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Co-cultivation of astroglial and neuronal primary cultures from rat brain
Brain Research, 366 (1986) 159-168
159
Elsevier BRE 11463
Co-Cultivation of Astroglial and Neuronal Primary Cultures from Rat Brain ELISABETH HANSSON
Institute of Neurobiology, Universityof GOteborg, G6teborg (Sweden) (Accepted June 18th, 1985)
Key words: kastroglia - - co-cultivation - - gliafibrillary acidic protein (GFAP) - - neuron - - neuron-specific enolase - primary brain culture
A technique is described for a two-cell co-cultivation system which permits in vitro evaluation of neuron-glia interactions. Primary astroglial enriched cultures from newborn rat cerebral hemispheres, striatum or cerebral cortex, grown for 3 days, were co-cultivated with primary neuron-containing cultures from 15- to 17-day rat embryo cerebral hemispheres, substantia nigra or brainstem, respectively, grown for 10 days on polylysine-coated surfaces. The neuronal cells were identified morphologically and immunohistochemically by antibodies to neuron-specific enolase. The two cultures were grown together for 7 days, separated by a U-formed 1 mm glassrod. The results show that neurons exert a morphogenetic effect on astroglial cells in the form of extension of cell processes. The coculture system allows investigation of potent local humoral interactions between astroglial cells and neurons.
INTRODUCTION Astroglial cells constitute a large volume of the brain 30. Due to their anatomical proximity to neurons and the synaptic regions, the cells might be involved in neuronal metabolic events. In fact, astroglial cells have been shown to act as K ÷ buffers20,25, at, to participate in uptake and metabolism of amino acid transmitters t,4,tl,13,t7,21,32,34-37, and to bear receptors for various neurotransmitters and neuromodulators 1639,22,4°. Consequently, it could be suggested that the cells play an important role for the functions of neurons. Dissociated primary brain culture is one model to obtain large quantities of metabolically active, nontransformed astroglial-like cells3,4,9,14,15,18,23,27,33. The cells show many morphological, biochemical and electrophysiological similarities with astroglia in situ 11,12,23,24,38. However, their degree of differentiation is low 1° and could be due to growth in the absence of neurons. Different ways have been tried to increase the differentiation of the cells: (1) addition to the culture medium of partially isolated protein or peptide factors from embryonic brain, factors which
have been shown to promote growth and morphological differentiation of astroglial cells39; and (2) treatment of the cultures with conditioned media, i.e. serum-free media from neuronal containing cultures8,28. Both models have proved interesting as they provided some morphological and biochemical differentiation of astroglial cells in culture. Differentiation of cells, however, is a complex process which requires interactions with specific substances in physiological concentrations at specific sequences. One way to circumvent the problems of identifying and isolating such factors and to probably increase differentiation of astroglial cells in culture would be to co-cultivate astroglial cultures with neuronal cultures. The cells should grow in the near vicinity of each other while remaining physically separated and allowing humoral transfer. Such a technique has been introduced previously with the co-culture of vascular endothelial cells and smooth muscle cells 5. In this paper one technique for co-cultivation of astroglial and neuronal cells is described. The paper focuses on the morphological effects on the astroglial culture.
Correspondence: E. Hansson, Institute of Neurobioiogy, University of G6teborg, P.O.B. 33 031, S-400 33 G6teborg, Sweden. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
160 MATERIALS AND METHODS
Cultivation of fetal brain tissue Cerebral hemispheres, substantia nigra or brainstem from 15 to 17-day-fetal rats (Sprague-Dawley strain, Anticimex, Sweden) were passed through an 80 a m nylon mesh into Eagle's minimum essential medium (Flow Laboratories, U.K.). The medium was supplied with extra substances to make up the following final composition: double concentrations of amino acids, 2 mM glutamine; 30 mM glucose, and quadruple concentrations of vitamins. In some experiments, 5/~g insulin/ml (Sigma Fine Chemicals, St. Louis, MO, U.S.A.) was added; 250,000 IU/liter penicillin, 0.5% streptomycin and 20% (v/v) fetal calf-serum (Gibco Bio Cult. Lab., U.K.) were also added, and the pH was set at 7.3. Material from one hemisphere, 6 substantia nigra or one brainstem, was seeded on top of the lid to one petri dish (35 mm in diameter) (Nunc, Denmark). The lids were coated with poly-L-lysine (Sigma Fine Chemicals, St. Louis, U.S.A.) at room temperature, 24 h before use, according to Pettmann and coworkers 29. Medium was changed only once after 3 days or after 3, 6 and 8
days (different experiments). After 3 or 6 days or after 3 and 6 days, the cultures were treated with 10-5 M cytosine-l-fl-D-arabinofuranoside (c-Ara) (Sigma) 7. After 10 days the lids were turned upside-down in the 50 mm petri dishes, prefilled with 6 ml medium. The lids were placed on U-formed glass-rods, 1 mm in diameter (Fig. 1).
Cultivation of primary cultures from newborn rat cerebral hemispheres, striatum and cerebral cortex These cultures were done according to Booher and Sensenbrenner 3, Schousboe et al. 33 and Hansson et al. 14,15,18. Co-cultivation of fetal and newborn brain cultures Ten days after seeding: (1) the fetal hemisphere cultures were placed on glass rods above 3-day-old primary astroglial-enriched hemisphere cultures from newborn brain; (2) the substantia nigra cultures were placed above 3-day-old primary atroglial-enriched striatum cultures; and (3) the brainstem cultures were placed above 3-day-old primary astroglial-enriched cerebral cortex cultures obtained from newborn rat (Fig. 1). The petri dishes were kept at C
.••
FETAL BRAIN PRIMARY CULTURE (CONTAINING NEURGNS)
o lmm
PRIMARY NEONATAL ASTROGLIAL CULTURE
f
Fig. 1. Schematic drawing of essential steps in the co-cultivation of fetal neuron-containing cultures and newborn astroglial-enriched cultures. Fetal cell material was cultivated for 10 days on poly-L-lysine-coatedlids of petri dishes (35 mm in diameter) placed in petri dishes (50 mm in diameter) (a). Primary cultures from newborn rat (b) were grown for 3 days, and the fetal culture was transferred to this latter culture upside-down and placed on a U-formed glass-rod 1 mm in diameter (c).
