful K channel system, which effectively controls the amount of presynaptic depolarization.     

Acute effects of endurance exercise on mitochondrial distribution and skeletal muscle morphology

Summary Biopsies of vastus lateralis from seven well-trained males were studied 1 month before and 15–30 min after a 100-km race. The distribution of interfibrillar mitochondria was analyzed to determine whether a long bout of exercise induced a redistribution of mitochondria. Capillary densities and mean fiber areas were also estimated. Capillary density and mean interfibrillar mitochondrial volume density were found to be significantly correlated with running time in the race. An earlier study on these biopsies found that the mean volume densities of interfibrillar and subsarcolemmal mitochondria did not change after a race, but the volume densities of lipid droplets and interfibrillar glycogen decreased significantly. In the present study, volume density of interfibrillar mitochondria [V_v(mi, fim)] before the race was highest with a value of 0.098±0.007 near the fiber border, and decreased progressively with distance to 0.045±0.004 at the fiber center. After the race, V_v(mi, fim) was unchanged at the fiber border, but was significantly higher (0.062±0.005) in the center of the fiber. This increase in mitochondrial volume density was attributable to the shrinkage of the fibers from consumption of energy stores, which was relatively greater for interfibrillar glycogen than for subsarcolemmal glycogen. Thus the primary effect of this extended bout of endurance exercise on vastus lateralis was the nearly complete depletion of the interfibrillar glycogen and lipids, but there was no evidence of an acute redistribution of mitochondria.Supported by a Postdoctoral Fellowship from the Swiss National Science Foundation     

General and strain-specific age changes at mouse limb neuromuscular junctions

Age changes at limb neuromuscular junctions (NMJs) of CB57 and BALB/C mice were investigated and compared with previous results in a hybrid strain, in order to identify common characteristics of neuromuscular transmission and to assess the prevalence of signs of denervation and disuse. Resting membrane potential decreased with age in soleus muscles with no change in passive membrane properties. Miniature end-plate potential, amplitude and quantal content were greatly increased, with no change in muscle fiber diameter or nerve terminal morphometry. The various observed patterns of age changes were not those of disuse or denervation. Although there were diverse age changes in spontaneous transmitter release in different mouse strains, increased transmitter release per unit length of nerve terminal in limb muscle was a notably consistent finding across strains. It is apparently less subject to epigenetic variation during aging than most other reported neuromuscular physiological parameters.     

The overall morphology of neuromuscular junctions as revealed by scanning electron microscopy

Summary Skeletal neuromuscular junctions (NMJs) of vertebrates were examined by scanning electron microscopy after removal of connective tissue components by HCl hydrolysis. In addition to the surface texture of NMJs, the subsynaptic organization of the sarcolemma was visualized in specimens in which nerve endings were detached from the muscle surface.A remarkable morphological variability between animal species was observed. The NMJs in the frog sartorius muscle consisted of longitudinal ribbon-like endings which fitted into a shallow synaptic gutter containing highly ordered cross-bands of junctional folds. The NMJs of the posterior latissimus dorsi muscle of the zebra finch were characterized by varicose swellings of the nerve endings which fitted into a round pit of the sarcolemma. NMJs in the sternothyroid muscle of the Chinese hamster consisted of thin ramified endings which were confined to an oval area on the muscle surface. The labyrinthine synaptic groove contained well-developed junctional folds without preferential spatial arrangement.The procedure used for the present study illustrates in great detail the terminal arborization of the motor nerve ending and the surface features of the subsynaptic sarcolemma. It may also allow quantitative study of the synaptic morphology of NMJs.     

