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Membrane structure in cultured astrocytes
Brain Research, 276 (1983) 31-41 Elsevier
31
Membrane Structure in Cultured Astrocytes DENNIS M. D. LANDIS and LORI A. WEINSTEIN Department of Neurology, Massachusetts GeneralHospital, Boston, MA 02114 (U.S.A.) (Accepted February 8th, 1983) Key words: astrocytes - - membrane structure - - freeze-fracture - - tissue culture - - glial fibrillary acidic protein
Astrocytes cultured from the brains of neonatal rat pups acquire at least two specializations of intramembrane particle distribution: 'assemblies' and gap junctions. The number and appearance of assemblies in the cultured astrocytes is not markedly influenced by the presence or absence of a collagen substrate, and the range of concentrations of assemblies in astrocytic membranes is fairly stable from 7 through 28 days in culture. The assemblies are not concentrated in apposition to the substrate, even though the astrocytic membranes containing the highest concentration of assemblies in vivo are apposed to basal lamina. Quantitative analysis shows that assemblies are not uniformly distributed over the plasmalemma of a single cell, raising the possibility that the nature of cells around an astrocytic process may influence its membrane composition in vitro. INTRODUCTION Astrocytic membranes in the mammalian central nervous system contain characteristic aggregates of small, uniform particles packed in orthogonal arrayl-3,lO,ll,13-15,22-27, 30,33. These particle aggregates, or 'assemblies', are densely packed in astrocytic membranes apposed to blood vessels, less numerous in astrocytic processes apposed to the cerebrospinal fluid at the surface of the brain, and least densely packed in astrocytic membranes investing neuronal processes. The composition of assemblies and their function are unknown26, 27. Assemblies can be identified only in freeze-fractured tissue; no corresponding structure is evident in thin-sectioned preparations. Within the brain, assemblies are found in the membranes of astrocytes and ependymal cells 7, but do not occur in neurons, oligodendrocytes, or endothelial cells. Assemblies occur in several types outside the central n e r v o u s system6,12,16,18A9,21,32, 38-41. Assemblies in these non-neuronal cells are like those in astrocytes in appearance and in the fact that they are concentrated in specific domains of the cell surface, except in the instance of hepatocytes 21. We have turned to tissue culture, with its potential for close control over cellular environment, in our effort to study the structure of assemblies and their role in astrocyte function. It has been shown that explant cultures of cerebellar cortex do manifest assemblies, 0006-8993/83/$03.00 (~ 1983 Elsevier Science Publishers B.V.
but these cultures were very heterogeneous35. In preliminary studies, we were unable to obtain useful numbers of assemblies in cultures of rodent C-6 glioma cells and in cultures of 3 human astrocytomas (ref. 24; see also ref. 17). Recently, a technique for establishing primary astrocyte cultures from 7-day rat cerebral hemispheres has been described 3. That system utilizes low-density plating to yield discrete colonies which differ markedly in cellular composition. T h o u g h astrocytes with many assemblies do appear, the system is contaminated by neurons, the number of assemblies is not stable over time in culture, and the number of assemblies in the membranes of one colony may vary markedly from that of another colony in the same culture. In order to obtain cultures with more uniform cellular composition and cellular differentiation, we have used secondary cultures, with techniques somewhat modified from those of McCarthy and deVellis 31. We present here the details of the culture methods, the cellular composition of the cultures, and the appearance and distribution of assemblies in the membranes of the cultured astrocytes. Some aspects of this study have been presented in brief form 2s. MATERIALS AND METHODS Tissue culture The tissue culture techniques are based on the ap-
32 proach devised by McCarthy and deVellis 31. Rat pups aged 2--4 days were stunned by concussion, decapitated, and the brain removed. The meninges, choroid plexus, and cerebellum were discarded. Cerebral hemispheres and bramstem were minced in L15 plating medium (composition as in Mains and Patterson 29 save that nerve growth factor was omitted), and then exposed to 0.25% trypsin for 25 rain at 37 °C with agitation. Dissociated cells were sedimented, and the cells collected from t0 brains were innoculated into four 75 cm 2 Falcon flasks each containing 20 ml of L 15-CO: growth medium (composition as in Mains and Patterson 29 save that rat serum, bovine serum albumin, nerve growth factor and Methocel were omitted) supplemented with 20% fetal calf serum. The medium was changed every 3 days. Cells were confluent at 7-10 days of culture. The flasks were sealed, placed on a rotary shaker and shaken 12-18 h at 37 °C to suspend oligodendrocytes and other cells growing over the confluent monolayer. The suspended cells were discarded, the flasks washed vigorously with the fresh media, and finally the cells of the monolayer were collected after brief exposure to 5 mM E D T A in Hank's balanced salt solution without divalent cations. The cells were sedimented, rinsed, resuspended, and innoculated into Falcon polystyrene tissue culture dishes (35 x 10 mm or 60 x 15 ram) at a concentration of 3 x 105 cells/cm 2. The cultures were fed every three days, and were usually confluent by 14 days. The results presented are based on 6 separate platings.
lmmunofluorescence Circular holes were cut in the center of 35 x 10 mm Falcon tissue culture dishes, and glass coverslips were sealed to the bottom of the dishes with paraffin. The portion of the coverslip exposed to the tissue culture medium was coated with collagen (Vitrogen100, Flow Laboratories). The cultures were prepared for immunofluorescence staining at 3, 4, 7, 14, 21 and 28 days of secondary culture. Cultures were rinsed briefly with fresh medium, and then fixed in 5% glacial acetic acid in ethanol for 15 min at - - 2 0 °C. The fixed cultures were rinsed in 0.12 M phosphate buffer and subsequent manipulations carried at 22 °C. Cells were incubated for 30 min in the presence of rabbit antisera to glial fibrillary acidic protein (graciously provided by Dr. D.
Dahl) at a dilution of 1:100. After washing in phosphate buffer, the cells were incubated in the presence of goat antisera raised against rabbit immunoglobulin and conjugated with rhodamine or fluorescein (Cappel Laboratories) for 30 min. After further washing, the cells were mounted in 1:1 glycerol:phosphate buffer and examined with epifluorescence optics. As a method control, cultures were prepared as above, except that primary antiserum was omitted.
Thin Section Electron Microscopy Square glass coverslips were placed in 60 x 15 mm tissue culture dishes, coated with collagen (Vitrogen100, Flow Laboratories), and the dishes were innoculated with cells collected from flasks. After the desired interval of culture, cells were fixed in 3% glutaraldehyde in 0.12 M sodium cacodylate buffer at pH 7.35 and 37 °C for 2-4 h. After post-fixation in 1% osmium tetroxide in 0.12 M sodium cacodylate buffer, most preparations were stained en-bloc with uranyl acetate for 4-16 h, dehydrated, and then embedded in epon.
