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A microtest plate assay of functional excitatory amino acid receptors
Journal of Neuroscience Methods. 28 (1988) 229-233 Elsevier
229
NSM 00941
A microtest plate assay of functional excitatory amino acid receptors N o m i E s h h a r , M o s h e L u s k y , P e e r D a v i d a n d V i v i a n I. T e i c h b e r g Department of Neurobiology, The Weizmann Institute of Science, Rehovot (Israel)
(Received 21 June 1988) (Revised 18 October 1988) (Accepted 21 October 1988)
Key words: Excitatory amino acid; Cultured chick retinal cell; Microtest plate assay
A microtest plate assay of functional excitatory amino acid receptors present on cultured chick embryo retinal cells has been developed. It is based on measurements of excitatory amino acid-mediated increase in 22Na+ influx into retinal cells adhering to each of the 96 wells of microtest plates. Dose-dependent responses to L-glutamate, kainate and N-methyl-D-aspartate but not to quisqualate can be measured. These responses are selectively inhibited by antagonists of excitatory amino acids. The assay is reliable, fast to perform and parsimonious in terms of the volume and thus of amount of the drug applied. It allows a single investigator to perform, in one day, measurements of the effects of known or putative glutamatergic ligands in more than 100 different conditions.
Introduction
The interactions of glutamatergic ligands, agonists and antagonists, with the m e m b r a n o u s excitatory amino acid receptors can be studied n o w a d a y s by a battery of electrophysiological and biochemical methods. The former include intracellular recordings under voltage clamp (McD o n a l d and Wojtowicz, 1982), single-channel recordings ( N o w a k et al., 1984) and use of m R N A injected Xenopus oocytes for the study of de n o v o synthesized receptors and their gating mechanisms (Yellen, 1984). The biochemical methods include binding techniques with various radioactive agonists, antagonists or modulators (Fagg and Foster, 1984), measurements of sodium (Luini et
Correspondence: V.I. Teichberg, Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel.
al., 1981) and calcium ion (Ichida et al., 1982; Wroblewski et al., 1985) fluxes, of cyclic G M P formation (Foster and Roberts, 1980; Garthwaite, 1982), of phosphatidylinositide turnover (Sladeczek et al., 1985; Nicoletti et al., 1986), and of the excitatory a m i n o acid-induced release of genuine [acetylcholine (Scatton and L e h m a n n , 1982), d o p a m i n e (Roberts and Anderson, 1979), yaminobutyric acid (Gallo et al., 1987)] or false [D-aspartate (McBean and Roberts 1981)] neurotransmitters from synaptosomes or brain slices. In spite of this relative profusion of techniques, n o n e of them is applicable for the simultaneous screening of a large n u m b e r of putative glutamatergic ligands (agonists, antagonists, modulators) particularly when time and a m o u n t of ligand are limited. We describe here the use of chick e m b r y o retinal cell cultures maintained in 96-well microtest plates for the assay of functional glutamate receptors via measurements of excitatory amino acid-induced 22Na+ influx.
0165-0270/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
23{)
Methods
Retinal cell cultures Retinas from chick embryos (8-11 days old) were removed and incubated at 3 7 ° C in Puck's medium containing 0.125% trypsin and 0.002% DNAse in order to dissociate the retinal tissue into cells. After 15 min, the medium was removed by decantation and substituted twice with a Dulbecco's modified Eagle medium ( D M E M ) containing 10% fetal calf serum. The sedimented cell mass was then triturated by several passages through fired Pasteur pipettes and centrifuged for 10 min at 1000 g in a desk centrifuge. The pelleted cells were washed with D M E M and recentrifuged as before. The cells were resuspended in D M E M containing 10% fetal calf serum and 0.2% antibiotics (50000 units penicillin, 50000/~g streptomycin, 5000 units nystatin/ml stock solution) and filtered through a nylon mesh to remove debris. Cell yields were 50 × 106 cells/retina. After counting the viable cells excluding Trypan blue ( > 95%), the cell suspension was diluted to a density of 15 x 1 0 6 cells/ml and 0.2 ml aliquots were distributed in each of the 96 wells of Costar microtiter plates (catalog no. 2590) consisting of 12 removable strips, each containing 8 flat-bottom wells. Before the addition of the cells, the wells were coated with 0.2 ml of a 0.02 m g / m l poly-Llysine (average mol. wt. 70,000) solution in water. The plates were incubated at 37 ° C under 5% CO 2 for 24-96 h.
surements were always carried out in quadruplicate, i.e. two assay conditions per strip. The effect of excitatory amino acid agonists was expressed as a stimulation index corresponding to the ratio between the radioactivity associated with cells exposed to the agonist and that of cells exposed to the same medium without agonist.
