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Potentiation of N-methyl-d-aspartate-evoked elevation of intracellular Ca2+ concentrations by exogenous glycine in cerebellar granule cells
European Journal of Pharmacology - Molecular Pharmacology Section, 266 (1994) 309-315
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© 1994 Elsevier Science B.V. All rights reserved 0922-4106/94/$07.00
EJPMOL 90585
Potentiation of N-methyl-D-aspartate-evoked elevation of intracellular Ca 2+ concentrations by exogenous glycine in cerebellar granule cells P e t e r B. S i m p s o n *, R.A. J o h n Challiss a n d S t e f a n R. N a h o r s k i Department of Cell Physiology and Pharmacology, P.O. Box 138, Medical Sciences Building, University of Leicester, Uniuersity Road, Leicester, LE1 9HN, UK Received 19 May 1993; accepted 2 November 1993)
The effect of glycine on the intracellular free Ca 2÷ c o n c e n t r a t i o n ([Ca2+])i response to N-methyl-D-aspartate (NMDA) was examined in small groups of cerebeilar granule cells loaded with fura 2. NMDA alone evoked a long-lasting monophasic [Ca2+] i plateau, which was abolished by removal of extracellular Ca 2+, or addition of the NMDA channel antagonist dizocilpine or the glycine site antagonist 5,7-dichlorokynurenic acid, virtually unaffected by the L-type Ca 2+ channel antagonist (-)-PN 202 791, and greatly, though variably, potentiated by addition of glycine. In the presence of glycine the response to NMDA was clearly biphasic. However, there was no consistent relationship between the magnitudes of the peak and plateau phases of the response, and their temporal relationship was also highly variable. The potentiation seen with exogenous glycine was highly dependent on plating density, which may be the result of higher levels of endogenous glycine in more dense cultures. Our results provide an explanation of the inconsistent findings previously reported by different groups on the potentiation of the [Ca2+]i response to NMDA by exogenous glycine. NMDA (N-Methyl-D-aspartate); Glycine; Ca 2+; Ca 2 + entry; Cerebellar granule cells; Cell culture
I. Introduction The neuronal glutamate receptor for which Nmethyl-D-aspartate (NMDA) is a specific agonist can be regulated by a complex array of modulators that exert their effects through distinct recognition loci within the r e c e p t o r / c h a n n e l structure (Wroblewski and Danysz, 1989). Of these, the glycine site, which has been shown to reside on the same protein as the glutamate recognition site (Moriyoshi et al., 1991), is by far the best characterized (Kemp and Leeson, 1993). Occupation of the glycine site is believed to be essential for receptor activation (Kleckner and Dingledine, 1988). Several groups have suggested that antagonists of the glycine site may be useful clinically in a variety of conditions in which the N M D A receptor is implicated, as they possess the anticonvulsant and anxiolytic properties commonly seen with N M D A antagonists, but display a substantially different behavioural profile and may lack the side effects of blockers of the N M D A receptor channel (Sheardown et al., 1989; Singh et al., 1990a,b; Kehne et al., 1991).
* Corresponding author. Tel.: (0533) 522920; Fax: (0533) 523996.
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It has been thought that in vivo the glycine site may normally be fully saturated, as glycine is present at micromolar levels in cerebrospinal fluid (Ferraro and Hare, 1985). However, direct measurement of the synaptic concentration is not yet technically possible, and experiments such as the ionophoresis of N M D A with and without exogenous glycine may result in tissue damage and subsequent release of glycine, thus rendering conclusions on normal glycine concentrations unreliable. There is evidence that at some cerebellar synapses the glycine site may not be normally saturated, as spontaneous synaptic activity in cerebellar granule cells in slice preparations, mediated via N M D A receptors, is only seen when exogenous glycine is perfused onto the ceils (D'Angelo et al., 1990). Also, intracerebellar injection of the glycine site agonist Dserine potentiates the effect of endogenous N M D A on cerebellar cyclic GMP levels (Wood et al., 1989). A high-affinity glycine uptake carrier is present in the cerebellum, mainly on Golgi cell axon terminals, which could regulate the synaptic concentration (Wilkin et al., 1981). Primary cultures of rat cerebellar granule cells have been reported to be a highly homogeneous preparation (Kingsbury et al., 1985) and to possess several types of
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glutamate receptor including those responsive to NMDA (Wroblewski et al., 1985; Cull-Candy et al., 1988). They therefore provide a useful model system for the investigation of the functional consequences of glutamate receptor activation. In the present study we have investigated NMDA receptor-mediated changes in intracellular free Ca 2÷ concentration ([Ca2÷] i) in small groups of cultured rat cerebellar granule cells, and the actions of glycine and glycine site antagonists on these responses.
2. Materials and methods
2.1. Materials Reagents of analytical grade and double-distilled water were used throughout. Basal modified Eagle's medium, foetal calf serum, penicillin/streptomycin and glutamine were from Gibco (UK). N-Methyl-D-aspartate, dizocilpine and poly(D-lysine) were from Sigma Chemical Co., UK. Glycine was from Fisons (UK). 5,7-Dichlorokynurenic acid and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were from Tocris Neuramin (UK). Isomers of PN 202 791 were a generous gift from Sandoz (Basel, Switzerland).