161 37 °C in a humidified atmosphere of 95% air and 5% carbon dioxide. Media were changed 3 times a week. The same media were used as for cultivation of fetal brain tissue.
Control experiments Control experiments were as follows: (1) co-cultivation of cerebral hemisphere cultures from newborn rat with poly-L-lysine-treated empty lids, separated by a U-shaped glass rod, 1 mm in diameter; (2) cultivation of fetal hemisphere cultures treated with polyL-lysine and cytosine arabinofuranoside 'upsidedown' in 50 mm diameter petri dishes with no culture in the latter. This experiment was performed to examine whether cells from the lid 'fall down' onto the bottom of the petri dish; (3) co-cultivation of fetal rat hemisphere culture grown in the absence of poly-Llysine with the hemisphere culture from newborn rat; and (4) co-cultivation of two cultures from cerebral hemispheres of newborn rat. One culture was treated with poly-L-lysine. Protein determinations The cultures were rinsed in glucose-saline at pH 7.4. The material was scraped off into 300/~l or 500/~l PBS, also at pH 7.4, (35 mm or 55 mm petri dishes, respectively). The cultures were homogenized (glass/glass). Part of the homogenates were solubilized in 1 M NaOH, and another part was centrifuged 85,000 g for 60 min. The supernatants contained soluble protein. Total and soluble protein was determined according to Lowry et al. 26. lmmunohistochemistry The cultures were fixed in freshly prepared 4% formaldehyde in 0.15 M cacodylate buffer, pH 7.2, for 60 min at room temperature. After 4 rinses for 10 min each in 0.01 M phosphate-buffered saline (PBS) at pH 7.4, the cultures were incubated either in the antibody-containing solutions or in the control solutions (200/A). The cultures were incubated for 30 min in antibodies against glial fibrillary acidic protein (GFAP, a-albumin) (1:100) (gift from Prof. A. Lowenthal, Belgium) or in antibodies against neuron-specific enolase (NSE) (1:100) (bought from Dakopatts, Copenhagen, Denmark). They were thereafter washed (3 x 10 min) and incubated in the goat antirabbit IgG conjugated with fluorescein isothiocya-
nate (FITC) or tetramethylrhodamine isothiocyanate (TRITC). The incubation was for 45 min in buffered saline at pH 7.4. The cells were examined unmounted in the fluorescence microscope after washings in PBS for 4 x 10 min.
Controls (1) Sera from non-immunized rabbits were used; (2) cultures were incubated as described but the specific antisera were omitted; (3) untreated cultures were used to assess the autofluorescence which was very low. Microscopy The fluorescence microscope used was a Zeiss photomicroscope III RS, equipped with a mercury lamp for epifluorescence. The excitation filters for FITC or rhodamine were exchangeable in a sliding arrangement which permitted rapid photography. Photographs were made using phase contrast on Kodak Tri-X-PAN, 27 DIN films. RESULTS
Morphological and immunohistochemical characterization of fetal cerebral hemisphere cultures, substan. tia nigra cultures and brainstem cultures The cerebral hemispheres from fetal rat were cultivated on poly-L-lysine-coated petri dishes with insulin added to the culture medium, this being changed 3 times a week. When these cultures were treated with cytosinearabinofuranoside on day 6, they contained polygonal cells at the time of observation on day 10. The soma of these cells had a diameter of approximately 16/~m, contained one central nucleus (diameter approximately 6/~m) with two or more nucleoli, and had processes extending from the cell. The cells stained with antibodies against gliofibrillary acidic protein (GFAP) (Fig. 2a), but did not stain with antibodies against NSE. Scattered among the polygonal cells were cells of different sizes with soma (10-25/~m in diameter) containing one central nucleus with only one visible nucleolus and a number of large processes extending from the soma. These latter cells constituted approximately 5-10% of the total cell number in the cultures. Soma, nucleus and processes stained with antibodies against NSE (Fig. 2b), but were not stained by
162 TABLE I
Cell-density and morphological differentiation of fetal, neuron-containing cultures and astroglial-enriched primary cultures from newborn rat after co-cultivation for 7 days Medium was changed 3 times a week. Fetal cultures taken from cerebral hemispheres, substantia nigra or locus coeruleus. Cell density, and morphological differentiation, i.e. extension of processes: +, low; + + , moderate; + + + , high. Fetal material was co-cultivated with newborn material from cerebral hemispheres, striatum or cerebral cortex, respectively.
Fetal cuhures treated with
Cell density in fetal cultures"
Poly< -lysine and 30 mM glucose Poly-~ -lysine and 30 mM glucose + insulin (5 !zg/ml) Polyq.-lysine and 30 mM glucose + l/) -~ M c-Ara on day 6 Polyq -lysine and 30 mM glucose + insulin + e-Ara (as above)
Differentiation of cells in fetal cultures
Cell density in primary cultures from newborn rat after co-cultivation
DifJerentiation of cells in primary cultures from newborn rat after co-cultivation
++
++
+++
++
+++
+
+++
+
+
++
+++
++
++
+++
++
+++
re-
C e l l s w i t h b e a t i n g cilia w e r e s e e n in u n f i x e d cul-
v e a l e d in d o u b l e - l a b e l l i n g e x p e r i m e n t s ; n o t s h o w n ) .
tures and they have been suggested previously to be
O t h e r cells in t h e c u l t u r e s w e r e f o u n d in m i n o r p r o -
e p e n d y m a l - l i k e 12. P h a s e - b r i g h t c e l l s , w i t h t h e a b i l i t y
portions.
to p h a g o c y t o s e I n d i a i n k , h a v e b e e n c h a r a c t e r i z e d as
antibodies
against
GFAP
(which
was further
Fig. 2.a: fetal hemisphere culture (10 days old) incubated with anti-GFAP and treated as described in the text. Most cells show a positive l-CllC'donIThc cells have long processcs, b: similar culture as in a. NSE-positivc cells can be seen 10-25 .um in diameter, with one cemral nucleus and scvcral large processcs. Similar and corresponding cells are exhibited in phase-contrast micrographs.
