Effects of physiological amounts of high- and low-rate chronic stimulation on fast-twitch muscle of the cat hindlimb. I. Speed- and force-related properties.

1. Long-term electrical stimulation was given during 4 or 8 wk to the peroneal nerve of deafferented hindlimbs in hemispinalized adult cats. Four different stimulation patterns were compared: 100-Hz bursts covering 5% of daily time (F1), 10-Hz bursts covering 5% of daily time (S1), pattern S1 plus added 100-Hz bursts during 0.5% of daily time (S1F2), and, finally, only the latter 100-Hz bursts (F2), again during 0.5% of daily time. 2. During the course of chronic stimulation, frequent noninvasive measurements were made of the twitch of the ankle dorsiflexors. In a terminal acute experiment under general anesthesia, performed after 4 or 8 wk of treatment, measurements were made of isometric contractile properties (speed, force) for one of the stimulated peroneal muscles, m. peroneus longus (PerL). Thereafter, the PerL muscle was removed for further histochemical/histological analysis. 3. Findings from chronically stimulated PerL muscles were compared with three kinds of control PerL muscles: 1) those from the contralateral (control) hindlimb of chronically treated animals, 2) those from the operated side of animals that had been deafferented and hemispinalized but not subjected to chronic stimulation, 3) those from normal animals that had not been subjected to chronic treatment. With respect to the presently studied parameters, the three kinds of control muscles rendered very similar results. 4. All the presently used patterns of chronic stimulation made the PerL muscles slower with respect to twitch contraction time, half-relaxation time, and tension-frequency relation. Patterns covering 5-5.5% of daily time (F1, S1, S1F2) also caused an increase in the percentage of fibers classified as 'slow' (type I) on basis of their staining for myosin adenosine triphosphatase (ATPase). 5. Among patterns covering 5% of daily time, the change in ATPase histochemistry and the degree of physiological slowing was at least as pronounced after chronic stimulation at 100 Hz (F1) as after treatment at 10 Hz (S1). The slowing produced by pattern S1 was not more pronounced than that caused by this pattern (10 Hz) plus an equal number of pulses at 100 Hz (S1F2). 6. The slowing produced by the presently used patterns of chronic stimulation took place within the initial 2-3 wk. 7. Patterns F1 and S1 caused a decrease in maximum tetanic force as well as in mean fiber diameter.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of physiological amounts of high- and low-rate chronic stimulation on fast-twitch muscle of the cat hindlimb. II. Endurance-related properties.

1. Long-term electrical stimulation was given to the peroneal nerve of deafferented hindlimbs in hemispinalized adult cats. The amount of stimulation covered 0.5-5.5% of total time per day, different in different animals. For some aspects of the present study, use was also made of cats subjected to "tonic" patterns of chronic stimulation (typically covering 50% of total time; 10, 16). 2. In a terminal acute experiment under general anesthesia, performed after 4 or 8 wk of long-term stimulation, one of the treated peroneal muscles (m. peroneus longus, PerL) was used for measurements of the resistance to contractile fatigue. The fatigue test consisted of 0.33-s bursts of motor-nerve stimulation at 40 Hz, repeated once a second for 4 min (6, 7). During this fatigue test, the evoked compound spikes of the muscle were recorded by electromyographic (EMG) techniques. Following the physiological procedures, PerL was removed for further histochemical analysis. In transverse sections, measurements of optical density were made in central regions of single fibers after staining for the activity of an oxidative enzyme, succinate dehydrogenase (core SDH). 3. Findings from chronically stimulated PerL muscles were compared with three kinds of control PerL muscles: 1) those contralateral to the stimulated ones, 2) those from the operated side of animals that had been deafferented and hemispinalized but not subjected to chronic stimulation, and 3) those from untreated normal animals. 4. Stimulation patterns covering both greater than or equal to 50% and 5-5.5% of daily time gave a marked improvement of fatigue resistance. Pulse rate seemed of little importance for these effects. The pattern covering only 0.5% of total daily time caused no increase of contractile endurance beyond that of normal muscles. 5. During the fatigue test of a control muscle (see above), the amplitude of the compound EMG spikes typically showed a marked decline. This "EMG depression" was effectively counteracted by all the present patterns of chronic stimulation, including the 0.5% pattern. 6. Fibers of chronically stimulated muscles became more similar to each other with respect to their density of core SDH staining. However, among muscles treated during 0.5-5.5% of total daily time, the degree and pattern of change in core SDH staining was not related to the amount and pattern of chronic stimulation or to the resulting degree of contractile endurance.

Fibre sizes and histochemical staining characteristics in normal and chronically stimulated fast muscle of cat.