Results
Fig. 1 shows the stimulation produced by 0.2 m M L-glutamate and 0.1 mM kainate on retinal cells removed from 9-day-old embryos and maintained in culture for 3-72 h: the effects of the excitatory amino acids, as expressed by the stimulation index, are very small 3 h after cell plating but reach significant values after 24 and 48 h. In the experiments with 72 h cultures, the average incorporation of 22Na+ is about 100 cpm in cells exposed for 5 rain to the radioactive medium without agonist and up to 1500 cpm in its presence at the highest concentration. If cells are plated at a density smaller than 3 x 106 cells/well and stimulated by an excitatory amino acid in the presence of 22Na+ ions, a proportionally smaller 16 X
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Assay of excitatory amino acid receptor function The presence of excitatory amino acid receptors on the cultured retinal cells was monitored via measurements of excitatory amino acid induced 22Na+ influx. For this purpose, the plates were washed twice with D M E M to remove all non-adhering ceils and exposed at t = 0 rain, to 0.1 ml of D M E M containing 0.25/~Ci of 2 2 N a + (Amersham) and an excitatory amino acid agonist, either alone or together with an antagonist. At t = 5 min, the radioactive medium was aspirated and discarded and the cells washed twice with DMEM. This washing solution was also discarded and 0.1 ml 1 M N a O H was added. The latter solution was removed and its radioactivity measured. All mea-
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H o u r s in c u l t u r e Fig. 1. In vitro development of the sensitivity of cultured chick embryo retinal cells to excitatory amino acids. Retinal cells were prepared from 9-day-old retinas, plated in microtest plates at the density of 3 x 106 cells/well and maintained in culture for up to 4 days. They were exposed for 5 min to a 22Na+ containing D M E M m e d i u m in the absence or presence of either 0.1 m M kainate (striped columns) or 0.2 m M Lglutamate (empty columns). The stimulation index is the ratio of the radioactivity associated with cells exposed to the agonist and that of ceils exposed to the same medium without agonist.
231
incorporation of radioactivity takes place. However, the plating of a larger number of cells per well does not improve the amount of radioactivity i n c o r p o r a t e d indicating that with 3 x 106 cells/well the maximal adhering cell density has been reached. The observed increase of the stimulation index as a function of time in culture is possibly a reflection of one or the combination of the following factors: the rate of recovery of the membrane potential following the trypsinization of the cell suspension, the rate of de novo synthesis of excitatory amino acid receptors and the timing of appearance of these receptors in the chick embryo. The first two factors may explain the finding that retinal cells are not sensitive to excitatory amino acids for several hours after plating while the third factor may account for the fact that day 10 retinal cells/48 h in culture are stimulated to approximately twice the level of stimulation as day 9 retinas/48 h in culture, whereas equal levels of stimulation are reached with cells of equal age (cells from day 9 e m b r y o / 7 2 h in culture are stimulated to the same extent as cells from day 10 e m b r y o / 4 8 h in culture). With cells maintained in culture for more than 4 days, a significant decrease in the apparent sensitivity to excitatory amino acids is observed and is correlated to a deterioration of the cultured cells and their detachment from the well walls. Fig. 2 shows dose-response curves obtained with 3 excitatory
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amino acids, kainate, L-glutamate and N-methylD-aspartate (NMDA), on day 9 retinal cells/48 h in culture. Their order of effectiveness as agonists is kainate > L-glutamate > N M D A . Quisqualate was also tested but was found to be, even at 10 mM, an extremely weak agonist. In order to ,<
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Fig. 2. Dependence of the stimulated 22Na+ influx on the concentration of kainate (I), [+-glutamate (i) and N-methylD-aspartate (~). Day 9 chick embryo retinal cells maintained 48 h in culture• Each point is the average of quadruplicate determinations.
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Fig. 4. Inhibition of the N-methyl-D-aspartate-stimulated 22Na+ influx by DL-aminosuberate (I) and 2-aminophosphonovalerate (O). Each point is the average of quadruplicate determinations. N-methyl-D-aspartate concentration: 0.5 mM.
