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Binding sites for [3H]dopamine and dopamine-antagonists on cultured astrocytes of rat striatum and spinal cord: An autoradiographic study
Neuroscience Letters, 65 (1986) 177-182 Elsevier Scientific Publishers Ireland Ltd.
177
NSL 03854 BINDING SITES FOR [3HIDOPAMINE AND DOPAMINE-ANTAGONISTS ON CULTURED ASTROCYTES OF RAT STRIATUM AND SPINAL CORD: AN AUTORADIOGRAPHIC STUDY
ELISABETH Ht~SLI and L. HOSLI* Department of Physiology, University of Basel, Vesalgasse 1, CH-4051 Basel (Switzerland)
(Received November 19th, 1985; Revised version received January 7th, 1986; Accepted January 10th, 1986)
Key words." binding - autoradiography - [3H]dopamine- Dt antagonist - D2 antagonist - tissue culture - striatum - spinal cord - rat
The cellular localization of binding sites for [3H]dopamirte, and dopamine-antagonists (D~ and D2) was studied in organotypic cultures of rat striatum and spinal cord by means of autoradiography. In both types of cultures, many astrocytes were labelled by [3H]dopamine, the Drantagonist [3H]cis-flupenthixol and the D2-antagonists [3H]domperidone and [3H]spiperone (10 -9 to 10-8 M). Addition of unlabelled dopamine and antagonists at high concentrations (10-6 to 10-4 M) inhibited or markedly reduced binding of the radioligands indicating 'specific' binding of the compounds. Our autoradiographic studies are consistent with biochemical investigations by other authors, suggesting that astrocytes possess receptors for dopamine. Biochemical studies have suggested that not only neurones but also glial cells possess adrenoceptors and histamine-receptors [2, 9, 10, 13, 14, 31, 34]. Electrophysiological and a u t o r a d i o g r a p h i c binding studies from o u r laboratory have added further evidence for the existence o f such receptors on cultured astrocytes o f rat central nervous system (CNS) [17, 18, 22, 23]. It was therefore o f great interest to investigate whether astrocytes also possess receptors for dopamine, another biogenic amine. Biochemical studies have revealed binding o f p H ] d o p a m i n e and [3H]haloperidol to h o m o g e n a t e s o f glial cells from bovine caudate nucleus [16]. Furthermore, a m a r k e d increase o f adenosine 3':5'-cyclic m o n o p h o s p h a t e ( c A M P ) by d o p a m i n e was f o u n d in primary cultures o f astroglial cells from rat striatum [15], whereas in cultures from rat brainstem, a brain region where few dopaminergic synapses exist [29], no increase in dopamine-evoked c A M P f o r m a t i o n could be detected [15]. Similarly, no dopamine-sensitive adenylate cyclase was observed in primary cultures o f glial cells from rat cortex [14] and mouse mesencephalon [8]. F r o m these findings it was suggested that glial cells from different regions o f the C N S might respond differentially to d o p a m i n e [15]. At least two subtypes o f d o p a m i n e receptors have been described in the m a m m a lian CNS: D r r e c e p t o r s which stimulate adenylate cyclase and D2-receptors which are *Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
178
linked in an inhibitory way to adenylate cyclase [25, 32]. In the present study an attempt was made to visualize binding sites for [3H]dopamine and D~- and D2-antagonists on astrocytes in organotypic cultures from rat striatum and spinal cord, two CNS regions where dopamine might act as transmitter [5, 11, 12, 29, 32]. Explant cultures were prepared from the striatum of 2- to 4-day-old rats and spinal cord of fetal rats (17-18 days in utero). The cultures were placed on collagen-coated coverslips and grown in Roller tubes for 15-37 days at 35°C [20]. For the binding studies, the cultures were rinsed for 1 h in several changes of Hank's solution. Then they were incubated either in Hank's solution (142 mM Na +) or in Na+-free solution (Na + being replaced by choline and Tris) containing [3H]dopamine (New England Nuclear (NEN); spec.act. 45.4 Ci/mmol), [3H]cis-flupenthixol (NEN; spec.act. 10.4 Ci/mmol), [3H]domperidone (NEN; spec.act. 32.2 Ci/mmol) or [3H]spiperone (NEN; spec.act. 24.3 Ci/mmol). Final concentration of the radioligands was 10 -9 and 10 -8 M and the incubation