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Basement membrane material and glial cells in spinal cord cultures of newborn rats
198
SHORT COMMUNICATIONS
Basement membrane material and glial cells in spinal cord cultures of newborn rats The morphological development and the onset of synapse formation of long term cultures of spinal cord of fetal rat and mouse have been described by several authorsl-4,7,L In the present investigation, we studied ultrastructural properties of glial cells in the outgrowth zone of spinal cord cultures of newborn rats. Special attention was paid to the formation of the basement membrane towards the collagen substrate. Explants from spinal cord were cultivated on collagen coated coverslips in a Maximov double coverslip assembly. The method of cultivation has been described previouslyL For electron microscopical studies the cultures were fixed on the coverslips in 2.5 ~ glutaraldehyde in a phosphate buffer (0.1 M, pH 7.4) for 15 min, postfixed in 1 ~ OsO4 in a phosphate buffer (0.1 M, pH 7.4) for 30 rain, dehydrated in ethanol and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate. Observations were made on cultures which were grown for 8-34 days in vitro. The migration of flattened, discus-like and spindle-shaped cells of possible mesodermal (pial, vascular) origin starts after 2-3 days in vitro. Small cells which resemble oligodendrocytes from the light microscopical appearance6, s are also among the first cells to migrate. These cells usually degenerate a short time after migration. This is in contrast to the behaviour of oligodendrocytes which migrate later, together with astrocytes and neurones. We usually observed a smaller number of oligodendrocytes in spinal cord cultures than in cultures of cerebellum and brain stem 4. Most of the glial
i~I ....
!~
Fig. 1. Astrocytic network in the zone of migration of a 22-day-old culture. Phase contrast, bar: 30/zm. Brain Research, 32 (1971) 198-202
A
B
C
D
Fig. 2. Outgrowth zone of a 34-day-old spinal cord culture. Sections cut perpendicular to the surface of the coverslip. A, Perikaryon of a glial cell in contact with a cell process. I, intercellular cleft of variable width; B, basement membrane material covering the collagen substrate (C). B, Thin layer of filaments very close to the apical plasma membrane (F). E, granulated endoplasmic reticulum containing osmiophilic material; D, dense inclusion bodies; M, mitocbondria; ~', local attachment to the basement membrane material (B) of a cell which partly overlaps another cell process. C, Bundle of filaments (F) forming a dense zone along the plasma membrane at places where the cells adhere to the basement membrane (~). D, Filamentous structures in basement membrane material at a higher magnification. Bar: 1 /~m.
B
A
¢
D
Fig. 3. Outgrowth zone of a spinal cord culture, 34 days in vitro (A, C, D). Sections cut parallel to the surface of the coverslip. A, Filament bundle (F) lying close to the plasma membrane and in the cytoplasm o f a glial cell. N, nucleus; P, cell processes. (Other abbreviations see Fig. 2.) B, Filament bundle at a higher magnification of another culture, 8 days in vitro. Glycogen granules in upper part of picture. C, Gap junction between two glial cells lying close to the explant. D, Cytoplasm of a glial cell with filament bundles (F), complicated profiles of mitochondria (M) and granulated endoplasmic reticulum (E). Bar: I ~m.
SHORT COMMUNICATIONS
201
cells which migrate after 4-6 days show the morphological features o f protoplasmic astrocytes but also few fibrillar astrocytes were observed. The cell bodies of the astrocytes vary considerably in shape and size. The cytoplasm of the perikaryon often contains granular inclusions and usually appears more transparent than the cytoplasm of neurones and oligodendrocytes. The irregular and relatively large nucleus is round or slightly elongated and often has more than one nucleolus. The processes usually form a dense network in the zone of migration (Fig. 1). The nerve cells, which usually do not migrate as far from the explant as the glial elements, are located mainly in the beveled and dense zones of the cultureL Electron microscopical observations have been made on cells in the outgrowth zones of the cultures. In sections cut perpendicular to the surface of the coverslip, flattened processes and perikarya of glial cells adhere to the collagen substrate. The cytoplasm of these cells contains one or several Golgi zones (Fig. 2A), round to elongated profiles of mitochondria, flattened cisterns of the granular endoplasmic reticulum and osmiophilic inclusions of variable size and shape. Glycogen granules are distributed diffusely throughout the cytoplasm. Their number varies considerably from section to section and from cell to cell. In thick processes, aggregates