2.2. Cell culture Cerebellar granule cells were prepared and cultured as described previously (Thangnipon et al., 1983; Whitham et al., 1991), a method which results in approximately 95% of the surviving cells being cerebellar
granule cells (Kingsbury et al., 1985; E.M. Whitham, R.A.J. Challiss and S.R. Nahorski, unpublished observations). Cells were plated out into 8-well multiwell plates (each well measuring 860 mm 2) in which had been placed glass cover-slips, precoated with poly(olysine), at a density of 3 × 106 cells/well, except where otherwise stated.
2.3. Measurement of [Ca 2 ÷]i Cerebellar granule cells were incubated with 2 /~M fura 2-AM in Krebs-Henseleit buffer (KHB) for 20 min at room temperature, washed, and left for 30 rain to allow fura 2-AM de-esterification to reach completion. [Ca2+] i was measured by standard epifluorescence microscopy using the Photon Technology International Deltascan system, as described previously (Simpson et al., 1993). Experiments were performed on groups of 5-8 cells cultured for up to 10 days in vitro (DIV), optically isolated by shutters fitted to the photomultiplier housing, which were continuously perfused with Mg2--free KHB (composition in mM: Na ÷ 143.3, K ÷ 5.9, Ca :÷ 1.3, C1- 125.9, H2PO 4 2.2, HCO 3 24.9, SOn2- 1.2, glucose 10; pH 7.4). Fluorescence ratios were not calibrated to [Ca 2÷] because of the variability in results which can be obtained using different calibration methods. However, the range of ratios found in these experiments are within the range closely proportional to [Ca2+]. Example traces are shown as are changes in the ratio of fluorescence at 340 nm to fluorescence at 380 nm (y-axis) against time (x-axis). Results are presented as mean ___S.E.M. IC50 values were calculated using the GraphPad program.
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Fig. 1. Potentiation of the response to N M D A by exogenous glycine. Example trace for 8 0 0 / z M N M D A in the absence and presence of 10 ~ M glycine. Note that a transient peak phase was present only after addition of glycine, and not in response to N M D A alone. (The 'blip' at ~ 200 s is a rarely found artefact of the method of agent addition used).
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3. Results
marked shift in the N M D A concentration-response curve (data not shown).
3.1. Glycine potentiation o f the N M D A response
3.2. NMDA-evoked Ca e + entry
T h e [Ca2+]i response to N M D A was concentrationdependent, and consisted of a long-lasting monophasic plateau (see Fig. 1), sometimes preceded by a rapidly decaying peak. The effect of exogenous glycine on the N M D A response was examined by adding 10 ~ M glycine, sufficient to fully saturate the glycine site on the receptor (Kleckner and Dingledine, 1988; Baron et al., 1990), after the plateau was established. A large, though variable, potentiation of the response was seen, and for all concentrations of N M D A tested the response in the presence of glycine was clearly biphasic, with a rapid transient increase in [Ca2+]i always appearing before the sustained plateau (Fig. 1). The potentiation was partially, though not completely, removed when perfusion of glycine was ended. Glycine had no significant effect on its own (data not shown). While the size of the response to N M D A was greatly increased by exogenous glyeine (Fig. 1), there was no
Significant responses to N M D A were apparent as early as 1 day(s) in vitro (DIV), and displayed no significant change in size over all DIV examined up to 10 DIV. Addition of 200/~M N M D A in the presence of 10 /~M glycine induced a peak increase in the 340/380 nm fluorescence ratio of 2.03 + 0.13, and a plateau increase of 1.99 + 0.11, basal ratio 1.22 +_ 0.02 (n = 97). There appeared to be no consistent relationship between peak size and plateau size, and the temporal relationship between the two phases of the response was highly variable (Fig. 2). Addition of the N M D A channel antagonist dizocilpine during the plateau phase of the response to 200 /~M N M D A caused a concentration-dependent decrease in the [Ca2+] i elevation, such that with 10 ~ M dizocilpine the response was completely abolished (101 + 4% inhibition), and a response to N M D A was essentially absent in nominally Ca2+-free KHB (data not shown), con-
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Fig. 2. Variability of response to NMDA. Example traces, from different groups of cells, of responses to 200 ~ M N M D A (10 /.tM glycine was present throughout the experiment). Peak and plateau phases were always detected, but these varied in size and in temporal separation.
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(+) ~
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time (s) Fig. 3. Effect of the isomers of PN 202 791 on the [Ca 2+] response to N M D A . Example traces, ( + ) - i s o m e r trace shifted vertically for ease of comparison. 1 /zM of the ( + ) - i s o m e r increased both the peak and plateau phases of the response to 200/xM N M D A in the presence of 1 0 / z M glycine, whereas no inhibition of the N M D A peak response and little inhibition of the plateau response was seen on addition of the (-)-isomer.
firming that the increase in [Ca2+]i is wholly dependent on Ca 2+ entry. A previous report has suggested that the peak phase of the [Ca2+]i elevation evoked by NMDA is due to Ca 2+ entry through L-type Ca 2+ channels (Courtney et al., 1990). However, this possibility was not actually fully investigated in that paper, so we examined the involvement of Ca 2+ channels in the NMDA response, using the ( + ) and (-)-isomers of the dihydopyridine PN 202 791, which are, respectively, an agonist and an antagonist of L-type channels, and also the N-type channel blocker J2-conotoxin. At a concentration (1 /zM) at which the (+)-isomer of PN 202 791 potenti-
100"
ated both phases of the NMDA response, the (-)-isomer had no effect on the NMDA-evoked peak and caused only a small (7 + 3%) inhibition of the plateau (Fig. 3). 1/zM 12 conotoxin did not inhibit either phase of the response (data not shown).
