High energy phosphates in a red and a white muscle of the rat

Summary Previously we reported that ATP as well as phosphoryl creatine decreased by 10% under ether anesthesia vs. barbiturate anesthesia in rat leg muscle. This decrease did not occur when the sciatic nerve was severed. We therefore suggested that the decrease of high energy phosphates under ether was due to an activation of muscle fibers, presumably of red ones.In this study this postulate is proved by measuring adenine nucleotides and phosphoryl creatine in a red and a white muscle, —m. pyramidalis and m. adductor magnus of the rat.The conditions compared are: 1. Barbiturate (Inactin) anesthesia, 2. Ether anesthesia, 3. Ether-anesthesia plus destroying the segments L 4/5 of the spinal cord, 4. Electrical stimulation under Inactin-anesthesia, and 5. Thermal contracture.Under Inactin anesthesia the ATP content is slightly lower in the red muscle (6230±177 nanomoles per gram fresh weight) than in the white (6860±122 nanomoles per gram fresh weight); the phosphoryl creatine contents do not differ significantly between the two muscles.Unter ether anesthesia the ATP content is lowered by 25% vs. Inactin anesthesia in the red muscle while the ATP content remained unchanged under ether in the white muscle.Phosphoryl creatine is lowered by 10% under ether vs. Inactin anesthesia in the red muscle, while it is unchanged in the white muscle.After destroying the segments L4/5 of the spinal cord ATP and phosphoryl creatine remain unchanged under ether vs. Inactin anesthesia in the red as well as in the white muscle.One minute tetanic contraction as produced by direct electrical stimulation results in a 35% decrease of phosphoryl creatine in both the red and the white muscle with matching increments of free creatine. Under the same condition neither ATP nor ADP are significantly affected in the white muscle. In the red muscle the total adenine nucleotide decreases by 11% due to a 16% fall of ATP wich is not stoichiometrically matched by a 16% increase of ADP.Thermal contracture (repeated freezing and thawing in situ) causes considerable decreases of ATP and of phosphoryl creatine in the red as well as in the white muscle.The results indicate that the known virtual disagreement with the Lohmann concept of the ATP and phosphoryl creatine decrements in the activated state is less prominent in red than in white muscle.The data are consistent with the postulate that a neurally mediated activation of red fibers occurs under ether anesthesia.     

Effect ofP CO2, bicarbonate and lactate on the isometric contractions of isolated soleus muscle of the rat

Summary Isometric contractions of isolated soleus muscles of 3 weeks old rats show a potentiation of the maximal twitch tension when theP _CO2 is increased, or when the [HCO ₃ ^– ] is increased with a reciprocal decrease in [Cl^–]. The reverse response is observed on decrease of theP _CO2 and on decrease of the [HCO ₃ ^– ] with a reciprocal increase in [Cl^–].The addition of lactate ions results in a potentiation of the maximal twitch, whether accompanied by a reciprocal decrease in [HCO ₃ ^– ] or in [Cl^–].The maximal tetanic tension is augmented by rising theP _CO2, by increasing the [HCO ₃ ^– ] with simultaneous lowering of [Cl^–], or by the addition of lactate ions.     

Acid hydrolase activity in red and white skeletal muscle of mice during a two-week period following exhausting exercise

