Robert H. Miller

A quantitative immunohistochemical study of macroglial cell development in the rat optic nerve: In vivo evidence for two distinct astrocyte lineages

We have shown previously that three antibodies--anti-galactocerebroside (GC), anti-glial fibrillary acidic protein (GFAP), and the A2B5 monoclonal antibody--can be used to help distinguish three classes of glial cells in the rat optic nerve: oligodendrocytes are GC+, GFAP-, almost all type-1 astrocytes are A2B5-, GFAP+, and almost all type-2 astrocytes are A2B5+, GFAP+. In the present study we have used these antibodies to examine the timing and sequence of the development of the three types of glial cells in vivo. We show that type-1 astrocytes first appear at embryonic Day 16 (E16), oligodendrocytes at birth (E21), and type-2 astrocytes between postnatal Days 7 and 10 (P7-10). Moreover, we demonstrate quantitatively that astrocytes in the optic nerve develop in two waves, with more than 95% of type-1 astrocytes developing before P15 and more than 95% of type-2 astrocytes developing after P15. Finally, we provide indirect evidence that type-2 astrocytes do not develop from type-1 astrocytes in vivo, supporting previous direct evidence that the two types of astrocytes develop from two serologically distinct precursor cells in vitro.

FIBROUS AND PROTOPLASMIC ASTROCYTES ARE BIOCHEMICALLY AND DEVELOPMENTALLY DISTINCT

We have studied semithin frozen sections of developing and adult rat central nervous system (CNS) by indirect immunofluorescence in order to determine the antigenic phenotype of protoplasmic and fibrous astrocytes. Using antibodies against glial fibrillary acidic protein (GFAP) to identify astrocytes, we show that the great majority of fibrous astrocytes in adult optic nerve are labeled by the monoclonal antibody A2B5, while the great majority of protoplasmic astrocytes in adult cerebral cortex are not. Astrocytes located at the periphery of the adult optic nerve that form the glial limiting membrane are more like protoplasmic astrocytes than fibrous astrocytes in that they strain relatively weakly with anti-GFAP antiserum and are A2B5-. In the developing rat optic nerve, protoplasmic-like astrocytes appear at least one week before the first fibrous astrocytes can be detected. Taken together with our previous observations on astrocytes in suspensions and cultures of developing rat optic nerve (Raff, M.C., E.R. Abney, J. Cohen, R. Lindsay, and M. Noble (1983) J. Neurosci. 3: 1289–1300; Raff, M.C., R.H. Miller, and M. Noble (1983) Nature 303: 390– 396), these results suggest that protoplasmic and fibrous astrocytes are distinct classes of glial cells that differ in their antigenic phenotype and developmental history, as well as in their morphology and location within the CNS.

Two glial cell lineages diverge prenatally in rat optic nerve

Three types of glial cells have been previously described in cultures of neonatal rat optic nerve--oligodendrocytes, type 1 astrocytes, and type 2 astrocytes--which can be distinguished using three different antibodies: antigalactocerebroside antibodies recognize oligodendrocytes; antibodies against glial fibrillary acidic protein recognize both types of astrocytes, while the A2B5 monoclonal antibody distinguishes between the two, binding to type 2 but not type 1 astrocytes. It was subsequently shown that oligodendrocytes and type 2 astrocytes, but not type 1 astrocytes, develop in cultures of 7 day optic nerve from a common, A2B5+ progenitor cell. In the present study, the distribution of rat neural antigen-2 (Ran-2), a cell-surface antigen defined by a monoclonal antibody, has been examined on optic nerve cells. It is demonstrated that, in contrast to A2B5, Ran-2 is present on type 1 but not type 2 astrocytes in optic nerve cultures. More importantly, it is shown that Ran-2 and A2B5 antibodies react with largely nonoverlapping populations of cells in cell suspensions of embryonic Day 17 (E17) and postnatal Day 1 (P1) optic nerve, and that the Ran-2+, A2B5- population contains type 1 astrocytes and their precursors while the A2B5+,Ran-2- population contains the progenitor cells for oligodendrocytes and type 2 astrocytes. These findings provide strong evidence that the glial cells of the rat optic nerve develop as two distinct lineages--one giving rise to type 1 astrocytes and the other to oligodendrocytes and type 2 astrocytes--and that the two lineages diverge as early as E17.

Evidence that migratory oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells are kept out of the rat retina by a barrier at the eye-end of the optic nerve.