3.3. Glycine site antagonists 5,7-Dichlorokynurenic acid is reported to be a potent and specific competitive antagonist of the glycine site, and to possess anticonvulsant properties in vivo (Baron et al., 1990, 1992). When added during the plateau phase of the response to 200 /zM NMDA, in
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Fig. 4. (a) Concentration-response curve for 5,7-dichlorokynurenic acid on N M D A response. 5,7-Dichlorokynurenic acid was added in increasing concentration during the plateau response to 200 /zM N M D A in the absence of exogenous glycine. The response was inhibited in a concentration-dependent manner, and at high concentrations of 5,7-dichlorokynurenic acid was completely abolished. (b) Addition of exogenous glycine can overcome the inhibitory effect of 5,7-dichlorokynurenic acid on NMDA-evoked increases in [Ca 2+ ]i- Example trace. 2 0 0 / z M N M D A was added in the absence of exogenous glycine, and a gradual-onset inhibition seen subsequent to addition of 100 n M 5,7-dichlorokynurenic acid. Exogenous glycine overcame the effect of 5,7-dichlorokynurenic acid in a concentration-dependent manner, and potentiated the response to N M D A as before (cf. Fig. 1).
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the absence of exogenous glycine, 5,7-dichlorokynurenic acid concentration-dependently decreased the [Ca2+] i elevation, with an IC50 of 8 nM (Fig. 4a). S u b m i c r o m o l a r c o n c e n t r a t i o n s of 5,7-dichlorokynurenic acid completely abolished the response to NMDA. Similar effects were seen using the a-amino3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, which is also active at the glycine site (Sheardown et al., 1989) (data not shown). The antagonism by 5,7-dichlorokynurenic acid of the [Ca2+] i elevation evoked by N M D A was overcome by exogenous glycine in a concentration-dependent manner (Fig. 4b). At high concentrations of glycine the potentiation seen in the presence of 5,7-dichlorokynurenic acid was similar to that previously described in its absence.
3. 4. Importance of plating density To examine whether the potentiation of the N M D A response by exogenous glycine was dependent on the density at which the cells were plated, we prepared culture plates from single cerebellar preparations, differing only in their plating density. The effects of stimulation by 200 /zM N M D A alone, and 200 /xM N M D A in the presence of 10 /xM exogenous glycine, were examined on cells from these cultures. The results of experiments on cells from a representative plating are shown in Fig. 5. There is a steep negative relationship between plating density and potentiation by exogenous glycine, with cells plated at high density having a significantly greater responsiveness to N M D A compared to cells plated at our normal density, without a correspondingly increased responsiveness to N M D A in the presence of glycine.
4. Discussion
Cerebellar granule cells are a widely used primary neuronal culture system, as they can be grown virtually devoid of other cell types and on this basis have been treated as a homogeneous preparation for investigations of [Ca2+] i homeostasis and other types of studies. However, most [CaZ+]i studies have involved population measurements, typically from several million cells (e.g., Holopainen et al., 1990; Courtney et al., 1990; Parks et al., 1991), so any variability in responsiveness between cells would not be detected. Also, in those experiments agonists were added into the bathing medium, rather than directly onto the cells, which may create a less precise time point for onset of action, decreasing the likelihood that any temporal variability in response would be detected even between cultures. In the work presented here, [Ca2+]i has been mea-
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Plating Density (xl0i cells / well) Fig. 5. Potentiation of the NMDA response by glycine is dependent on plating density. Direct comparison of the responsiveness to NMDA (black bars), and to NMDA in the presence of 10 /~M glycine (grey bars), for 3 different densities of plating. Results from one representative series of experiments are shown. Experimental procedure was as seen in Fig. 1. Increasing plating density caused increasing response to NMDA alone, but not increasing response to NMDA in the presence of 10 ~M exogenousglycine.