Freeze-fracture The cultured cells were freeze-fractured with a Balzers complementary replication device, using techniques described by Pauli et al. 34, Yee et al. 43, and Cohen and Pumplin9. Glass coverslips were cut into 4.5 mm squares, placed on the bottom of a 60 x 15 mm Petri dish, and each coated with collagen. The dish was inoculated with cells as described above. After the desired interval of culture, either all the squares were fixed by flooding the dish with fixative, or selected squares were removed with sterile technique and plunged immediately into fixative. The fixative contained 3% glutaraldehyde and 0.12 M sodium cacodylate buffer adjusted to pH 7.35. After 1-3 b in fixative (initial temperature 37 °C, allowed to cool to 22 °C), the cells were rinsed in 0.12 M sodium cacodylate buffer. The cells were then soaked in 12.5% glycerol in 0.06 M sodium cacodylate buffer for 30 rain and then in 25% glycerol in 0.06 M sodium cacodylate buffer for an additional one half hour. Double-height gold or copper specimen supports were coated with polyvinyl alcohol and then sandwiched with the coverslips and frozen by immersion in melting monochlorodifluoromethane (Freon 22). Freeze-fracturing was performed in a Balzers freeze-
33 fracture apparatus at - - 1 1 0 °C and in a 2--4 x 10-7 torr vacuum with a c o m p l e m e n t a r y replica device, and the fractured cells were replicated i m m e d i a t e l y , without etching.
minicomputer. The area of the m e m b r a n e fracture surface and the n u m b e r of assemblies were determined three times by a single observer. OBSERVATIONS
Morphometric techniques Freeze-fracture replicas were p h o t o g r a p h e d in a J E O L 100 CX electron microscope at a p r i m a r y magnification of 27,000-33,000 and enlarged p h o t o graphically. Magnification and photographic enlargement were measured with micrographs of grating replicas. The area of m e m b r a n e fracture surfaces was measured with a sonic digitizer (Graf-Pen) interfaced with a Digital E q u i p m e n t C o r p o r a t i o n PDP-8
Phase contrast and immunofluorescence Immediately after inoculation of the tissue culture dishes with cells suspended from the flask monolayers most cells were single, but clumps of cells were also present. The secondary cultures b e c a m e confluent between 10 and 14 days. During this initial growth interval, cells a p p e a r e d h e t e r o g e n e o u s (Fig. 1). A f t e r 21 and 28 days of culture, some tissue cul-
Fig. 1. Secondary culture at 21 days. In phase contrast, cells in the monolayer range from epitheloid to spindle-shaped (acetic acid/ethanol fixation; 80 x). Fig. 2. Secondary culture at 21 days, containing ceils with GFAP immunoreactivity. The field is the same as that of Fig. 1 (acetic acid/ethanol fixation; 80 x ). Fig. 3. Secondary culture at 21 days. In this portion of the culture, phase-dark rounded cells are present above the monolayer. Some give rise to narrow, straight processes which branch at acute angles (acetic acid/ethanol fixation; 80 x). Fig. 4. Secondary culture at 21 days, containing cells with GFAP immunoreactivity. The field is the same as that of Fig. 3, but the plane of focus is in the monolayer. Cells containing GFAP immunoreactivity are below the rounded cells, but the rounded cells and their processes are not stained (acetic acid/ethanol fixation; 80 x ).
34 ture dishes contained a population of round cells above the confluent layer (Fig. 3). There was no obvious difference in cell morphology and cell density on the surface of the glass coverslips as compared to the plastic dish surface. Glial fibrillary acidic protein (GFAP) immunoreactivity was detected as early as 3 days in secondary culture, even in rounded cells which had not yet begun to spread over the collagen-coated substrate. Over the first 4 weeks in culture, there was no dramatic change in the proportion of cells demonstrating GFAP immunoreactivity. In different platings, the proportion of cells with GFAP immunoreactivity ranged between 40% and 70% of cells present (Figs. 2, 4). The distribution of these cells was strikingly non-uniform. In some regions on the tissue culture dish, immunoreactive cells made up virtually all the
cells present, while in nearby regions of the same petri dish, only scattered cells within a field would contain GFAP immunoreactivity. The morphology of cells with G F A P immunoreactivity ranged from stellate with well-defined, radiating processes to nearly epithelioid with broad, flat expanses of cytoplasm. Cells in regions of the culture which did not manifest G F A P immunoreactivity tended to be more homogeneous in terms of nuclear and cytoplasmic morphology, and often appeared epithelioid. Through 21 days of culture, the GFAP positive cells did not become more homogeneous in their cytoplasmic morphology. At 21 and 28 days of culture, some tissue culture dishes included a population of cells which appeared in phase contrast to be round, phase-dark, and above the levels of the confluent monolayer (Fig. 3). The cells tended to occur in clusters, and were uniformly
Fig. 5. Abundant intermediate filaments in an astrocyte at 28 days. The nucleus is at the lower right, and several lysosomesare evident in the upper right (25,000x).
35 G F A P negative (Fig. 4). The monolayer below these cells, however, invariably consisted of nearly confluent GFAP-positive cells.
Thin-section electron microscopy Cultures were prepared for thin-section electron microscopy at 7, 14, 21, and 28 days of culture, and were sectioned in a plane parallel to the surface of the glass coverslip substrate. Many flattened cells in these preparations contained complicated skeins of intermediate filaments, richly interwoven (Fig. 5). These filaments in the perikaryal region of flattened cells also extended to fill small diameter cell processes. Some of the cells in older cultures which contained numerous filaments also contained a large number of pleomorphic lysosomal inclusions. These lysosomes were highly variable in their appearance, and often contained lamellar structures suggestive of ingested and densely packed lipid. Many of the flattened, filament-containing cells established gap junctions with neighboring cells. Such junctions were often encountered where a process of one cell indented the cytoplasm of an adjacent cell. Thin sections above the level of flattened cells constituting the monolayer of the culture occasionally revealed profiles of the rounded cells. These rounded cells had comparatively electron-dense cytoplasm, and in the perikaryal region manifested large amounts of rough endoplasmic reticulum and free ribosomes. They were relatively devoid of intermediate filaments, and did not contain the abundant lysosomai inclusions present in some of the flattened cells. Processes arising from these rounded cells often contained large numbers of microtubules, and several vesicle-filled mounds were encountered on the surfaces of the rounded cells.