232
establish the pharmacological specificity of the Na + influx produced in chick retinal ceils by the excitatory amino acid agonists, the effects of the latter were measured in the presence of glutamatergic antagonists. The profiles of inhibition of the responses of L-glutamate, kainate and N M D A produced by these antagonists are displayed in Fig. 3. Antagonists of the kainate response such as secobarbital and 3-hydroxy-2-quinoxalinecarboxylic acid (Teichberg et al., 1984; Erez et al., 1985) were also tested and found to reduce the kamateinduced 22Na+ influx by 50% and 70%, respectively. Fig. 4 shows the inhibition of the NMDAinduced Z2Na+ influx produced by increasing concentrations of 2-aminophosphonovalerate and 2aminosuberate. The results obtained establish that the chick embryo retinal receptors for excitatory amino acids display the same pharmacological specificity as the amphibian and mammalian receptors (Watkins and Evans, 1981).
Discussion
The 22Na+ influx assay of functional excitatory amino acid receptors is based on the property of excitatory amino acids to depolarize neurons by increasing their membrane permeability to sodium ions (Puil, 1981). Theoretically, the increased traffic of sodium ions, induced by excitatory amino acids across the neuronal membrane, can be monitored in neuronal cell cultures, using 22Na+ as tracer ions, provided that the following 3 requirements are fulfilled: (1) the resting membrane permeability to sodium ions must be small; (2) the cultured neurons must express excitatory amino acid receptors on their surface; and (3) the intracellular compartments accessible to sodium ions must be large. Practically, the chick embryo retinal cells in culture fulfill these requirements. After 48-72 h in culture, the retinal cells express on their surface receptors sensitive to kainate, Lglutamate and N M D A but not to quisqualate. Their resting membrane permeability to sodium ions is relatively small since the 22Na+ influx can be increased more than 10-fold by kainate. When the retinal cells are plated at the density of 3 x 10 6 cells/well, the intracellular compartments accessi-
ble to 22Na+ ions allow the incorporation of up to 1500 cpm. This result is very close to the theoretical value derived from calculations based on the assumptions that all the plated cells express glutamatergic receptors on their surface, are spherical, have an average diameter of 5 /,m and are packed in a monolayer on the bottom and sides of the well. For the practical purpose of assaying glutamate receptors, the level of incorporation of 22Na+ ions obtained with 3 X 10 6 cells/well prohibits the plating of cells at a lower density. This restriction represents in fact the major drawback of the assay since it imposes a time limit for the performance of the assay as retinal cells cannot be maintained in a viable state for more than 4 days in culture at a high density. However, within this time limit, the 22Na+ influx assay exhibits advantages of reliability, ease and rapidity of execution and drug parsimony. The 22Na+ influx assay is reliable since the pharmacological properties displayed by the excitatory amino acid receptors on chick embryo retinal cells are similar to those described for amphibian and mammalian receptors (Watkins and Evans, 1981). It is important to stress nevertheless that quisqualate did not produce any significant 22Na+ influx in retinal cells in spite of the fact that the latter are sensitive to its neurotoxic effects (Olney, 1988). It is possible that the effects of quisqualate in the chick embryo retina are mediated by the activation of a metabotropic receptor (Nicoletti et al., 1986: Sugiyama et al., 1987). Another point concerns the rather high N M D A concentrations needed to elicit a 22Na~ influx. We attribute this result to the presence in the culture medium of magnesium ions which are well known to antagonize the N M D A response (Nowak et al., 1984). The 22Na+ influx assay is easy and fast to perform since it allows a single investigator to perform, in one day, measurements of the effects of known or putative glutamatergic ligands in more than 100 different conditions. It is parsimonious in terms of the volume (0.1 ml/well) and thus of amount of drug applied. We have already applied this microtest plate assay in one of the instances imposing strict requirements both on the quantity of ligand available for the test (small), on
233 the number of tests (large) and on the screening t i m e ( s h o r t ) w h i c h is t h e s e l e c t i o n o f h y b r i d o m a s secreting monoclonal antibodies directed against excitatory amino acid receptors (Eshhar and T e i c h b e r g , to b e p u b l i s h e d ) . A future application could now be the use of c h i c k e m b r y o r e t i n a l cells, c u l t u r e d i n m i c r o t e s t plates, in an automated assay of functional excitatory amino acid receptors.
Acknowledgements This work was supported by a grant from Makor Chemicals+ V.I.T. holds the Louis and Florence Katz-Cohen professorial chair in neuropharmacology.
