European Journal of Neuroscience, Vol. 8, pp. 2129-2136, 1996 @I European Neuroscience Association

Comparative Antagonism of Kainate-activated Kainate and AMPA Receptors in Hippocampal Neurons

Ana V. Paternainl, Angeles Vicentel, Elsebet 0. Nielsen2 and Juan Lermal 'Department of Neural Plasticity, lnstituto Cajal, Consejo Superior de lnvestigaciones CientRicas, Av. Doctor Arce 37, 28002-Madrid, Spain *Neurosearch A/S, DK-2600 Glostrup, Denmark

Keywords: kainate-selective receptor, NSI 02, NS394, CNQX, cyclothiazide, immunocytochemistry

Abstract Native kainate receptors expressed by cultured hippocampal cells were studied in the whole-cell configuration of the patch-clamp technique by using a fast perfusion system. About 80% of the neurons expressed kainate receptors independently of the time in culture (0-4 days), which coincided with the number of cells immunoreactive for a monoclonal antibody against the GluR5/6/7 subunits. Three types of cells were considered: neurons in which the rapid application of kainate induced a rapidly desensitizing current, cells in which kainate induced a more slowly rising, non-desensitizing, response and those in which a mixture of both responses was apparent. Steady responses induced by 300 pM kainate were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in a dose-dependent manner (ICs0 = 0.92 pM). CNQX was less potent in blocking transient kainate- induced responses (IC50 = 6.1 pM). Responses to kainate, whether steady or transient, were also inhibited by NS102, showing poor selectivity for the transient response (IC50 = 4.1 and 2.2 pM respectively). The new a- amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor antagonist NS394 was very potent in inhibiting steady kainate-induced currents (ICs0 = 0.45 pM), but was even more effective in preventing peak responses (IC50 = 0.1 3 pM). In contrast, cyclothiazide did not affect transient kainate-induced responses but did potentiate current induced by activation of AMPA receptors by AMPA or kainate. These results demonstrate the lack of complete selectivity amongst some available competitive antagonists for AMPA and kainate receptors, and indicate that kainate receptors expressed by hippocampal cells lack the cyclothiazide modulatory site present at AMPA receptors. In addition, the present data support the idea that low-affinity kainate binding sites in the brain correspond to receptor channels selectively activated by kainate.

Introduction In the central nervous system, the neurotransmitter glutamate is released at most excitatory synapses (Mayer and Westbrook, 1987; Collingridge and Lester, 1989), where it is recognized by different types of glutamate receptors. Distinct receptors having different pharmacological and biophysical properties may coexist in the same synaptic contact (Bekkers and Stevens, 1989; Jones and Baughman, 1991). Besides the subunits that make up NMDA and a-amino-3- hydroxy-5-methyl-4-isoxazole (AMPA) receptors, cloning experi- ments have provided evidence of the existence of two structurally distinct subunit types postulated to form non-NMDA, non-AMPA receptors in the mammalian brain. These have been named GluR5, GluR6 and GluR7, of which GluR6 forms homomeric channels activated by kainate but not by AMPA, and KAl and KA2, which do not form functional channels when expressed alone but bind kainate with high affinity (for review see Hollmann and Heinemann, 1994). The KAl and KA2 subunits are ca'pable of combining with GluR5 and GluR6, forming heteromeric receptors with emergent properties (Herb et al., 1992; Sakimura et al., 1992; Sommer et al., 1992). In sifu hybridization and immunocytochemical studies have

revealed that kainate receptor subunits are expressed by most central nervous system neurons, but the functional significance of kainate receptors is still not known.