163 macrophages 12. Small phase-dark cells with one or two processes have been suggested to be oligoblasts 12. Even mesenchymal-like cells 12 were observed. The age of the tissue, the amount of material seeded, and the treatment of the cultures, were all critical in order to obtain the characteristics of the cultures described. In the absence of poly-L-lysine, no NSEpositive cells were found. If poly-L-lysine coating is utilized while insulin and cytosinearabinofuranoside are omitted, the morphology of the cultures was different from that de-
scribed above. The cells were densely packed. The 10-day-old cultures consisted of round or polygonal cells having a central nucleus with two or more nucleoli, but having only few or no visible processes. They stained with antibodies against GFAP but few stained for NSE. Cells with beating cilia, phagocytotic cells, mesenchyrnal-like cells and oligoblasts occurred as above. Very few cells were seen in cultures treated with c-Ara on day 3 or on days 3 and 6. After treatment with c-Ara on day 6 only, the cultures contained more cells. When insulin was added to the culture medium at every change but no c-Ara, the cultures
164
Fig. 3.a: primary cerebral hemisphere cultures co-cultivated for 7 days with fetal brain cultures, as described in the text and in Fig. 1. Most cells are polygonal with large processes extending from the soma. b: similar culture as in a, cultivated f o r l 0 days (no co-cultivation). The cells are polygonal and spindle-shaped with few or no visible processes. The culture is more dense than in a.
165 became very dense and only few processes were visible on the GFAP-positive cells. When the cultures were fed only once, i.e. on day 3 after seeding, very few NSE-positive cells were seen. Feeding every second day, i.e. 3 times a week, seemed necessary for the NSE positive cells to survive (Table I). Cultures from substantia nigra and brainstem showed the best morphology as outlined above with extensive processes on the GFAP-positive cells and many NSE-positive cells when treated with poly-L-lysine, 30 mM glucose, insulin, c-Ara on day 6 and with medium changed 3 times a week (Table I).
positive cells were seen in the primary cultures from newborn rat. After 7 days of co-cultivation, the GFAP-positive cells had even more processes extending from the cell soma and even branching (Table I, Fig. 3). No NSE-positive cells were seen. Total and water-soluble proteins were less in cultures from newborn rat after co-cultivation with fetal cultures containing NSE-positive cells (Table II). However, the cultures reached confluence at 6 - 7 days, whether co-cultivated or not. The morphology of fetal hemisphere cultures with NSE-positive cells did not change after co-cultivation.
Co-cultivation
Control experiments
Morphology o f primary cerebral hemisphere cultures f r o m newborn rat after co-cultivation with fetal cerebral hemisphere cultures
Prior to co-cultivation the primary hemisphere cultures from newborn rat (3 days old) contained scattered round or polygonal cells with central nuclei and with few processes. The cultures were not confluent and different cell types could not be separated, although there was a weak reaction in most cells after fluorescent-antibody labelling against GFAP. After 4 days of co-cultivation with fetal hemisphere cultures containing NSE-stainable cells, the cultures (totally 7 days old) from newborn rat had become confluent, although not dense. Most cells were polygonal, (diameter = 16 ~m) contained one central nucleus (6 g m in diameter) and had processes extending from the cell soma. These cells bound antibodies to GFAP. In addition, cells with beating cilia, phase-bright cells, phagocytotic cells, mesenchymallike cells and phase-dark cells were present. No NSE-
(1) When cerebral hemisphere cultures from newborn rat were grown under a poly-L-lysine-coated lid without cultured cells, the morphology of the cerebral cells beneath was similar to that of a primary hemisphere culture grown as earlier describedl4,t8 up to 10 days. Thus, the upside-down lid conditions did not affect cell growth or morphology. (2) Scattered cells were found on the bottom of the petri dish when fetal hemisphere cultures had been grown upside-down for 7 days. No NSE-positive cells were identified. The total protein amount was less than 1/~g/petri dish. The results show that there is extremely low contamination from the 'upper' to the 'lower' culture. (3) and (4). When the 'upper' cultures were devoid of treatment with poly-L-lysine or when cultures from newborn rat were used, the morphology of the 'lower' newborn brain culture was similar to that under item (1) above, or as earlier described. Therefore, it seems that NSE-positive cells are necessary in the fetal culture for the morphological alterations to take place.
TABLE II Total and water-soluble protein in primary cerebral hemisphere cultures co-cultivated for 4 and 7 days with or without fetal brain cultures containing NSE-positive cells
Mean + S.E.M.; number of cultures = 5. Statistical analysis: Student's t-test. Days of cultivation
Co-cultivation
No co-cultivation
Total protein a
Soluble protein a
Total protein a
Soluble protein a
7 10
91.8 _+4.5** 221.2 _+6.7**
51.4 _+5.4** 123.4 _+6.0**
125.3 _+3.7 295.0 + 4.1
68.5 + 2.4 157.8 + 3.8
* P < 0.05; ** P < 0.01; compared to total or soluble protein values, respectively, of data from poly-L-lysineand cytosinearabinofuranoside omission. a Protein measured asflg/petri dish (50 mm diameter).