The activities of -glucuronidase, -N-acetylglucosaminidase, arylsulphatase, ribonuclease,p-nitrophenylphosphatase, and malate dehydrogenase together with protein content were assayed from representative mixed (m. rectus femoris), predominantly red (proximal heads ofm. vastus lateralis, m.v. medius andm. v. intermedius), and predominantly white (distal head ofm. vastus lateralis) muscle homogenates of mice during a two-week period following one single exposure to exhausting intermittent running on a treadmill. The activities of cathepsin D and -glycerophosphatase were assayed from mixed muscle only. In all three muscle types, particularly in red muscle, the activities of -glucuronidase, -N-acetylglucosaminidase, arylsulphatase, and ribonuclease progressively increased between one to five days after the exercise; thereafter the activities began to decrease, being near the control values 15 days after the exercise. In mixed muscle, cathepsin D activity increased. No corresponding changes were observed in the activities of acid phosphatases.The time course of the activity changes closely resembled that earlier found to be caused by ischaemia in rabbit muscles. It is tentatively concluded that the two treatments, exhaustive exercise and temporary ischaemia, cause similar cell injuries, and that the lysosomal system involved seems to function similarly in the post-stress recovery of the fibres from these injuries.Supported by grants from the Ministry of Education (Finland) and the Emil Aaltonen Foundation     

Characterization and control of muscle response to electrical stimulation

The maintenance of upright posture in neurologically intact human subjects is mediated by two major nervous pathways. The first, leading from the cerebral cortex through the spinal cord to motor neurons, activates muscles which produce postural movements. The second, leading from various sensory organs to higher centers, provides sensory feedback regarding the postural state. The path through the spinal cord is no longer intact in victims of spinal cord injury and loss of normal control of muscle activity results. Functional neuromuscular stimulation (FNS) has been shown as a feasible method for obtaining muscle contraction in paraplegic and has been proposed as a means for control of antero-posterior sway to make upright posture possible for these individuals. Before muscle can be controlled through the use of FNS, the response of muscle to electrical stimulation must be understood. In past studies, linear control theory has been applied to the analysis of this response and to the testing of various controllers. The aim of this study was to demonstrate some control issues in FNS using linear control theory, as it applies to electrical stimulation of muscle for stabilization of posture. The linearity of the muscle response was improved through closed-loop control using pole compensation techniques. The excess phase shift of the system due to the time delay in the muscle response, however, limits the ability to increase the open-loop gain in order to obtain improved performance. A suggestion for further study is the application of this methodology for uses in posture control.     

The response of adult and developing rat plantaris muscle to overload

The effect of overload on the rat plantaris muscle was studied in animals of different ages. Overload was induced by removal of gastrocnemius and soleus muscles. As expected, when the operation was carried out in adults, the plantaris muscle became heavier and stronger. These changes occured within 30 days after the operation. In animals in which the operation was carried out 1–12 days after birth and the muscle examined 6–20 weeks later, different results were obtained. In the group operated at 1–9 days of age, the muscles developed a lower maximal twitch and tetanic tension than the contralateral plantaris muscle. There was no difference in the time to peak or muscle weight between the overloaded and the contralateral muscles. Similar changes were observed in animals where the overload was induced at 11 or 12 days of age except for the weight which was significantly higher than that of the control plantaris muscles. The number of slow fibres increased in animals where overload was induced 11–12 days postnatally or in adults, but not when muscles were overloaded at 9 days of age. The possible reasons for the different response of adult and neonatal muscles to overload are discussed.     

Continuous monitoring of extracellular lactate concentration by microdialysis lactography for the study of rat muscle metabolism in vivo

A method is described for the measurement and on-line monitoring of muscular extracellular lactate concentration in both anaesthetized and freely moving rats. This method is based on microdialysis sampling and lactic dehydrogenase-catalysed nicotinamide adenine dinucleotide, reduced (NADH)-fluorescence detection techniques. In vivo calibration revealed a resting extracellular lactate concentration of 1.92±0.13 mmol/l (± SEM) in the gastrocnemius muscle of adult male Wistar rats (n=6), while the average whole-blood lactate level was 0.76±0.12 mmol/l (± SEM). This measured extracellular lactate concentration was 1.73-times higher than that deduced from the arterial lactate concentration. Blocking glycolysis with iodoacetate reduced the extracellular lactate concentration to 52±6% (± SEM, n= 4) of the resting level. The extracellular lactate concentration in rat gastrocnemius muscle had increased to significantly (P0.05) different levels, 2.4±0.03 (± SEM) or 4.0±0.55 (± SEM) times the control value, 1 h after aortic clamping (n=3) or cardiac arrest (n=3), respectively. Stimulation of the sciatic nerve induced elevations of the extracellular lactate concentration in the tibialis anterior muscle which were linearly related to the recorded isometric force-time integral. We also monitored on-line the changes in extracellular lactate concentration in the tibialis anterior muscle of a swimming rat. Our results indicate that microdialysis lactate reflects also intracellular metabolism. Lactography may be a useful alternative to biopsies and nuclear magnetic resonance spectroscopy in clinical medicine and physiology for the monitoring of metabolism in vivo.     