The development of specific agonists and antagonists for NMDA receptors has made it possible to determine many of the processes in which these receptors are involved. However, separation of kainate and AMPA receptors in native cells has been difficult since both receptors are activated by the same collection of exogenous agonists, including kainate, domoate and quisqualate. For instance, kainate activates AMPA receptors, inducing a large, non-desensitizing response in central neurons. Such a response has been considered as being mediated by kainate receptors. However, we have previously presented evidence that hippocampal neurons express a class of glutamate receptors which are activated by kainate but not by AMPA (Lerma et al., 1993). These glutamate receptors coexist with AMPA receptors and show fast activation4esensitization kinetics, which is compatible with involvement in fast neurotransmission. Responses induced by kainate in hippocampal neurons at an age when AMPA receptors are not evident resemble currents produced when cloned

Correspondence to: Dr J. Lerma, as above

Received 21 March 1996, accepted 30 May 1996

2 130 Pharmacology of kainate receptors

kainate receptor subunits are expressed in mammalian cell lines. Recently, a combination of whole-cell patch-clamp recording and analysis of mRNA harvested from single identified hippocampal neurons using the polymerase chain reaction has revealed that native kainate receptors are most probably made up of GluR6 subunits (Ruano ef al., 1995).

Native kainate receptor channels are not well characterized from a functional or a pharmacological point of view. We have analysed the sensitivity of kainate and AMPA receptors to drugs which are antagonists or modulators of AMPA receptors to get an insight into the pharmacological and functional properties of these glutamate receptors. Similarly, we studied the action on kainate-induced responses of a selective displacer of low-affinity [3H]kainate binding in cortical neurons, NS 102 (5-nitro-6,7,8,9-tetrahydrobenzo(G)indole- 2,3,-dione-3-oxime) (Johansen et al., 1993). Our results show a lack of selectivity for competitive antagonism of non-NMDA glutamate receptors and evidence for the absence of the benzothiadiazide modulatory site in neuronal kainate receptors.

Materials and methods Hippocampal cultures Hippocampal neurons were mechanically dissociated from 17- to 18- day rat embryos after treatment with trypsin I (Sigma; 0.12 mg/ml, I5 min, 37°C) and seeded onto 35 mm Petri dishes previously coated with pOly-D-lySine (5 mg/ml) and laminin (4 pg/ml). The initial density was adjusted to lo3 cells/mm2. Cells were incubated in Dulbecco's modified Eagle medium supplemented with transfemn (0.1 mg/ml), insulin (5 mg/ml), putrescine (100 pM), progesterone (20 nM), Se02 (30 nM), ovalbumin (0.1%), glucose (3.3 mM), sodium pyruvate (1 mM), glutamine (4 mM) and antibiotics in a humidified incubator at 37°C and 5% CO2. Some cultures were prepared using a serum-free basal medium (NeurobasalTM, Gibco BRL) supplemented with B27 medium supplement for neurons (Gibco BRL; Brewer et al., 1993), 25 pM 2-mercaptoethanol and 0.5 mM glutamine. Neurobasal medium improved culture viability but no differences in results concerning glutamate receptors were observed.

Recordings and perfusion procedures Electrophysiological experiments were carried out up to 4 days after plating. Membrane currents were recorded at a membrane potential of -70 mV, using the whole-cell configuration of the patch-clamp technique (Hamill ef al., 1981) and a List EPC-7 amplifier. Series resistance during whole-cell recording was 8-20 MQ. However, as series resistance compensation did not notably improve the response amplitude or speed, it was not used. Currents were filtered at 1 kHz (two-pole Butterworth filter, -12 dB/octave) and transferred at a sampling rate of 2.5 kHz to a personal computer for analysis and display purposes using pClamp software (Axon Instruments, Foster City, CA). The cells were rapidly perfused using a linear array of six to eight glass tubes placed 200-300 pm from the soma. Ringer's solution with and without agonist flowed from adjacent barrels. Solution changes were achieved by laterally displacing the whole perfusion array using a motorized device controlled via a personal computer (Lerma, 1992).