166 Co-cultivation of substantia nigra cultures from fetal rat with striatum cultures from newborn rat, or co-cultivation of brainstem cultures from fetal rat with cerebral cortex cultures from newborn rat Most of the cells in the striatum and cerebral cortex cultures stained with fluorochrome-labelled GFAP-antibodies. However, no NSE-positive cells could be found. The GFAP-positive cells had large processes extending from their somas, and the cultures morphologically appeared differentiated (Table I). DISCUSSION Astroglia exerts a positive influence upon neuronal development and migration in situ (see ref. 31). Even in culture systems, a glial monolayer affects neuronal morphology 6. Such effects must be mediated by biochemical interactions between the astroglia and neurons. It therefore seems probable that neurons influence astroglial maturation. Fetal cerebral hemisphere, substantia nigra and brainstem primary cultures were cultivated on polyL-lysine29-coated surfaces and treated with the mitogenic inhibitor cytosinearabinofuranoside7 to depress astroglial growth. These cultures contained neuronal cells, which were stained with fluorochrome-labelled antibodies to nerve-specific enolase (NSE), a unique neuronal protein 2. The number of neuronal cells (approximately 5-10% of total cell number) was low in comparison to results from other laboratories s.29, and this difference could be due to technical reasons. It might be observed that the GFAP-positive astroglial-like cells in the fetal brain cultures had long processes and showed a differentiated appearance on a morphological basis. Interestingly, these long processes of the GFAP-positive cells were present only when NSE-positive cells could be identified. When the neuron-containing fetal cultures had been grown 1 mm apart from the astroglial enriched culture for 4-5 days, the cells in the latter culture adopted a differentiated appearance from a morphological point of view, with the extension of long pro-
cesses. The processes became even more pronounced after 7 days of co-cultivation. The specificity of the results were ensured by control experiments, demonstrating that the effects were obtained only in the presence of neuronal-like cells. The time sequence for cultivating and then co-cultivating the cultures was critical for the morphological differentiation of the astroglial cells, as were also the substances added to the incubation medium. Thus, 10-day-old neuroncontaining fetal cultures and 3-day-old astroglial-enriched newborn cultures should be co-cultured. In addition, insulin should be added to the incubation media of the fetal, neuron-containing cultures, and they should be treated with c-Ara on day 6 and have their medium changed 3 times a week. It seems that neuronal cells from one brain region, e.g. cerebral hemispheres, influence the morphological differentiation of astroglial cells from the same brain region. Neurons even seem to influence astroglial cells in brain regions constituting a natural projection area for the neuronal pathways, e.g. substantia nigra neurons vs striatum and brainstem neurons vs cerebral cortex. The use of an appropriate co-cultivation system might be an attempt to restore in vitro some of the direct interactions which may occur between cells, without losing the benifits of easy manipulations of cell and medium components and their isolation for biochemical analyses. The effects of co-cultivation on some biochemical parameters of the astroglial-enriched cultures will be shown in a subsequent paper.
ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Medical Research Council (Project B84-12X06812-01, B85-12X-06812-02, K85--12P-7308-01), from ~ k e Wiberg's Foundation, from Torsten and Ragnar S6derberg's Foundation and from the University of G6teborg. The technical assistance of Tomas Machek is highly appreciated. The antibodies against GFAp were kindly provided by Professor A. Lowenthat of Antwerp.
167 REFERENCES 1 Balcar, V.J., Borg, J. and Mandel, P., High-affinity uptake of L-glutamate and L-aspartate by glial cells, J. Neurochem., 28 (1977) 87-93. 2 Bock, E. and Dissing, J., Demonstration of enolase activity connected to the brain specific 14-3-2, Scand. J. Immunol., 4 (1975) 31-36. 3 Booher, J. and Sensenbrenner, M., Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures, Neurobiology, 2 (1972) 97-105. 4 Borg, J., Ramaharobandro, N., Mark, J. and Mandel, P., Changes in the uptake of GABA and taurine during neuronal and glial maturation, J. Neurochem., 34 (1980) 1113-1122. 5 Davies, P. and Kerr, C., Co-cultivation of vascular endothelial and smooth muscle cells using microcarrier techniques, Exp. Cell Res., 141 (1982)455-459. 6 Denis-Donini, S., Glowinski, J. and Prochiantz, A., Glial heterogeneity may define the three-dimensional shape of mouse mesencephalic dopaminergic neurones, Nature (London), 307 (1984) 641-643. 7 Dichter, M.A., Rat cortical neurons in cell culture; culture methods, cell morphology, electrophysiology, and synapse formation, Brain Research, 149 (1978) 279-293. 8 Drejer, J., Meier, E. and Schousboe, A., Novel neuron-related regulatory mechanisms for astrocytic glutamate and GABA high-affinity uptake, Neurosci. Lett., 37 (1983) 301-306. 9 Hallermayer, K., Harmening, C. and Hamprecht, B., Cellular localization and regulation of glutamine synthetase in primary cultures of brain cells from newborn mice, J. Neurochem., 37 (1981) 43-52. 10 Hansson, E., Primary Astroglial Cultures. Aspects of Morphology, Biochemistry and Transmitter Metabolism, Thesis, G6teborg, 1982. 11 Hansson, E., Enzyme activities of monoamine oxidase, cathechol-O-methyltransferase and y-aminobutyric acid transaminase in primary astroglial cultures and adult rat brain from different brain regions, Neurochem. Res., 9 (1984) 45-47. 12 Hansson, E., Cellular composition of a cerebral hemisphere primary culture, Neurochem. Res., 9 (1984) 153-172. 13 Hansson, E., Isacsson, H. and Sellstr6m, ~., Some characteristics of dopamine and GABA transport in primary cultures of astroglial cells, Acta Physiol. Scand., 121 (1984) 333-341. 14 Hansson, E., R6nnb~ick, L., Lowenthal, A., Noppe, M., Ailing, C., Karlsson, B. and Sellstrfm, ~., Brain primary culture - - a characterization (part II), Brain Research, 231 (1982) 173-183. 15 Hansson, E,, R6nnb~ick, L., Persson, L.I., Lowenthal, A., Noppe, M., Ailing, C. and Karlsson, B., Cellular composition of primary cultures from cerebral cortex, striatum, hippocampus, brain stem and cerebellum, Brain Research, 300 (1984) 9-18. 16 Hansson, E,, Rfnnb~ick, L. and Sellstr0m, A., Is there a 'dopaminergic glial cell'? Neurochem. Res., 9 (1984) 679-689. 17 Hansson, E. and Sellstr6m., /~., MAO, COMT, and GABA-T activities in primary astroglial cultures, J. Neurochem., 40 (1983) 220-225.
