Changes in the muscle fibre extracellular action potentials in long-lasting (fatiguing) activity

The dependence of extracellular action potentials (ECAPs) of single frog muscle fibres on intracellular action potentials (ICAPs) was studied during long-lasting (fatiguing) activity. The conduction velocity, peak-to-peak amplitude and amplitudes of the separate phases of the first and second ICAP time derivatives decreased during long-lasting activity. The phases of the first and second ICAP space derivatives also decreased in amplitude and lengthened. ECAPs near the membrane were similar in shape and proportional in amplitude to (formula; see text) when recording at a distance from both the end of the fibre and the point of stimulation. At long radial distances, the amplitudes of the separate ECAP phases depended on the amplitude and length of the corresponding phases of (formula; see text). Thus the decrease in ECAP amplitude during long-lasting activity at long radial distances was less than at points close to the muscle fibre membrane. The consequences of these findings for the changes in electromyograms recorded by needle or superficial electrodes during long-lasting (fatiguing) activity are discussed.     

Differential changes in myoelectric characteristics of slow and fast fatigable frog muscle fibres during long-lasting activity.

The electrical activity of different muscle fibre types during fatigue at varying stimulation frequency and fibre stretch was studied. Extracellular action potentials (ECAPs) were recorded from isolated frog muscle fibres at initial length and stretched by 15%, 25% and 35% and stimulated for 180 s by suprathreshold pulses with frequencies of 5, 6.7 and 10Hz. The changes in ECAP negative phase duration (T(0)), propagation velocity of excitation (PV), potential power spectrum and its median frequency (MDF) were analysed for the period of uninterrupted activity (endurance time, ET). Slow (SMF) and fast (FMF) fatigable muscle fibre types were distinguished by the rate of PV decrease during ET. With the increase of stimulation frequency and fibre stretch, the rate of ECAP parameter changes increased and was larger in FMF, but this proportion was reversed with stretching over 25% and 10Hz stimulation. In both fibre types the power spectrum shift to lower frequencies during continuous activity was more pronounced with higher stimulation frequency. In FMFs the rates of MDF changes were positively and more strongly correlated with the rates of PV changes, whilst in SMFs the inverse correlation between the rates of changes of MDF and T(0) was stronger. The results indicate specific adaptation of slow and fast fatigable muscle fibres to stretch and activation frequency due to the differences in their membrane processes.     

Spectral and time domain characteristics of single muscle fibre action potentials during continuous activity extracted from model considerations

A model of the muscle fibre extracellular action potentials (ECAPs) calculation using experimentally recorded intracellular action potentials (ICAPs) has been applied to investigate the effect of repetitive stimulation on the electrical activity of isolated frog muscle fibres. The ECAPs were calculated both at small (0.01 mm) and at large (5 mm) radial distances to the fibre axis, and their relationship with the original ICAP parameters has been inferred. Fourier transformation of the calculated ECAPs in order to obtain the spectral characteristics and to trace out their behaviour during continuous fibre activity was performed. Stimulation frequency dependence on the ECAP time characteristics and on the shift of the maximum spectral density towards low frequencies at small and large radial distance were observed. The spectral density peak frequency is propagation velocity (PV)-dependent. The advantage of the presented method over the available experimental extracellular recording techniques from isolated muscle fibers is the possibility to show the effect of continuous muscle fibre activity on the parameters of the ECAPs and their spectral characteristics at large radial distance, which is not experimentally accessible. Our results are in agreement with those experimentally obtained. The results from the model prove the role of changes in PV of excitation along the muscle fibres (representing the last link in the complex organized motor system) in the development of fatigue. Received: 24 July 1997 / Accepted in revised form: 2 July 1998     

Extracellular potentials of single active muscle fibres: Effects of finite fibre length