sured in small groups of 5 - 8 granule cells, and agents were added via a multi-line perfusion pipette positioned close to the cells in view. Although all groups of cells tested displayed a robust [Ca2+]i response to addition of NMDA, significant variability between groups was found with respect to the magnitude and pattern of the [Ca2+]i elevation. Peak and plateau sizes varied in an apparently unconnected manner, and the temporal relationship between these two phases also displayed considerable variability. Clearly further study of individual or small groups of cells will enable the variations in responsiveness present in cultured granule cells to be more completely described. While ligand binding experiments have demonstrated complex interactions between the glycine and N M D A binding sites (Monaghan et al., 1988; Grimwood et al., 1993), glycine potentiated the response to N M D A without markedly shifting the concentrationresponse curve in our hands, in accordance with previous functional reports (Johnson and Ascher, 1987; Parks et al., 1991). The response to N M D A in the presence of glycine was always biphasic in our experiments. This is in agreement with some previous reports (Burgoyne et al., 1988; Courtney et al., 1990), but several groups have repor,ed only a long-lasting plateau response (Holopainen et al., 1989; Parks et al., 1991; Baron et al., 1992; Irving et al., 1992). This does not appear to correspond to any consistent difference in the method of application of agonist, presence or absence of exogenous glycine, temperature at which experiments were performed, or number of cells used per experiment. Courtney et al. (1990) suggested that the
314 transient peak phase may be due to activation of L-type voltage-operated Ca 2+ channels. However, our own experiments indicate that the p e a k is not significantly decreased by maximally effective concentrations of an L-type, or indeed an N-type, channel blocker. T h e [Ca2+] i response to N M D A could be almost completely abolished by removal of extracellular Ca 2+, or addition of the channel antagonist dizocilpine, indicating that while the [Ca2+] i elevation may be partly due to release from intracellular stores (Simpson et al., 1993), it is d e p e n d e n t on the entry of Ca 2+, which in our hands appears to occur wholly t h r o u g h the N M D A r e c e p t o r / i o n channel complex. T h e N M D A response was also abolished by addition of the glycine site-specific antagonist 5,7-dichlorokynurenic acid, confirming that glycine is a co-agonist, rather than simply a modulator, of the N M D A receptor in these cells. 5,7-dichlorokynurenic acid had an ICs0 of 8 n M for inhibition of the [Ca2+]i response to N M D A , indicating that it is a m o r e p o t e n t functional antagonist of the glycine site than was suggested by its ability to inhibit [3H]-glycine binding ( K i = 70 nM) (Baron et al., 1992). In contrast to several previous reports (Courtney et al., 1990; H o l o p a i n e n et al., 1990; Ciardo and Meldolesi, 1991) but in a g r e e m e n t with o t h e r [Ca2+]i (Parks et al., 1991; Baron et al., 1992) and electrophysiological (Van der Valk et al., 1991) experiments, addition of exogenous glycine evoked a large t h o u g h variable potentiation of the response to N M D A in cerebellar granule cells in our hands, indicating that the glycine site was not saturated. The variability in the effect of exogenous glycine within cultures plated at 3 × 106 cells/well may be due to differences in glycine concentration, due to the characteristic aggregations of cells f o u n d in cerebellar granule cell cultures. T h e discrepancy between our findings and those of some other groups can be explained by the results presented here which d e m o n s t r a t e the importance of plating density on the potentiation induced by exogenous glycine. T h e response to N M D A alone was significantly higher in cultures plated more densely than in our standard density cultures. This was not due to increased maximal responsiveness to N M D A , as there is no significant change in the response to N M D A in the presence of 10 /~M glycine with changing density. T h e increased [Ca2+] i elevation evoked by application of N M D A alone is likely to be due to a higher m e a n concentration of glycine in the m o r e densely plated cultures. A l t h o u g h the different m e t h o d s of plating used make direct comparisons of densities difficult, it seems probable that other groups who have d e m o n s t r a t e d little or no potentiation by exogenous glycine were employing more densely plated cultures than ourselves. T h e steep relationship we have d e m o n s t r a t e d between granule cell plating density and glycine potentia-
tion provides an explanation of the inconsistent findings on the effects of addition of N M D A , and N M D A in the presence of exogenous glycine, on granule cells from different groups present in the literature. T h e study of small numbers of cells in our experiments, rather than large populations, and the use of a direct perfusion system, rather than addition of agonists into the bathing medium, have enabled us to detect considerable heterogeneity in the pattern of N M D A responsiveness in cultured granule cells.
Acknowledgements This work was supported by the provision of a Prize Studentship in Toxicology to P.B.S. and programme support to S.R.N. by the Wellcome Trust.
References Baron, B.M., B.L. Harrison, F.P. Miller, I.A. McDonald, F.G. Salituro, C.J. Schmidt, S.M. Sorensen, H.S. White and M.G. Palfreyman, 1990, Activity of 5,7-dichlorokynurenic acid, a potent antagonist at the N-Methyl-o-aspartate receptor-associated glycine binding site, Mol. Pharmacol. 38, 554. Baron, B.M., B.L. Harrison, I.A. McDonald, B.S. Meldrum, M.G. Palfreyman, F.G. Salituro, B.W. Siegel, A.L. Slone, J.P. Turner and H.S. White, 1992, Potent indole- and quinoline-containing N-methyl-o-aspartate antagonists acting at the strychnine-insensitive glycine binding site, J. Pharmacol. Exp. Ther. 262, 947. Burgoyne, R., I.A. Pearce and M. Cambray-Deakin, 1988, N-methylD-aspartate raises cytosolic calcium in rat cerebellar granule cells in culture, Neurosci. Lett. 91, 47. Ciardo, A. and J. Meldolesi, 1991, Regulation of intracellular calcium in cerebellar granule neurons: effects of depolarisation and of glutamatergic and cholinergic stimulation, J. Neurochem. 56, 184. Courtney, M.J., J.J. Lambert and D.G. Nicholls, 1990, The interactions between plasma membrane depolarisation and glutamate receptor activation in the regulation of cytoplasmic free calcium in cultured cerebellar granule cells, J. Neurosci. 10, 3873. Cull-Candy, S.G., J.R. Howe and D.C. Ogden, 1988, Noise and single channels activated by excitatory amino acids in rat cerebellar granule neurones, J. Physiol. (Lond.) 400, 189. D'Angelo, E., P. Rossi, and J. Garthwaite, 1990, Dual-component NMDA receptor currents at a single central synapse, Nature, 346, 467. Ferraro, T.N. and T.A. Hare, 1985, Free and conjugated amino acids in human CSF: Influence of age and sex, Brain Res. 338, 53. Grimwood, S., G.J.C. Wilde, A.C. Foster, 1993, Interactions between the glutamate and glycine recognition sites of the N-Methyl-Daspartate receptor from rat brain, as revealed from radioligand binding studies, J. Neurochem. 60, 1729. Holopainen, I., M. Louve, M.O.K. Enkvist and K.E.O. Akerman, 1990, Coupling of glutamatergic receptors to changes in intracellular Ca2+ in rat cerebellar granule cells in primary culture, J. Neurosci. Res. 25, 187. Irving, A.J., G.L. Collingridge and J.G. Schofield, 1992, L-Glutamate and acetylcholine mobilise Ca 2+ from the same intracellular pool in cerebellar granule cells using transduction mechanisms with different Caz+ sensitivities, Cell Calcium 13, 293. Johnson, J.W. and P. Ascher, 1987, Glycine potentiates the NMDA response in cultured mouse brain neurones, Nature 325, 529.