time ranged between 10 and 30 min. Incubation in Na+-free medium was done at 35°C, but in Na+-containing solution the temperature was 0°C to block active transport mechanisms, The monoamine oxidase inhibitor pargyline (10 6 M) and ascorbic acid (1 #g/ml) were added to the preincubation and incubation media (final pH 7.3). To provide estimates of 'specific' binding of the radioligands, unlabelled dopamine, domperidone, spiperone and cisflupenthixol were added to the preincubation and incubation media at high concentrations (10 -6 to 10 -4 M). After the incubation, the cultures were dipped twice in ice-cold phosphate buffer, fixed for 30 min in glutaraldehyde vapour (50°C), rinsed quickly in ice-cold phosphate buffer and distilled water. The cultures were dried in a gentle stream of cold air, covered with Ilford L4 emulsion by the loop technique, exposed for 1-3 months at 4°C and developed with Kodak D19 developer. The morphological properties of the various cell types in organotypic cultures of rat CNS have been described in a previous publication [21]. Whereas neurones usually remain in the dense zones or at the edge of the explants, glial cells also migrate into the outgrowth zone where they form a dense network with outgrowing neurites. Staining the cultures with anti-glial fibrillary acidic protein (GFAP) [6] has shown that the majority of cells in the outgrowth zone are GFAP-positive and thus can be identified as astrocytes. After incubation of striatal and spinal cord cultures with [3H]dopamine which binds to D1- and D2-receptors [25, 32], a great number of astrocytes were labelled by this biogenic amine. Silver grains were distributed over the soma and processes of the cells (Fig. 1A, B). The labelling by dopamine seems to reflect binding only, since biochemical [15] and autoradiographic [19] studies have shown that glial cells do not actively take up [3H]dopamine. The thioxantene neuroleptic [3H]cis-flupenthixol has been described to bind only to Dwreceptors provided that unlabelled spiperone which is known to label D2-and serotonin2-receptors is added to the incubation medium [4, 7, 24]. After incubation with [3H]cis-flupenthixol, in the presence of spiperone (10 -6 M), the majority of astrocytes in striatal and spinal cord cultures showed heavy labelling by this compound (Fig. 1C).
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Fig. 1. Binding sites for [3H]dopamine,and Dr and D2-antagonistson astrocytes. A: glial cells in the outgrowth zone of a spinal cord culture showing binding of [3H]dopamine(10-s M; Na÷-containing medium; culture 20 days in vitro). B: dark-field illumination autoradiograph of astrocytes in a striatal culture. The cells are labelled over the soma and processes by [3H]dopamine (10-s M; Na+-free medium; culture 32 days in vitro). C: intensely labelled astrocytes after incubation with the Drantagonist [3H]cis-flupenthixol (10-8 M; Na+-free medium; culture 28 days in vitro). D: astrocytes which show moderate binding of the D2-antagonist [3H]spiperone 00 -8 M; Na+-containing medium; culture 28 days in vitro). Bars: A, C, D = 50 #m; B = 30/zm. As D2-antagonist we have used [3H]domperidone a n d [3H]spiperone. [3H]Domperid o n e has been f o u n d to selectively label D2-receptors in various areas o f the C N S [1, 3, 28], whereas [3H]spiperone also binds to serotonin-receptors [4, 7, 26, 27, 30, 33]. To avoid b i n d i n g o f [3H]spiperone to serotonin-receptors, k e t a n s e r i n - a n S2a n t a g o n i s t - was a d d e d to the i n c u b a t i o n m e d i u m at a c o n c e n t r a t i o n o f 10 -6 M. After i n c u b a t i o n o f the cultures with [3H]domperidone or [3H]spiperone, m a n y astrocytes in striatal a n d spinal cord cultures were labelled over the cell b o d y a n d proc-
180 r
B
F"
-
m
Fig. 2. Bindingsites for [3H]dopamineand [3H]domperidoneon neurones. A: dark-fieldilluminationautoradiograph of spinal neurones which are intenselylabelledby [3H]dopamine(10 s M; Na+-freemedium; culture 28 days in vitro). B: group of striatal neurones showing binding sites for the D2-antagonist [3H]domperidone(10 8 M; Na+-containing medium; culture 32 days in vitro). Bars: A= 30/~m; B = 50 //m.