Qualitative freeze-fracture studies The plane of fracture in most instances propagated through the plasma membranes of the flattened cells constituting the confluent monolayer. A potential hazard of the technique is that the plasma membrane adjacent to the substrate might be preferentially fractured (or missed). However, many cells were found in which the plane of fracture went from one plasma membrane across the attenuated cytoplasm to the other membrane, and so we certainly encountered fractures through the substrate and through the su-
perficial membrane. The plane of fracture rarely exposed the membrane structure of rounded cells above the confluent monolayer, and virtually all the observations made in this portion of the study are restricted to the flattened cells. Many of the cultured cells had assemblies in their plasma membranes, arrayed nearly uniformly across the cell surface. The particles constituting the assemblies were packed in orthogonal array, and were associated with the cytoplasmic half of the fractured membrane (Fig. 7), while complementary pits were present on the extracellular half of the membrane. The particles constituting gap junctions were also present in the plasma membranes of many of the cells with assemblies (Fig. 8); the particles were associated with the cytoplasmic half of the fractured membrane, and there were complementary pits on the extracellular membrane half. The particles of gap junctions were present in hexagonal packing and in unordered patterns. Whenever it was possible to examine the membrane structure of both astrocytes forming a gap junction, both were found to contain assemblies. Some of the cells in the cultures had elaborated tight junctions with adjacent cells. Many of these tight junctions had associated gap junctions, but assemblies were never recognized in the membrane of a cell participating in a tight junction. There was a distinct tendency for cells with tight junctions to occur in clusters. Cells with assemblies in the plasma membranes, cells with tight junctions and no assemblies, and cells with no evident specialization of intramembrane particle distribution all had variable numbers of caveolae. These were evident as shallow hemispherical indentations without associated particle aggregates (the lack of the particles serves to distinguish them from the invaginations of coated pits). Cross-fractures through the perikaryal cytoplasm and nuclear membranes were infrequent, but served to expose the usual array of cytoplasmic organelles (Fig. 6). Astrocytes in the cerebellar cortex and cerebral cortex elaborate a junction composed of large, pleomorphic particles which we have provisionally described as a 'polygonal particle junction'26.27. We were unable to recognize certain examples of this junctional specialization in the cultured astrocytes. Individual constituent particles might have been pre-
Fig. t~. Freeze-fractured astrocvtc, 28 days. I-he plane ol lracture has exposed the extraccllular hall of the plasma m e m b r a n e (E), and has cross-fractured the perikaryon to reveal the nucleus (N), mitoehondria (M), lysosomes (I.), and cross-fractured filaments (arrows). Imprints of assemblies on the tractured m e m b r a n e were clearly visible at higher magnification (27,1/00×). Fig. 7. Assemblies on the cytoplasmic half of lracturcd astroeytie m e m b r a n e at 7 days of secondary culture. The constituenl particles are packed in square or rectangular aggregates with a center-to-center spacing of 4-6 nm (208,000>
37
Distribution of the Values for the Number of Assemblies per Square Micron Measured
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sent, but were never packed into a recognizable aggregate.
Quantitative freeze-fracture studies To determine whether the number of assemblies per unit membrane surface area is uniform across the cell surface, many non-overlapping membrane regions were photographed and the number of assemblies counted in two cells from a 7-day culture (Fig. 9). In this counting, and throughout the study, an assembly was defined as having at least 4 constituent particles. In 64 fields of cell A (total area examined 359 p m 2) the counts ranged between 1 and 5 assemblies//~m 2. In 25 fields of cell B (total area examined 139 pm2), the counts ranged between 4 and 8 assemblies//~m 2. We conclude from the lack of uniformity that counting a few fields in a given cell would not provide a reliable estimate of the number of assemblies/pm 2 over the entire cell surface. In order to determine whether the number of assemblies in cell membranes changed with duration of culture, dishes from one plating were fixed at 7, 14,
21, and 28 days of subculture (Fig. 10). Cells containing many assemblies were present throughout the first 4 weeks. The cultures became confluent at 10-14 days. We could not detect a significant change in the number of assemblies/pm 2 over the first 7-28 days in culture. The highest concentration of assemblies in astrocytes in vivo occurs in processes apposed to the basal lamina of blood vessels. It seemed possible that the rate of growth and differentiation might vary with the presence or absence of a collage substrate. Accordingly, we prepared a plating in the usual manner except that collagen was not added to the glass coverslips. The appearance in phase contrast of these secondary cultures was indistinguishable from that of :ontrol platings. A b o u t 60% of the cells had G F A P immunoreactivity throughout the last 3 weeks of cultures. The secondary cultures were confluent at 10-14 days. The range in the values obtained for the concentrations of assemblies in cells at 7 through 28 days was similar to that of the control cultures (Fig. 10).
38
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Fig. 10. Histogram of the number of cells with particular numbers of assemblies//~m2, at several different durations of secondary culture, in the presence and in the absence of a collagen substrate. The numbers in parenthesis indicate the total area of cells examined at a given age, the average number of assemblies, and the standard deviation.
DISCUSSION
Astrocytes isolated from the brains of neonatal rat pups and cultured with techniques patterned after those described by McCarthy and deVellis31 acquire both assemblies and gap junctions. The constituent particles of assemblies appear to be the same in vitro
and in viva. The distribution of assemblies over the surface of cultured astrocytes is not uniform, but there is no obvious concentration near the substrate or in the vicinity of adjacent cells. The proportion of cells with glial fibriUary acidic protein immunoreactivity is fairly constant over the second through fourth weeks in culture, and the concentration of as-