The effect of anesthesia on hemodynamics and renal function in the rat

Although many renal physiologic studies in the rat have utilized pentobarbital anesthetized animals, there has been no systematic evaluation of the effects of barbiturate anesthesia on renal function. We therefore compared systemic hemodynamic and renal function parameters in conscious (C) and pentobarbital anesthetized (A) rats. Cardiac index and renal blood flow as assessed by microspheres were greater in C rats while systemic and renal vascular resistance were less in C rats. Although basal glomerular filtration rate was similar in both groups of animals, glomerular filtration rate following hypotonic volume expansion was greater in C than A rats. During hypotonic volume expansion, distal delivery as estimated by the sum of free water and sodium clearances was greater in C rats. In contrast, function of the distal nephron as assessed by maximum fractional free water clearance during hypotonic volume expansion and by solute free water reabsorption at maximum levels of osmolar clearance were comparable in C and A rats. Collecting duct water permeability as assessed by the response to exogenous vasopressin was similar in C and A rats. Following an exogenous sodium chloride load, C rats excreted 16% more of the sodium. No differences in measured whole blood or plasma volume were present when C and A rats were compared.We conclude that pentobarbital does not impair basal glomerular filtration rate, distal nephron function or the water permeability response of the collecting duct. Pentobarbital does result in alterations in systemic and renal hemodynamics which may contribute to the failure of A rats to normally excrete a saline load.     

The influence of changes in the stimulation pattern on force and fusion in motor units of the rat medial gastrocnemius muscle

The effects of irregularity in the pattern of stimulation on the tension produced by motor units in the rat medial gastrocnemius muscle were investigated. The effects of decreasing as well as increasing the interpulse intervals were observed for each motor unit in tetani fused to different degrees. For each motor-unit type, it was found that the effects of these changes depended on the extent of tetanic fusion. Decreasing the interpulse interval produced an increase in tension during the tetanus: the more fused the profile of tetanus, the smaller the tension increase. Increasing the interpulse interval resulted in a decrease in tetanic tension. This effect was most prominent when the tetanic fusion index was approximately 0.75. This phenomenon resulted from the prolongation in relaxation when tetanic fusion increased, thereby preventing a decrease in tension when the interpulse interval increased. We also investigated the effects of introducing a short interpulse interval (”doublet”) at the beginning of the stimulation. The doublets produced increased tetanic tension with a more fused profile. However, the doublet enhanced the sensitivity of the tetanus to increases in interpulse interval and decreased its sensitivity to decreases in interpulse intervals. Slow-twitch motor units appeared to be significantly less sensitive to both increases and decreases in interpulse interval than fast-twitch units. This suggests that slow-twitch units are better suited for producing long-lasting contractions with a constant tension level. Conversely, the high sensibility of fast-twitch units to changes in stimulation frequency enhances their participation in regulation of tension of the muscular contraction. Received: 25 March 1998 / Accepted: 24 March 1999     