18 Hansson, E., Sellstr6m, A., Persson, L.I. and ROnnb~ick, L., Brain primary culture - - a characterization, Brain Research, 188 (1980) 233-246. 19 Henri, F.A., Anderson, D.J. and Sellstr6m, /~., Possible relationship between glial cells, dopamine and the effects of antipsychotic drugs, Nature (London), 266 (1977) 637-638. 20 Henri, F.A., Haljam~ie, H. and Hamberger, A., Glial cell function: active control of extracellular K+ concentration, Brain Research, 43 (1972) 437-443. 21 Henn, F.A. and Hamberger, A., Glial cell function: uptake of transmitter substances, Proc. Natl. Acad. Sci. U.S.A., 68 (1971) 2686-2690. 22 Hertz, L., Baldwin, F. and Schousboe, A., Serotonin receptors on astrocytes in primary cultures: effects of methysergide and fluoxetine, Can. J. Physiol. Pharmacol., 57 (1979) 223-226. 23 Hertz, L., Schousboe, A., Boechler, N., Mukerji, S. and Fedoroff, S., Kinetic characteristics of the glutamate uptake into normal astrocytes in culture, Neurochem. Res., 3 (1978) 1-14. 24 Kimelberg, H.K., Bowman, C., Biddlecome, S. and Bourke, R.S., Cation transport and membrane potential of primary astroglial cultures from neonatal rat brains, Brain Research, 177 (1979) 533-550. 25 Kuffler, S.W. and Nicholls, J.G., The physiology in neuroglial cells, Ergebn. Physiol. Biol. Chem. Exp. Pharmakol., 57 (1966) 1-90. 26 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 27 McCarthy, K. and de Vellis, J., Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue, J. Cell Biol., 85 (1980) 890-902. 28 Peterson, A. and Walum, E., Growth and morphology of neuronal cell lines cultures in perfusion, In Vitro, 19 (1983) 875-880. 29 Pettmann, B., Louis, J.C. and Sensenbrenner, M., Morphological and biochemical maturation of neurones cultured in the absence of glial cells, Nature (London), 281 (1979) 378-380. 30 Pope, A., Neuroglia: quantitative aspects. In E. Schoffeniels, G. Franck, D.B. Tower and L. Hertz (Eds.), Dynamic Properties of Gila Cells, Pergamon Press, Oxford, 1978, pp. 13-20. 31 Rakic, P. and Goldman-Rakic, Development and modifiability of the cerebral cortex, Neurosci. Res. Progr. Bull., 20 (1982) 433-438. 32 Roberts, P.J. and Keen, P., [14C]glutamate uptake and compartmentation in glia of rat dorsal sensory ganglion, J. Neurochem., 23 (1974) 201-209. 33 Schousboe, A., Fosmark, H. and Formby, B., Effect of serum withdrawal on Na+-K÷ ATPase activity in astrocytes cultured from dissociated brain hemispheres, J. Neurochem., 26 (1976) 1053-1055. 34 Schousboe, A., Hertz, L. and Svenneby, G., Uptake and metabolism of GABA in astrocytes cultured from dissociated mouse brain hemispheres, Neurochem. Res., 2 (1977) 217-229. 35 Schousboe, A., Svenneby, G. and Hertz, L., Uptake and metabolism of glutamate in astrocytes cultured from dissociated mouse brain hemispheres, J. Neurochem., 29 (1977) 999-1005. 36 Schrier, B.K. and Thompson, E.J., On the role of glial cells
168 in the mammalian nervous system. Uptake, excretion, and metabolism of putative neurotransmitters by cultured glial tumor cells, J. Biol. Chem., 249 (1974) 1769-1780. 37 Sellstr6m,/~. and Hamberger, A., Neuronal and glial systems for y-aminobutyric acid transport. J. Neurochem., 24 (1975) 847-852. 38 Sensenbrenner, M., Dissociated brain cells in primary cultures. In S. Fedoroff and L. Hertz (Eds.), Cell, Tissue, and Organ Cultures in Neurobiology, Academic Press, New York, 1977, pp. 191-213. 39 Sensenbrenner, M., Labourdette, G., Delaunoy, J.P., Pettmann, B., Devilliers, G., Moonen, G. and Bock, E..
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159
Elsevier BRE 11463
Co-Cultivation of Astroglial and Neuronal Primary Cultures from Rat Brain ELISABETH HANSSON
Institute of Neurobiology, Universityof GOteborg, G6teborg (Sweden) (Accepted June 18th, 1985)
Key words: kastroglia - - co-cultivation - - gliafibrillary acidic protein (GFAP) - - neuron - - neuron-specific enolase - primary brain culture
A technique is described for a two-cell co-cultivation system which permits in vitro evaluation of neuron-glia interactions. Primary astroglial enriched cultures from newborn rat cerebral hemispheres, striatum or cerebral cortex, grown for 3 days, were co-cultivated with primary neuron-containing cultures from 15- to 17-day rat embryo cerebral hemispheres, substantia nigra or brainstem, respectively, grown for 10 days on polylysine-coated surfaces. The neuronal cells were identified morphologically and immunohistochemically by antibodies to neuron-specific enolase. The two cultures were grown together for 7 days, separated by a U-formed 1 mm glassrod. The results show that neurons exert a morphogenetic effect on astroglial cells in the form of extension of cell processes. The coculture system allows investigation of potent local humoral interactions between astroglial cells and neurons.
INTRODUCTION Astroglial cells constitute a large volume of the brain 30. Due to their anatomical proximity to neurons and the synaptic regions, the cells might be involved in neuronal metabolic events. In fact, astroglial cells have been shown to act as K ÷ buffers20,25, at, to participate in uptake and metabolism of amino acid transmitters t,4,tl,13,t7,21,32,34-37, and to bear receptors for various neurotransmitters and neuromodulators 1639,22,4°. Consequently, it could be suggested that the cells play an important role for the functions of neurons. Dissociated primary brain culture is one model to obtain large quantities of metabolically active, nontransformed astroglial-like cells3,4,9,14,15,18,23,27,33. The cells show many morphological, biochemical and electrophysiological similarities with astroglia in situ 11,12,23,24,38. However, their degree of differentiation is low 1° and could be due to growth in the absence of neurons. Different ways have been tried to increase the differentiation of the cells: (1) addition to the culture medium of partially isolated protein or peptide factors from embryonic brain, factors which
have been shown to promote growth and morphological differentiation of astroglial cells39; and (2) treatment of the cultures with conditioned media, i.e. serum-free media from neuronal containing cultures8,28. Both models have proved interesting as they provided some morphological and biochemical differentiation of astroglial cells in culture. Differentiation of cells, however, is a complex process which requires interactions with specific substances in physiological concentrations at specific sequences. One way to circumvent the problems of identifying and isolating such factors and to probably increase differentiation of astroglial cells in culture would be to co-cultivate astroglial cultures with neuronal cultures. The cells should grow in the near vicinity of each other while remaining physically separated and allowing humoral transfer. Such a technique has been introduced previously with the co-culture of vascular endothelial cells and smooth muscle cells 5. In this paper one technique for co-cultivation of astroglial and neuronal cells is described. The paper focuses on the morphological effects on the astroglial culture.