39 semblies in astrocytic membranes also appears to be fairly constant over that interval. About 40--60% of the cells in the subculture contain glial fibrillary acidic protein (GFAP) immunoreactivity, a reliable marker for astrocytes in vitro 4,5,8,20,36,37,42. Often the cell with GFAP immunoreactivity were clustered in the confluent monolayer, as if they arose from one or a few precursors in that region. Presumably, other flattened cells in the culture are fibroblasts, meningeal cells, and endothelial cells. After cells have reached confluency in flask cultures, and occasionally after 21-28 days of secondary culture in tissue culture dishes, there appears a sparse population of rounded cells above the monolayer. These cells do not contain GFAP immunoreactivity, but the cells below them in the monolayer are usually GFAP-positive. Thin-section electron microscopy shows that the cytoplasm of the rounded cells is comparatively electron-dense and that it contains few intermediate filaments. It seems likely that these cells represent oligodendrocytes or oligodendroglial precursors, as suggested by McCarthy and deVellis 31. Their association with GFAP immunoreactive cells in the monolayer below is consistent with the possibility that a single precursor cell gives rise to both astrocytes and oligodendrocytes. We have presumed that cultured cells which manifest assemblies in their plasma membrane when freeze-fractured are astrocytes or astrocyte precursors. This is based partly on the fact that in postnatal rodent brain, only astrocytes and ependymal cells contain assemblies1-3,7,10.11A3-15,22-27,30,33. Furthermore, the shapes of the cells containing assemblies in cultures resemble those of the GFAP-immunoreactive cells and the cells found with thin-section techniques to contain abundant filaments. Finally, we encountered instances in which the cross-fractured cytoplasm of cells contained many filaments while the fractured membrane of the same cell contained assemblies. One of the hallmarks of astrocyte morphology in vivo is that their membrane composition varies over the cell surfaces11.22,26.27. Astrocytic processes apposed to the basal lamina of vascular structures contain numerous assemblies. Processes of the glial limitans apposed to the basal lamina at the surface of the brain contain fewer, but nonetheless abundant assemblies. Astrocytic processes investing neuronal
structures manifest few assemblies, and these tend to be in the vicinity of astrocytic gap junctions. In vitro, however, assemblies were not concentrated in apposition to the culture substrate. In many cells we were able to examine both the membrane apposed to the substrate and the membrane facing the medium, and the concentration of assemblies was similar. We cultured astrocytes with and without a collagen substrate, and did not find any obvious difference in the pattern of assemblies in the astrocytic membranes. We do not entirely discount a role of basal lamina in membrane differentiation, though, because fibroblasts in the culture probably lay down collagen whether or not we supply a collagen substrate. One advantage of the culture system is that we were able to examine comparatively vast expanses of the membrane from a single cell. Quantitative analysis revealed that assemblies were not uniformly distributed over the cell surface, as in vivo, but we could not detect a preferential distribution in proximity to adjacent cells. Because assemblies are not uniformly distributed, a very large portion of the cell surface must be examined to arrive at a reliable estimate of the concentration averaged across the entire cell. This limitation has to be carefully considered in designing studies to detect perturbations of assembly number with various culture conditions. It is also clear that the concentration of assemblies in astrocytic membranes varies from cell to cell within a culture dish, and from dish to dish within a plating. This may prove an advantageous feature, since one may be able to determine whether microheterogeneity in cellular environment is reflected in the composition of the astrocytic membrane. ACKNOWLEDGEMENTS We wish to thank Dr. S. C. Landis and Dr. K. Sweadner for valuable advice and instruction in tissue culture technique. Ms. Doreen McDowell prepared the media used in this study. Dr. D. Dahl graciously provided the antisera to glial fibrillary acidic protein. Mr. L. Cherkas and Ms. D. Jackson provided excellent technical assistance. The computerized morphometric methods were devised by Dr. J. Halperin and Dr. A. John Petkau gave valuable advice on appropriate statistical methods. The investigation was supported by a Teacher-Investigator award to D M D L and NS 15573.
40 REFERENCES 1 Anders, J. J. and Brightman, M. W., Assemblies of particles in the cell membranes of developing, mature~ and reactive astrocytes, J. Neurocytol., 8 (1979) 777-795. 2 Anders, J. J. and Brightman, M. W., Orthogonal assemblies of intramembranous particles - - an attribute of the astrocyte. In Glial and Neuronal Cell Biology, Alan R. Liss, New York, 1981, pp. 21-35. 3 Anders, J. J. and Brightman, M. W., Particle assemblies in astrocytic plasma membrane are rearranged by various agents in vitro and cold injury in vivo, J. Neurocytol., 11 (1982) 1009-1029. 4 Antanitus, D. S., Choi, B. H. and Lapham, L. W., Immunofluorescence staining of astrocytes in vitro using antiserum to glial fibrillary acidic protein, Brain Research, 89 (1975) 363-367. 5 Bock, E., Moiler, M., Nissen, C. and Sensenbrenner, M., Glial fibrillary acidic protein in astroglial cell cultures derived from newborn rat brain, FEBS Lett., 83 11977) 207-211. 6 Bordi, C. and Perrelet, A., Orthogonal arrays of particles in plasma membrane of the gastric parietal cell, Anat. Rec., 192 (1978) 297-304. 7 Brightman, M. W., Prescott, L. and Reese, T. S., Intercellular junctions of special ependyma. In K. M. Knigge, D. E. Scott, M. Kobayashi and S. Ishii (Eds.), Brain Endocrine Interaction, H. The Ventricular System, Karger, Basel, 1975, pp. 146-165. 8 Chiu, F.-C., Norton, W. T. and Fields, K. L., The cytoskeleton of primary astrocytes in culture contains actin, glial fibrillary acidic protein, and the fibroblast type filament protein, vimentin, J. Neurochem., 37 (1979) 147-155. 9 Cohen, S. A. and Pumplin, D. W., Clusters of intramembrane particles associated with binding sites for alpha-bungarotoxin in cultured chick myotubes, J. Cell Biol., 82 11979) 494--516. 10 Dermietzel, R., Visualization by freeze-fracturing of regular structure in glial cell membranes, Naturwissenschaften, 60 11973) 208. 11 Dermietzel, R., Junctions in the central nervous system of the cat. III. Gap junctions and membrane-associated orthogonal particle complexes (MOPC) in astrocytic membranes, Cell Tissue Res., 149 (1974) 121-135. 12 Elfvin, L. G. and Forsman, C., The ultrastructure of junctions between satellite cells and mammalian sympathetic ganglia as revealed by freeze-etching, J. Ultrastruct. Res., 63 11978) 261-274. 13 Gulley, R. L., Landis, D. M. D. and Reese, T. S., Internal organization of membranes at end-bulbs of Held in the anteroventral cochlear nucleus, J. comp. Neurol., 180 (1978) 707-742. 14 Hanna, R. B., Hirano, A. and Pappas, G. D., Membrane specializations of dendrite spines and glia in the weaver mouse cerebellum: a freeze-fracture study, J. Cell Biol., 68 (1976) 403-410. 15 Hatton, J. D. and Ellisman, M. H., The distribution of orthogonal arrays and their relationship to intercellular junctions in neuroglia of the freeze-fractured hypothalamo-neurohypophysial system, Cell Tissue Res., 215 (1981) 309-323. 16 Heuser, J. E., Reese, T. S. and Landis, D. M. D., Functional changes in frog neuromuscular junction studied with freeze-fracture, J. Neurocytol., 3 (1974) 109-131.