esses (Fig. I D). Besides gliai cells many striatai and spinal neurones showed binding sites for the radioligands. A relatively large number of neurones were intensely labelled by [3H]dopamine (Fig. 2A), the D2-antagonists [3H]domperidone (Fig. 2B) and [3H]spiperone (not illustrated). Neurones also revealed binding of the Dr-antagonist [3H]cisflupenthixol, although the labelling was less intense than by the other radioligands. There was no obvious difference in binding of the radioligands in Na+-containing at 0°C or in Na+-free incubation media at 35°C (Fig. 1A, B). Binding of [3H]dopamine, [3H]domperidone, [3H]spiperone and [3H]cis-flupenthixol was markedly reduced or inhibited by unlabelled dopamine, domperidone, spiperone and c/s-flupenthixol, respectively, at high concentrations (10 -6 to 10 a M ) suggesting 'specific' binding of the radioligands. From our autoradiographic binding studies as well as from biochemical investigations by other authors [15, 16], it is suggested that astrocytes possess receptors for dopamine in addition to adrenergic and histamine-receptors. Little is as yet known of the functional role of dopamine-receptors on glial cells. To obtain information on the physiological properties of these receptors, electrophysiological studies of the effect of dopamine and antagonists on the membrane potential of cultured astrocytes are in progress in our laboratory. We are grateful to Dr. S. Bischoff, Ciba-Geigy Ltd. Basel for valuable advice, to Miss Ch. Gysin for skilful technical assistance and to Mr. M. Wymann for photographic work. We also acknowledge Janssen Pharmaceutica Beerse/Belgium for the gift of unlabelled domperidone, spiperone and ketanserin and H. Lundbeck A/S,
181
Copenhagen for a sample of unlabelled
cis-(Z)-flupenthixol.
1 Arluison, M., Martres, M.P. and Sokoloff, P., High-resolution radioautographic study of dopamine binding sites in the rat neostriatum using 3H-domperidone, J. Neural: Transm., 18 (1983) 9-24. 2 Barovsky, K. and Brooker, G., (-)-(~25I)-Iodopindolol, a new highly selective radioiodinated ~-adrenergic receptor antagonist: measurement of r-receptors on intact rat astrocytoma cells, J. Cyclic Nucleotide Res., 6 (1980) 297-307. 3 Baudry, M., Martres, M.P. and Schwarz, J.C., 3H-Domperidone: a selective ligand for dopamine receptors, Naunyn-Schmiedeberg's Arch. Pharmacol., 308 (1979) 231-237. 4 Bischoff, S., Bittiger, H. and Krauss, J., In vivo (3H) spiperone binding to the rat hippocampal formation: involvement of dopamine receptors, Eur. J. Pharmacol., 68 (1980) 305-315. 5 Blessing, W.W. and Chalmers, J.P., Direct projection of catecholamine (presumably dopamine)-conraining neurons from hypothalamus to spinal cord, Neurosci. Lett., 11 (1979) 35-40. 6 Bologa, L., Bisconte, J.C., Joubert, R., Marangos, P.J., Derbin, C., Rioux, R. and Herschkowitz, N., Accelerated differentiation of oligodendrocytes in neuronal-rich embryonic mouse brain cell cultures, Brain Res., 252 (1982) 129 136. 