315 Kehne, J.H., T.C. McCloskey, B.M. Baron, E.M. Chi, B.L Harrison, J.P. Whitten and M.G. Palfreyman, 1991, NMDA receptor complex antagonists have potential anxiolytic effects as measured with separation-induced ultrasonic vocalizations, Eur. J. Pharmacol. 193, 283. Kemp, J.A. and P.D. Leeson, 1993, The glycine site of the NMDA receptor - five years on, Trends Pharmacol. Sci. 14, 20. Kingsbury, A., V. Gallo, P. Woodham and R. Balazs, 1985 , Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum supplemented medium, Dev. Brian Res. 17, 17. Kleckner, N.W., and R. Dingledine, 1988, Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes, Science 241,835. Monaghan, D.T., H.J. Olverman, L. Nguyen, J.C. Watkins, and C.W. Cotman, 1988, Two classes of N-methyl-D-aspartate recognition sites: Differential distribution and differential regulation by glycine, Proc. Natl. Acad. Sci. USA 85, 9836. Moriyoshi, K., M. Masu, T. Ishii, R. Shigemoto, N. Mizuno and S. Nakanishi, 1991, Molecular cloning and characterization of the rat NMDA receptor, Nature 354, 31. Parks, T.N., L.D. Artman, N. Alasti and E.F. Nemeth, 1991, Modulation of N-methyl-D-aspartate receptor-mediated increases in cytosolic calcium in cultured rat cerebellar granule cells, Brain Res. 552, 13. Sheardown, M.J., J. Drejer, L.H. Jensen, C.E. Stidsen and T. Honore, 1989, A potent antagonist of the strychnine insensitive glycine receptor has anticonvulsant properties, European J. Pharmacol. 174, 197. Simpson, P.B., R.A.J. Challiss and S.R. Nahorski, 1993, Involvement of intracellular stores in the Ca 2+ responses to N-methyl-Daspartate and depolarization in cerebellar granule cells, J. Neurochem. 61,760. Singh, L., A.E. Donald, A.C. Foster, P.H. Hutson, L.L. Iversen, S.D. Iversen, J.A. Kemp, P.D. Leeson, G.R. Marshall, R.J. Oles, T. Priestley, L. Thorn, M.D. Tricklebank, C.A. Vass and B.J.
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© 1994 Elsevier Science B.V. All rights reserved 0922-4106/94/$07.00
EJPMOL 90585
Potentiation of N-methyl-D-aspartate-evoked elevation of intracellular Ca 2+ concentrations by exogenous glycine in cerebellar granule cells P e t e r B. S i m p s o n *, R.A. J o h n Challiss a n d S t e f a n R. N a h o r s k i Department of Cell Physiology and Pharmacology, P.O. Box 138, Medical Sciences Building, University of Leicester, Uniuersity Road, Leicester, LE1 9HN, UK Received 19 May 1993; accepted 2 November 1993)
The effect of glycine on the intracellular free Ca 2÷ c o n c e n t r a t i o n ([Ca2+])i response to N-methyl-D-aspartate (NMDA) was examined in small groups of cerebeilar granule cells loaded with fura 2. NMDA alone evoked a long-lasting monophasic [Ca2+] i plateau, which was abolished by removal of extracellular Ca 2+, or addition of the NMDA channel antagonist dizocilpine or the glycine site antagonist 5,7-dichlorokynurenic acid, virtually unaffected by the L-type Ca 2+ channel antagonist (-)-PN 202 791, and greatly, though variably, potentiated by addition of glycine. In the presence of glycine the response to NMDA was clearly biphasic. However, there was no consistent relationship between the magnitudes of the peak and plateau phases of the response, and their temporal relationship was also highly variable. The potentiation seen with exogenous glycine was highly dependent on plating density, which may be the result of higher levels of endogenous glycine in more dense cultures. Our results provide an explanation of the inconsistent findings previously reported by different groups on the potentiation of the [Ca2+]i response to NMDA by exogenous glycine. NMDA (N-Methyl-D-aspartate); Glycine; Ca 2+; Ca 2 + entry; Cerebellar granule cells; Cell culture
I. Introduction The neuronal glutamate receptor for which Nmethyl-D-aspartate (NMDA) is a specific agonist can be regulated by a complex array of modulators that exert their effects through distinct recognition loci within the r e c e p t o r / c h a n n e l structure (Wroblewski and Danysz, 1989). Of these, the glycine site, which has been shown to reside on the same protein as the glutamate recognition site (Moriyoshi et al., 1991), is by far the best characterized (Kemp and Leeson, 1993). Occupation of the glycine site is believed to be essential for receptor activation (Kleckner and Dingledine, 1988). Several groups have suggested that antagonists of the glycine site may be useful clinically in a variety of conditions in which the N M D A receptor is implicated, as they possess the anticonvulsant and anxiolytic properties commonly seen with N M D A antagonists, but display a substantially different behavioural profile and may lack the side effects of blockers of the N M D A receptor channel (Sheardown et al., 1989; Singh et al., 1990a,b; Kehne et al., 1991).