References Erez, U., Frenk, H.. Goldberg, O., Cohen, A. and Teichberg, V.I. (1985) Anticonvulsant properties of 3-hydroxy-2quinoxalinecarboxylic acid, a newly found antagonist of excitatory amino acids, Eur. J. Pharmacol., 110: 31-39. Foster, A.C. and Fagg, G.E. (1984) Acidic amino acid binding sites in mammalian neuronal membranes: their characteristics and relationship to synaptic receptors, Brain Res. Rev., 7: 103-164. Foster, G.A. and Roberts, P.J. (1980) Pharmacology of excitatory amino acid receptors mediating the stimulation of rat cerebellar cyclic GMP levels, Life Sci., 27: 215-221. Gallo, V., Suergiu, R., Giovannini, C. and Levi, G. (1987) Glutamate receptor subtypes in cultured cerebellar neurons: modulation of glutamate and v-aminobutyric release, J. Neurochem., 49: 1801-1808. Garthwaite, J. (1982) Excitatory amino acid receptors and guanosine 3',5'-cyclic monophosphate in incubated slices of immature and adult rat cerebellum, Neuroscience, 7: 2491-2497. Ichida, S., Tokunaga, H., Moriyama, M., Oda, Y., Tanaka, S. and Kita, T. (1982) Effects of neurotransmitter candidates on 45Ca uptake by cortical slices of rate brain: stimulatory effect of L-glutamic acid, Brain Res.+ 248: 305-311. Luini, A., Goldberg, O. and Teichberg+ V.I. (1981) Distinct pharmacological properties of excitatory amino acid recep-
tors in the rate striatum: study by Na + efflux assay, Proc. Natl. Acad. Sci. U.S.A., 78: 3250-3254. MacDonald, J.F. and Wojtowicz, J.M. (1982) The effect of L-glutamate and its analogues upon the membrane conductance of central murine neurones in culture. Can. J. Physiol. Pharmacol., 60: 282-296. McBean, G.J. and Roberts, P.J. (1981) Glutamate preferring receptors regulate the release of D-3H-aspartate from rat hippocampal slices, Nature (Lond.) 291: 593-594. Nicoletti, F., Wroblewski, J.T., Novelli, A., Alho, H., Guidotti. A. and Costa, E. (1986) The activation of inositol phospholipid metabolism as a signal transducing system for excitatory amino acids in primary cultures of cerebellar granule cells, J. Neurosci., 6: 1905-1911. Nowak, L+, Bregestovski, P.+ Ascher, P., Herbet, A. and Prochiantz, A. (1984) Magnesium gates glutamate-activated channels in mouse central neurones, Nature (Lond.) 307: 462-463. Olney, J.W. (1988) Endogenous excitotoxins and neuropathological disorders. In D. Lodge (Ed.), Excitatory Amino Acids in Health and Disease, Wiley, New York, pp. 337-351. Puil, S. (1981) S-Glutamate: its interactions with spinal neurons, Brain Res. Rev., 3: 229-433. Roberts, P.J. and Anderson, S.D. (1979) Effects of glutamate and related amino acids on 3H-dopamine release from rat striatum: an in vitro model for glutamate actions, J. Neurochem., 32: 1539-1545. Scatton, B. and Lehmann, J. (1982) N-MethyI-D-aspartate type receptors mediate striatal 3H-acetylcholine release evoked by excitatory amino acids, Nature (Lond.) 297: 422-424. Sladeczek, F., Pin, J.P., Recasens, M., Bockaert, J. and Weiss, S. (1985) Glutamate stimulates inositol phosphate formation in striatal neurones, Nature (Lond.), 317: 717-718. Sugiyama, H., Ito, I. and Hirono, C. (1987) A new type of glutamate receptor linked to inositol phospholipid metabolism, Nature (Lond.), 325: 531-533. Teichberg, V.I,, Tal, N., Goldberg, O. and Luini, A. (1984) Barbiturates, alcohols and the CNS excitatory, neurotransmission: specific effects on the kainate and quisqualate receptors+ Brain Res., 291: 285-292. Watkins, J.C. and Evans, R.H. (1981) Excitatory amino acid transmitters, Annu. Rev. Pharmacol. Toxicol., 21: 165-204. Wroblewski, J.T., Nicoletti, F. and Costa, E. (1985l Different coupling of excitatory amino acid receptors with Ca 2+ channels in primary cultures of cerebellar granule cells, Neuropharmacology, 24: 919-921. Yellen, G. (1984) Channels from genes: the oocyte as an expression system, Trends Neurosci., 7: 457-458.