The effects of anesthesia on the excretion of an isotonic saline load in the rat

The ability to excrete a volume of isotonic saline equal to 10% of body weight infused over 60 min, was examined in awake rats and in rats anesthetized with 1 of the 2 agents most commonly used in renal clearance studies, Inactin or Nembutal. Rats anesthetized with Inactin excreted significantly less of the infused sodium during the period of infusion and in the 120-min post-infusion periods as compared to Nembutal-anesthetized rats or awake rats. Following saline infusion, there was a significantly greater decrease in serum protein concentration (25.5±4.7%) in rats anesthetized with Inactin, compared to that observed in the awake or Nembutal-treated rats. In a separate group of saline-infused awake rats, induction of anesthesia with Inactin resulted in a significant increase in hematocrit and a decrease in serum protein concentration. These studies suggest that Inactin anesthesia decreases the ability of the kidney to excrete a saline load and that, in studies of sodium excretion in the rat, especially if volume expansion is to be part of the experimental protocol, Nembutal rather than Inactin may be the anesthetic of choice.These studies were performed while Drs. Knight and Frankfurt were Fellows in Nephrology of Baylor College of Medicine, and were supported in part by a Clinical Investigatorship award to Dr. Weinman and an Associate Investigatorship award to Dr. Frankfurt from the Veterans Administration. This work was presented in part at the Combined Session, Southern Section, American Federation for Clinical Research and the Southern Society for Clinical Investigation, New Orleans, Louisiana, January 27–29, 1977, and has appeared in abstract form in Clin. Res.25, 61 A (1977)     

Rapid increase in the number of androgen receptors following electrical stimulation of the rat muscle

Summary The changes in the number of androgen receptors in rat gastrocnemius muscle following muscle contraction caused by electrical stimulation were investigated. The gastrocnemius muscle of one leg, which was selected at random for each rat, was stimulated with needle type electrodes, e.g. for 2 s at 10 V and 100 Hz, with 5-s intervals. The contralateral leg was left unstimulated as a control. One set consisted of ten such stimuli, followed by 5-min rest. Three sets every 2 days caused a statistically significant increase in muscle mass, the increase being about 2.5% after the 3rd day of stimulation, 4.4% after the 5th day, 5.9% after the 13th day and 8.3% after the 27th day compared with each control muscle (P< 0.001 in each case). The protein content also increased but the water content did not change. Stimulation over 4 weeks induced an increase in the area of the cross-section of the muscle fibres to about 30076 more than that of the control muscles, though the total muscle fibre numbers were slightly, but significantly, reduced. Electromechanical properties supported the development of the muscle by stimulation, because the maximal isometric tetanic force and peak twitch force markedly increased in the stimulated muscle. The androgen receptors in the muscle cytosol fraction were determined by means of a binding assay involving [³H]methyltrieno-lone, which is an analogue of testosterone, the number having rapidly increased in the stimulated leg, when compared with that in the control leg, by about 25% after the 3rd day. The increase then slowed down, reaching a plateau after the 5th-day of stimulation. The receptor dissociation constants for [³H]methyltrienolone remained unchanged, i.e. approximately 0.3 to 0.4 nmol throughout the experimental period. These findings suggested that a rapid increase in the number of androgen receptors occurred as an early event for a practical increase in muscle mass and thus it may have contributed in part to the triggering of muscle hypertrophy by enhancing the muscle sensitivity to androgen.     

Fatigue and recovery of phosphorus metabolites and pH during stimulation of rat skeletal muscle: an evoked electromyography and in vivo31P-nuclear magnetic resonance spectroscopy study