Experimental solutions The normal external solution was (mM): NaCl, 160; KCI, 2.5; CaCI2, 1.8; MgCI2, 2; glucose, 10; HEPES, 10 (pH 7.4, 330 mOsm). Pipettes were filled with the following solution (mM): caesium methane sulphonate, 140; CsCI, 5; CaCI2, 0.5; MgC12, 5; ATP-Mg, 4; EGTA,

10; HEPES, 10 (pH 7.4, 314 mOsm). NS102 and NS394 (8-methyl- 5-(4-nitrophenyl)-6,7,8,9-tetrahydro- 1 H-pyrrolo[3,2-h]isoquinoline- 2.3-dione-3-oxime) were prepared in 10 mM stock solutions in dimethyl sulphoxide (DMSO). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) was prepared at 50 mM in DMSO and stored at 4°C until used. The final concentration of DMSO applied to the cells was 60.2%. Since, at this concentration, DMSO slightly decreased kainate- activated responses, it was included in control solutions at the same concentrations present in test tubes. Cyclothiazide was dissolved at 50 mM stock solutions in absolute ethanol. Agonists were dissolved in distilled water as 50 mh4 stock solutions. Kainate, AMPA and CNQX were obtained from Tocris Neuramin (Bristol, UK). NS 102 was kindly supplied by Dr J. Drejer (NeuroSearch, Glostrup, Denmark). Cyclothiazide was generously supplied by Dr M. Casaiia (Lilly Espafia, Madrid, Spain). Salts were obtained from Sigma or Merck.

lmmunocytochemistry Cultured cells were fixed by incubation in 4% paraformaldehyde solution (20 min) and washed with PBS and treated with Triton X-100 (0.1%, 20 min). Petri dishes containing cultured cells were divided into two compartments with the aid of an immunopen (Calbiochem, La Jolla, CA). One half was incubated with and the other without (control) anti-GluR5/6/7 (Pharmingen 4F5, San Diego, CA; dilution 1:300) monoclonal antibody for 1 h at room temperature. After washing with PBS, the cells were incubated for 1 h wiih a biotinylated sheep anti-mouse IgM (Amersham, 1:300), followed by incubation with Texas red-conjugated streptavidin (Amersham). Preparations were examined on an Axiophot microscope (Zeiss, Germany) equipped with phase-contrast optics and a UV light source.

Membrane preparation and binding assays The [3H]kainate binding assays were performed on membrane prepara- tions from the cerebral cortex isolated from adult male Wistar rats. The methods have been described in detail by Johansen et al. (1993). In brief, the standard assay was performed at 2 nM ['Hlkainate in 50 mM Tris-HCI buffer, whereas the low-affinity binding assay was performed at 20 nM [3H]kainate in Tris-HC1 buffer containing 20 mM CaCI2. The high-affinity [3H]kainate binding was estimated by subtracting the amount of low-affinity binding from the total specific binding in the standard assay. To measure non-specific binding, L-glutamate was added at a final concentration of 0.6 mM. Displace- ment of [3H]AMPA binding was performed using the method previ- ously described by Honore and Nielsen (1985). In this assay the samples were incubated with 5 nM [3H]AMPA in Tris-HCI buffer (30 mM, pH 7.4) containing 2.5 mM CaCI2 and 100 mM potassium thiocyanate for 30 min at 0°C. Estimates of binding parameters were calculated as described in Johansen et al. (1993).

Resu I ts Kainate receptors in hippocampal cells. The responses to kainate mediated by activation of kainate receptors consisted of a rapidly desensitizing inward current (Fig. IA; Lerma et al., 1993). These responses were more often seen early in cultures than after some days. To determine whether the expression of kainate receptors changed during in vitro differentiation, we studied hippocampal cells from freshly dissociated neurons (4 h after plating) over 4 days in culture. Electrophysiological responses to rapid application of kainate showed three forms. The first consisted of a rapidly and completely desensitizing inward current (Fig. 1 A), the typical response mediated by kainate receptors. The second was a

Pharmacology of kainate receptors 2 13 1

Kainate Kainate AM=- B

Kainate A

C

70 -1 = OnlyTransient I . - h

5 60

5 50 0 g 40

2 30

z

u) -

L

5 20 10

0 0 1 2 3 4

Days in Culture (~32) ( ~ 1 5 7 ) ( ~ 1 5 2 ) (n=83) (~38)