SummaryThere is evidence that oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells migrate along the developing rat optic nerve from the chiasm toward the eye before differentiating into oligodendrocytes that myelinate the retinal ganglion cell axons in the nerve. Why, then, do these progenitor cells not migrate into the eye, differentiate into oligodendrocytes and myelinate the nerve fibre layer of the retina? Myelination would opacify the neural retina and thereby severely impair vision. Here we provide evidence that there is a barrier at the eye-end of the rat optic nerve that prevents the migration of O-2A progenitor cells into the retina. Our findings in the rat support a previous hypothesis that such a barrier keeps myelin-forming glial cells out of the human retina.

EFFECTS OF NEONATAL TRANSECTION ON GLIAL-CELL DEVELOPMENT IN THE RAT OPTIC-NERVE - EVIDENCE THAT THE OLIGODENDROCYTE-TYPE-2 ASTROCYTE CELL LINEAGE DEPENDS ON AXONS FOR ITS SURVIVAL

SummaryWe have previously provided evidence that the rat optic nerve contains three types of macroglial cells that develop as two distinct lineages: one lineage comprises type 1 astrocytes, which develop before birth, while the other comprises oligodendrocytes and type 2 astrocytes, which develop after birth from a common, bipotential glial progenitor cell. In the present study we have examined the influence of axons on the development of these two glial cell lineages by cutting the optic nerve at birth so that the retinal ganglion cell axons in the nerve degenerate. Using antibodies to distinguish the different types of glial cells in suspensions and semithin frozen sections of cut and uncut optic nerves, we show that neonatal transection results in a striking decrease in the total number of oligodendrocytes, type 2 astrocytes and their progenitor cells but has much less effect on the number of type 1 astrocytes. Since the [3H]thymidine labelling indices of oligodendrocytes and their progenitor cells were not significantly decreased in cut nerves, our results suggest that the progenitor cells and/or their progeny die in large numbers following neonatal nerve transection. We conclude that axons are required for the survival of cells of the oligodendrocyte-type 2 astrocyte lineage, at least during postnatal development.

A Novel Type of Glial Cell Associated with Nodes of Ranvier in Rat Optic Nerve

Using Golgi impregnation and intracellular injection of horseradish peroxidase, we show that the adult rat optic nerve contains two distinct types of astrocyte‐like glial cells: one has mainly radially oriented processes that terminate on blood vessels or on the pial surface; the other has mainly longitudinally oriented processes that associate with, and often terminate at, nodes of Ranvier, but do not end on blood vessels or the pial surface. The sequence of appearance of the two types of glial cells in the developing nerve, taken together with previous immunocytochemical findings, suggests that these cells may correspond to the two types of astrocytes previously described in cultures of perinatal optic nerve cells—those with mainly radially oriented processes corresponding to type‐1 astrocytes and those with mainly longitudinally oriented processes corresponding to type‐2 astrocytes. To our knowledge, this is the first description of a class of central nervous system (CNS) glial cell whose processes are primarily associated with nodes of Ranvier.

Radially oriented astrocytes in the normal adult rat spinal cord.

Glial cell organization in the adult rat spinal cord was studied using a modified Golgi technique and anti-GFAP immunofluorescence. Gray matter astrocytes appeared to be a homogeneous population, while in white matter, two morphologically distinct astrocyte subpopulations were seen. One astrocyte had the morphological characteristics of classically described fibrous astrocytes. However, the predominant astrocyte was a radially oriented cell which appeared to span the white matter from the pial surface to the gray-white interface.

A4: an antigenic marker of neural tube-derived cells

SummaryThe A4 monoclonal antibody was originally found to bind to the surface of the majority of neurons in rat CNS cultures, but not to PNS neurons or non-neural cells. It was subsequently shown to bind to immature oligodendrocytes and their precursor cells but not to the most mature oligodendrocytes. In the present study, we have used immunofluorescence assays on cell suspensions and cultures and on semi-thin, frozen tissue sections to show that protoplasmic and fibrous astrocytes and most ependymal cells are also A4+. Taken together, these results suggest that in adult rats A4 is expressed exclusively by cells of the CNS and that all cell types derived from the neural tube are A4+, at least at some time in their development. While neurons, astrocytes and ependymal cells continue to express the antigen in adults, most oligodendrocytes appear to lose it as they mature. The finding that macrophages in CNS cell suspensions and cultures are A4− suggests that microglial cells are not derived from the neural tube.