7 Bruinjnk, A., Lichtensteiger, W. and Schlumpf, M., Pre- and postnatal ontogeny and characterization of dopaminergic D2, serotonergic $2, and spirodecanone binding sites in rat forebrain, J. Neurochem., 40 (1983) 1227-1235. 8 Chneiweiss, H., Prochiantz, A., Glowinski, J. and Premont, J., Biogenic amine-sensitive adenylate cyclases in primary culture of neuronal or glial cells from mesencephalon, Brain Res., 302 (1983) 363370. 9 Clark, R.B. and Perkins, J.P., Regulation of adenosine 3':5'-cyclic monophosphate concentration in cultured human astrocytoma cells by catecholamines and histamine, Proc. Natl. Acad. Sci. USA, 68 (1971) 2757-2760. 10 Clark, R.B., Su, Y.F., Ortmann, R., Cubbedu, X.L., Johnson, G.L. and Perkins, J.P., Factors influencing the effect of hormones on the accumulation of cyclic AMP in cultured human astrocytoma cells, Metabolism, 24 (1975) 343-358. 11 Commissiong, J.W. and Sedgwick, E.M., On the presence of dopamine in the mammalian spinal cord, Br. J. Pharmacol., 51 (1974) 118-119. 12 Dupelj, M. and Geber, J., Dopamine as a possible neurotransmitter in the spinal cord, Neuropharmacology, 20 (1981) 145-148. 13 Ebersolt, C., Perez, M. and Bockaert, J., ~ And ~2 adrenergic receptors in mouse brain astrocytes from primary cultures, J. Neurosci. Res., 6 (1981) 643-652. 14 Ebersolt, C., Perez, M. and Bockaert, J., Neuronal, glial and meningeal localizations of neurotransmitter-sensitive adenylate cyclases in cerebral cortex of mice, Brain Res., 213 (1981) 139-150. 15 Hansson, E., R6nnb/ick, L. and Sellstr6m, A., Is there a 'dopaminergic glial cell'?, Neurochem. Res., 9 (1984) 679-689. 16 Henn, F.A., Anderson, D.J. and SeUstr6m, ,&., Possible relationship between glial cells, dopamine and the effects of antipsychotic drugs, Nature (London), 266 (1977) 637-638. 17 H6sli, E. and H6sli, L., Evidence for the existence of ~- and fl-adrenoceptors on neurones and glial cells of cultured rat central nervous system - an autoradiographic study, Neuroscience, 7 (1982) 28732881. 18 H6sli, E. and H6sli, L., Autoradiographic localization of binding sites for (3H)histamine and H~- and H2-antagonists on cultured neurones and glial cells, Neuroscience, 13 (1984) 863-870. 19 H6sli, E., Bucher, U.M. and H6sli, L., Uptake of 3H-noradrenaline and 3H-5-hydroxytryptamine in cultured rat brainstem, Experientia, 31 (1975) 354-356. 20 H6sli, E., M6hler, H., Richards, J.G. and H6sli, L., Autoradiographic localization of binding sites for (3H)~-aminobutyrate, (3H)muscimol, (+)-(3H)bicuculline methiodide and (3H)flunitrazepam in cultures of rat cerebellum and spinal cord, Neuroscience, 5 (1980) 1657-1665. 21 H6sli, L. and H6sli, E., Action and uptake of neurotransmitters in CNS tissue culture, Rev. Physiol. Biochem. Pharmacol., 81 (1978) 135-188.