229
NSM 00941
A microtest plate assay of functional excitatory amino acid receptors N o m i E s h h a r , M o s h e L u s k y , P e e r D a v i d a n d V i v i a n I. T e i c h b e r g Department of Neurobiology, The Weizmann Institute of Science, Rehovot (Israel)
(Received 21 June 1988) (Revised 18 October 1988) (Accepted 21 October 1988)
Key words: Excitatory amino acid; Cultured chick retinal cell; Microtest plate assay
A microtest plate assay of functional excitatory amino acid receptors present on cultured chick embryo retinal cells has been developed. It is based on measurements of excitatory amino acid-mediated increase in 22Na+ influx into retinal cells adhering to each of the 96 wells of microtest plates. Dose-dependent responses to L-glutamate, kainate and N-methyl-D-aspartate but not to quisqualate can be measured. These responses are selectively inhibited by antagonists of excitatory amino acids. The assay is reliable, fast to perform and parsimonious in terms of the volume and thus of amount of the drug applied. It allows a single investigator to perform, in one day, measurements of the effects of known or putative glutamatergic ligands in more than 100 different conditions.
Introduction
The interactions of glutamatergic ligands, agonists and antagonists, with the m e m b r a n o u s excitatory amino acid receptors can be studied n o w a d a y s by a battery of electrophysiological and biochemical methods. The former include intracellular recordings under voltage clamp (McD o n a l d and Wojtowicz, 1982), single-channel recordings ( N o w a k et al., 1984) and use of m R N A injected Xenopus oocytes for the study of de n o v o synthesized receptors and their gating mechanisms (Yellen, 1984). The biochemical methods include binding techniques with various radioactive agonists, antagonists or modulators (Fagg and Foster, 1984), measurements of sodium (Luini et
Correspondence: V.I. Teichberg, Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel.
al., 1981) and calcium ion (Ichida et al., 1982; Wroblewski et al., 1985) fluxes, of cyclic G M P formation (Foster and Roberts, 1980; Garthwaite, 1982), of phosphatidylinositide turnover (Sladeczek et al., 1985; Nicoletti et al., 1986), and of the excitatory a m i n o acid-induced release of genuine [acetylcholine (Scatton and L e h m a n n , 1982), d o p a m i n e (Roberts and Anderson, 1979), yaminobutyric acid (Gallo et al., 1987)] or false [D-aspartate (McBean and Roberts 1981)] neurotransmitters from synaptosomes or brain slices. In spite of this relative profusion of techniques, n o n e of them is applicable for the simultaneous screening of a large n u m b e r of putative glutamatergic ligands (agonists, antagonists, modulators) particularly when time and a m o u n t of ligand are limited. We describe here the use of chick e m b r y o retinal cell cultures maintained in 96-well microtest plates for the assay of functional glutamate receptors via measurements of excitatory amino acid-induced 22Na+ influx.
0165-0270/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
23{)
Methods
Retinal cell cultures Retinas from chick embryos (8-11 days old) were removed and incubated at 3 7 ° C in Puck's medium containing 0.125% trypsin and 0.002% DNAse in order to dissociate the retinal tissue into cells. After 15 min, the medium was removed by decantation and substituted twice with a Dulbecco's modified Eagle medium ( D M E M ) containing 10% fetal calf serum. The sedimented cell mass was then triturated by several passages through fired Pasteur pipettes and centrifuged for 10 min at 1000 g in a desk centrifuge. The pelleted cells were washed with D M E M and recentrifuged as before. The cells were resuspended in D M E M containing 10% fetal calf serum and 0.2% antibiotics (50000 units penicillin, 50000/~g streptomycin, 5000 units nystatin/ml stock solution) and filtered through a nylon mesh to remove debris. Cell yields were 50 × 106 cells/retina. After counting the viable cells excluding Trypan blue ( > 95%), the cell suspension was diluted to a density of 15 x 1 0 6 cells/ml and 0.2 ml aliquots were distributed in each of the 96 wells of Costar microtiter plates (catalog no. 2590) consisting of 12 removable strips, each containing 8 flat-bottom wells. Before the addition of the cells, the wells were coated with 0.2 ml of a 0.02 m g / m l poly-Llysine (average mol. wt. 70,000) solution in water. The plates were incubated at 37 ° C under 5% CO 2 for 24-96 h.
surements were always carried out in quadruplicate, i.e. two assay conditions per strip. The effect of excitatory amino acid agonists was expressed as a stimulation index corresponding to the ratio between the radioactivity associated with cells exposed to the agonist and that of cells exposed to the same medium without agonist.
