Myelinogenesis in Optic Nerve

Morphological, autoradiographic, and biochemical methods were used to study the time of appearance, distribution, and nature of sulfated constituents in the developing rat optic nerve. Electron microscope studies showed that myelination begins (6 days postnatal) shortly after the appearance of oligodendroglia (5 days postnatal). Over the ensuing 3 wk, myelination increased rapidly. During the 1st postnatal wk, mucopolysaccharides and glycoproteins were labeled with ~S and autoradiographs showed grains over arachnoidal cells, astroglia, and the glia limitans. These results indicated that astroglia synthesize sulfated mucopolysaccharides of the glia limitans. After the onset of myelination, however, the major portion of laSS]sulfate was incorporated into sulfatide. Autoradiographs showed a shift of radioactive grains from astroglia and arachnoidal cells to myelin, indicating that actively myelinating oligodendroglia incorporate [35S]sulfate into myelin sulfatide; there was a concomitant increase in the activity of cerebroside sulfotransferase. In addition, the increasing amounts of proteolipid protein and myelin basic protein corresponded with the morphological appearance of myelin. These results point to a strict correlation between the structural and biochemical changes occurring during myelination. This system provides a useful model for studies designed to evaluate the effects of various perturbations on the process of myelination. The process of myelination in the central nervous system (CNS) has been studied morphologically (50, 51, 53) or biochemically (32, 33, 37), but in only one study have the two approaches been combined (25). Morphological investigations can define the onset and, to some extent, estimate the rate of myelination in specific single pathways. However, biochemical studies have usually been done on CNS tissue containing several asynchronously myelinating systems, so that it has been difficult to relate biochemical findings with morphological observations. We have studied the development of rat optic nerve, a system in which there is a single anatomical pathway being myelinated. This investigation is part of a larger body of work designed to define parameters of gliogenesis (50, 51) and myelinogenesis in a specific myelinating system. In the study of gliogenesis in rat optic nerve (50, 51), [aH]thymidine autoradiography showed that astroglia and oligodendroglia are derived from astroblasts and oligodendroblasts, respectively, and not from undifferentiated glioblasts. Astrocytes are formed throughout late embryonic and postna6 0 4 THE JOURNAL OF CELL BIOLOGY 9 VOLUME 72, 1977 9 p a g e s 6 0 4 6 1 6 on O cber 0, 2017 jcb.rress.org D ow nladed fom tal development, while oligodendroglia begin their final division at 5 days postnatal, i.e., a day or two before the onset of myelination (50, 51). Using biochemical and high resolution autoradiographic methods, the present study focuses on the changes in the pattern of incorporation and the cellular distribution of sulfate before and after the onset of myelination. At 2 days postnatal, sulfate is incorporated into glycoproteins and mucopolysaccharides, whereas at 12 and 16 days the 35S appears predominantly in sulfatide. High resolution autoradiography shows a corresponding change in grain density from arachnoidal elements and glia iimitans to myelin sheaths. These findings are related to changes in the activity of cerebroside sulfotransferase and the amounts of proteolipid protein and myelin basic protein. The purpose of these studies is twofold: first, to provide quantitative data on both gliogenesis and myelination in a single system; second, to establish a framework which can be used to study pathological processes in which myelination is affected (24). M A T E R I A L S A N D M E T H O D S Sprague-Dawley rats (Charles River Farms, Boston, Mass.) were obtained between 7 and 10 days postnatal. For pups of younger ages, 13-day gestation mothers were obtained and, on the day of delivery, each litter was reduced to 10 pups. The adult animals were fed ad lib. on a standard laboratory rodent diet. [~S]Sodium sulfate (Na2SO4) and [asS]phosphadenosine phosphosulfate (PAPS) were obtained from New England Nuclear (Boston, Mass.). Imidazole was obtained from Calbiochem (San Diego, Calif.); galactocerebroside from Supelco, Inc. (Bellefonte, Pa.); and Triton X-100 from Schwarz/Mann Div., Becton, Dickinson & Co. (Orangeburg, N. Y.). All solvents were reagent grade. Histology and Autoradiography For microscope examination, specimens were fixed by immersion or by intra-aortic perfusion with the following fixative solution: 3% glutaraldehyde, 1.5% formaldehyde, 0.6% acrolein, 0.6% dimethyl sulfoxide, and 0.001% calcium chloride in 0.1 M cacodylate buffer (pH 7.3). Animals were sacrificed 48 h after injection of isotope. After overnight fixation at 4~ the intracranial portion of the optic nerve was removed, and rinsed in 0.1 M cacodylate buffer (pH 7.3) with 10% sucrose. The specimens were postfixed in 2% solution of buffered osmium tetroxide for 90 min at room temperature, dehydrated in a graded series of ethanols, and embedded in Epon 812, Sections for light microscopy were cut at 1 /.tin and stained with toluidine blue; thin sections were stained with uranyl acetate and lead citrate. For autoradiography, thick (1.5 p.m) sections were dipped in Kodak NTB-2; thin sections (pale gold) were coated with Ilford L4 emulsion (Ilford Ltd., Ilford, Essex, England) (purple interference color) which was maintained at 40~ and diluted 1:5 with water. Exposure took place in light-tight boxes at 4~ for 10-80 days. Light and electron microscope autoradiographs were developed in Kodak D19, stained with toluidine blue or uranyl acetate-lead citrate, respectively, and examined in a Zeiss photomicroscope (Carl Zeiss, Inc., New York) or an AEI 801 electron microscope.