of glycogen granules are only rarely observed. Cytoplasmic filaments (4-6 nm in diameter) are mainly confined to a thin rim very close to the plasma membrane (Figs. 2B, 3A). Bundles of thicker filaments (8-9 nm in diameter) are also observed within the cytoplasm or near the membrane (Figs. 2C, 3B). In sections cut parallel to the surface of the coverslip, the glial cells often contain rather complicated profiles of mitochondria (Fig. 3D). The bundles of filaments are much thicker than those observed in perpendicular sections (Fig. 3A, D). The granular endoplasmic reticulum consists of rounded cisterns containing an osmiophilic granular substance. The processes and perikarya of most of these cells are separated by an intercellular cleft of 10-40 nm (Fig. 2B). Only in few areas the width of the intercellular clefts seems to be less than 10 nm (Fig. 3A). Typical gap junctions have not been observed between cells in the peripheral areas of the outgrowth zones, however, a small number has been found between glial cells located close to the explant (Fig. 3C). As was described by Guillery et al. 3,4 basement membranes (basal lamina) have been observed at the basal layer of the explant and between neural elements and newly formed collagen from meningeal or vascular sources. In our cultures basement membrane material was also observed between migrating cells and the collagen substrate in the outgrowth zones (Fig. 2A, B) and in parts of the collagen substrate where glial cells have passed before (Fig. 2D). No basement membrane material was found in the cell free regions of the cultures. The basement membrane consists of filaments (3-6 nm) which run mainly parallel to the surface of the collagen substrate. Dense zones in the glial cytoplasm and an increased density and thickening of the basement membrane are observed in some regions where the plasma membrane of the cells is in contact with the basement membrane. These local contact regions resemble 'half-desmosomes' which have been observed in epithelia. In Fig. 2B one of Brain Research, 32 (1971) 198-202
202
SHORT COMMUNICATIONS
these 'half-desmosomes' is illustrated. Fig. 2C shows a basement membrane which is folded and partially detached from the plasma membrane of the glial cell. In studies of cultured spinal cord of fetal animals no basement membrane material was described between cells and the collagen substrate in the outgrowth zones 3,4. Our observation that basement membranes are also formed between glial cells and the collagen substrate in the outgrowth zones might indicate that tissue of newborn animals has a greater capacity to produce basement membrane material in this area, however, it might also be due to the different staining procedures used (uranyl acetate instead of lead citrate only )3,4.The finding that bundles of filaments with a diameter of 8-9 nm were observed in many glial cells suggests that the majority of these cells might be immature astrocytes (for references see ref. 4). Division of Fundamental Research, Sandoz Ltd., 4000 Basle and Department of Neurophysiology, Neurological Clinic of the University of Basle, 4051 Basle (Switzerland)
J. R. W O L F F * E. HOSLI L. HOSLI
1 BUNGE, M. B., BUNGE, R. P., AND PETERSON, E. R., The onset of synapse formation in spinal cord cultures as studied by electron microscopy, Brain Research, 6 (1967) 728-749. 2 BUNGE, R. P., BUNGE, M. B., AND PETERSON, E. R., An electron microscope study of cultured rat spinal cord, J. Cell Biol., 24 (1965) 163-191. 3 GUILLERY, R. W., SOBKOWICZ, H. M., AND SCOTT, G. L., Light and electron microscopical observations of the ventral horn and ventral root in long term cultures of the spinal cord of the fetal mouse, J. comp. Neurol., 134 (1968) 433476. 4 GUILLERY, R. W., SOBKOWlCZ, H. M., AND SCOTT, G. L., Relationships between glial and neuronal elements in the development of long term cultures of the spinal cord of the fetal mouse, J. comp. Neurol., 140 (1970) 1-34. 5 H~)SLI, E., AND H0SLI, L., Acetylcholinesterase in cultured rat spinal cord, Brain Research, 30 (1971) 193-197. 60KAMOTO, M., Observations on neurons and neuroglia from the area of the reticular formation in tissue culture, Z. Zellforsch., 47 (1958) 269-287. 7 PETERSON, E. R., CRAIN, S. M., AND MURRAY, M. R., Differentiation and prolonged maintenance of bioelectrically active spinal cord cultures (rat, chick and human), Z. Zellforsch., 66 (1965) 130154. 8 POMERAT, C. M., AND COSTERO, 1., Tissue cultures of cat cerebellum, Amer. J. Anat., 99 (1956) 211-247. 9 SOBKOWICZ, H. M., GUILLERY, R. W., AND BORNSTEIN, M. B., Neuronal organization in long term cultures of the spinal cord of the fetal mouse, J. comp. Neurol., 132 (1968) 365-396. (Accepted June 9th, 1971)
* Permanent address: II. Anatomisches Institut, Freie UniversitS.t, K~Snigin-Luise Str. 15, Berlin (G.F.R.)