182 22 H6sli, L., H6sli, E., Zehntner, C., Lehmann, R. and Lutz, T.W., Evidence for the existence of ~- and fl-adrenoceptors on cultured glial cells - an electrophysiological study, Neuroscience, 7 (1982) 2867 2872. 23 H6sli, L., H6sli, E., Schneider, U. and Wiget, W., Evidence for the existence of histamine H~- and H2-receptors on astrocytes of cultured rat central nervous system, Neurosci. Lett., 48 (1984) 287-291. 24 Hyttel, J., Preferential labelling of adenylate cyclase coupled dopamine receptors with thioxanthene neuroleptics, In M. Kohsaka (Ed.), Advances in Dopamine Research, Advances in the Biosciences, Vol. 37, Pergamon Press Oxford, 1982, pp. 147-152. 25 Kebabian, J.W. and Calne, D.B., Multiple receptors for dopamine, Nature (London), 277 (1979) 9396. 26 Klemm, N., Murrin, L.C. and Kuhar, M.J., Neuroleptic and dopamine receptors: autoradiographic localization of [3H]spiperone in rat brain, Brain Res., 169 (1979) 1-9. 27 Leysen, J.E., Serotoninergic receptors in brain tissue: properties and identification of various 3H-ligand binding sites in vitro, J. Physiol. (Paris), 77 (1981) 351-362. 28 Martres, M.P., Baudry, M. and Schwartz, J.C., Characterization of 3H-domperidone binding on striatal dopamine receptors, Life Sci., 23 (1978) 1781 1784. 29 McGeer, P.L., Eccles, J.C. and McGeer, E., Catecholamine neurons. In P.L. McGeer, J.C. Eccles and E. McGeer (Eds.), Molecular Neurobiology of The Mammalian Brain, Plenum Press, New York-London. 1978, pp. 233-271. 30 Palacios, J.M., Niehoff, D.L. and Kuhar, M.J., [3H]Spiperone binding sites in brain: autoradiographic localization of multiple receptors, Brain Res., 213 (1981) 277-289. 31 Schubert, D., Tarikas, H. and LaCorbi+re, M., Neurotransmitter regulation of adenosine Y,5'-monophosphate in clonal nerve, glia, and muscle cell lines, Science, 192 (1976) 471~472. 32 Stool J.C. and Kebabian, J.W., Two dopamine receptors: biochemistry, physiology and pharmacology, Life Sci., 35 (1984) 2281-2296. 33 Taylor, J.E. and DeFeudis, F.V., Inhibition of (3H)spiperone binding to 5-HT2-receptors of rat cerebral cortex by the calcium antagonists verapamil and D600, Eur. J. Pharmacol., 106 (1985) 215-216. 34 VanCalker, D., Miiller, M. and Hamprecht, B., Adrenergic ~- and fl-receptors expressed by the same cell type in primary culture of perinatal mouse brain, J. Neurochem., 30 (1978) 713-718.
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NSL 03854 BINDING SITES FOR [3HIDOPAMINE AND DOPAMINE-ANTAGONISTS ON CULTURED ASTROCYTES OF RAT STRIATUM AND SPINAL CORD: AN AUTORADIOGRAPHIC STUDY
ELISABETH Ht~SLI and L. HOSLI* Department of Physiology, University of Basel, Vesalgasse 1, CH-4051 Basel (Switzerland)
(Received November 19th, 1985; Revised version received January 7th, 1986; Accepted January 10th, 1986)
Key words." binding - autoradiography - [3H]dopamine- Dt antagonist - D2 antagonist - tissue culture - striatum - spinal cord - rat
The cellular localization of binding sites for [3H]dopamirte, and dopamine-antagonists (D~ and D2) was studied in organotypic cultures of rat striatum and spinal cord by means of autoradiography. In both types of cultures, many astrocytes were labelled by [3H]dopamine, the Drantagonist [3H]cis-flupenthixol and the D2-antagonists [3H]domperidone and [3H]spiperone (10 -9 to 10-8 M). Addition of unlabelled dopamine and antagonists at high concentrations (10-6 to 10-4 M) inhibited or markedly reduced binding of the radioligands indicating 'specific' binding of the compounds. Our autoradiographic studies are consistent with biochemical investigations by other authors, suggesting that astrocytes possess receptors for dopamine. Biochemical studies have suggested that not only neurones but also glial cells possess adrenoceptors and histamine-receptors [2, 9, 10, 13, 14, 31, 34]. Electrophysiological and a u t o r a d i o g r a p h i c binding studies from o u r laboratory have added further evidence for the existence o f such receptors on cultured astrocytes o f rat central nervous system (CNS) [17, 18, 22, 23]. It was therefore o f great interest to investigate whether astrocytes also possess receptors for dopamine, another biogenic amine. Biochemical studies have revealed binding o f p H ] d o p a m i n e and [3H]haloperidol to h o m o g e n a t e s o f glial cells from bovine caudate nucleus [16]. Furthermore, a m a r k e d increase o f adenosine 3':5'-cyclic m o n o p h o s p h a t e ( c A M P ) by d o p a m i n e was f o u n d in primary cultures o f astroglial cells from rat striatum [15], whereas in cultures from rat brainstem, a brain region where few dopaminergic synapses exist [29], no increase in dopamine-evoked c A M P f o r m a t i o n could be detected [15]. Similarly, no dopamine-sensitive adenylate cyclase was observed in primary cultures o f glial cells from rat cortex [14] and mouse mesencephalon [8]. F r o m these findings it was suggested that glial cells from different regions o f the C N S might respond differentially to d o p a m i n e [15]. At least two subtypes o f d o p a m i n e receptors have been described in the m a m m a lian CNS: D r r e c e p t o r s which stimulate adenylate cyclase and D2-receptors which are *Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
178
linked in an inhibitory way to adenylate cyclase [25, 32]. In the present study an attempt was made to visualize binding sites for [3H]dopamine and D~- and D2-antagonists on astrocytes in organotypic cultures from rat striatum and spinal cord, two CNS regions where dopamine might act as transmitter [5, 11, 12, 29, 32]. Explant cultures were prepared from the striatum of 2- to 4-day-old rats and spinal cord of fetal rats (17-18 days in utero). The cultures were placed on collagen-coated coverslips and grown in Roller tubes for 15-37 days at 35°C [20]. For the binding studies, the cultures were rinsed for 1 h in several changes of Hank's solution. Then they were incubated either in Hank's solution (142 mM Na +) or in Na+-free solution (Na + being replaced by choline and Tris) containing [3H]dopamine (New England Nuclear (NEN); spec.act. 45.4 Ci/mmol), [3H]cis-flupenthixol (NEN; spec.act. 10.4 Ci/mmol), [3H]domperidone (NEN; spec.act. 32.2 Ci/mmol) or [3H]spiperone (NEN; spec.act. 24.3 Ci/mmol). Final concentration of the radioligands was 10 -9 and 10 -8 M and the incubation time ranged between 10 and 30 min. Incubation in Na+-free medium was done at 35°C, but in Na+-containing solution the temperature was 0°C to block active transport mechanisms, The monoamine oxidase inhibitor pargyline (10 6 M) and ascorbic acid (1 #g/ml) were added to the preincubation and incubation media (final pH 7.3). To provide estimates of 'specific' binding of the radioligands, unlabelled dopamine, domperidone, spiperone and cisflupenthixol were added to the preincubation and incubation media at high concentrations (10 -6 to 10 -4 M). After the incubation, the cultures were dipped twice in ice-cold phosphate buffer, fixed for 30 min in glutaraldehyde vapour (50°C), rinsed quickly in ice-cold phosphate buffer and distilled water. The cultures were dried in a gentle stream of cold air, covered with Ilford L4 emulsion by the loop technique, exposed for 1-3 months at 4°C and developed with Kodak D19 developer. The morphological properties of the various cell types in organotypic cultures of rat CNS have been described in a previous publication [21]. Whereas neurones usually remain in the dense zones or at the edge of the explants, glial cells also migrate into the outgrowth zone where they form a dense network with outgrowing neurites. Staining the cultures with anti-glial fibrillary acidic protein (GFAP) [6] has shown that the majority of cells in the outgrowth zone are GFAP-positive and thus can be identified as astrocytes. After incubation of striatal and spinal cord cultures with [3H]dopamine which binds to D1- and D2-receptors [25, 32], a great number of astrocytes were labelled by this biogenic amine. Silver grains were distributed over the soma and processes of the cells (Fig. 1A, B). The labelling by dopamine seems to reflect binding only, since biochemical [15] and autoradiographic [19] studies have shown that glial cells do not actively take up [3H]dopamine. The thioxantene neuroleptic [3H]cis-flupenthixol has been described to bind only to Dwreceptors provided that unlabelled spiperone which is known to label D2-and serotonin2-receptors is added to the incubation medium [4, 7, 24]. After incubation with [3H]cis-flupenthixol, in the presence of spiperone (10 -6 M), the majority of astrocytes in striatal and spinal cord cultures showed heavy labelling by this compound (Fig. 1C).