Correspondence: E. Hansson, Institute of Neurobioiogy, University of G6teborg, P.O.B. 33 031, S-400 33 G6teborg, Sweden. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
160 MATERIALS AND METHODS
Cultivation of fetal brain tissue Cerebral hemispheres, substantia nigra or brainstem from 15 to 17-day-fetal rats (Sprague-Dawley strain, Anticimex, Sweden) were passed through an 80 a m nylon mesh into Eagle's minimum essential medium (Flow Laboratories, U.K.). The medium was supplied with extra substances to make up the following final composition: double concentrations of amino acids, 2 mM glutamine; 30 mM glucose, and quadruple concentrations of vitamins. In some experiments, 5/~g insulin/ml (Sigma Fine Chemicals, St. Louis, MO, U.S.A.) was added; 250,000 IU/liter penicillin, 0.5% streptomycin and 20% (v/v) fetal calf-serum (Gibco Bio Cult. Lab., U.K.) were also added, and the pH was set at 7.3. Material from one hemisphere, 6 substantia nigra or one brainstem, was seeded on top of the lid to one petri dish (35 mm in diameter) (Nunc, Denmark). The lids were coated with poly-L-lysine (Sigma Fine Chemicals, St. Louis, U.S.A.) at room temperature, 24 h before use, according to Pettmann and coworkers 29. Medium was changed only once after 3 days or after 3, 6 and 8
days (different experiments). After 3 or 6 days or after 3 and 6 days, the cultures were treated with 10-5 M cytosine-l-fl-D-arabinofuranoside (c-Ara) (Sigma) 7. After 10 days the lids were turned upside-down in the 50 mm petri dishes, prefilled with 6 ml medium. The lids were placed on U-formed glass-rods, 1 mm in diameter (Fig. 1).
Cultivation of primary cultures from newborn rat cerebral hemispheres, striatum and cerebral cortex These cultures were done according to Booher and Sensenbrenner 3, Schousboe et al. 33 and Hansson et al. 14,15,18. Co-cultivation of fetal and newborn brain cultures Ten days after seeding: (1) the fetal hemisphere cultures were placed on glass rods above 3-day-old primary astroglial-enriched hemisphere cultures from newborn brain; (2) the substantia nigra cultures were placed above 3-day-old primary atroglial-enriched striatum cultures; and (3) the brainstem cultures were placed above 3-day-old primary astroglial-enriched cerebral cortex cultures obtained from newborn rat (Fig. 1). The petri dishes were kept at C
.••
FETAL BRAIN PRIMARY CULTURE (CONTAINING NEURGNS)
o lmm
PRIMARY NEONATAL ASTROGLIAL CULTURE
f
Fig. 1. Schematic drawing of essential steps in the co-cultivation of fetal neuron-containing cultures and newborn astroglial-enriched cultures. Fetal cell material was cultivated for 10 days on poly-L-lysine-coatedlids of petri dishes (35 mm in diameter) placed in petri dishes (50 mm in diameter) (a). Primary cultures from newborn rat (b) were grown for 3 days, and the fetal culture was transferred to this latter culture upside-down and placed on a U-formed glass-rod 1 mm in diameter (c).
161 37 °C in a humidified atmosphere of 95% air and 5% carbon dioxide. Media were changed 3 times a week. The same media were used as for cultivation of fetal brain tissue.
Control experiments Control experiments were as follows: (1) co-cultivation of cerebral hemisphere cultures from newborn rat with poly-L-lysine-treated empty lids, separated by a U-shaped glass rod, 1 mm in diameter; (2) cultivation of fetal hemisphere cultures treated with polyL-lysine and cytosine arabinofuranoside 'upsidedown' in 50 mm diameter petri dishes with no culture in the latter. This experiment was performed to examine whether cells from the lid 'fall down' onto the bottom of the petri dish; (3) co-cultivation of fetal rat hemisphere culture grown in the absence of poly-Llysine with the hemisphere culture from newborn rat; and (4) co-cultivation of two cultures from cerebral hemispheres of newborn rat. One culture was treated with poly-L-lysine. Protein determinations The cultures were rinsed in glucose-saline at pH 7.4. The material was scraped off into 300/~l or 500/~l PBS, also at pH 7.4, (35 mm or 55 mm petri dishes, respectively). The cultures were homogenized (glass/glass). Part of the homogenates were solubilized in 1 M NaOH, and another part was centrifuged 85,000 g for 60 min. The supernatants contained soluble protein. Total and soluble protein was determined according to Lowry et al. 26. lmmunohistochemistry The cultures were fixed in freshly prepared 4% formaldehyde in 0.15 M cacodylate buffer, pH 7.2, for 60 min at room temperature. After 4 rinses for 10 min each in 0.01 M phosphate-buffered saline (PBS) at pH 7.4, the cultures were incubated either in the antibody-containing solutions or in the control solutions (200/A). The cultures were incubated for 30 min in antibodies against glial fibrillary acidic protein (GFAP, a-albumin) (1:100) (gift from Prof. A. Lowenthal, Belgium) or in antibodies against neuron-specific enolase (NSE) (1:100) (bought from Dakopatts, Copenhagen, Denmark). They were thereafter washed (3 x 10 min) and incubated in the goat antirabbit IgG conjugated with fluorescein isothiocya-
nate (FITC) or tetramethylrhodamine isothiocyanate (TRITC). The incubation was for 45 min in buffered saline at pH 7.4. The cells were examined unmounted in the fluorescence microscope after washings in PBS for 4 x 10 min.