* Corresponding author. Tel.: (0533) 522920; Fax: (0533) 523996.
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It has been thought that in vivo the glycine site may normally be fully saturated, as glycine is present at micromolar levels in cerebrospinal fluid (Ferraro and Hare, 1985). However, direct measurement of the synaptic concentration is not yet technically possible, and experiments such as the ionophoresis of N M D A with and without exogenous glycine may result in tissue damage and subsequent release of glycine, thus rendering conclusions on normal glycine concentrations unreliable. There is evidence that at some cerebellar synapses the glycine site may not be normally saturated, as spontaneous synaptic activity in cerebellar granule cells in slice preparations, mediated via N M D A receptors, is only seen when exogenous glycine is perfused onto the ceils (D'Angelo et al., 1990). Also, intracerebellar injection of the glycine site agonist Dserine potentiates the effect of endogenous N M D A on cerebellar cyclic GMP levels (Wood et al., 1989). A high-affinity glycine uptake carrier is present in the cerebellum, mainly on Golgi cell axon terminals, which could regulate the synaptic concentration (Wilkin et al., 1981). Primary cultures of rat cerebellar granule cells have been reported to be a highly homogeneous preparation (Kingsbury et al., 1985) and to possess several types of
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glutamate receptor including those responsive to NMDA (Wroblewski et al., 1985; Cull-Candy et al., 1988). They therefore provide a useful model system for the investigation of the functional consequences of glutamate receptor activation. In the present study we have investigated NMDA receptor-mediated changes in intracellular free Ca 2÷ concentration ([Ca2÷] i) in small groups of cultured rat cerebellar granule cells, and the actions of glycine and glycine site antagonists on these responses.
2. Materials and methods
2.1. Materials Reagents of analytical grade and double-distilled water were used throughout. Basal modified Eagle's medium, foetal calf serum, penicillin/streptomycin and glutamine were from Gibco (UK). N-Methyl-D-aspartate, dizocilpine and poly(D-lysine) were from Sigma Chemical Co., UK. Glycine was from Fisons (UK). 5,7-Dichlorokynurenic acid and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were from Tocris Neuramin (UK). Isomers of PN 202 791 were a generous gift from Sandoz (Basel, Switzerland).
2.2. Cell culture Cerebellar granule cells were prepared and cultured as described previously (Thangnipon et al., 1983; Whitham et al., 1991), a method which results in approximately 95% of the surviving cells being cerebellar
granule cells (Kingsbury et al., 1985; E.M. Whitham, R.A.J. Challiss and S.R. Nahorski, unpublished observations). Cells were plated out into 8-well multiwell plates (each well measuring 860 mm 2) in which had been placed glass cover-slips, precoated with poly(olysine), at a density of 3 × 106 cells/well, except where otherwise stated.
2.3. Measurement of [Ca 2 ÷]i Cerebellar granule cells were incubated with 2 /~M fura 2-AM in Krebs-Henseleit buffer (KHB) for 20 min at room temperature, washed, and left for 30 rain to allow fura 2-AM de-esterification to reach completion. [Ca2+] i was measured by standard epifluorescence microscopy using the Photon Technology International Deltascan system, as described previously (Simpson et al., 1993). Experiments were performed on groups of 5-8 cells cultured for up to 10 days in vitro (DIV), optically isolated by shutters fitted to the photomultiplier housing, which were continuously perfused with Mg2--free KHB (composition in mM: Na ÷ 143.3, K ÷ 5.9, Ca :÷ 1.3, C1- 125.9, H2PO 4 2.2, HCO 3 24.9, SOn2- 1.2, glucose 10; pH 7.4). Fluorescence ratios were not calibrated to [Ca 2÷] because of the variability in results which can be obtained using different calibration methods. However, the range of ratios found in these experiments are within the range closely proportional to [Ca2+]. Example traces are shown as are changes in the ratio of fluorescence at 340 nm to fluorescence at 380 nm (y-axis) against time (x-axis). Results are presented as mean ___S.E.M. IC50 values were calculated using the GraphPad program.
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Fig. 1. Potentiation of the response to N M D A by exogenous glycine. Example trace for 8 0 0 / z M N M D A in the absence and presence of 10 ~ M glycine. Note that a transient peak phase was present only after addition of glycine, and not in response to N M D A alone. (The 'blip' at ~ 200 s is a rarely found artefact of the method of agent addition used).
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3. Results
marked shift in the N M D A concentration-response curve (data not shown).