The extracellular action potentials (ECAPs) of single active muscle fibres immersed an isotropic volume conductor were investigated. The origination of excitation in the motor end-plate and its reaching the fibre end were taken into consideration. It was explained why at short radial distances the ECAPs over the fibre at points close to the end were similar in shape to the first time derivative and at points close to the motor end-plate-to the first time derivative of the intracellular action potential (ICAP) taken with minus sign. The fibre end changed the ECAP which would be recorded if the fibre was infinite and this change called pure termination potential (PTP) was a biphase positive-negative potential, proportional to the first time derivative of the ICAP at points close to the membrane and over the very end. With increasing the radial and axial distances PTP decreases in amplitude. Taking into account the PTP, the genesis of the terminal positive phase of the ECAPs (Gydikov and Kosarov 1972a, b) can be explained. The onset of the fibre or the motor end-plate also changed the potential which would be recorded if the fibre was infinite. This change was given the term of pure onset potential (POP)-a biphase negative-positive potential, proportional to the first time derivative of the ICAP taken with minus sign at a point close to the membrane and over the motor end-plate. With increasing the radial and the axial distance POP decreased in amplitude.Close to the membrane PTP and POP were commensurable with the potential of an infinite fibre only at points close to the ends or to the motor end-plate. At long radial distances they were commensurable with the potential of an infinite fibre for all axial distances.     

Extracellular action potentials of skeletal muscle fibre affected by 4-aminopyridine: a model study

A new specially designed analytical function approximating the intracellular action potentials (ICAPs) for calculation of the extracellular potentials (ECAPs) at various radial and axial distances from the active fibre is proposed. 4-Aminopyridine (4-AP) was used to obtain ICAPs with a prolonged repolarization phase in order to investigate the influence of changes in ICAP shape on the ECAPs. From the experimentally recorded ICAPs before and after treatment of frog skeletal muscle fibres with 4-AP, approximated by the new function, the ECAPs were calculated applying the linesource model in a finite fibre. Using this function allowed calculation of the ECAPs at distances not accessible for the experimental recordings. The total ionic current (I _i) during the action potential was calculated using the cable equation. Our results showed that the ratio of the first positive to the negative phases of the ECAPs of treated fibres increased at large radial distances (3000 m and more) and the terminal positive phase was asymmetric with an abrupt initial deflection followed by a slow inverse deflection. The calculated ECAPs at various axial distances from the fibre end (cylindrical and conical part) and at radial distances from the fibre membrane ranging from 0 to 5000 m, corresponded in shape to the experimentally recorded potentials of untreated and 4-AP-treated muscle fibres.     

The radial distribution of succinate dehydrogenase activity in porcine muscle fibres

Summary A microscope photometer with a computer-controlled scanning stage was used to map the distribution of succinate dehydrogenase (SDH) activity in transverse sections of skeletal muscle fibres of pigs from 9 to 29 weeks of age. Absorbance due to SDH activity was measured in successive concentric zones that converged on the central axis of the muscle fibre. In all fibres, there was less SDH activity in the axis of the fibre than in the periphery of the fibre. In fibres with strong adenosine triphosphatase (ATPase) activity and weak overall SDH activity, the radial gradient of SDH activity remained constant as fibres grew in cross-sectional area. However, in fibres with weak ATPase and strong SDH and in fibres with strong ATPase and strong SDH, the radial gradient of SDH activity increased as fibres grew larger. Changes in radial gradients were due to both decreased SDH activity in the axis and to increased SDH activity in the periphery. In all three fibre types, the overall SDH activity per fibre increased with age.     

Effect of local SHF-irradiation of the rat foot on impulse activity in the tibial nerve

In experiments on rats the effects of 2.45 GHz microwave radiation on impulse activity of the afferent structures were studied in single afferent fibres of n. tibialis. Microwaves of both low and high intensity (from 0.04 to 100 mW/cm2) were shown to modify the impulse activity of the afferents which was mainly displayed by the increase in a current average frequency of impulse activity.     

Metabolic and physiologic characteristics of skeletal muscle determine its response to clenbuterol treatment

beta-Adrenoceptor agonists are reported to induce skeletal muscle hypertrophy and hence serve as valuable adjunct to the treatment of wasting disorders. In the present study, we attempted to find out whether metabolic and physiologic characteristics of fibres are important in determining skeletal muscle response to clenbuterol (an adrenergic receptor agonist) therapy, as proposed in the treatment of wasting disorders. The treatment of mice with clenbuterol (2 mg/kg body wt for 30 days) resulted in skeletal muscle hypertrophy, more common amongst fast-twitch glycolytic fibres/muscle, with increase in body mass and a parallel rise in muscle mass to body mass ratio. Measurement of fibre diameters in soleus (rich in slow-twitch oxidative fibres), ALD or anterior latissimus dorsi (with a predominance of fast-twitch glycolytic fibres) and gastrocnemius (a mixed-type of muscle) from clenbuterol-treated mice for 30 days revealed noticeable increase in the per cent population of narrow slow-twitch fibre and a corresponding decline in white-type or fast-twitch glycolytic fibres in gastrocnemius and ALD. As revealed by counting of muscle cells in soleus, narrow red fibres declined with corresponding increase in white-type glycolytic fibres population. A significant decline in the succinic dehydrogenase activity was observed, thereby suggesting abnormality in oxidative activity of skeletal muscles in response to clenbuterol therapy.     

