FINE STRUCTURE OF AUTONOMIC NERVES AFTER VITAL STAINING WITH METHYLENE BLUE

Autonomic nerves in the rat, mouse, guinea‐pig and cat were vitally stained with methylene blue and examined electron‐microscopically after fixation in potassium permanganate. Dense patches in the axoplasm and swelling of mitochondria were found in the stained axons. By controlling the pH and saturating with oxygen, only cholinergic axons were stained at pH 6.5–7.0 whereas at pH 5.0–5.3 both cholinergic and adrenergic axons stained. Similar results were obtained by immersion in a bath at controlled temperature as with close arterial perfusion of dye into pelvic viscera. The intermediate pH at which adrenergic axons begin to stain could not be determined. However, as a means of identifying cholinergic axons, particularly those sectioned at a level where no agranular vesicles occur, M B staining is unreliable because not all cholinergic axons respond. In sympathectomized rat iris lacking adrenergic axons altogether, some of the persisting cholinergic axons remained unstained. Experiments with M B thiocyanate and M B chloride in phosphate‐citrate or succinate‐HCl buffer indicated that purity of dyestuff was less important than pH control. Methods for electron‐microscopical demonstration of acetylcholinesterase are more useful for distinguishing cholinergic axons especially if adrenergic nerve vesicles are I reserved in the same preparation by the initial fixation. Copyright © 1969 Wiley‐Liss, Inc.