1. Normal and chronically stimulated peroneus longus muscles of the cat's hind limb were studied with respect to fibre size and staining properties for myofibrillar (myosin) adenosine triphosphatase (ATPase) and succinate dehydrogenase (SDH) activity. The intensity of staining for SDH activity was measured by microphotometry from the central portions of the muscle fibres ('core-SDH staining'). For comparison, histochemical properties were also studied in non-stimulated soleus muscles. 2. On account of the pH sensitivity of their myofibrillar ATPase, about 18% of the fibres in normal peroneus longus muscles were classified as type I, and about half of the remainder as II A and II B respectively. 3. In the normal peroneus longus muscles, the mean diameter of single muscle fibres generally varied between about 25 and 75 micron, whereby the average size of type I less than type II. 4. In the normal peroneus longus muscles the staining intensity for core SDH varied over a wide range. The average heaviness of staining was clearly ranked in the order type I greater than type II A greater than type II B. 5. Chronic stimulation was given to the deafferented common peroneal nerve by aid of a portable and remotely controlled mini-stimulator. The stimulation was delivered in 'tonic' patterns (greater than or equal to 50% of total time taken up by activity) of 'fast' (20 or 40 Hz) or 'slow' (5 or 10 Hz) rates. 6. Prior to the period of long-term stimulation, the cats had been subjected to a dorsal rhizotomy and hemispinalization on the ipsilateral (left) side. In the absence of chronic stimulation, these operations had no evident effects on the sizes or staining properties of peroneus longus fibres. 7. After 8 weeks of treatment with tonic patterns of stimulation, the fibres of peroneus longus muscles clearly became more similar to each other with respect to their diameter as well as their staining for ATPase and SDH activity. With respect to ATPase staining, however, the chronically stimulated peroneus longus fibres had become more similar to non-stimulated soleus fibres than to non-stimulated type I fibres of peroneus longus. With respect to the staining for core SDH, the chronically stimulated fibres all became similar to normal II A fibres of peroneus longus. The 'fast' and 'slow' patterns of chronic stimulation had the same effects on the staining properties. 8. Chronically stimulated peroneus longus muscles showed a decrease in fibre diameter which corresponded, roughly, to the concomitant decrease in muscle weight.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative cytochemical analysis of cytochrome oxidase and succinate dehydrogenase activity in spinal neurons.

This paper describes cytophotometric determinations of cytochrome oxidase (COX) and succinate dehydrogenase (SDH) activities in neurons in 3 areas of the spinal motor column of the teleost fish Brachydanio rerio (the Zebrafish). Purpose of this investigation was to analyse the correlation between the oxidative metabolic capacity of motoneurons with their activity patterns. The spatial organization of the spinal cord of the zebrafish allows such an analysis, because the motoneurons which innervate different muscle fiber types (slow tonic red and fast phasic white, respectively) occupy separate areas of the motor column (Van Raamsdonk et al. 1983). We analysed the COX and SDH activities on serially sectioned neurons, We found large variations in the ratio of COX/SDH activity: the ratio was high for large neurons (in the "white" area) and low for small neurons (in the "red" area). These findings were contrary our expectations, because COX as well as SDH activity have been proposed as indicators for neuronal activity if both activities are reliable indicators, then their ratio should be constant. In addition, COX and SDH activities were analysed on serially sectioned anterior horn neurons of the cat spinal cord. In contrast to the situation in fish, we observed a statistically significant proportionality between COX and SDH activities. We conclude that the histochemical reactions for COX or SDH activity have no general validity as markers for the same type of neuronal activity.

Soma size and oxidative enzyme activity in normal and chronically stimulated motoneurones of the cat's spinal cord.

In normal adult cats we measured the density of staining for the activity of succinate dehydrogenase (SDH staining) in ventral horn cells of different sizes. The measurements were restricted to that part of the lumbar ventral horn (L6-L7) which is known to contain motoneurones of the peroneal nerve. A statistically significant tendency was found for the SDH staining to be denser in smaller than in larger neurones within the size range of a motoneurones (soma diameter greater than 40 microns). These results are consistent with recently published evidence for ventral horn cells of rats and qualitatively similar relationships between size and SDH staining have also been observed among skeletal muscle fibres (confirmed for mixed muscle of cat in present study). In hindlimb muscles, size as well as SDH staining are known to be markedly activity-dependent. We tested whether this is the case for peroneal motoneurones as well by analyzing the effects of chronic nerve stimulation on the properties of neurones within the appropriate region of the ventral horn. Prior to the final acute experiment, these cats had been subjected to a left-side dorsal rhizotomy and hemispinalization. By aid of a portable mini-stimulator, the left-side common peroneal nerve was activated by repetitive pulses during 50% of total time per day (intra-activity rate: 10, 20 or 40 Hz). After 8 weeks of such treatment, cell sizes as well as the densities of SDH staining showed hardly any differences between peroneal ventral horn cells of the experimental and control sides of the spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatotopic relations between spinal motoneurones and muscle fibres of the cat's musculus peroneus longus.