Voltage-clamp of cut-end skeletal muscle fibre: a diffusion experiment

Membrane potential and current were studied in cut end fibres of frog skeletal muscle under current and voltage clamp conditions, by the double sucrose gap technique. Similar action potentials were recorded under current clamp conditions with either the microelectrode or the double sucrose gap techniques. Under voltage clamp conditions, the control of the membrane potential was maintained adequately. The early current was sensitive to both TTX and external Na concentration suggesting that the current was carried by Na ions. Sodium current (INa) was subsequently analysed using the Hodgkin-Huxley formulae. INa half-activation and inactivation occurred at -34 mV and -60 mV, respectively. Na-rich solution applied internally by diffusion through cut ends produced a reduction of INa associated with a shift of the sodium current reversal potential (VNa) towards more negative membrane potentials. This suggested that the sodium electromotive force was reduced by the increase in internal Na content of the fibre. Iodate applied externally changed neither the activation nor the inactivation time courses of INa, but reduced the peak current. Conversely, internally applied by diffusion from the cut end of skeletal muscle fibre, iodate slowed down the time course of INa inactivation and decreased the current peak. In conclusion, the double sucrose gap technique adapted to cut end frog skeletal muscle fibre allows a satisfactory analysis of INa.     

Skeletal muscle abnormalities associated with occupational exposure to mercury vapours

There is scarce information on the possible effects of chronic exposure to mercury on skeletal muscle. Dental personnel are frequently exposed to inhalation of metallic mercury vapours. The skeletal muscle of five technicians and one dentist (females, age 36-55) was studied. All of them presented symptoms of chronic mercury poisoning. Needle biopsy was taken from the quadriceps femoris muscle and samples were prepared for light microscope histochemistry and for transmission electron microscopy. Selective atrophy of type IIB muscle fibres was found in patients, and in one of them there was fibre grouping. Most of the muscles showed increased fibre area per capillary. Atrophy was confirmed by the ultrastructural study, demonstrating increase of intermyofibrillar spaces, loss of myofibrils or complete disappearance in some fibres, and sarcolemmal foldings. Splitting of the fibres was also found. Some capillaries were altered, showing endothelial infoldings into the lumen, thickened basement membrane and partial or total occlusion. The alterations found in muscle may be secondary to nerve damage, to ischemia caused by capillary lesion and/or to a direct effect of mercury on muscle fibre proteins.     

Influence of 2.45 GHz microwave radiation on enzyme activity

Summary The in vitro activity of acetylcholinesterase and creatine phosphokinase was determined during in vitro exposure to 2.45 GHz microwave radiation. The enzyme activities were examined during exposure to microwave radiation at specific absorption rates (SAR) of 1, 10, 50, and 100 mW/g. These specific absorption rates had no effect on the activity of either enzyme when the temperature of the control and exposed samples were similar. These data demonstrate that the activity of these two enzymes is not affected by microwave radiation at the SARs and frequency employed in this study.     

Biochemical verification of quantitative histochemical analysis of succinate dehydrogenase activity in skeletal muscle fibres