³¹P-nuclear magnetic resonance spectroscopy and evoked electromyography were applied to rat skeletal muscle to examine the mechanism of muscle fatigue and the recovery of muscle phosphorus metabolites and pH during fatigue. When the sciatic nerve was electrically stimulated at 1 Hz, the contraction force of the gastrocnemius muscle decreased gradually to 46% of the maximal force, accompanied by a decrease in phosphocreatine (PCr) and a corresponding increase in inorganic phosphate (P_i) and diprotonated inorganic phosphate (H₂PO₄ ^–). Neither the amplitudes of compound muscle action potentials (CMAP) nor muscle pH changed significantly. At 10-Hz stimulation, contraction force rapidly decreased to 26% of maximal force, accompanied by a decrease in PCr and increases in P_i and H₂PO₄ ^–. Muscle pH decreased for a few minutes, then gradually recovered during continued stimulation. The amplitude of the CMAP also decreased for a few minutes and then reached steady values. At 100-Hz stimulation, the contraction force decreased to 6% of the maximal force and there was a decrease in the amplitude of the CMAP. However, the changes in the phosphorus metabolites and pH were transient and recovered to the control value during the stimulation. These results indicated that fatigue at 1 and 100-Hz stimulation was mainly caused by the change in phosphorus metabolite concentrations and electrical failure, respectively, and that fatigue at 10-Hz stimulation might have been due to both of the these factors. These results also indicated that electrical failure might have been the cause of the recovery of the phosphorus metabolites and pH during 100-Hz stimulation and of pH during 10-Hz stimulation.     

Do muscle afferents contribute to the cervical response evoked by electrical stimulation of the median nerve in man?

Summary The possible contribution of low threshold muscle afferents to the postsynaptic component (N₁₃) of the cervical response evoked by electrical stimulation of the median nerve (MN) was investigated in normal subjects. Electroneurographic (ENG) and electromyographic (EMG) correlates of the reflex motoneuronal discharge (RMND) were recorded simultaneously. A. No reflex activity could be elicited by stimulation of the MN at the wrist, at least in the resting subjects, while well developed ENG (P₂ efferent volley) and EMG (H reflex) monosynaptic responses occurred following stimulation of the MN at the elbow at suitable strengths. In neither case could a surface correlate of interneuronal activity evoked by muscle afferents be demonstrated. B. Recruitment curves showed that at stimulus intensities above maximal for the H reflex both P₂ and H responses started to decrease until they completely disappeared, while N₁₃ showed further enhancement. C. Subthreshold conditioning stimulation of the MN enhanced both P₂ and H responses, while vibratory muscle stimuli provoked a clearcut suppression of these two responses. In contrast, N₁₃ was completely unaffected by either manuvre. D. No cervical evoked activity could be detected following tendon tapping of the anterior forearm muscles in spite of the appearance of well developed cortical responses and the ENG and EMG correlates of the T reflex. E. Conditioning volleys elicited by tendon taps of the anterior forearm muscles suppressed both P₂ and H responses following stimulation of the MN at the elbow without affecting the related N₁₃ component. F. Conditioning supramaximal stimulation of the MN at the wrist suppressed the N₁₃ component of the cervical response evoked by stimulation of the MN at the elbow without affecting the related reflex responses. No component chronologically related to the RMND could be recorded at the posterior neck region during suppression of N₁₃, thus ruling out the possibility that failure to detect the RMND (as well as its interneuronal concomitants) with cervical electrodes is due to a masking effect of the N₁₃ component. G. Conditioning tendon taps of anterior forearm muscles provoked a clearcut reduction of the primary cortical response to finger stimulation without affecting the postsynaptic component of the related cervical response. It is concluded that neither segmental (motoneuronal or interneuronal in origin) nor ascending postsynaptic impulses generated in the spinal cord by stimulation of low threshold muscle afferents contribute to N₁₃, the latter being probably due to activation of both short and long axoned spinal neurons by cutaneous afferents.     

Spatial distribution of muscle fibers in a lumbrical muscle of the rat

The spatial distributions of two differnt populations of muscle fibers were measured in cross-sections taken from the mid-belly of adult 4DL muscles. Muscle fibers belonging to a single motor unit (identified by glycogen depletion) were distributed randomly in most muscles. Muscle fibers which contained slow myosin (identified immunohistochemically) were distributed nonrandomly, being evenly distributed throughout most of the muscle cross-section, but excluded from the edge of the muscle. Interpreted from a developmental perspective, the results are consistent with the proposals that slow myosin-containing fibers in the adult represent the original population of primary myotubes, and that the adult pattern of motor unit fiber type is achieved by synapse elimination from mismatched fibers rather than by conversion of fiber type. © 1993 Wiley-Liss, Inc.     