17 Hogan, J. C. and Manuelidis, L., Intramembranc particle distribution and lectin binding of glioblastoma cells after long term subculture, Acta neuropath., 36 (1976) 199-208. 18 Humbert, F., Pricam, C., Perrelet, A. and Orci, 1,., Specific plasma membrane differentiations in the cells of the kidney collecting tubules, J. Uhrastruct. Res., 52 (1975) 13-2/I. 19 Inoue, S. and Hogg, J. C., Freeze-etch study of the tracheal epithelium of normal guinea pigs with particular reference to intercellular junctions, J. Ultrastruct. Res., 61 11977) 89-99. 20 Kozak, L. P., Dahl, D. and Bignami, A., Glial fibrillary acidic protein in reaggregating and monolayer cultures of fetal mouse cerebral hemispheres, Brain Research. 150 (1978) 631-637. 21 Kreutziger, G. O., Freeze-etching of intercellular junctions of mouse liver. In Proceedings o f the 26th Meeting of the Electron Microscopy Society of America, Claitors Publishing Division, Baton-Rouge, 1968, pp. 234. 22 Landis, D. M. D. and Reese, T. S., Arrays of particles in freeze-fractured astrocytic membranes, J. Cell Biol., 60 (1974) 316-320. 23 Landis, D. M. D. and Reese, T. S., Structure of the Purkinje cell membrane in staggerer and weaver mutant mice, J. comp. Neurol., 171 (1977) 247-260. 24 Landis, D. M. D. and Reese, T. S., Further studies of astrocyte membrane structure, Neurology, 30 (1980) 406. 25 Landis, D. M. D. and Reese, T. S., Astrocyte membrane structure: changes after circulatory arrest, J. Cell Biol., 88 (1981) 660-663. 26 Landis, D. M. D. and Reese, T. S., Membrane structure in mammalian astrocytes: A review of freeze-fracture studies in adult, developing, reactive and cultured astrocytes, J. exp. Biol., 95 (t981) 35-48. 27 Landis, D. M. D. and Reese, T. S., Regional organization of membrane structure in astrocytes, Neuroscience, 7 (1982) 937-950. 28 Landis, D. M. D., Reese, T. S., Sweadner, K. J. and Weinstein, L. A., Membrane structure in developing, mature, and cultured astrocytes, Soc. Neurosci. Abstr., 6 (1980) 325. 29 Mains, R. E. and Patterson, P. H., Primary cultures of dissociated sympathetic neurons. I. Establishment of longterm growth in culture and studies of differentiated properties, J. Cell Biol., 59 (1973) 329-345. 30 Massa, P. T. and Mugnaini, E., Cell junctions and intramembrane particles of astrocytes and oligodendrocytes: a freeze-fracture study, Neuroscience, 7 (1982) 52,3-538. 31 McCarthy, K. D. and deVellis, J., Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue, J. Cell Biol., 85 (1980) 890-902. 32 McNutt, S., Ultrastructure of the myocardial sarcolemma, Circulat. Res., 37 (1975) 1-13. 33 Nabeshima, S., Reese, T. S., Landis, D. M. D. and Brightman, M. W., Junctions in the meninges and marginal glia, J. comp. Neurol., 164 (1975) 127-170. 34 Pauli, B. U., Weinstein, R. S., Soble, L. W. and Alroy, J.. Freeze-fracture of monolayer cultures, J. Cell Biol., 72 (1977) 763-769. 35 Prescott, L. and Brightman, M. W., A technique for the freeze-fracture of tissue culture, J. Cell Sci.. 3111 (1978) 34-43. 36 Pruss, R. M., Thy-I antigen on astrocytes in long-term cultures of rat central nervous system, Nature (Lond.), 2811 (1979) 688-689.
41 37 Raff, M. C., Fields, K. L., Hakomori, S.-l., Mirsky, R., Pruss, R. C. and Winter, J., Cell-type specific markers for distinguishing and studying neurons and the major classes of glial cells in culture, Brain Research, 174 (1979) 283-308. 38 Rash, J. E. and Ellisman, M. H., Studies of excitable membranes. I: Macromolecular specializations of the neuromuscular junction and the non-junctional sarcolemma, J. Cell Biol., 63 (1974) 567-586. 39 Rash, J. E., Staehelin, L. A. and Ellisman, M. H., Rectangular arrays of particles on freeze-cleaved plasma membranes are not gap junctions, Exp. Cell Res., 86 (1974) 187-190. 40 Smith, D. S., Bearwald, R. J. and Hart, M. A., The distribution of orthogonal assemblies and other intercalated par-
ticles in frog sartorius and rabbit sacrospinalis muscle, T/ssue Cell, 7 (1975) 369-382. 41 Staehelin, L. A., Three types of gap junctions interconnecting intestinal epithelial cells visualized by freeze-etching, Proc. nat. Acad. Sci. U.S.A., 69 (1972) 1318-1321. 42 Steig, P. E., Kimelborg, H. K., Mazurkiewicz, J. E. and Banker, G. A., Distribution of glial fibrillary acidic protein and fibronectin in primary astroglial cultures from rat brain, Brain Research, 199 (1980) 493-500. 43 Yee, A. G., Fischbach, G. D. and Karnovsky, M. J., Clusters of intramembranous particles on cultured myotubes at sites that are highly sensitive to acetylcholine, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 3004--3008.
31
Membrane Structure in Cultured Astrocytes DENNIS M. D. LANDIS and LORI A. WEINSTEIN Department of Neurology, Massachusetts GeneralHospital, Boston, MA 02114 (U.S.A.) (Accepted February 8th, 1983) Key words: astrocytes - - membrane structure - - freeze-fracture - - tissue culture - - glial fibrillary acidic protein
Astrocytes cultured from the brains of neonatal rat pups acquire at least two specializations of intramembrane particle distribution: 'assemblies' and gap junctions. The number and appearance of assemblies in the cultured astrocytes is not markedly influenced by the presence or absence of a collagen substrate, and the range of concentrations of assemblies in astrocytic membranes is fairly stable from 7 through 28 days in culture. The assemblies are not concentrated in apposition to the substrate, even though the astrocytic membranes containing the highest concentration of assemblies in vivo are apposed to basal lamina. Quantitative analysis shows that assemblies are not uniformly distributed over the plasmalemma of a single cell, raising the possibility that the nature of cells around an astrocytic process may influence its membrane composition in vitro. INTRODUCTION Astrocytic membranes in the mammalian central nervous system contain characteristic aggregates of small, uniform particles packed in orthogonal arrayl-3,lO,ll,13-15,22-27, 30,33. These particle aggregates, or 'assemblies', are densely packed in astrocytic membranes apposed to blood vessels, less numerous in astrocytic processes apposed to the cerebrospinal fluid at the surface of the brain, and least densely packed in astrocytic membranes investing neuronal processes. The composition of assemblies and their function are unknown26, 27. Assemblies can be identified only in freeze-fractured tissue; no corresponding structure is evident in thin-sectioned preparations. Within the brain, assemblies are found in the membranes of astrocytes and ependymal cells 7, but do not occur in neurons, oligodendrocytes, or endothelial cells. Assemblies occur in several types outside the central n e r v o u s system6,12,16,18A9,21,32, 38-41. Assemblies in these non-neuronal cells are like those in astrocytes in appearance and in the fact that they are concentrated in specific domains of the cell surface, except in the instance of hepatocytes 21. We have turned to tissue culture, with its potential for close control over cellular environment, in our effort to study the structure of assemblies and their role in astrocyte function. It has been shown that explant cultures of cerebellar cortex do manifest assemblies, 0006-8993/83/$03.00 (~ 1983 Elsevier Science Publishers B.V.
