Kay L. Fields

The cytoskeleton of primary astrocytes in culture contains actin, glial fibrillary acidic protein, and the fibroblast-type filament protein, vimentin.

Abstract: Primary astrocytes were cultured from the forebrains of 1‐day‐old rats. Immunofluorescence microscopy showed that approximately 80% of the cells were positive for glial fibrillary acidic protein (GFAP) and >80% were stained with an antiserum to the molecular weight 58,000 fibroblast intermediate filament protein (vimentin). Gel electrophoresis of Triton‐insoluble cytoskeleton preparations from these cultures revealed three major bands having molecular weights of 58,000, 51,000, and 42,000, together with some prominent lower‐molecular‐weight species. The protein of molecular weight 51,000 was not present in preparations from fibroblasts. Each of the three major astrocyte proteins was subjected to limited proteolysis, while two of the proteins were cleaved by cyanogen bromide. The electrophoretic peptide patterns of the 58,000 protein were similar to those of vimentin isolated from NIL‐8 fibroblasts, and the patterns of the 51,000 protein were similar to those of GFAP isolated from rat spinal cord. The patterns of the protein of molecular weight 42,000 resembled those of muscle actin. Rocket immunoelectrophoresis showed that the 51,000 astrocyte protein reacted with an antiserum to bovine GFAP, but the 58,000 and 42,000 proteins failed to react. We conclude that the major proteins of cytoskeleton preparations from cultured primary astrocytes are vimentin (58,000), GFAP (51,000), and actin (42,000), and that our data show no obvious structural relationship among them.

Distribution of phosphoprotein p19 in rat brain during ontogeny: stage-specific expression in neurons and glia

p19 is an evolutionarily highly conserved 19-kDa cytosolic protein that undergoes hormonally regulated phosphorylation in a variety of mammalian cells. Its expression is abundant in brain and testis and is developmentally regulated. Here we have used immunocytochemistry to define the cell types expressing p19 in the rat CNS during pre- and postnatal development. p19-like immunoreactivity appears in young postmitotic neurons in the mantle zone of the neural tube on embryonic day 12-13. Subsequently, it is abundant in most, if not all, early immature forms of both neurons and glia and declines to undetectable levels in fully differentiated cells. In adult brain, strong p19-like immunoreactivity remains detectable in selective regions, primarily where production of glia and neurons is known to persist, such as the subventricular zone of olfactory bulb and lateral ventricle, and the dentate gyrus. The abundance of p19 mRNA, determined by Northern blot analysis of selected brain regions, parallels the distribution of p19 assessed by immunocytochemistry, suggesting that control of p19 expression is pretranslational. Together with previous findings on the transient expression of p19 during spermatogenesis, the present data suggest that expression of p19 occurs in a number of cell lineages in a differentiation stage-dependent manner. In brain, p19 represents a new marker that may prove valuable for defining immature cell populations.

The long term culture of bulk-isolated bovine oligodendroglia from adult brain

Oligodendroglia isolated from adult bovine brain by the method of Farooq et al. could be plated on polylysine-coated plastic dishes with an efficiency of 55-80%, and maintained in culture for as long as 4 months. The addition of cytosine arabinoside to the nutrient medium resulted in cultures that were approximately 90% oligodendroglia and 10% large fibroblasts. From 50 g of white matter 100-160 X 10(6) oligodendroglia, containing approximately 6-10 mg protein, could be obtained in culture. These small round cells started to send out processes at 5 days in vitro and by 2 weeks they formed an extensive network of processes. By immunofluorescence, all cells of this morphology were positive for galactocerebroside (GC) and myelin basic protein (MBP), and negative for glial filament protein and fibronectin. Most of the large flat cells were positive for fibronectin and negative for GC, MBP and glial filament protein. As the cultures aged the oligodendroglia tended to clump and blebs formed on the surface of both perikarya and processes. By 4 months they showed evidence of degeneration and detached from the substrate. Electron microscope examination showed that the cells had the appearance typical of oligodendroglia in situ. The somata were round to elliptical, with eccentrically placed nuclei, and were larger than freshly isolated cells. They grew directly on the substrate or on the surface of the fibroblasts. In older cultures the cells formed tight nests. The somata were enveloped by sheets of oligodendrocyte cytoplasm, sometimes having a myelin-like appearance. Gap junctions and small desmosomes were seen between oligodendroglial processes and between oligodendroglia and fibroblasts. The cytoplasm was characterized by a prominent Golgi apparatus, many mitochondria and lysosomes, scattered rough endoplasmic reticulum, free ribosomes, frequent centrioles and an abundance of microtubules. In cells from older cultures large vacuoles were common, and rarely they had multilamellar walls with alternating major and minor dense lines resembling myelin.

A subset of Schwann cells in peripheral nerves contain a 50-kDa protein antigenically related to astrocyte intermediate filaments.