Motor and sensory innervation of muscle spindles in the neonatal rat

Summary Neural and muscular elements of three muscle spindles from the soleus muscles of 4-day-old rats were reconstructed by electron microscopy of skip-serial transverse ultrathin sections. Each spindle contained four encapsulated intrafusal fibers, including a minimum of one bag₁, one bag₂ and one chain fiber. The fibers were innervated by unmyelinated motor and sensory axons. The primary and secondary afferents approached the spindles as single axons and terminated on the central region of the intrafusal fibers. Single profiles of terminal axons occupied the sites of sensory neuromuscular junctions, similar to adult sensory endings. No morphological features suggested retraction of afferents from 4-day postnatal spindles. Motor axons approached spindles tightly packed in bundles of 5–20 axons and terminated in the juxtaequatorial and polar regions of both bag and chain fibers. Multiple profiles of terminal axons were visible for each intrafusal motor ending. More motor axons innervated 4-day postnatal spindles and a greater number of axon terminals were visible in immature intrafusal motor endings than in adult spindles. The data suggest that postnatal maturation of motor innervation to intrafusal fibers involves the elimination of supernumerary motor nerve inputs. Synapse elimination in the development of the fusimotor system might represent a mechanism whereby individual axons adjust the number of spindles they innervate.     

Β-Glucuronidase activity in trained red and white skeletal muscle of mice

Summary We studied the effects of prolonged running exercise (5 days a week, 1.5 h per day at a speed of 17.6 m/min) on the activity of some acid hydrolases (-glucuronidase, -N-acetylglucosaminidase, acid phosphatase and cathepsin D) and three enzymes of energy metabolism (cytochrome c oxidase, lactate dehydrogenase and creatine kinase) in the distal and in the proximal, the predominantly white and red parts, respectively, of the vastus lateralis-muscle from mice. The acid hydrolase activity levels were 1.24–1.69 higher in untrained red muscle compared to untrained white muscle. The light training applied increased the activity of -glucuronidase in both red and white muscle. No other significant training effects were observed in the enzyme activities measured.     

Jaw muscle response to stimulation of type II somatosensory afferents of limbs in the rat

Convergence of various afferent inputs onto brainstem neurones may play an important role in the regulation of trigeminal motor activity. In particular, previous studies suggest that, besides sensory inputs arising from the orofacial region, extratrigeminal information may modulate jaw muscle function. In the present study the actions exerted on masseter and digastric muscles by the activation of somatosensory afferents coming from fore- and hind limbs were examined. The electromyographic activity (EMG) of masseter and digastric muscles was recorded in 20 anaesthetised rats, and EMG responses to single and paired electrical stimulation of common radial and sciatic nerves, at a threshold intensity for the activation of group II afferent fibres, were studied. The stimulation induced an excitatory response in both masseter and digastric muscles bilaterally. Ipsi- and contralateral radial nerve stimulation evoked masseter responses at latencies of 13.8±2.4 ms and of 18.0±2.6 ms, respectively, and digastric responses 1.6±0.4 ms later. Ipsi- and contralateral sciatic nerve stimulation elicited masseter responses at latencies of 21.4±2.6 ms and of 23.3±2.0 ms, respectively, and digastric responses 2.0±0.2 ms later. The same masseter and digastric motor units were excited by both radial and sciatic nerve stimulation; this suggests a convergence of somatosensory inputs arising from fore- and hind limbs on the same pool of masseter and digastric motoneurones. Paired stimulation of the two nerves did not induce any summation of the responses; this finding suggests that the two inputs, reaching a common relay station, could give rise either to occlusion or to inhibitory interactions. Spinotrigeminal relationship evidenced in this study may be involved in the coordination of jaw and limb movements. Electronic Publication