Controls (1) Sera from non-immunized rabbits were used; (2) cultures were incubated as described but the specific antisera were omitted; (3) untreated cultures were used to assess the autofluorescence which was very low. Microscopy The fluorescence microscope used was a Zeiss photomicroscope III RS, equipped with a mercury lamp for epifluorescence. The excitation filters for FITC or rhodamine were exchangeable in a sliding arrangement which permitted rapid photography. Photographs were made using phase contrast on Kodak Tri-X-PAN, 27 DIN films. RESULTS
Morphological and immunohistochemical characterization of fetal cerebral hemisphere cultures, substan. tia nigra cultures and brainstem cultures The cerebral hemispheres from fetal rat were cultivated on poly-L-lysine-coated petri dishes with insulin added to the culture medium, this being changed 3 times a week. When these cultures were treated with cytosinearabinofuranoside on day 6, they contained polygonal cells at the time of observation on day 10. The soma of these cells had a diameter of approximately 16/~m, contained one central nucleus (diameter approximately 6/~m) with two or more nucleoli, and had processes extending from the cell. The cells stained with antibodies against gliofibrillary acidic protein (GFAP) (Fig. 2a), but did not stain with antibodies against NSE. Scattered among the polygonal cells were cells of different sizes with soma (10-25/~m in diameter) containing one central nucleus with only one visible nucleolus and a number of large processes extending from the soma. These latter cells constituted approximately 5-10% of the total cell number in the cultures. Soma, nucleus and processes stained with antibodies against NSE (Fig. 2b), but were not stained by
162 TABLE I
Cell-density and morphological differentiation of fetal, neuron-containing cultures and astroglial-enriched primary cultures from newborn rat after co-cultivation for 7 days Medium was changed 3 times a week. Fetal cultures taken from cerebral hemispheres, substantia nigra or locus coeruleus. Cell density, and morphological differentiation, i.e. extension of processes: +, low; + + , moderate; + + + , high. Fetal material was co-cultivated with newborn material from cerebral hemispheres, striatum or cerebral cortex, respectively.
Fetal cuhures treated with
Cell density in fetal cultures"
Poly< -lysine and 30 mM glucose Poly-~ -lysine and 30 mM glucose + insulin (5 !zg/ml) Polyq.-lysine and 30 mM glucose + l/) -~ M c-Ara on day 6 Polyq -lysine and 30 mM glucose + insulin + e-Ara (as above)
Differentiation of cells in fetal cultures
Cell density in primary cultures from newborn rat after co-cultivation
DifJerentiation of cells in primary cultures from newborn rat after co-cultivation
++
++
+++
++
+++
+
+++
+
+
++
+++
++
++
+++
++
+++
re-
C e l l s w i t h b e a t i n g cilia w e r e s e e n in u n f i x e d cul-
v e a l e d in d o u b l e - l a b e l l i n g e x p e r i m e n t s ; n o t s h o w n ) .
tures and they have been suggested previously to be
O t h e r cells in t h e c u l t u r e s w e r e f o u n d in m i n o r p r o -
e p e n d y m a l - l i k e 12. P h a s e - b r i g h t c e l l s , w i t h t h e a b i l i t y
portions.
to p h a g o c y t o s e I n d i a i n k , h a v e b e e n c h a r a c t e r i z e d as
antibodies
against
GFAP
(which
was further
Fig. 2.a: fetal hemisphere culture (10 days old) incubated with anti-GFAP and treated as described in the text. Most cells show a positive l-CllC'donIThc cells have long processcs, b: similar culture as in a. NSE-positivc cells can be seen 10-25 .um in diameter, with one cemral nucleus and scvcral large processcs. Similar and corresponding cells are exhibited in phase-contrast micrographs.
163 macrophages 12. Small phase-dark cells with one or two processes have been suggested to be oligoblasts 12. Even mesenchymal-like cells 12 were observed. The age of the tissue, the amount of material seeded, and the treatment of the cultures, were all critical in order to obtain the characteristics of the cultures described. In the absence of poly-L-lysine, no NSEpositive cells were found. If poly-L-lysine coating is utilized while insulin and cytosinearabinofuranoside are omitted, the morphology of the cultures was different from that de-
scribed above. The cells were densely packed. The 10-day-old cultures consisted of round or polygonal cells having a central nucleus with two or more nucleoli, but having only few or no visible processes. They stained with antibodies against GFAP but few stained for NSE. Cells with beating cilia, phagocytotic cells, mesenchyrnal-like cells and oligoblasts occurred as above. Very few cells were seen in cultures treated with c-Ara on day 3 or on days 3 and 6. After treatment with c-Ara on day 6 only, the cultures contained more cells. When insulin was added to the culture medium at every change but no c-Ara, the cultures
164
Fig. 3.a: primary cerebral hemisphere cultures co-cultivated for 7 days with fetal brain cultures, as described in the text and in Fig. 1. Most cells are polygonal with large processes extending from the soma. b: similar culture as in a, cultivated f o r l 0 days (no co-cultivation). The cells are polygonal and spindle-shaped with few or no visible processes. The culture is more dense than in a.
165 became very dense and only few processes were visible on the GFAP-positive cells. When the cultures were fed only once, i.e. on day 3 after seeding, very few NSE-positive cells were seen. Feeding every second day, i.e. 3 times a week, seemed necessary for the NSE positive cells to survive (Table I). Cultures from substantia nigra and brainstem showed the best morphology as outlined above with extensive processes on the GFAP-positive cells and many NSE-positive cells when treated with poly-L-lysine, 30 mM glucose, insulin, c-Ara on day 6 and with medium changed 3 times a week (Table I).
positive cells were seen in the primary cultures from newborn rat. After 7 days of co-cultivation, the GFAP-positive cells had even more processes extending from the cell soma and even branching (Table I, Fig. 3). No NSE-positive cells were seen. Total and water-soluble proteins were less in cultures from newborn rat after co-cultivation with fetal cultures containing NSE-positive cells (Table II). However, the cultures reached confluence at 6 - 7 days, whether co-cultivated or not. The morphology of fetal hemisphere cultures with NSE-positive cells did not change after co-cultivation.