Antisera raised to the astrocyte intermediate filament structural protein stained elements in the peripheral nerves of several species. These elements were not associated with myelinated nerve fibers, were more common in splenic and vagus nerves than in the sciatic nerve, and persisted after nerve transection. In teased nerve preparations antigen-positive cells appeared to be the Schwann cells that surround small diameter, unmyelinated axons. Absorption of the antiserum with purified rat spinal cord 50-kDa protein or with bovine splenic nerve cytoskeletal extract blocked the reaction with CNS astrocyte processes or with PNS nerve fibers. Immunoblots of cytoskeletal preparations of bovine splenic nerve or rat sciatic nerve showed that the antigen from peripheral nerves comigrated at 50 kDa with antigen from bovine or rat spinal cord or cultured rat astrocytes. The CNS and PNS 50-kDa proteins from bovine tissues were subjected to limited digestion with Staphylococcus aureus protease V8. After separation on SDS-gels, antigenic peptides were detected by immunoblotting. The pattern of antigenic peptides for the CNS and PNS proteins were identical. We conclude that Schwann cells associated with nonmyelinated axons contain a cytoskeletal protein that is the same size and has the same peptide map as the major structural protein of astrocyte intermediate filaments.

Characterization of opioid receptors in cultured neurons.

The appearance of μ‐, δ‐, and k‐opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high‐affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (‐)‐[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax= 210 ± 40 fmol/mg protein); this density is approximately two‐thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the μ‐specific ligand [3H]D‐Ala2,N‐Me‐Phe4,Gly‐ol5‐enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 ± 0.1 nM and a density (Bmax) of 160 ± 20 fmol/mg of protein. Specific binding of (‐)‐[3H]bremazocine under conditions in which μ‐ and δ‐receptor binding was suppressed (k‐receptor labeling conditions) occurred to an apparent single class of sites (KD= 2 ± 1 nM;Bmax= 40 ± 15 fmol/mg of protein). There was no detectable binding of the selective δ‐ligand [3H]D‐Pen2,d‐Pen5‐enkephalin. Thus, cultured striatal neurons expressed μ‐ and k‐receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not δ‐receptors.

Ultrastructure and immunocytochemistry of rat Schwann cells and fibroblasts in vitro

The ultrastructure of dissociated rat sciatic nerve Schwann cells and fibroblasts has been examined and correlated at the morphologic and immunocytochemical levels. In agreement with previous studies, the two cell types were readily distinguishable morphologically on the basis of cell shape and size but no unique structural feature could be found. Schwann cells in this system essentially lacked basal laminae but did show some membrane specializations reminiscent of the in vivo situation. Fibroblasts were exceedingly rich in actin webs beneath the plasmalemma. Immunocytochemically, only Schwann cells stained with anti-Ran-1 antisera and fibroblasts only with anti-Thy-1. Both staining patterns were exclusively associated with the surface membrane. The findings provide a basis for future studies with this system to which neuronal elements may be added in an attempt to initiate myelinative events.

Chapter 11 Cell Type-Specific Antigens of Cells of the Central and Peripheral Nervous System

Publisher Summary The most basic level of differentiation of the nervous system is the determination of distinctive neuronal, astrocytic, and oligodendroglial cell types. Immunological techniques are used to distinguish these cell types in tissue culture, where many of the clues of gross location and cellular pattern formation of in vivo material are completely missing. This chapter reviews the markers that are specific for Schwann cells, neurons, astrocytes, or oligodendroglia in cultures from the central and peripheral nervous system. It considers the Thy-1 antigen in some detail and presents the evidence that it is a differentiation antigen of neurons. It also describes the practical use of cell surface antigens in preparing pure populations of Schwann cells. The use of brain-specific antigens as markers is only one application of immunological techniques to the study of the nervous system. Schwann cells can be recognized by the presence of a surface antigen Ran-1 or by several other antisera. Fibroblasts, which contaminate cultures of Schwann cells, can be recognized by surface LETS protein, or the glycoprotein Thy-1. Antisera cytotoxic for cells with Thy-1 antigen can be used to obtain pure Schwann cell cultures.

Schwann cells cultured from adult rats contain a cytoskeletal protein related to astrocyte filaments

Cells that bound antibody to the astrocyte intermediate filament protein were cultured from adult rat sciatic nerve. The antigen was intracellular, finely filamentous, and formed perinuclear caps in response to colchicine, all properties of intermediate filaments. Cytoskeletal proteins of these cultures were separated by SDS-gel electrophoresis, transferred to nitrocellulose paper, and shown to bind the glial-specific antiserum to a protein of 50,000 daltons. All the cells that bound this serum had a Schwann cell surface antigen, Ran-1, whereas fibroblasts from the nerve had Thy-1 surface antigen and did not contain the astrocyte filament antigen. These results prove that some Schwann cells from adult nerve, in contrast to fibroblasts or immature Schwann cells, have an intermediate filament protein that shares antigenic determinants with, or may be identical to, the astrocyte filament protein.

Neuronal and Glial Surface Antigens on Cells in Culture

This review will emphasize cell surface antigenic markers and practically ignore work with most of the described cytoplasmic antigens, simply because this field is growing so fast that it is impractical to try to cover all the new antigens. The emphasis here is on antigens that have been used with success in cultures of the nervous system.

The study of Schwann cells using antigenic markers

Following on from Rhona Mirskys recent article on cell-type-specific antigenic markers Kay Fields provides here a more detailed account of their application in the study of Schwann cell differentiation and function.