Results
Fig. 1 shows the stimulation produced by 0.2 m M L-glutamate and 0.1 mM kainate on retinal cells removed from 9-day-old embryos and maintained in culture for 3-72 h: the effects of the excitatory amino acids, as expressed by the stimulation index, are very small 3 h after cell plating but reach significant values after 24 and 48 h. In the experiments with 72 h cultures, the average incorporation of 22Na+ is about 100 cpm in cells exposed for 5 rain to the radioactive medium without agonist and up to 1500 cpm in its presence at the highest concentration. If cells are plated at a density smaller than 3 x 106 cells/well and stimulated by an excitatory amino acid in the presence of 22Na+ ions, a proportionally smaller 16 X
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Assay of excitatory amino acid receptor function The presence of excitatory amino acid receptors on the cultured retinal cells was monitored via measurements of excitatory amino acid induced 22Na+ influx. For this purpose, the plates were washed twice with D M E M to remove all non-adhering ceils and exposed at t = 0 rain, to 0.1 ml of D M E M containing 0.25/~Ci of 2 2 N a + (Amersham) and an excitatory amino acid agonist, either alone or together with an antagonist. At t = 5 min, the radioactive medium was aspirated and discarded and the cells washed twice with DMEM. This washing solution was also discarded and 0.1 ml 1 M N a O H was added. The latter solution was removed and its radioactivity measured. All mea-
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H o u r s in c u l t u r e Fig. 1. In vitro development of the sensitivity of cultured chick embryo retinal cells to excitatory amino acids. Retinal cells were prepared from 9-day-old retinas, plated in microtest plates at the density of 3 x 106 cells/well and maintained in culture for up to 4 days. They were exposed for 5 min to a 22Na+ containing D M E M m e d i u m in the absence or presence of either 0.1 m M kainate (striped columns) or 0.2 m M Lglutamate (empty columns). The stimulation index is the ratio of the radioactivity associated with cells exposed to the agonist and that of ceils exposed to the same medium without agonist.
231
incorporation of radioactivity takes place. However, the plating of a larger number of cells per well does not improve the amount of radioactivity i n c o r p o r a t e d indicating that with 3 x 106 cells/well the maximal adhering cell density has been reached. The observed increase of the stimulation index as a function of time in culture is possibly a reflection of one or the combination of the following factors: the rate of recovery of the membrane potential following the trypsinization of the cell suspension, the rate of de novo synthesis of excitatory amino acid receptors and the timing of appearance of these receptors in the chick embryo. The first two factors may explain the finding that retinal cells are not sensitive to excitatory amino acids for several hours after plating while the third factor may account for the fact that day 10 retinal cells/48 h in culture are stimulated to approximately twice the level of stimulation as day 9 retinas/48 h in culture, whereas equal levels of stimulation are reached with cells of equal age (cells from day 9 e m b r y o / 7 2 h in culture are stimulated to the same extent as cells from day 10 e m b r y o / 4 8 h in culture). With cells maintained in culture for more than 4 days, a significant decrease in the apparent sensitivity to excitatory amino acids is observed and is correlated to a deterioration of the cultured cells and their detachment from the well walls. Fig. 2 shows dose-response curves obtained with 3 excitatory
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amino acids, kainate, L-glutamate and N-methylD-aspartate (NMDA), on day 9 retinal cells/48 h in culture. Their order of effectiveness as agonists is kainate > L-glutamate > N M D A . Quisqualate was also tested but was found to be, even at 10 mM, an extremely weak agonist. In order to ,<
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Fig. 2. Dependence of the stimulated 22Na+ influx on the concentration of kainate (I), [+-glutamate (i) and N-methylD-aspartate (~). Day 9 chick embryo retinal cells maintained 48 h in culture• Each point is the average of quadruplicate determinations.