Co-cultivation
Control experiments
Morphology o f primary cerebral hemisphere cultures f r o m newborn rat after co-cultivation with fetal cerebral hemisphere cultures
Prior to co-cultivation the primary hemisphere cultures from newborn rat (3 days old) contained scattered round or polygonal cells with central nuclei and with few processes. The cultures were not confluent and different cell types could not be separated, although there was a weak reaction in most cells after fluorescent-antibody labelling against GFAP. After 4 days of co-cultivation with fetal hemisphere cultures containing NSE-stainable cells, the cultures (totally 7 days old) from newborn rat had become confluent, although not dense. Most cells were polygonal, (diameter = 16 ~m) contained one central nucleus (6 g m in diameter) and had processes extending from the cell soma. These cells bound antibodies to GFAP. In addition, cells with beating cilia, phase-bright cells, phagocytotic cells, mesenchymallike cells and phase-dark cells were present. No NSE-
(1) When cerebral hemisphere cultures from newborn rat were grown under a poly-L-lysine-coated lid without cultured cells, the morphology of the cerebral cells beneath was similar to that of a primary hemisphere culture grown as earlier describedl4,t8 up to 10 days. Thus, the upside-down lid conditions did not affect cell growth or morphology. (2) Scattered cells were found on the bottom of the petri dish when fetal hemisphere cultures had been grown upside-down for 7 days. No NSE-positive cells were identified. The total protein amount was less than 1/~g/petri dish. The results show that there is extremely low contamination from the 'upper' to the 'lower' culture. (3) and (4). When the 'upper' cultures were devoid of treatment with poly-L-lysine or when cultures from newborn rat were used, the morphology of the 'lower' newborn brain culture was similar to that under item (1) above, or as earlier described. Therefore, it seems that NSE-positive cells are necessary in the fetal culture for the morphological alterations to take place.
TABLE II Total and water-soluble protein in primary cerebral hemisphere cultures co-cultivated for 4 and 7 days with or without fetal brain cultures containing NSE-positive cells
Mean + S.E.M.; number of cultures = 5. Statistical analysis: Student's t-test. Days of cultivation
Co-cultivation
No co-cultivation
Total protein a
Soluble protein a
Total protein a
Soluble protein a
7 10
91.8 _+4.5** 221.2 _+6.7**
51.4 _+5.4** 123.4 _+6.0**
125.3 _+3.7 295.0 + 4.1
68.5 + 2.4 157.8 + 3.8
* P < 0.05; ** P < 0.01; compared to total or soluble protein values, respectively, of data from poly-L-lysineand cytosinearabinofuranoside omission. a Protein measured asflg/petri dish (50 mm diameter).
166 Co-cultivation of substantia nigra cultures from fetal rat with striatum cultures from newborn rat, or co-cultivation of brainstem cultures from fetal rat with cerebral cortex cultures from newborn rat Most of the cells in the striatum and cerebral cortex cultures stained with fluorochrome-labelled GFAP-antibodies. However, no NSE-positive cells could be found. The GFAP-positive cells had large processes extending from their somas, and the cultures morphologically appeared differentiated (Table I). DISCUSSION Astroglia exerts a positive influence upon neuronal development and migration in situ (see ref. 31). Even in culture systems, a glial monolayer affects neuronal morphology 6. Such effects must be mediated by biochemical interactions between the astroglia and neurons. It therefore seems probable that neurons influence astroglial maturation. Fetal cerebral hemisphere, substantia nigra and brainstem primary cultures were cultivated on polyL-lysine29-coated surfaces and treated with the mitogenic inhibitor cytosinearabinofuranoside7 to depress astroglial growth. These cultures contained neuronal cells, which were stained with fluorochrome-labelled antibodies to nerve-specific enolase (NSE), a unique neuronal protein 2. The number of neuronal cells (approximately 5-10% of total cell number) was low in comparison to results from other laboratories s.29, and this difference could be due to technical reasons. It might be observed that the GFAP-positive astroglial-like cells in the fetal brain cultures had long processes and showed a differentiated appearance on a morphological basis. Interestingly, these long processes of the GFAP-positive cells were present only when NSE-positive cells could be identified. When the neuron-containing fetal cultures had been grown 1 mm apart from the astroglial enriched culture for 4-5 days, the cells in the latter culture adopted a differentiated appearance from a morphological point of view, with the extension of long pro-
cesses. The processes became even more pronounced after 7 days of co-cultivation. The specificity of the results were ensured by control experiments, demonstrating that the effects were obtained only in the presence of neuronal-like cells. The time sequence for cultivating and then co-cultivating the cultures was critical for the morphological differentiation of the astroglial cells, as were also the substances added to the incubation medium. Thus, 10-day-old neuroncontaining fetal cultures and 3-day-old astroglial-enriched newborn cultures should be co-cultured. In addition, insulin should be added to the incubation media of the fetal, neuron-containing cultures, and they should be treated with c-Ara on day 6 and have their medium changed 3 times a week. It seems that neuronal cells from one brain region, e.g. cerebral hemispheres, influence the morphological differentiation of astroglial cells from the same brain region. Neurons even seem to influence astroglial cells in brain regions constituting a natural projection area for the neuronal pathways, e.g. substantia nigra neurons vs striatum and brainstem neurons vs cerebral cortex. The use of an appropriate co-cultivation system might be an attempt to restore in vitro some of the direct interactions which may occur between cells, without losing the benifits of easy manipulations of cell and medium components and their isolation for biochemical analyses. The effects of co-cultivation on some biochemical parameters of the astroglial-enriched cultures will be shown in a subsequent paper.
ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Medical Research Council (Project B84-12X06812-01, B85-12X-06812-02, K85--12P-7308-01), from ~ k e Wiberg's Foundation, from Torsten and Ragnar S6derberg's Foundation and from the University of G6teborg. The technical assistance of Tomas Machek is highly appreciated. The antibodies against GFAp were kindly provided by Professor A. Lowenthat of Antwerp.
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