FIG. 1. Identification of kainate receptor-mediated responses. (A) Current activated by the rapid perfusion (indicated by the bar above the records) of kainate in a cell exclusively expressing kainate receptors. Note the rapid and complete desensitization. In cells expressing AMPA receptors, responses to kainate were steady (B, left). In these cells, the existence of kainate receptors was revealed after desensitizing AMPA receptors with a high concentration of S-AMPA (200 1M; the initial response is truncated) and rapidly changing to a kainate-containing solution (B, right). The peak response (arrow) at the beginning of kainate perfusion revealed the presence of kainate receptors. The subsequent slowly developing current represented the activation by kainate of AMPA receptors as they recovered from the desensitized state. (C) Frequency of hippocampal neurons expressing kainate receptors. Bars are the percentage of cells showing only transient currents, and both transient and steady currents (mixed pattern) at different culture ages. The numbers in brackets at the bottom are the numbers of cells studied electrophysiologically at each time point.

steady response to kainate with no evidence of desensitization (e.g. Fig. 3A), while the third was a mixed pattern as the initial peak response declined to a steady current, resembling partial desensitiza- tion. Selective desensitization of AMPA receptors in a high concentra- tion of AMPA and the subsequent jump to a kainate-containing solution demonstrated that the kainate-induced steady current was due to AMPA receptor activation. This method served to unmask the presence of kainate receptors from the larger steady current induced by kainate in cells also expressing AMPA receptors (Fig. IB). In Figure lC, the histogram shows the percentage of cells that showed a transient response to kainate (pure and mixed patterns). As can be seen, the percentage of cells expressing exclusively kainate receptors decreased during the culture period (from 41% at day 0 to 8% at day 4), while the number of cells having AMPA receptors (i.e. steady currents) increased with time in culture (from 45% to 63%). The presence of kainate receptors in hippocampal cells, independent of the presence of AMPA receptors, was fairly constant throughout the

culture period, however. Transient kainate-activated currents were demonstrated in 85% of the cells recorded 4 h after plating, and in a slightly lower percentage of cells (71%) in cultures after 4 days. The amplitude of peak current was fairly constant through development in culture, with a mean value of 74 ? 6.4 pA (mean f SEM, n = 47; range 60-92 PA) and was not different in cells also expressing AMPA receptors (64 ? 5 PA, n = 72; range 55-72 PA). In contrast, kainate-induced steady current increased with the age in culture [from 52 ? 11 pA in cells at day 0 (n = 10) to 148 ? 40 pA at day 4 (n = 13)]. Altogether these results suggest that expression of kainate receptors is not developmentally regulated, at least under in virro conditions.

The presence of kainate receptors in our cultures was also assessed by immunocytochemistry using a monoclonal antibody against the kainate receptor subunits GluR5, 6 and 7. As can be seen in Figure 2A, most of the cells in culture were labelled by the antibody. Young cells were poorly developed morphologically, but labelling was uniformly distributed in the soma and short neurites. Labelling was somewhat less intense in more developed cells, being concentrated in both thick neurites and fine processes. The number of cells labelled by this antibody was similar in young (4 h) cultures (75%, n = 365) and older (4 days) cultures (80%, n = 241), which agrees well with the number of cells exhibiting functional responses in electrophysiological experiments (Fig. 2B).

Pharmacology of kainate-activated responses To determine the potency of different glutamate receptor antagonists on kainate receptors, the degree of antagonism was evaluated in cells responding exclusively with desensitizing responses upon rapid perfusion of kainate. The same analysis was repeated on cells responding with a steady current to the perfusion of kainate, i.e. on AMPA receptor-mediated responses. Figure 3 illustrates the action of the prototypical competitive antagonist of AMPA receptors CNQX (Honor6 et al., 1988) on cells exhibiting either type of response. The dose-inhibition curve for the action of CNQX on steady responses induced by 300 pM kainate revealed an ICso of 0.92 pM. However, although less potently, CNQX also inhibited kainate-induced responses in cells with no AMPA receptors. Half-inhibition was obtained at a concentration of 6.1 pM (Fig. 3B, C).