Brain Research, 32 (1971) 198-202
SHORT COMMUNICATIONS
Basement membrane material and glial cells in spinal cord cultures of newborn rats The morphological development and the onset of synapse formation of long term cultures of spinal cord of fetal rat and mouse have been described by several authorsl-4,7,L In the present investigation, we studied ultrastructural properties of glial cells in the outgrowth zone of spinal cord cultures of newborn rats. Special attention was paid to the formation of the basement membrane towards the collagen substrate. Explants from spinal cord were cultivated on collagen coated coverslips in a Maximov double coverslip assembly. The method of cultivation has been described previouslyL For electron microscopical studies the cultures were fixed on the coverslips in 2.5 ~ glutaraldehyde in a phosphate buffer (0.1 M, pH 7.4) for 15 min, postfixed in 1 ~ OsO4 in a phosphate buffer (0.1 M, pH 7.4) for 30 rain, dehydrated in ethanol and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate. Observations were made on cultures which were grown for 8-34 days in vitro. The migration of flattened, discus-like and spindle-shaped cells of possible mesodermal (pial, vascular) origin starts after 2-3 days in vitro. Small cells which resemble oligodendrocytes from the light microscopical appearance6, s are also among the first cells to migrate. These cells usually degenerate a short time after migration. This is in contrast to the behaviour of oligodendrocytes which migrate later, together with astrocytes and neurones. We usually observed a smaller number of oligodendrocytes in spinal cord cultures than in cultures of cerebellum and brain stem 4. Most of the glial
i~I ....
!~
Fig. 1. Astrocytic network in the zone of migration of a 22-day-old culture. Phase contrast, bar: 30/zm. Brain Research, 32 (1971) 198-202
A
B
C
D
Fig. 2. Outgrowth zone of a 34-day-old spinal cord culture. Sections cut perpendicular to the surface of the coverslip. A, Perikaryon of a glial cell in contact with a cell process. I, intercellular cleft of variable width; B, basement membrane material covering the collagen substrate (C). B, Thin layer of filaments very close to the apical plasma membrane (F). E, granulated endoplasmic reticulum containing osmiophilic material; D, dense inclusion bodies; M, mitocbondria; ~', local attachment to the basement membrane material (B) of a cell which partly overlaps another cell process. C, Bundle of filaments (F) forming a dense zone along the plasma membrane at places where the cells adhere to the basement membrane (~). D, Filamentous structures in basement membrane material at a higher magnification. Bar: 1 /~m.
B
A
¢
D
Fig. 3. Outgrowth zone of a spinal cord culture, 34 days in vitro (A, C, D). Sections cut parallel to the surface of the coverslip. A, Filament bundle (F) lying close to the plasma membrane and in the cytoplasm o f a glial cell. N, nucleus; P, cell processes. (Other abbreviations see Fig. 2.) B, Filament bundle at a higher magnification of another culture, 8 days in vitro. Glycogen granules in upper part of picture. C, Gap junction between two glial cells lying close to the explant. D, Cytoplasm of a glial cell with filament bundles (F), complicated profiles of mitochondria (M) and granulated endoplasmic reticulum (E). Bar: I ~m.
SHORT COMMUNICATIONS
201
cells which migrate after 4-6 days show the morphological features o f protoplasmic astrocytes but also few fibrillar astrocytes were observed. The cell bodies of the astrocytes vary considerably in shape and size. The cytoplasm of the perikaryon often contains granular inclusions and usually appears more transparent than the cytoplasm of neurones and oligodendrocytes. The irregular and relatively large nucleus is round or slightly elongated and often has more than one nucleolus. The processes usually form a dense network in the zone of migration (Fig. 1). The nerve cells, which usually do not migrate as far from the explant as the glial elements, are located mainly in the beveled and dense zones of the cultureL Electron microscopical observations have been made on cells in the outgrowth zones of the cultures. In sections cut perpendicular to the surface of the coverslip, flattened processes and perikarya of glial cells adhere to the collagen substrate. The cytoplasm of these cells contains one or several Golgi zones (Fig. 2A), round to elongated profiles of mitochondria, flattened cisterns of the granular endoplasmic reticulum and osmiophilic inclusions of variable size and shape. Glycogen granules are distributed diffusely throughout the cytoplasm. Their number varies considerably from section to section and from cell to cell. In thick processes, aggregates of glycogen granules are only rarely observed. Cytoplasmic filaments (4-6 nm in diameter) are mainly confined to a thin rim very close to the plasma membrane (Figs. 2B, 3A). Bundles of thicker filaments (8-9 nm in diameter) are also observed within the cytoplasm or near the membrane (Figs. 2C, 3B). In sections cut parallel to the surface of the coverslip, the glial cells often contain rather complicated profiles of mitochondria (Fig. 3D). The bundles of filaments are much thicker than those observed in perpendicular sections (Fig. 3A, D). The granular endoplasmic reticulum consists of rounded cisterns containing an osmiophilic granular substance. The processes and perikarya of most of these cells are separated by an intercellular cleft of 10-40 nm (Fig. 2B). Only in few areas the width of the intercellular clefts seems to be less than 10 nm (Fig. 3A). Typical gap junctions have not been observed between cells in the peripheral areas of the outgrowth zones, however, a small number has been found between glial cells located close to the explant (Fig. 3C). As was described by Guillery et al. 3,4 basement membranes (basal lamina) have been observed at the basal layer of the explant and between neural elements and newly formed collagen from meningeal or vascular sources. In our cultures basement membrane material was also observed between migrating cells and the collagen substrate in the outgrowth zones (Fig. 2A, B) and in parts of the collagen substrate where glial cells have passed before (Fig. 2D). No basement membrane material was found in the cell free regions of the cultures. The basement membrane consists of filaments (3-6 nm) which run mainly parallel to the surface of the collagen substrate. Dense zones in the glial cytoplasm and an increased density and thickening of the basement membrane are observed in some regions where the plasma membrane of the cells is in contact with the basement membrane. These local contact regions resemble 'half-desmosomes' which have been observed in epithelia. In Fig. 2B one of Brain Research, 32 (1971) 198-202
202
SHORT COMMUNICATIONS
these 'half-desmosomes' is illustrated. Fig. 2C shows a basement membrane which is folded and partially detached from the plasma membrane of the glial cell. In studies of cultured spinal cord of fetal animals no basement membrane material was described between cells and the collagen substrate in the outgrowth zones 3,4. Our observation that basement membranes are also formed between glial cells and the collagen substrate in the outgrowth zones might indicate that tissue of newborn animals has a greater capacity to produce basement membrane material in this area, however, it might also be due to the different staining procedures used (uranyl acetate instead of lead citrate only )3,4.The finding that bundles of filaments with a diameter of 8-9 nm were observed in many glial cells suggests that the majority of these cells might be immature astrocytes (for references see ref. 4). Division of Fundamental Research, Sandoz Ltd., 4000 Basle and Department of Neurophysiology, Neurological Clinic of the University of Basle, 4051 Basle (Switzerland)
J. R. W O L F F * E. HOSLI L. HOSLI
1 BUNGE, M. B., BUNGE, R. P., AND PETERSON, E. R., The onset of synapse formation in spinal cord cultures as studied by electron microscopy, Brain Research, 6 (1967) 728-749. 2 BUNGE, R. P., BUNGE, M. B., AND PETERSON, E. R., An electron microscope study of cultured rat spinal cord, J. Cell Biol., 24 (1965) 163-191. 3 GUILLERY, R. W., SOBKOWICZ, H. M., AND SCOTT, G. L., Light and electron microscopical observations of the ventral horn and ventral root in long term cultures of the spinal cord of the fetal mouse, J. comp. Neurol., 134 (1968) 433476. 4 GUILLERY, R. W., SOBKOWlCZ, H. M., AND SCOTT, G. L., Relationships between glial and neuronal elements in the development of long term cultures of the spinal cord of the fetal mouse, J. comp. Neurol., 140 (1970) 1-34. 5 H~)SLI, E., AND H0SLI, L., Acetylcholinesterase in cultured rat spinal cord, Brain Research, 30 (1971) 193-197. 60KAMOTO, M., Observations on neurons and neuroglia from the area of the reticular formation in tissue culture, Z. Zellforsch., 47 (1958) 269-287. 7 PETERSON, E. R., CRAIN, S. M., AND MURRAY, M. R., Differentiation and prolonged maintenance of bioelectrically active spinal cord cultures (rat, chick and human), Z. Zellforsch., 66 (1965) 130154. 8 POMERAT, C. M., AND COSTERO, 1., Tissue cultures of cat cerebellum, Amer. J. Anat., 99 (1956) 211-247. 9 SOBKOWICZ, H. M., GUILLERY, R. W., AND BORNSTEIN, M. B., Neuronal organization in long term cultures of the spinal cord of the fetal mouse, J. comp. Neurol., 132 (1968) 365-396. (Accepted June 9th, 1971)
* Permanent address: II. Anatomisches Institut, Freie UniversitS.t, K~Snigin-Luise Str. 15, Berlin (G.F.R.)
Brain Research, 32 (1971) 198-202