The effects of exercise training of different intensities on neuromuscular junction morphology

Summary Little is known about the effects of exercise training on neuromuscular junction morphology in skeletal muscle. The objectives of this investigation were: 1) to determine if exercise training would elicit changes in neuromuscular junction morphology, 2) to determine if exercise training of different intensities would evoke specific changes in neuromuscular junction morphology, and 3) to determine whether changes in neuromuscular junction structure occur independently of changes in muscle fibre type and size. Twenty-four age and size matched male Sprague-Dawley rats were randomly assigned to three groups: high-intensity trained (HIT), low-intensity trained (LIT), or untrained. Neuromuscular junction morphology of the soleus muscle was determined via immunofluorescent staining. Presynaptic acetylcholine vesicles were visualized with SV-2 antibody in conjunction with fluorescein isothiocyanate labelled secondary antibody. Postsynaptic acetylcholine receptors were identified with rhodamine labelled -bungarotoxin. Laser scanning microscopy was used to produce images of synapses, which were used to quantitate the following: total area of SV-2 and -bungarotoxin staining, density of acetylcholine vesicles and receptors, structural complexity, and synaptic coupling. To visualize nerve terminal branching, a smaller number of neuromuscular junctions were stained with C-2 antibody, which reacts with a neurofilament epitope, in conjunction with fluorescein isothiocyanate labelled secondary antibody. Total length of branching, number of branches, average length of branches, and ratio of secondary to primary branches per neuromuscular junction were determined. Citrate synthase activity, fibre type composition and fibre cross-sectional areas of the soleus muscle were assessed to determine the presence of a training effect in that muscle. Results indicate that training did induce hypertrophy of the neuromuscular junction that was independent of muscle hypertorphy. Although the HIT and LIT groups exhibited similar hypertrophic responses of the neuromuscular junction, the HIT group displayed more dispersed synapses than the LIT group. Neither exercise training program, however, resulted in altered densities of acetylcholine vesicles or receptors, nor did training significantly change synaptic coupling. Nerve terminal branching was also affected by exercise training. Neuromuscular junctions from the HIT group demonstrated a greater total length of branching, average length per branch, and number of finer, or secondary, branches than those of the LIT group.     

Noise analysis of acetylcholine induced current at neuromuscular junctions of the mouse diaphragm

Miniature end-plate currents (mepcs) and membrane noise elicited by acetylcholine (ACh) iontophoresis were investigated at neuromuscular junctions of the mouse diaphragm. All the experiments were performed at a holding potential of -70 mV at a temperature of 19 degrees C. The equilibrium potential of the ACh response was estimated to be near zero; the mepcs displayed a peak amplitude of 2.46 +/- 0.13 nA (mean +/- S.E.) and relaxed exponentially with a time constant to 1.63 +/- 0.11 msec. Single ACh-activated channels had a conductance of 26.5 +/- 1.5 pS and a mean life time of 1.69 +/0- 0.13 msec.     

Effects of aerobic endurance training status and specificity on oxygen uptake kinetics during maximal exercise

The main purpose of this study was to analyze the effects of exercise mode, training status and specificity on the oxygen uptake (O₂) kinetics during maximal exercise performed in treadmill running and cycle ergometry. Seven runners (R), nine cyclists (C), nine triathletes (T) and eleven untrained subjects (U), performed the following tests on different days on a motorized treadmill and on a cycle ergometer: (1) incremental tests in order to determine the maximal oxygen uptake (O_2max) and the intensity associated with the achievement of O_2max (IO_2max); and (2) constant work-rate running and cycling exercises to exhaustion at IO_2max to determine the effective time constant of the O₂ response (O₂). Values for O_2max obtained on the treadmill and cycle ergometer [R=68.8 (6.3) and 62.0 (5.0); C=60.5 (8.0) and 67.6 (7.6); T=64.5 (4.8) and 61.0 (4.1); U=43.5 (7.0) and 36.7 (5.6); respectively] were higher for the group with specific training in the modality. The U group showed the lowest values for O_2max, regardless of exercise mode. Differences in O₂ (seconds) were found only for the U group in relation to the trained groups [R=31.6 (10.5) and 40.9 (13.6); C=28.5 (5.8) and 32.7 (5.7); T=32.5 (5.6) and 40.7 (7.5); U=52.7 (8.5) and 62.2 (15.3); for the treadmill and cycle ergometer, respectively]; no effects of exercise mode were found in any of the groups. It is concluded that O₂ during the exercise performed at IO_2max is dependent on the training status, but not dependent on the exercise mode and specificity of training. Moreover, the transfer of the training effects on O₂ between both exercise modes may be higher compared with O_2max.