NS102 has been described as a glutamate receptor antagonist which, in cortical membranes, completely inhibits [3H]kainate binding to its low-affinity site whilst essentially showing no effect on high- affinity r3H]kainate binding (Table 1). In addition, NS102 is a weak inhibitor of r3H]AMPA binding (Johansen er al., 1993). NS102 blocked transient kainate-induced responses in hippocampal cells in a dose-dependent and reversible manner (Fig. 4A; calculated ICSO = 2.2 pM). NS102 was also active at AMPA receptors, as evaluated on steady kainate-induced currents, but was, however, less potent (the ICSO for steady responses was 4.1 pM). At 10 pM (close to the limit of solubility), NS 102 was unable to completely inhibit either response (Fig. 4C), and 23.7 2 4.7% (mean ? SEM; n = 4) and 31.6 -C 5.1% (n = 7) of the control peak and steady currents respectively remained when 100 pM kainate was used to induced responses.

Another competitive antagonist of AMPA receptors has been synthesized, NS394. In the binding assay, this compound was about ten times more potent in displacing [3H]AMPA binding than low- affinity r3H]kainate binding to cortical membranes (Table 1). In cultured cells, NS394 potently antagonized both steady and transient responses induced by kainate in a dose-dependent and reversible manner (Fig. 5). However, this compound was somewhat more efficient on transient (ICsO = 0.13 pM) than on steady currents (ICSo = 0.45 pM), indicating poor selectivity in discriminating

2132 Pharmacology of kainate receptors

A B

- 100 pm

1 U E l e c t r o p h y s F l -a n l o o W lmrnunocytochemistry

0 1 3 4 Days in Culture

FIG. 2. (A) Immunocytochemical detection of GluR5/6/7 subunits in hippocampal cultures. Panels a and c are phase-contrast micrographs of low-density hippocampal cultures 4 h (a) and 3 days (c) after plating. Immunofluorescence microscopy shows that GluR5/6/7 staining is uniformly distributed in the perikaryon and neurites in younger cells (b), whereas in more developed cells there are cases in which it is more abundant in thick processes rather than cell bodies (d) (arrowheads). (B) Comparison of frequency of cells observed to respond with rapidly densensitizing responses to kainate (electrophysiology) and cells positively labelled by the aGluR5/6/7 antibody (immunocytochemistry) during the developmental time in culture. Immunocytochemical data were calculated from 365, 458, 347 and 241 counted cells 0 (4 h) 1, 3 and 4 days after plating respectively, pooled from three culture preparations. Numbers of cells studied electrophysiologically are as in Figure 1C. The interrupted line represents the average, irrespective of time in culture. Vertical lines represent the SEM.

Kainate A

B mate QM 1 X M 3 x M

C 1 '

0.01 0.1 1 l o loo 1000 CNQX (pM)

FIG. 3. Inhibitory effect of CNQX on AMPA receptor-mediated (A) and kainate receptor-mediated (B) responses. To allow receptor equilibration with the antagonist, the antagonist was introduced for 1 min before receptor activation. (C) Dose-inhibition curves for CNQX of transient and steady kainate-induced responses. The points represent the mean 2 SEM (in most cases the symbol exceeds the error bar size) obtained in three to seven neurons. In this and Figures 4 and 5 , solid lines represent the fitting of experimental data to the equation:

I00

1 + [AnrdlCso]" R =

where R is the current response, Anra is the concentration of antagonist, IC5, is the half-maximal inhibitory concentration and n is a curve slope coefficient. Curves were fitted to single points pooled from different neurons, leaving the two parameters n and IC,, free for iterations. Values of IC50 were 0.92 and 6.1 pM for AMPA and kainate receptors respectively. Slope coefficients were 1.1 and 1.0.

