The immunohistochemical localization oftwo myelin-specific proteins-basic protein (BP) and proteolipid protein (PLP)-was compared during the process of myelination. Although both proteins were present in oligodendrocytes, (i) neither protein was observed in oligodendrocytes not already closely associated with nerve fibers exhibiting a fluorescent coating; (ii) in any discrete anatomical area oligodendrocytes were positive for BP before PLP was visible; and (iii) as myelination progressed, immunoreactivity for BP in oligodendrocytes appeared to decrease and simultaneously PLP immunofluorescence became visible in this cell type. During the period of active myelination, fibers exhibited a distinct varicose appearance. As myelination progressed, the myelin sheath increased in thickness and these varicosities became less prominent, eventually completely disappearing. Therefore, the nature and the appearance ofvaricosities can be used as an index ofthe relative stage ofmaturation ofmyelin in an individual fiber. In general, PLP appeared in fibers at a later stage ofmaturation than did BP based on the above criteria. However, in a relatively small number offine fibers PLP was observed at a very early stage. In fully mature myelin, very large fibers were frequently more intensely fluorescent for BP than PLP, whereas fine myelinated fibers were more intensely stained for PLP. These observations are consistent with the following interpretations. (i) Substantial differentiation of oligodendrocytes occurs prior to appearance of either of these proteins by immunofluorescence. (ii) BP is added to the myelin sheath prior to PLP and there appears to be a shift in priority of synthesis from BP to PLP in individual oligodendrocytes during the process of myelination. (iii) Very small fibers often contain low concentrations ofBP relative to PLP, and conversely, very large fibers may contain a high concentration of BP relative to PLP. Thus, the relative concentration of these proteins in myelin appears not to be constant but may vary as a function of the size of the myelinated fiber.Proteolipid protein (PLP) and basic protein (BP)-the major structural proteins of central nervous system myelin-account for 50% and 30%, respectively, of the total myelin membrane proteins ofrat brain (1-2). The limited solubility of myelin PLP in aqueous solution has posed a persistent problem in the purification of PLP for further biochemical and immunological characterization. Techniques for the isolation ofan antigenically active preparation of myelin PLP have been developed in this laboratory (3). Specific precipitating antibodies to homogeneous preparations ofboth PLP and BP isolated from rat brain myelin have been produced. The purity and specificity ofthese antisera have been established by immunodiffusion and cross immunoadsorption (3,4). The antisera to PLP and BP were used for the immunohistochemical localization of these proteins in the central nervous system. The results of the immunohistochemical studies in adult brain established ...

Antisera to highly purified basic protein (BP) from rat and chicken brain were prepared and their purity and specificity demonstrated by double immunodiffusion and cross-immunoadsorption. These antisera were used for immunohistochemical localization of BP in the brains of adult and developing rat and chick. Myelin basic protein was exclusively localized to myelin or the myelin forming elements of the CNS. It was present in high concentrations in white matter and absent in areas free of myelin. Neuronal parikarya and dendrites were negative as were axons cut in cross section and at Nodes of Ranvier. The latter was best observed in cross sections of human spinal cord demonstrating also the immunoreactivity of the antibodies with human BP. The internodal distance in a fine (1.5 micrometer) rat cortical fiber was determined to be approximately 45 micrometers. Myelin basic protein was shown to extend into cranial roots, in contrast to myelin proteolipid protein which abruptly lose fluorescence as the nerves emerged from the brain. During development, BP was first observed on the fourteenth day of incubation in chick and at birth in the rat. The protein appeared in oligodendrocytes and in association with fibers near these cells. Fluorescent processes were frequently observed connecting the oligodendrocytes with the fibers. As myelination progressed, the intensity of the immunohistochemical reaction decreased in the oligodendrocytes while the brightness in fibers increase. Eventually, the oligodendrocytes became undetectable. Fibers with immature myelin exhibited a beaded or varicosed appearance with the highest concentration of immunofluorescence in the outer portion of the varicosities. The varicosities were postulated to represent dilations in the newly forming sheath between intervals of compaction along the axon undergoin myelination. These dilations might represent areas of increased cytoplasmic volume which could serve as channels for transport and/or storage sites for myelin proteins prior to incorporation into the membrane. The varicosities became less prominent with the thickening of the myelin sheath and mature myelinated fibers became smooth. The process of synthesis of BP, transport of the protein to the varicosed fibers, and maturation of the myelin sheath was seen to progress in a more or less caudal to rostral direction as myelination of the CNS takes place. In the rat, this was accomplished over approximately a 30-day period starting near the time of birth. In the chick, most of the myelination was accomplished in the three or four days immediately before hatching. At this time, innumerable oligodendrocytes were observed producing BP simultaneously in the major white fiber tracts. It is postulated that in chick some degree of oligodendrocytic cell death occurs normally during myelination.

Rabbit anti-bovine myo-inositol-1-phosphate synthase was used to examine the distribution of that enzyme in perfused and immersion-fixed bovine brain and testis. In brain, intense and specific staining was found in the walls of all the vascular elements including cerebral capillaries. The remainder of brain parenchyma exhibited only low levels of background staining. In testis, an organ rich in the enzyme, blood vessels showed no specific staining. Instead, the enzyme was found in the seminiferous epithelium of the seminiferous tubules, perhaps localized in spermatozoa. To confirm the brain finding, the activity of myo-inositol-1-phosphate synthase was measured in bovine brain microvessel preparations and brain pial vessels. In these preparations the activity of the enzyme was found on average to be 7 and 22 times enriched over that in whole brain, respectively. The activities of two other enzymes of inositol metabolism, myo-inosose reductase and myo-inositol-1-phosphatase, were also examined for their distribution in brain. Those enzymes were found to be generally distributed. The surprising finding of a vascular localization of myo-inositol-1-phosphate synthase in brain raises new questions about the mechanism by which myo-inositol is concentrated to such high cellular levels in the principal substance of that organ.

Abstract— A homogeneous preparation of proteolipid protein (PLP) from rat brain myelin was isolated by preparative gel electrophoresis in sodium dodecyl sulfate and chemically characterized. The results of amino acid and N‐terminal amino acid analyses are reported. The same preparation of myelin PLP was used to produce specific precipitating antibodies. Rabbit and goat antisera to myelin PLP each gave a single precipitin line with purified PLP dissolved in Triton X‐100. Under identical conditions, no precipitation was observed with antiserum to myelin basic protein or with control serum. Immunofluorescence localization employing antiserum to PLP demonstrated bright specific fluorescence restricted to the myelin sheaths of axons in all anatomical areas of the rat brain examined. Neuronal cell bodies and their dendrites were completely negative with respect to the presence of proteolipid protein. PLP could not be localized in the cell bodies or fibrous processes in any of the glial elements in the adult rat brain. However, myelin PLP was clearly visible in the cytoplasm and processes of actively myelinating oligodendrocytes in the corpus callosum in the brains of 10‐day‐old rats.