but these cultures were very heterogeneous35. In preliminary studies, we were unable to obtain useful numbers of assemblies in cultures of rodent C-6 glioma cells and in cultures of 3 human astrocytomas (ref. 24; see also ref. 17). Recently, a technique for establishing primary astrocyte cultures from 7-day rat cerebral hemispheres has been described 3. That system utilizes low-density plating to yield discrete colonies which differ markedly in cellular composition. T h o u g h astrocytes with many assemblies do appear, the system is contaminated by neurons, the number of assemblies is not stable over time in culture, and the number of assemblies in the membranes of one colony may vary markedly from that of another colony in the same culture. In order to obtain cultures with more uniform cellular composition and cellular differentiation, we have used secondary cultures, with techniques somewhat modified from those of McCarthy and deVellis 31. We present here the details of the culture methods, the cellular composition of the cultures, and the appearance and distribution of assemblies in the membranes of the cultured astrocytes. Some aspects of this study have been presented in brief form 2s. MATERIALS AND METHODS Tissue culture The tissue culture techniques are based on the ap-
32 proach devised by McCarthy and deVellis 31. Rat pups aged 2--4 days were stunned by concussion, decapitated, and the brain removed. The meninges, choroid plexus, and cerebellum were discarded. Cerebral hemispheres and bramstem were minced in L15 plating medium (composition as in Mains and Patterson 29 save that nerve growth factor was omitted), and then exposed to 0.25% trypsin for 25 rain at 37 °C with agitation. Dissociated cells were sedimented, and the cells collected from t0 brains were innoculated into four 75 cm 2 Falcon flasks each containing 20 ml of L 15-CO: growth medium (composition as in Mains and Patterson 29 save that rat serum, bovine serum albumin, nerve growth factor and Methocel were omitted) supplemented with 20% fetal calf serum. The medium was changed every 3 days. Cells were confluent at 7-10 days of culture. The flasks were sealed, placed on a rotary shaker and shaken 12-18 h at 37 °C to suspend oligodendrocytes and other cells growing over the confluent monolayer. The suspended cells were discarded, the flasks washed vigorously with the fresh media, and finally the cells of the monolayer were collected after brief exposure to 5 mM E D T A in Hank's balanced salt solution without divalent cations. The cells were sedimented, rinsed, resuspended, and innoculated into Falcon polystyrene tissue culture dishes (35 x 10 mm or 60 x 15 ram) at a concentration of 3 x 105 cells/cm 2. The cultures were fed every three days, and were usually confluent by 14 days. The results presented are based on 6 separate platings.
lmmunofluorescence Circular holes were cut in the center of 35 x 10 mm Falcon tissue culture dishes, and glass coverslips were sealed to the bottom of the dishes with paraffin. The portion of the coverslip exposed to the tissue culture medium was coated with collagen (Vitrogen100, Flow Laboratories). The cultures were prepared for immunofluorescence staining at 3, 4, 7, 14, 21 and 28 days of secondary culture. Cultures were rinsed briefly with fresh medium, and then fixed in 5% glacial acetic acid in ethanol for 15 min at - - 2 0 °C. The fixed cultures were rinsed in 0.12 M phosphate buffer and subsequent manipulations carried at 22 °C. Cells were incubated for 30 min in the presence of rabbit antisera to glial fibrillary acidic protein (graciously provided by Dr. D.
Dahl) at a dilution of 1:100. After washing in phosphate buffer, the cells were incubated in the presence of goat antisera raised against rabbit immunoglobulin and conjugated with rhodamine or fluorescein (Cappel Laboratories) for 30 min. After further washing, the cells were mounted in 1:1 glycerol:phosphate buffer and examined with epifluorescence optics. As a method control, cultures were prepared as above, except that primary antiserum was omitted.
Thin Section Electron Microscopy Square glass coverslips were placed in 60 x 15 mm tissue culture dishes, coated with collagen (Vitrogen100, Flow Laboratories), and the dishes were innoculated with cells collected from flasks. After the desired interval of culture, cells were fixed in 3% glutaraldehyde in 0.12 M sodium cacodylate buffer at pH 7.35 and 37 °C for 2-4 h. After post-fixation in 1% osmium tetroxide in 0.12 M sodium cacodylate buffer, most preparations were stained en-bloc with uranyl acetate for 4-16 h, dehydrated, and then embedded in epon.
Freeze-fracture The cultured cells were freeze-fractured with a Balzers complementary replication device, using techniques described by Pauli et al. 34, Yee et al. 43, and Cohen and Pumplin9. Glass coverslips were cut into 4.5 mm squares, placed on the bottom of a 60 x 15 mm Petri dish, and each coated with collagen. The dish was inoculated with cells as described above. After the desired interval of culture, either all the squares were fixed by flooding the dish with fixative, or selected squares were removed with sterile technique and plunged immediately into fixative. The fixative contained 3% glutaraldehyde and 0.12 M sodium cacodylate buffer adjusted to pH 7.35. After 1-3 b in fixative (initial temperature 37 °C, allowed to cool to 22 °C), the cells were rinsed in 0.12 M sodium cacodylate buffer. The cells were then soaked in 12.5% glycerol in 0.06 M sodium cacodylate buffer for 30 rain and then in 25% glycerol in 0.06 M sodium cacodylate buffer for an additional one half hour. Double-height gold or copper specimen supports were coated with polyvinyl alcohol and then sandwiched with the coverslips and frozen by immersion in melting monochlorodifluoromethane (Freon 22). Freeze-fracturing was performed in a Balzers freeze-
33 fracture apparatus at - - 1 1 0 °C and in a 2--4 x 10-7 torr vacuum with a c o m p l e m e n t a r y replica device, and the fractured cells were replicated i m m e d i a t e l y , without etching.