Summary The purpose of this investigation was to examine critically the validity of a computerized quantitative microphotometric histochemical technique for the determination of succinate dehydrogenase (SDH) activity in skeletal muscle fibres. Sections from the anterior costal diaphragm were removed from Fischer-344 rats (n = 12) and assayed histochemically to determine SDH activity. The SDH activity in individual muscle fibres was computed using a computerized microphotometric histochemical technique which involves measurement of the optical density of deposited diformazan derived from nitroblue tetrazolium within the fibres. To validate the histochemical technique, whole muscle SDH activities were calculated from the histochemical procedure and were compared to SDH activities determined from whole muscle homogenates via a standard quantitative biochemical assay. The mean within-day variability of the computerized microphotometric histochemical technique of determining SDH activity was 6% (range = 0.5–10.9%) for an area containing ~50 fibres and 6.1% (range = 1.05–14.9%) for an individual muscle fibre. Similarly, the mean between-day variability of the microphotometric histochemical technique of determining SDH activity was 5.9% (range = 2.6–13.9%) for an area containing ~50 fibres and 6.6% (range = 2.2–13.9%) for an individual muscle fibre. The inter-class correlation coefficient between biochemically determined SDH activity and histochemically determined SDH activity was r = 0.83 (p < 0.05). Collectively, these data demonstrate that the quantitative histochemical technique of Blanco et al. (1988) is both valid and reliable in the determination of SDH activity in skeletal muscle fibres.     

Carbonic anhydrase activity in mammalian skeletal and cardiac muscle.

1. The presence of extravascular carbonic anhydrase activity in skeletal muscle, and its absence from cardiac muscle, were demonstrated in the rat. 2. The activity in skeletal muscle is approximately correlated with the proportion of dark fibres present in the middle fibre bundles.     

Slow and fast fatigable frog muscle fibres: electrophysiological and histochemical characteristics

Continuous activity of isolated frog gastrocnemius muscle fibres provoked by repetitive stimulation of 5 Hz was used as an experimental model for fatigue development in different fibre types. Parameter changes of the elicited intracellular action potentials and mechanical twitches during the period of uninterrupted activity were used as criteria for fatigue evaluation. Slow fatigable muscle fibre (SMF) and fast fatigable muscle fibre (FMF) types were distinguished depending on the duration of their uninterrupted activity, which was significantly longer in SMFs than in FMFs. The normalized changes of action potential amplitude and duration were significantly smaller in FMFs than in SMFs. The average twitch force and velocity of contraction and relaxation were significantly higher in FMFs than in SMFs. Myosin ATPase (mATPase) and succinate dehydrogenase activity were studied by histochemical assessment in order to validate the fibre type classification based on their electrophysiological characteristics. Based on the relative mATPase reactivity, the fibres of the studied muscle were classified as one of five different types (1-2, 2, 2-3, 3 and tonic). Smaller sized fibres (tonic and type 3) expressed higher succinate dehydrogenase activity than larger sized fibres (type 1-2, 2), which is related to the fatigue resistance. The differences between fatigue development in SMFs and FMFs during continuous activity were associated with fibre-type specific mATPase and succinate dehydrogenase activity.     

Microwave facilitation of methylatropine antagonism of central cholinomimetic drug effects

Pilocarpine-induced hypothermia and oxotremorine-induced tremors in mice are central cholinomimetic drug effects that are readily blocked by the muscarinic antagonist atropine. However, the quaternary ammonium derivative of atropine, methylatropine, is unable to block these cholinomimetic drug effects by virture of its inability to penetrate the blood-brain barrier (BBB) and blood-cerebral spinal fluid barrier (B-CSFB). Dose-response curves for pilocarpine and oxotremorine effects are not appreciably affected either by pretreatment with methylatropine (1.0 mg/kg) or by exposure to moderate-level microwave irradiation (2.45 GHz, 23.7 W/kg, CW, 10-min exposure). However, in mice receiving both the methylatropine pretreatment and microwave irradiation, the dose-response curves for both pilocarpine and oxotremorine effects were significantly shifted to the right, signifying a central anticholinergic action by methylatropine. These data indicate that a single acute exposure to a thermogenic level of microwave irradiation facilitates methylatropine antagonism of centrally mediated cholinomimetic drug effects. One possible explanation for this observation is that microwave radiation may enhance passage of quaternary ammonium compounds like methylatropine across the BBB and B-CSFB.     

Influence of environmental temperature and energy intake on skeletal muscle respiratory enzymes and morphology

Influence of a cold (10 degrees C) or warm (35 degrees C) environment and a high or low level of energy intake on respiratory enzyme activities has been investigated in porcine skeletal muscle. Scanning microdensitometry was used to measure the reaction products from mitochondrial enzymes in individual slow- and fast-twitch muscle fibres. A cold environment was found to increase the activity of succinate dehydrogenase in both types of muscle fibre (P less than 0.001 for dark fibres, P less than 0.01 for light fibres) from young growing animals. Enzyme activity was also increased in animals on a low compared with a high energy intake (P less than 0.01) when living at 10 degrees C but not at 35 degrees C. Similar findings were obtained for NADH diaphorase and cytochrome oxidase aa3. The numbers of slow-twitch muscle fibres also increased after exposure to cold (P less than 0.01) and as a result of a low energy intake (P less than 0.01). These results are similar to those obtained in other species after exercise or as a result of peripheral arterial insufficiency. The extent to which they could be related to local tissue hypoxia or to changes in metabolic hormones is discussed.     