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232
establish the pharmacological specificity of the Na + influx produced in chick retinal ceils by the excitatory amino acid agonists, the effects of the latter were measured in the presence of glutamatergic antagonists. The profiles of inhibition of the responses of L-glutamate, kainate and N M D A produced by these antagonists are displayed in Fig. 3. Antagonists of the kainate response such as secobarbital and 3-hydroxy-2-quinoxalinecarboxylic acid (Teichberg et al., 1984; Erez et al., 1985) were also tested and found to reduce the kamateinduced 22Na+ influx by 50% and 70%, respectively. Fig. 4 shows the inhibition of the NMDAinduced Z2Na+ influx produced by increasing concentrations of 2-aminophosphonovalerate and 2aminosuberate. The results obtained establish that the chick embryo retinal receptors for excitatory amino acids display the same pharmacological specificity as the amphibian and mammalian receptors (Watkins and Evans, 1981).
Discussion
The 22Na+ influx assay of functional excitatory amino acid receptors is based on the property of excitatory amino acids to depolarize neurons by increasing their membrane permeability to sodium ions (Puil, 1981). Theoretically, the increased traffic of sodium ions, induced by excitatory amino acids across the neuronal membrane, can be monitored in neuronal cell cultures, using 22Na+ as tracer ions, provided that the following 3 requirements are fulfilled: (1) the resting membrane permeability to sodium ions must be small; (2) the cultured neurons must express excitatory amino acid receptors on their surface; and (3) the intracellular compartments accessible to sodium ions must be large. Practically, the chick embryo retinal cells in culture fulfill these requirements. After 48-72 h in culture, the retinal cells express on their surface receptors sensitive to kainate, Lglutamate and N M D A but not to quisqualate. Their resting membrane permeability to sodium ions is relatively small since the 22Na+ influx can be increased more than 10-fold by kainate. When the retinal cells are plated at the density of 3 x 10 6 cells/well, the intracellular compartments accessi-
ble to 22Na+ ions allow the incorporation of up to 1500 cpm. This result is very close to the theoretical value derived from calculations based on the assumptions that all the plated cells express glutamatergic receptors on their surface, are spherical, have an average diameter of 5 /,m and are packed in a monolayer on the bottom and sides of the well. For the practical purpose of assaying glutamate receptors, the level of incorporation of 22Na+ ions obtained with 3 X 10 6 cells/well prohibits the plating of cells at a lower density. This restriction represents in fact the major drawback of the assay since it imposes a time limit for the performance of the assay as retinal cells cannot be maintained in a viable state for more than 4 days in culture at a high density. However, within this time limit, the 22Na+ influx assay exhibits advantages of reliability, ease and rapidity of execution and drug parsimony. The 22Na+ influx assay is reliable since the pharmacological properties displayed by the excitatory amino acid receptors on chick embryo retinal cells are similar to those described for amphibian and mammalian receptors (Watkins and Evans, 1981). It is important to stress nevertheless that quisqualate did not produce any significant 22Na+ influx in retinal cells in spite of the fact that the latter are sensitive to its neurotoxic effects (Olney, 1988). It is possible that the effects of quisqualate in the chick embryo retina are mediated by the activation of a metabotropic receptor (Nicoletti et al., 1986: Sugiyama et al., 1987). Another point concerns the rather high N M D A concentrations needed to elicit a 22Na~ influx. We attribute this result to the presence in the culture medium of magnesium ions which are well known to antagonize the N M D A response (Nowak et al., 1984). The 22Na+ influx assay is easy and fast to perform since it allows a single investigator to perform, in one day, measurements of the effects of known or putative glutamatergic ligands in more than 100 different conditions. It is parsimonious in terms of the volume (0.1 ml/well) and thus of amount of drug applied. We have already applied this microtest plate assay in one of the instances imposing strict requirements both on the quantity of ligand available for the test (small), on
233 the number of tests (large) and on the screening t i m e ( s h o r t ) w h i c h is t h e s e l e c t i o n o f h y b r i d o m a s secreting monoclonal antibodies directed against excitatory amino acid receptors (Eshhar and T e i c h b e r g , to b e p u b l i s h e d ) . A future application could now be the use of c h i c k e m b r y o r e t i n a l cells, c u l t u r e d i n m i c r o t e s t plates, in an automated assay of functional excitatory amino acid receptors.
Acknowledgements This work was supported by a grant from Makor Chemicals+ V.I.T. holds the Louis and Florence Katz-Cohen professorial chair in neuropharmacology.