3.1. Glycine potentiation o f the N M D A response
3.2. NMDA-evoked Ca e + entry
T h e [Ca2+]i response to N M D A was concentrationdependent, and consisted of a long-lasting monophasic plateau (see Fig. 1), sometimes preceded by a rapidly decaying peak. The effect of exogenous glycine on the N M D A response was examined by adding 10 ~ M glycine, sufficient to fully saturate the glycine site on the receptor (Kleckner and Dingledine, 1988; Baron et al., 1990), after the plateau was established. A large, though variable, potentiation of the response was seen, and for all concentrations of N M D A tested the response in the presence of glycine was clearly biphasic, with a rapid transient increase in [Ca2+]i always appearing before the sustained plateau (Fig. 1). The potentiation was partially, though not completely, removed when perfusion of glycine was ended. Glycine had no significant effect on its own (data not shown). While the size of the response to N M D A was greatly increased by exogenous glyeine (Fig. 1), there was no
Significant responses to N M D A were apparent as early as 1 day(s) in vitro (DIV), and displayed no significant change in size over all DIV examined up to 10 DIV. Addition of 200/~M N M D A in the presence of 10 /~M glycine induced a peak increase in the 340/380 nm fluorescence ratio of 2.03 + 0.13, and a plateau increase of 1.99 + 0.11, basal ratio 1.22 +_ 0.02 (n = 97). There appeared to be no consistent relationship between peak size and plateau size, and the temporal relationship between the two phases of the response was highly variable (Fig. 2). Addition of the N M D A channel antagonist dizocilpine during the plateau phase of the response to 200 /~M N M D A caused a concentration-dependent decrease in the [Ca2+] i elevation, such that with 10 ~ M dizocilpine the response was completely abolished (101 + 4% inhibition), and a response to N M D A was essentially absent in nominally Ca2+-free KHB (data not shown), con-
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time (s) Fig. 3. Effect of the isomers of PN 202 791 on the [Ca 2+] response to N M D A . Example traces, ( + ) - i s o m e r trace shifted vertically for ease of comparison. 1 /zM of the ( + ) - i s o m e r increased both the peak and plateau phases of the response to 200/xM N M D A in the presence of 1 0 / z M glycine, whereas no inhibition of the N M D A peak response and little inhibition of the plateau response was seen on addition of the (-)-isomer.
firming that the increase in [Ca2+]i is wholly dependent on Ca 2+ entry. A previous report has suggested that the peak phase of the [Ca2+]i elevation evoked by NMDA is due to Ca 2+ entry through L-type Ca 2+ channels (Courtney et al., 1990). However, this possibility was not actually fully investigated in that paper, so we examined the involvement of Ca 2+ channels in the NMDA response, using the ( + ) and (-)-isomers of the dihydopyridine PN 202 791, which are, respectively, an agonist and an antagonist of L-type channels, and also the N-type channel blocker J2-conotoxin. At a concentration (1 /zM) at which the (+)-isomer of PN 202 791 potenti-
100"
ated both phases of the NMDA response, the (-)-isomer had no effect on the NMDA-evoked peak and caused only a small (7 + 3%) inhibition of the plateau (Fig. 3). 1/zM 12 conotoxin did not inhibit either phase of the response (data not shown).
3.3. Glycine site antagonists 5,7-Dichlorokynurenic acid is reported to be a potent and specific competitive antagonist of the glycine site, and to possess anticonvulsant properties in vivo (Baron et al., 1990, 1992). When added during the plateau phase of the response to 200 /zM NMDA, in
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Fig. 4. (a) Concentration-response curve for 5,7-dichlorokynurenic acid on N M D A response. 5,7-Dichlorokynurenic acid was added in increasing concentration during the plateau response to 200 /zM N M D A in the absence of exogenous glycine. The response was inhibited in a concentration-dependent manner, and at high concentrations of 5,7-dichlorokynurenic acid was completely abolished. (b) Addition of exogenous glycine can overcome the inhibitory effect of 5,7-dichlorokynurenic acid on NMDA-evoked increases in [Ca 2+ ]i- Example trace. 2 0 0 / z M N M D A was added in the absence of exogenous glycine, and a gradual-onset inhibition seen subsequent to addition of 100 n M 5,7-dichlorokynurenic acid. Exogenous glycine overcame the effect of 5,7-dichlorokynurenic acid in a concentration-dependent manner, and potentiated the response to N M D A as before (cf. Fig. 1).
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the absence of exogenous glycine, 5,7-dichlorokynurenic acid concentration-dependently decreased the [Ca2+] i elevation, with an IC50 of 8 nM (Fig. 4a). S u b m i c r o m o l a r c o n c e n t r a t i o n s of 5,7-dichlorokynurenic acid completely abolished the response to NMDA. Similar effects were seen using the a-amino3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, which is also active at the glycine site (Sheardown et al., 1989) (data not shown). The antagonism by 5,7-dichlorokynurenic acid of the [Ca2+] i elevation evoked by N M D A was overcome by exogenous glycine in a concentration-dependent manner (Fig. 4b). At high concentrations of glycine the potentiation seen in the presence of 5,7-dichlorokynurenic acid was similar to that previously described in its absence.
3. 4. Importance of plating density To examine whether the potentiation of the N M D A response by exogenous glycine was dependent on the density at which the cells were plated, we prepared culture plates from single cerebellar preparations, differing only in their plating density. The effects of stimulation by 200 /zM N M D A alone, and 200 /xM N M D A in the presence of 10 /xM exogenous glycine, were examined on cells from these cultures. The results of experiments on cells from a representative plating are shown in Fig. 5. There is a steep negative relationship between plating density and potentiation by exogenous glycine, with cells plated at high density having a significantly greater responsiveness to N M D A compared to cells plated at our normal density, without a correspondingly increased responsiveness to N M D A in the presence of glycine.
