Mammalian central nervous system axolemma: histochemical evidence for axonal plasma membrane origin of bovine and rat axolemma-enriched membrane fractions

Abstract

Purified myelinated axons were isolated from fresh bovine corpus callosum and rat brainstem and osmotically shocked to strip myelin and axolemma from axon filaments. Two axolemma-enriched fractions, myelin, and a pellet of myelin-free axons were harvested by discontinuous sucrose centrifugation of the shocked myelinated axons. By electron microscopy the axolemma-enriched fraction was composed of trilaminar membrane vesicles (diameter 50–1000 nm) and linear membrane fragments; myelin fragments were seldom seen but some mitochondria) membrane contamination was evident. The classical myelin stain Luxol Fast Blue never counter-stained the myelin-free axons, but did stain myelin, myelinated axons, and to a lesser extent, both axolemmal fractions. The staining of the axolemma-enriched fraction was amorphous and seemed incommensurate with the level of myelin contamination. Acetylcholinesterase staining of the axolemma-enriched fraction counterstained with Luxol Fast Blue revealed a number of acetylcholinesterase-positive membrane vesicles by light microscopy. Occasional enzyme-positive vesicles were also evident in the myelin while myelin-free axons showed virtually no Luxol Fast Blue stain but were heavily stained for the enzyme. Higher resolution histochemical staining by electron microscopy confirmed the light microscopy, showing acetylcholinest erase reaction product lining the axolemma of bovine and rat CNS neurons. Myelin, mitochondria, and other diverse membranes showed no reaction product. About 30% of bovine corpus callosal axons showed some acetylcholinesterase staining but only 3% of the total lengths of all the bovine CNS axolemma showed reaction product. Positive acetylcholinesterase staining could also be demonstrated in a small proportion of the bovine axolemma-enriched membranes, with reaction product lining the membrane fragments and captured within their vesicles. The percentage of vesicles that were stained closely agreed with the percentage of axolemma) length stained in the whole white matter. Since acetylcholinesterase staining was highly localized to the axonal plasma membrane, the acetylcholinesterase-positive membrane fragments in the axolemma-enriched membranes could only originate from the axolemma. We conclude on the basis of the operational behavior of these axolemmal vesicles that many of the membranes found in these fractions are derived from the axonal plasma membrane.