The cat's m.peroneus longus was analyzed with respect to the somatotopic relation between the rostro-caudal site of emergence of ventral root filaments (i.e. rostro-caudal site of motoneurones) and the intramuscular distribution of innervation. Rostro-caudally distinct fractions of ventral roots were stimulated repetitively in order to deplete their respective muscle fibres of glycogen. The intramuscular position of glycogen-depleted fibres was analyzed in transverse sections from different proximo-distal levels. At each level, depleted muscle fibres were dispersed across the whole muscle. No consistent relation was found between the spinal site of origin of a ventral root filament and the proximo-distal distribution of its fibres within the pennate muscle. A significant and evident tendency was found, however, for rostral root filaments (i.e. rostral motoneurones) to innervate a greater number of muscle fibres in anterior than in posterior muscle portions. For caudal root filaments, the opposite pattern of innervation was observed.

Development of immunohistochemical characteristics of intrafusal fibres in normal and de-efferented rat muscle spindles.

Intrafusal muscle fibres in adult muscle spindles differ in their myosin composition. After selective motor denervation intrafusal muscle fibres develop mature ultrastructural characteristics. In order to evaluate the role of fusimotor innervation on the maturation of the myosin composition of intrafusal muscle fibres we have examined with immunohistochemical techniques i) the postnatal development of muscle spindles in new-born rats and in 7-21 day old rats; ii) muscle spindles in the EDL of 21-day-old rats de-efferented at birth. For the characterization of myosins in intrafusal fibres we used three myosin antisera: antipectoral myosin, antiheart myosin and antiheart myosin adsorbed with muscle powder from the soleus muscle of guinea pig. We show in this study that during development intrafusal fibres change immunoreactivity and that in the absence of motor innervation bag fibres do not fully develop the myosin characteristics of control spindles. We conclude that the maturation of bag1 and bag2 fibres apparently requires next to the inductive influence of sensory axon terminals the presence and activity of fusimotor axons.

Immunohistochemical differences in myosin composition among intrafusal muscle fibres.

Mammalian intrafusal fibre types (nuclear chain, nuclear bag1 and nuclear bag2 fibres) are known to differ in their ultrastructure, intensity of the myofibrillar histochemical ATP-ase reaction, type of innervation and time course of contraction. The present study concerns the myosin composition of these intrafusal fibre types in the soleus muscle (mouse) and the extensor digitorum longus muscle (rat). We used an immunohistochemical method with three myosin antisera raised in rabbits: anti chicken pectoral myosin, anti chicken heart myosin (1) and anti chicken heart myosin (2) (= anti chicken heart myosin (1) adsorbed with muscle powder from soleus muscle of guinea pig). The results showed that three intrafusal fibre types differed in their myosin composition. A comparison of intrafusal fibre types with extrafusal fibre types for the histochemical myofibrillar ATP-ase reactivity and the reactivity with myosin antisera showed a resemblance of nuclear chain fibres with extrafusal type II fibres and a difference between nuclear bag1 and nuclear bag2 fibres and all other fibre types.

Effect of temperature and ouabain on th Na+--K+ activated membrane ATPase and electrogenic ionic pump of the golden hamster and mouse diaphragm.

The activity of membrane Na+--K+-ATPase and electrogenic ionic pump was estimated by biochemical and electrophysiological techniques on diaphragm muscles of mouse and non-hibernating golden hamster between 5 degrees C to 37 degrees C. The electrogenic capacity of ionic pump (measured as maximum hyperpolarization after adding 5 nmol/l K+ to Na+-enriched muscle) was highest at 37 degrees C for the mouse and between 25--30 degrees C for the golden hamster. At lower temperatures (15 degrees C--5 degrees C), the hyperpolarization after adding K+ reversed to depolarization between 15 degrees C and 10 degrees C in the case of the mouse. In the hamster, the slight hyperpolarization persisted even at 5 degrees C. With decreasing temperature, the activity of membrane ATPase of mouse and hamster membrane fraction decreased in both cases to the same extent between 37--25 degrees C. Within the temperature range between 10 degrees C and 5 degrees C the activity of this enzyme of hamster preparations was about 2.4 times higher than in the case of the mouse. In the mouse Na+-enriched diaphragm, ouabain (10(-4) mol/l) decreased the resting membrane potential (RMP) in a K+-free solution at 20 degrees C by about 5 mV, but in the golden hamster preparation, ouabain in the same concentration increased RMP of Na+-enriched diaphragm fibres by more than 5 mV. The activity of membrane ATPase of hamsters was increased by 10(-4) mol/l ouabain more than 2.5 times in a K+-free reaction medium, whereas in the case of the mouse no change of enzyme activity was observed. This indicates that ouabain can be substituted for potassium as an activator of membrane ATPase in the sarcolemma of golden hamster muscle fibres.

The alpha-glucan-uridine diphosphate glucosyltransferase reation for the identification of glycogen-depleted muscle fibers.

This paper decribes the use of the alpha-glucan uridine di-phosphate glucosyl transferase reaction for enhancing the contrast between glycogen depleted and non-depleted muscle fibers in the periodic acid schiff (PAS) reaction. Muscle fiber glycogen was depleted by prolonged repetitive stimulation of single motor units of the extensor digitorum longus muscle from the rat.