4. Discussion
Cerebellar granule cells are a widely used primary neuronal culture system, as they can be grown virtually devoid of other cell types and on this basis have been treated as a homogeneous preparation for investigations of [Ca2+] i homeostasis and other types of studies. However, most [CaZ+]i studies have involved population measurements, typically from several million cells (e.g., Holopainen et al., 1990; Courtney et al., 1990; Parks et al., 1991), so any variability in responsiveness between cells would not be detected. Also, in those experiments agonists were added into the bathing medium, rather than directly onto the cells, which may create a less precise time point for onset of action, decreasing the likelihood that any temporal variability in response would be detected even between cultures. In the work presented here, [Ca2+]i has been mea-
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Plating Density (xl0i cells / well) Fig. 5. Potentiation of the NMDA response by glycine is dependent on plating density. Direct comparison of the responsiveness to NMDA (black bars), and to NMDA in the presence of 10 /~M glycine (grey bars), for 3 different densities of plating. Results from one representative series of experiments are shown. Experimental procedure was as seen in Fig. 1. Increasing plating density caused increasing response to NMDA alone, but not increasing response to NMDA in the presence of 10 ~M exogenousglycine.
sured in small groups of 5 - 8 granule cells, and agents were added via a multi-line perfusion pipette positioned close to the cells in view. Although all groups of cells tested displayed a robust [Ca2+]i response to addition of NMDA, significant variability between groups was found with respect to the magnitude and pattern of the [Ca2+]i elevation. Peak and plateau sizes varied in an apparently unconnected manner, and the temporal relationship between these two phases also displayed considerable variability. Clearly further study of individual or small groups of cells will enable the variations in responsiveness present in cultured granule cells to be more completely described. While ligand binding experiments have demonstrated complex interactions between the glycine and N M D A binding sites (Monaghan et al., 1988; Grimwood et al., 1993), glycine potentiated the response to N M D A without markedly shifting the concentrationresponse curve in our hands, in accordance with previous functional reports (Johnson and Ascher, 1987; Parks et al., 1991). The response to N M D A in the presence of glycine was always biphasic in our experiments. This is in agreement with some previous reports (Burgoyne et al., 1988; Courtney et al., 1990), but several groups have repor,ed only a long-lasting plateau response (Holopainen et al., 1989; Parks et al., 1991; Baron et al., 1992; Irving et al., 1992). This does not appear to correspond to any consistent difference in the method of application of agonist, presence or absence of exogenous glycine, temperature at which experiments were performed, or number of cells used per experiment. Courtney et al. (1990) suggested that the
314 transient peak phase may be due to activation of L-type voltage-operated Ca 2+ channels. However, our own experiments indicate that the p e a k is not significantly decreased by maximally effective concentrations of an L-type, or indeed an N-type, channel blocker. T h e [Ca2+] i response to N M D A could be almost completely abolished by removal of extracellular Ca 2+, or addition of the channel antagonist dizocilpine, indicating that while the [Ca2+] i elevation may be partly due to release from intracellular stores (Simpson et al., 1993), it is d e p e n d e n t on the entry of Ca 2+, which in our hands appears to occur wholly t h r o u g h the N M D A r e c e p t o r / i o n channel complex. T h e N M D A response was also abolished by addition of the glycine site-specific antagonist 5,7-dichlorokynurenic acid, confirming that glycine is a co-agonist, rather than simply a modulator, of the N M D A receptor in these cells. 5,7-dichlorokynurenic acid had an ICs0 of 8 n M for inhibition of the [Ca2+]i response to N M D A , indicating that it is a m o r e p o t e n t functional antagonist of the glycine site than was suggested by its ability to inhibit [3H]-glycine binding ( K i = 70 nM) (Baron et al., 1992). In contrast to several previous reports (Courtney et al., 1990; H o l o p a i n e n et al., 1990; Ciardo and Meldolesi, 1991) but in a g r e e m e n t with o t h e r [Ca2+]i (Parks et al., 1991; Baron et al., 1992) and electrophysiological (Van der Valk et al., 1991) experiments, addition of exogenous glycine evoked a large t h o u g h variable potentiation of the response to N M D A in cerebellar granule cells in our hands, indicating that the glycine site was not saturated. The variability in the effect of exogenous glycine within cultures plated at 3 × 106 cells/well may be due to differences in glycine concentration, due to the characteristic aggregations of cells f o u n d in cerebellar granule cell cultures. T h e discrepancy between our findings and those of some other groups can be explained by the results presented here which d e m o n s t r a t e the importance of plating density on the potentiation induced by exogenous glycine. T h e response to N M D A alone was significantly higher in cultures plated more densely than in our standard density cultures. This was not due to increased maximal responsiveness to N M D A , as there is no significant change in the response to N M D A in the presence of 10 /~M glycine with changing density. T h e increased [Ca2+] i elevation evoked by application of N M D A alone is likely to be due to a higher m e a n concentration of glycine in the m o r e densely plated cultures. A l t h o u g h the different m e t h o d s of plating used make direct comparisons of densities difficult, it seems probable that other groups who have d e m o n s t r a t e d little or no potentiation by exogenous glycine were employing more densely plated cultures than ourselves. T h e steep relationship we have d e m o n s t r a t e d between granule cell plating density and glycine potentia-
tion provides an explanation of the inconsistent findings on the effects of addition of N M D A , and N M D A in the presence of exogenous glycine, on granule cells from different groups present in the literature. T h e study of small numbers of cells in our experiments, rather than large populations, and the use of a direct perfusion system, rather than addition of agonists into the bathing medium, have enabled us to detect considerable heterogeneity in the pattern of N M D A responsiveness in cultured granule cells.
Acknowledgements This work was supported by the provision of a Prize Studentship in Toxicology to P.B.S. and programme support to S.R.N. by the Wellcome Trust.
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