179
Fig. 1. Binding sites for [3H]dopamine,and Dr and D2-antagonistson astrocytes. A: glial cells in the outgrowth zone of a spinal cord culture showing binding of [3H]dopamine(10-s M; Na÷-containing medium; culture 20 days in vitro). B: dark-field illumination autoradiograph of astrocytes in a striatal culture. The cells are labelled over the soma and processes by [3H]dopamine (10-s M; Na+-free medium; culture 32 days in vitro). C: intensely labelled astrocytes after incubation with the Drantagonist [3H]cis-flupenthixol (10-8 M; Na+-free medium; culture 28 days in vitro). D: astrocytes which show moderate binding of the D2-antagonist [3H]spiperone 00 -8 M; Na+-containing medium; culture 28 days in vitro). Bars: A, C, D = 50 #m; B = 30/zm. As D2-antagonist we have used [3H]domperidone a n d [3H]spiperone. [3H]Domperid o n e has been f o u n d to selectively label D2-receptors in various areas o f the C N S [1, 3, 28], whereas [3H]spiperone also binds to serotonin-receptors [4, 7, 26, 27, 30, 33]. To avoid b i n d i n g o f [3H]spiperone to serotonin-receptors, k e t a n s e r i n - a n S2a n t a g o n i s t - was a d d e d to the i n c u b a t i o n m e d i u m at a c o n c e n t r a t i o n o f 10 -6 M. After i n c u b a t i o n o f the cultures with [3H]domperidone or [3H]spiperone, m a n y astrocytes in striatal a n d spinal cord cultures were labelled over the cell b o d y a n d proc-
180 r
B
F"
-
m
Fig. 2. Bindingsites for [3H]dopamineand [3H]domperidoneon neurones. A: dark-fieldilluminationautoradiograph of spinal neurones which are intenselylabelledby [3H]dopamine(10 s M; Na+-freemedium; culture 28 days in vitro). B: group of striatal neurones showing binding sites for the D2-antagonist [3H]domperidone(10 8 M; Na+-containing medium; culture 32 days in vitro). Bars: A= 30/~m; B = 50 //m.
esses (Fig. I D). Besides gliai cells many striatai and spinal neurones showed binding sites for the radioligands. A relatively large number of neurones were intensely labelled by [3H]dopamine (Fig. 2A), the D2-antagonists [3H]domperidone (Fig. 2B) and [3H]spiperone (not illustrated). Neurones also revealed binding of the Dr-antagonist [3H]cisflupenthixol, although the labelling was less intense than by the other radioligands. There was no obvious difference in binding of the radioligands in Na+-containing at 0°C or in Na+-free incubation media at 35°C (Fig. 1A, B). Binding of [3H]dopamine, [3H]domperidone, [3H]spiperone and [3H]cis-flupenthixol was markedly reduced or inhibited by unlabelled dopamine, domperidone, spiperone and c/s-flupenthixol, respectively, at high concentrations (10 -6 to 10 a M ) suggesting 'specific' binding of the radioligands. From our autoradiographic binding studies as well as from biochemical investigations by other authors [15, 16], it is suggested that astrocytes possess receptors for dopamine in addition to adrenergic and histamine-receptors. Little is as yet known of the functional role of dopamine-receptors on glial cells. To obtain information on the physiological properties of these receptors, electrophysiological studies of the effect of dopamine and antagonists on the membrane potential of cultured astrocytes are in progress in our laboratory. We are grateful to Dr. S. Bischoff, Ciba-Geigy Ltd. Basel for valuable advice, to Miss Ch. Gysin for skilful technical assistance and to Mr. M. Wymann for photographic work. We also acknowledge Janssen Pharmaceutica Beerse/Belgium for the gift of unlabelled domperidone, spiperone and ketanserin and H. Lundbeck A/S,
181
Copenhagen for a sample of unlabelled
cis-(Z)-flupenthixol.
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