A histochemical analysis of mammalian oxidative skeletal muscle fibres using the enzymes of energetic metabolism

Summary Some histochemical parameters of the three main fibre types of rat vastus lateralis muscle were studied. Succinic dehydrogenase (SDH), creatine kinase (CK), sarcotubular ATPase (SR-ATPase) and mitochondrial ATPase activities were demonstrated in serial sections. The three fibre types, recognised by the distribution pattern of SDH activity, all show high CK activity. However, red Type I oxidative fibres when examined for ATPase and ATPase dependent CK activity, show distinct heterogeneity revealing sub-populations within the same homogeneous fibre type. Three distinct patterns were recognised in the red Type I fibres depending on the distribution of the final reaction product. The present histochemical evidence confirms the fact that subdivision of mammalian skeletal muscle into three fibre types is only approximate and probably more than three types exist.     

Microwave Facilitation of Methylnaltrexone Antagonism of Morphine-Induced Analgesia in Mice

Exposure to low dose microwave irradiation has been postulated to influence effects of centrally active pharmacological agents. In this investigation, the analgesic dose, 50% (ADSO) for morphine in the mouse tail flick test was significantly increased by pretreatment with the centrally active opiate receptor blocker naltrexone, and the dose-response curve ms shifted significantly to the right. On the other hand, the AD50 and dose-response curve for morphine were not influenced by either the peripherally active opiate receptor blocker methylnaltrexone alone or microwave irradiation (2.45 GHz, 20 mW/cm², CW, 10 min) alone. However, following both methylnaltrexone pretreatment and microwave exposure, there was both a significant increase in the AD50 as well as a significant rightward shift of the dose-response curve, indicating a competitive antagonism of morphine by methylnaltrexone. One possible explanation for this microwave-drug interaction is that microwave energy might facilitate entry of methylnaltrexone into the central nervous system.     

Localization of lactate dehydrogenase isozymes in human muscle tissues by the mixed aggregation immunocytochemical technique.

Lactate dehydrogenase (LDH) isozyme composition and localization was determined in sections of skeletal, heart and smooth muscle by the mixed aggregation immunocytochemical method using first antibody directed against purified human LDH-A4 (M4) or LDH-B4 (H4) followed by the enzymes LDH-A4 and LDH-B4, respectively. An even distribution of the two monomers in all fibres was seen with heart muscle and smooth muscle. Heart muscle had a low concentration of A-monomers and a high concentration of B-monomers, whereas the smooth muscle had equal concentrations of the two monomers. In contrast, skeletal muscle from m. quadriceps femoris was found to be composed of two muscle fibre types, one containing mainly A-, the other mainly B-monomers. On the basis of succinate dehydrogenase activity it was shown that the red (type 1) fibres contain mainly B-monomers and the white (type 2) fibres mainly A-monomers of LDH.     

Effect of microwave radiation on the permeability of carbonic anhydrase loaded unilamellar liposomes

The influence of 2.45 GHz microwave exposure (6 mW/g) on the diffusion processes in enzyme-loaded unilamellar liposomes as bioreactors was studied. The enzyme carbonic anhydrase (CA) was entrapped into cationic unilamellar vesicles. Previous kinetic experiments showed a very low self-diffusion rate of the substrate p-nitrophenyl acetate (PNPA) across intact liposome bilayer. A twofold increase in the diffusion rate of PNPA through the lipid bilayer was observed after 120 min of microwave radiation compared to temperature control samples. The microwave effect was time dependent. The enzyme activity, as a function of increased diffusion of PNPA, rises over 120 min from 22.3% to 80%. The increase in stearylamine concentration reduces the enzyme activity from 80% to 65% at 120 min. No enzyme leakage was observed. © 1994 Wiley-Liss, Inc.