Pharmacology of kainate receptors 2133

C 1201 - Kainate

100 uM Recoverv

A

Steady _]

0.01 0.1 1 10 100 0.5 s NS-102 (pM) -

FIG. 4. Inhibitory effect of NS 102 on AMPA and kainate receptors in hippocampal neurons. NS 102 reversibly antagonized both kainate receptor-mediated (A) and AMPA receptor-mediated (B) responses. The antagonist was perfused from 1 min before kainate to allow receptor equilibration. (C) Dose-inhibition curves (calculated as described in legend to Figure 3) revealed ICSO values of 2.2 and 4.1 pM for kainate and AMPA receptors respectively. Slope coefficients were 0.8 and 0.7. Points represent the mean t SEM for three to nine neurons.

A Kainate 300 pM C

0.01 0.1 1 10 100

NS- 394 (pM) 4 E m s

FIG 5. Inhibition of kainate-induced responses in hippocampal neurons by NS394. (A) In the presence of kiunate, the concentration of NS394 was increased until complete block was attained. (B) Effect of NS394 on transient kamate-induced responses. The antagonist was perfused from 1 min before kainate to allow receptor equilibration Analysis of dose-inhibition curves (C) (as described in the legend to Figure 3) revealed ICSO values of 0.45 and 0.13 pM for AMPA and kainate receptors respectively. Slope coefficients were 1.2 and 1.0. Points represent the mean 2 SEM of data for three to ten neurons.

functional receptors. For comparison, pharmacological parameters for each of these compounds, calculated from kainate- and AMPA receptor-mediated responses, are summarized in Table 1 together with parameters obtained from binding experiments in cortical membranes.

AMPA receptors have an allosteric modulatory site which is recognized by benzothiadiazides (diazoxide, cyclothiazide) (It0 et al., 1990; Yamada and Rothman, 1992; Zorumski ef al., 1993). Some non-competitive antagonists of AMPA receptors, like the 2,3-benzo- diazepine GYIU 53655, seem to specifically interact with this or an overlapping site (Zorumski et al., 1993; Palmer and Lodge, 1993; but see Desai et al., 1995; Partin and Mayer, 1996). We have recently shown that this compound unmasks smaller kainate receptor-mediated currents by preventing the activation of AMPA receptors (Patemain et al., 1995). Application of cyclothiazide abolished the desensitization of AMPA receptors when activated by AMPA, potentiating the AMPA receptor-mediated response not only when activated by AMPA but

also by kainate (Fig. 6A, B). However, cyclothiazide was completely ineffective on native kainate receptors at concentrations producing a nearly saturating effect on AMPA receptors. At high concentrations (50-100 pM), cyclothiazide produced a slight decrease (5-10%) rather than a potentiation of the response mediated by kainate receptors. This result demonstrates that kainate receptors do not have an allosteric site similar to the AMPA receptors, allowing specific antagonism of the receptor via this site.

Discussion

Consistent with binding data, we have shown that NS102, a presum- ably selective inhibitor of kainate receptors (Johansen et al., 1993), is much less potent on AMPA receptor-mediated responses than any other antagonist (Table 1). However, NS102 was not very potent in antagonizing kainate receptors. NS394 was approximately twice as

21 34 Pharmacology of kainate receptors

A B S-AMPA 200 PM Kainate 100 pM

+CTZ 100 PM Control +CTZ 100 pM . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Control

400 ms

100 PA

400 ms

FIG 6. Effect of cyclothiazide on AMPA and kainate receptors. AMPA receptors were activated by S-AMPA (A) or kainate (B). Note the large increase in both responses in the presence of cyclothiazide. (C) Kainate receptor-mediated responses were not affected by cyclothiazide up to concentrations of 30 pM. To allow receptor equilibration, in all cases cyclothiazide was perfused for 2 rnin before agonist perfusion.