minicomputer. The area of the m e m b r a n e fracture surface and the n u m b e r of assemblies were determined three times by a single observer. OBSERVATIONS
Morphometric techniques Freeze-fracture replicas were p h o t o g r a p h e d in a J E O L 100 CX electron microscope at a p r i m a r y magnification of 27,000-33,000 and enlarged p h o t o graphically. Magnification and photographic enlargement were measured with micrographs of grating replicas. The area of m e m b r a n e fracture surfaces was measured with a sonic digitizer (Graf-Pen) interfaced with a Digital E q u i p m e n t C o r p o r a t i o n PDP-8
Phase contrast and immunofluorescence Immediately after inoculation of the tissue culture dishes with cells suspended from the flask monolayers most cells were single, but clumps of cells were also present. The secondary cultures b e c a m e confluent between 10 and 14 days. During this initial growth interval, cells a p p e a r e d h e t e r o g e n e o u s (Fig. 1). A f t e r 21 and 28 days of culture, some tissue cul-
Fig. 1. Secondary culture at 21 days. In phase contrast, cells in the monolayer range from epitheloid to spindle-shaped (acetic acid/ethanol fixation; 80 x). Fig. 2. Secondary culture at 21 days, containing ceils with GFAP immunoreactivity. The field is the same as that of Fig. 1 (acetic acid/ethanol fixation; 80 x ). Fig. 3. Secondary culture at 21 days. In this portion of the culture, phase-dark rounded cells are present above the monolayer. Some give rise to narrow, straight processes which branch at acute angles (acetic acid/ethanol fixation; 80 x). Fig. 4. Secondary culture at 21 days, containing cells with GFAP immunoreactivity. The field is the same as that of Fig. 3, but the plane of focus is in the monolayer. Cells containing GFAP immunoreactivity are below the rounded cells, but the rounded cells and their processes are not stained (acetic acid/ethanol fixation; 80 x ).
34 ture dishes contained a population of round cells above the confluent layer (Fig. 3). There was no obvious difference in cell morphology and cell density on the surface of the glass coverslips as compared to the plastic dish surface. Glial fibrillary acidic protein (GFAP) immunoreactivity was detected as early as 3 days in secondary culture, even in rounded cells which had not yet begun to spread over the collagen-coated substrate. Over the first 4 weeks in culture, there was no dramatic change in the proportion of cells demonstrating GFAP immunoreactivity. In different platings, the proportion of cells with GFAP immunoreactivity ranged between 40% and 70% of cells present (Figs. 2, 4). The distribution of these cells was strikingly non-uniform. In some regions on the tissue culture dish, immunoreactive cells made up virtually all the
cells present, while in nearby regions of the same petri dish, only scattered cells within a field would contain GFAP immunoreactivity. The morphology of cells with G F A P immunoreactivity ranged from stellate with well-defined, radiating processes to nearly epithelioid with broad, flat expanses of cytoplasm. Cells in regions of the culture which did not manifest G F A P immunoreactivity tended to be more homogeneous in terms of nuclear and cytoplasmic morphology, and often appeared epithelioid. Through 21 days of culture, the GFAP positive cells did not become more homogeneous in their cytoplasmic morphology. At 21 and 28 days of culture, some tissue culture dishes included a population of cells which appeared in phase contrast to be round, phase-dark, and above the levels of the confluent monolayer (Fig. 3). The cells tended to occur in clusters, and were uniformly
Fig. 5. Abundant intermediate filaments in an astrocyte at 28 days. The nucleus is at the lower right, and several lysosomesare evident in the upper right (25,000x).
35 G F A P negative (Fig. 4). The monolayer below these cells, however, invariably consisted of nearly confluent GFAP-positive cells.
Thin-section electron microscopy Cultures were prepared for thin-section electron microscopy at 7, 14, 21, and 28 days of culture, and were sectioned in a plane parallel to the surface of the glass coverslip substrate. Many flattened cells in these preparations contained complicated skeins of intermediate filaments, richly interwoven (Fig. 5). These filaments in the perikaryal region of flattened cells also extended to fill small diameter cell processes. Some of the cells in older cultures which contained numerous filaments also contained a large number of pleomorphic lysosomal inclusions. These lysosomes were highly variable in their appearance, and often contained lamellar structures suggestive of ingested and densely packed lipid. Many of the flattened, filament-containing cells established gap junctions with neighboring cells. Such junctions were often encountered where a process of one cell indented the cytoplasm of an adjacent cell. Thin sections above the level of flattened cells constituting the monolayer of the culture occasionally revealed profiles of the rounded cells. These rounded cells had comparatively electron-dense cytoplasm, and in the perikaryal region manifested large amounts of rough endoplasmic reticulum and free ribosomes. They were relatively devoid of intermediate filaments, and did not contain the abundant lysosomai inclusions present in some of the flattened cells. Processes arising from these rounded cells often contained large numbers of microtubules, and several vesicle-filled mounds were encountered on the surfaces of the rounded cells.
Qualitative freeze-fracture studies The plane of fracture in most instances propagated through the plasma membranes of the flattened cells constituting the confluent monolayer. A potential hazard of the technique is that the plasma membrane adjacent to the substrate might be preferentially fractured (or missed). However, many cells were found in which the plane of fracture went from one plasma membrane across the attenuated cytoplasm to the other membrane, and so we certainly encountered fractures through the substrate and through the su-
perficial membrane. The plane of fracture rarely exposed the membrane structure of rounded cells above the confluent monolayer, and virtually all the observations made in this portion of the study are restricted to the flattened cells. Many of the cultured cells had assemblies in their plasma membranes, arrayed nearly uniformly across the cell surface. The particles constituting the assemblies were packed in orthogonal array, and were associated with the cytoplasmic half of the fractured membrane (Fig. 7), while complementary pits were present on the extracellular half of the membrane. The particles constituting gap junctions were also present in the plasma membranes of many of the cells with assemblies (Fig. 8); the particles were associated with the cytoplasmic half of the fractured membrane, and there were complementary pits on the extracellular membrane half. The particles of gap junctions were present in hexagonal packing and in unordered patterns. Whenever it was possible to examine the membrane structure of both astrocytes forming a gap junction, both were found to contain assemblies. Some of the cells in the cultures had elaborated tight junctions with adjacent cells. Many of these tight junctions had associated gap junctions, but assemblies were never recognized in the membrane of a cell participating in a tight junction. There was a distinct tendency for cells with tight junctions to occur in clusters. Cells with assemblies in the plasma membranes, cells with tight junctions and no assemblies, and cells with no evident specialization of intramembrane particle distribution all had variable numbers of caveolae. These were evident as shallow hemispherical indentations without associated particle aggregates (the lack of the particles serves to distinguish them from the invaginations of coated pits). Cross-fractures through the perikaryal cytoplasm and nuclear membranes were infrequent, but served to expose the usual array of cytoplasmic organelles (Fig. 6). Astrocytes in the cerebellar cortex and cerebral cortex elaborate a junction composed of large, pleomorphic particles which we have provisionally described as a 'polygonal particle junction'26.27. We were unable to recognize certain examples of this junctional specialization in the cultured astrocytes. Individual constituent particles might have been pre-
Fig. t~. Freeze-fractured astrocvtc, 28 days. I-he plane ol lracture has exposed the extraccllular hall of the plasma m e m b r a n e (E), and has cross-fractured the perikaryon to reveal the nucleus (N), mitoehondria (M), lysosomes (I.), and cross-fractured filaments (arrows). Imprints of assemblies on the tractured m e m b r a n e were clearly visible at higher magnification (27,1/00×). Fig. 7. Assemblies on the cytoplasmic half of lracturcd astroeytie m e m b r a n e at 7 days of secondary culture. The constituenl particles are packed in square or rectangular aggregates with a center-to-center spacing of 4-6 nm (208,000>
37
Distribution of the Values for the Number of Assemblies per Square Micron Measured
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sent, but were never packed into a recognizable aggregate.
