Frozen Muscle Sections Research Articles

EFFECTS OF SHORT AND LONG TERM EXERCISE ON INTRACELLULAR GLYCOGEN AND FAT IN PIGEON PECTORALIS

In an attempt to determine the functional role of the component broad white and narrow red fibre types during activity, the pectroalis muscle of the adult pigeon (Columba livia) was electrically stimulated in situ after anaesthetizing the birds with sodium pentobarbital. For examination of the effect of short term exercise, the pectoral muscle of one side was stimulated through the innervating brachial plexus, using an electronic stimulator for 1 hr, and a biopsy sample was taken from the stimulated muscle. The stimulation was resumed for 17 hr more with additional doses of anaesthesia, and a muscle sample was taken after the decapitation of the birds in the long term experiment. Muscle samples from non-stimulated pigeons under identical conditions served as control. Fixed and frozen sections were made from the samples and were stained with Periodic Acid-Schiff's reagent for the demonstration of glycogen, while additional frozen muscle sections were stained with Fettrot 7B according to Pearse, A.G.E. (1960) for the demonstration of neutral lipids in the specimen. Thus histochemical examinations were made to study effects of short and long term exercises on energy stores in skeletal muscles. Short-term stimulation resulted in a selective depletion of intracellular glycogen stores from white fibre populations whereas prolonged long term stimulation resulted in the resynthesis of glycogen within these fibres and a concomitant reduction in the intracellular fat and glycogen reserves in most red fibres. It is postulated that during flight the white fibres indulge only in brief bursts of intense phasic activity such as in quick take-off, rapid accelerations or sudden manoeuvres and utilize glycogen as fuel whereas the red fibres perform sustained activity as in cruising flight and metabolize chiefly fat.

Fact and artifact in the histochemical procedure for myofibrillar ATPase

A recent quantitative biochemical study has shown that skeletal muscle mitochondria have high ATPase activity under the same conditions that are employed in the histochemical procedure for myofibrillar (actomyosin) ATPase. However, mitochondrial staining is not observed when this histochemical procedure is applied to frozen sections of skeletal muscle. The present investigation was performed to resolve this discrepancy. Frozen sections of muscle were incubated in the presence of inorganic phosphate (in place of ATP) and then treated sequentially with calcium chloride, cobalt chloride, and ammonium sulfide to determine whether inorganic phosphate binds to specific sarcoplasmic constituents. Binding of phosphate did occur and, furthermore, it was localized to the myofibrils and not at all to the mitochondria. It is concluded that the hydrolysis of ATP in the histochemical reaction can result from ATPase activity of myofibrils, mitochondria, or both. Since the released inorganic phosphate binds to the myofibril, the localization of the reaction product does not necessarily reveal all sites of enzymatic activity. Thus the histochemical procedure does not reflect myofibrillar ATPase activity exclusively, and one therefore cannot use this technique to infer that individual muscle fibers are physiologically fast or slow according to the intensity of the ATPase staining of their myofibrils.