References Erez, U., Frenk, H.. Goldberg, O., Cohen, A. and Teichberg, V.I. (1985) Anticonvulsant properties of 3-hydroxy-2quinoxalinecarboxylic acid, a newly found antagonist of excitatory amino acids, Eur. J. Pharmacol., 110: 31-39. Foster, A.C. and Fagg, G.E. (1984) Acidic amino acid binding sites in mammalian neuronal membranes: their characteristics and relationship to synaptic receptors, Brain Res. Rev., 7: 103-164. Foster, G.A. and Roberts, P.J. (1980) Pharmacology of excitatory amino acid receptors mediating the stimulation of rat cerebellar cyclic GMP levels, Life Sci., 27: 215-221. Gallo, V., Suergiu, R., Giovannini, C. and Levi, G. (1987) Glutamate receptor subtypes in cultured cerebellar neurons: modulation of glutamate and v-aminobutyric release, J. Neurochem., 49: 1801-1808. Garthwaite, J. (1982) Excitatory amino acid receptors and guanosine 3',5'-cyclic monophosphate in incubated slices of immature and adult rat cerebellum, Neuroscience, 7: 2491-2497. Ichida, S., Tokunaga, H., Moriyama, M., Oda, Y., Tanaka, S. and Kita, T. (1982) Effects of neurotransmitter candidates on 45Ca uptake by cortical slices of rate brain: stimulatory effect of L-glutamic acid, Brain Res.+ 248: 305-311. Luini, A., Goldberg, O. and Teichberg+ V.I. (1981) Distinct pharmacological properties of excitatory amino acid recep-
tors in the rate striatum: study by Na + efflux assay, Proc. Natl. Acad. Sci. U.S.A., 78: 3250-3254. MacDonald, J.F. and Wojtowicz, J.M. (1982) The effect of L-glutamate and its analogues upon the membrane conductance of central murine neurones in culture. Can. J. Physiol. Pharmacol., 60: 282-296. McBean, G.J. and Roberts, P.J. (1981) Glutamate preferring receptors regulate the release of D-3H-aspartate from rat hippocampal slices, Nature (Lond.) 291: 593-594. Nicoletti, F., Wroblewski, J.T., Novelli, A., Alho, H., Guidotti. A. and Costa, E. (1986) The activation of inositol phospholipid metabolism as a signal transducing system for excitatory amino acids in primary cultures of cerebellar granule cells, J. Neurosci., 6: 1905-1911. Nowak, L+, Bregestovski, P.+ Ascher, P., Herbet, A. and Prochiantz, A. (1984) Magnesium gates glutamate-activated channels in mouse central neurones, Nature (Lond.) 307: 462-463. Olney, J.W. (1988) Endogenous excitotoxins and neuropathological disorders. In D. Lodge (Ed.), Excitatory Amino Acids in Health and Disease, Wiley, New York, pp. 337-351. Puil, S. (1981) S-Glutamate: its interactions with spinal neurons, Brain Res. Rev., 3: 229-433. Roberts, P.J. and Anderson, S.D. (1979) Effects of glutamate and related amino acids on 3H-dopamine release from rat striatum: an in vitro model for glutamate actions, J. Neurochem., 32: 1539-1545. Scatton, B. and Lehmann, J. (1982) N-MethyI-D-aspartate type receptors mediate striatal 3H-acetylcholine release evoked by excitatory amino acids, Nature (Lond.) 297: 422-424. Sladeczek, F., Pin, J.P., Recasens, M., Bockaert, J. and Weiss, S. (1985) Glutamate stimulates inositol phosphate formation in striatal neurones, Nature (Lond.), 317: 717-718. Sugiyama, H., Ito, I. and Hirono, C. (1987) A new type of glutamate receptor linked to inositol phospholipid metabolism, Nature (Lond.), 325: 531-533. Teichberg, V.I,, Tal, N., Goldberg, O. and Luini, A. (1984) Barbiturates, alcohols and the CNS excitatory, neurotransmission: specific effects on the kainate and quisqualate receptors+ Brain Res., 291: 285-292. Watkins, J.C. and Evans, R.H. (1981) Excitatory amino acid transmitters, Annu. Rev. Pharmacol. Toxicol., 21: 165-204. Wroblewski, J.T., Nicoletti, F. and Costa, E. (1985l Different coupling of excitatory amino acid receptors with Ca 2+ channels in primary cultures of cerebellar granule cells, Neuropharmacology, 24: 919-921. Yellen, G. (1984) Channels from genes: the oocyte as an expression system, Trends Neurosci., 7: 457-458.