Quantitative freeze-fracture studies To determine whether the number of assemblies per unit membrane surface area is uniform across the cell surface, many non-overlapping membrane regions were photographed and the number of assemblies counted in two cells from a 7-day culture (Fig. 9). In this counting, and throughout the study, an assembly was defined as having at least 4 constituent particles. In 64 fields of cell A (total area examined 359 p m 2) the counts ranged between 1 and 5 assemblies//~m 2. In 25 fields of cell B (total area examined 139 pm2), the counts ranged between 4 and 8 assemblies//~m 2. We conclude from the lack of uniformity that counting a few fields in a given cell would not provide a reliable estimate of the number of assemblies/pm 2 over the entire cell surface. In order to determine whether the number of assemblies in cell membranes changed with duration of culture, dishes from one plating were fixed at 7, 14,
21, and 28 days of subculture (Fig. 10). Cells containing many assemblies were present throughout the first 4 weeks. The cultures became confluent at 10-14 days. We could not detect a significant change in the number of assemblies/pm 2 over the first 7-28 days in culture. The highest concentration of assemblies in astrocytes in vivo occurs in processes apposed to the basal lamina of blood vessels. It seemed possible that the rate of growth and differentiation might vary with the presence or absence of a collage substrate. Accordingly, we prepared a plating in the usual manner except that collagen was not added to the glass coverslips. The appearance in phase contrast of these secondary cultures was indistinguishable from that of :ontrol platings. A b o u t 60% of the cells had G F A P immunoreactivity throughout the last 3 weeks of cultures. The secondary cultures were confluent at 10-14 days. The range in the values obtained for the concentrations of assemblies in cells at 7 through 28 days was similar to that of the control cultures (Fig. 10).
38
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DISCUSSION
Astrocytes isolated from the brains of neonatal rat pups and cultured with techniques patterned after those described by McCarthy and deVellis31 acquire both assemblies and gap junctions. The constituent particles of assemblies appear to be the same in vitro
and in viva. The distribution of assemblies over the surface of cultured astrocytes is not uniform, but there is no obvious concentration near the substrate or in the vicinity of adjacent cells. The proportion of cells with glial fibriUary acidic protein immunoreactivity is fairly constant over the second through fourth weeks in culture, and the concentration of as-
39 semblies in astrocytic membranes also appears to be fairly constant over that interval. About 40--60% of the cells in the subculture contain glial fibrillary acidic protein (GFAP) immunoreactivity, a reliable marker for astrocytes in vitro 4,5,8,20,36,37,42. Often the cell with GFAP immunoreactivity were clustered in the confluent monolayer, as if they arose from one or a few precursors in that region. Presumably, other flattened cells in the culture are fibroblasts, meningeal cells, and endothelial cells. After cells have reached confluency in flask cultures, and occasionally after 21-28 days of secondary culture in tissue culture dishes, there appears a sparse population of rounded cells above the monolayer. These cells do not contain GFAP immunoreactivity, but the cells below them in the monolayer are usually GFAP-positive. Thin-section electron microscopy shows that the cytoplasm of the rounded cells is comparatively electron-dense and that it contains few intermediate filaments. It seems likely that these cells represent oligodendrocytes or oligodendroglial precursors, as suggested by McCarthy and deVellis 31. Their association with GFAP immunoreactive cells in the monolayer below is consistent with the possibility that a single precursor cell gives rise to both astrocytes and oligodendrocytes. We have presumed that cultured cells which manifest assemblies in their plasma membrane when freeze-fractured are astrocytes or astrocyte precursors. This is based partly on the fact that in postnatal rodent brain, only astrocytes and ependymal cells contain assemblies1-3,7,10.11A3-15,22-27,30,33. Furthermore, the shapes of the cells containing assemblies in cultures resemble those of the GFAP-immunoreactive cells and the cells found with thin-section techniques to contain abundant filaments. Finally, we encountered instances in which the cross-fractured cytoplasm of cells contained many filaments while the fractured membrane of the same cell contained assemblies. One of the hallmarks of astrocyte morphology in vivo is that their membrane composition varies over the cell surfaces11.22,26.27. Astrocytic processes apposed to the basal lamina of vascular structures contain numerous assemblies. Processes of the glial limitans apposed to the basal lamina at the surface of the brain contain fewer, but nonetheless abundant assemblies. Astrocytic processes investing neuronal
structures manifest few assemblies, and these tend to be in the vicinity of astrocytic gap junctions. In vitro, however, assemblies were not concentrated in apposition to the culture substrate. In many cells we were able to examine both the membrane apposed to the substrate and the membrane facing the medium, and the concentration of assemblies was similar. We cultured astrocytes with and without a collagen substrate, and did not find any obvious difference in the pattern of assemblies in the astrocytic membranes. We do not entirely discount a role of basal lamina in membrane differentiation, though, because fibroblasts in the culture probably lay down collagen whether or not we supply a collagen substrate. One advantage of the culture system is that we were able to examine comparatively vast expanses of the membrane from a single cell. Quantitative analysis revealed that assemblies were not uniformly distributed over the cell surface, as in vivo, but we could not detect a preferential distribution in proximity to adjacent cells. Because assemblies are not uniformly distributed, a very large portion of the cell surface must be examined to arrive at a reliable estimate of the concentration averaged across the entire cell. This limitation has to be carefully considered in designing studies to detect perturbations of assembly number with various culture conditions. It is also clear that the concentration of assemblies in astrocytic membranes varies from cell to cell within a culture dish, and from dish to dish within a plating. This may prove an advantageous feature, since one may be able to determine whether microheterogeneity in cellular environment is reflected in the composition of the astrocytic membrane. ACKNOWLEDGEMENTS We wish to thank Dr. S. C. Landis and Dr. K. Sweadner for valuable advice and instruction in tissue culture technique. Ms. Doreen McDowell prepared the media used in this study. Dr. D. Dahl graciously provided the antisera to glial fibrillary acidic protein. Mr. L. Cherkas and Ms. D. Jackson provided excellent technical assistance. The computerized morphometric methods were devised by Dr. J. Halperin and Dr. A. John Petkau gave valuable advice on appropriate statistical methods. The investigation was supported by a Teacher-Investigator award to D M D L and NS 15573.
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