TABLE 1. Inhibition of kainate- and AMPA-induced responses in cultured hippocampal neurons and AMPA and kainate binding in cortical membranes by excitatory amino acid antagonists

AMPA receptors Kainate receptors

[3H]AMPA [ 3H] kainate [3H]kainate Functional bindingC Functional low-affinityc high-affinityc

IC50' n K , IC50 G o a n K, K ,

1.6 ? 0.4 CNQX 0.92 1.1 0.41 0.34 2 0.03 6.1 NS394 0.45 1.2 0.20 0.13 ? 0.01 0.13 1 .0 0.50 ? 0.07 18.3 ? 2.5 NS 102 4.1 0.7 2.9 7.2 2 1.9 2.2 0.8 0.63 ? 0.10 > 10

1 .0 0.75 2 0.11

"IC50 and slope coefficients (n) were calculated by curve fitting to dose-inhibition curves, taking peak responses as mediated by kainate receptors and steady currents as representative of AMPA receptor-mediated activity. bInhibition constants (K , ) for functional receptors were calculated by the Cheng-Prusoff equation from the ICso values obtained for steady responses (EC50 for kainate = 240 pM: Lerma el al., 1993). 'Data are mean ? SEM values for three separate experiments. Except for NS394, data are from Johansen et al. (1993). All values are expressed in pM.

potent as CNQX in inhibiting AMPA receptor-mediated responses (Ki = 0.2 and 0.41 pM respectively). However, CNQX appeared to be much more selective for AMPA receptors than NS394. Although data for kainate receptors were calculated in non-equilibrium condi- tions (see below) and direct comparison of the ICsO may be misleading, it was clear that CNQX was much less effective in preventing kainate- induced transient responses than was NS394 (6.1 and 0.13 pM respectively). This provides an example in which selectivity and potency appear unrelated. Unfortunately, neither of these compounds proved to be useful to adequately separate AMPA and kainate receptors in a functional assay.

For a long time, binding experiments have differentiated at least two binding sites for [3H]kainate in nervous tissue. One of them shows high affinity with a KD in the low nanomolar range (-5 nM), and the other showed lower affinity, with a KD of -5CL60 nM (for review see Young and Fagg, 1990). In addition, there should be a very low-affinity site (KD in the pM range) corresponding to AMPA receptors rather than kainate receptors, since kainate interacts with [3H]AMPA binding (Honor6 et al., 1988) and activates AMPA receptors. After binding experiments carried out on recombinant glutamate receptors, it became apparent that high-affinity kainate sites may correspond to receptors formed by KAl and KA2 subunits, whereas low-affinity sites for kainate would be related to GluR5-7 subunits (Hollmann and Heinemann, 1994). However, in the absence

of a direct demonstration, this remains just a reasonable hypothesis, inasmuch as the concentrations of agonist required to activate func- tional receptors are usually much higher than the KD values determined from binding experiments.

From our data it was obvious that CNQX has some antagonistic activity on functional kainate receptors. This result is not surprising since it has been shown that CNQX also interacts with a low-affinity [3H]kainate binding site in brain membranes (Johansen el al., 1993). In contrast to AMPA receptors, antagonism of kainate receptors could not be calculated at equilibrium since rapid desensitization left essentially no steady current. Consequently, the Cheng-Prusoff for- mula to calculate the inhibition constant (&) could not be applied to our measurements (Cheng and Prusoff, 1973). However, to test the idea that low-affinity kainate binding sites correspond to functional kainate receptors it would still be very interesting to compare the values obtained by the present study, from electrophysiological responses and from binding data. For comparison purposes, one can estimate the half-maximal inhibitory concentrations by rearranging the Cheng-Prusoff formula and using the K, values calculated in equilibrium binding experiments (Table 1):

KA e1C50 = K i ( + )

Pharmacology of kainate receptors 2 135

where Ki is the inhibition constant from binding experiments, KA is the concentration we have used to calculate dose-inhibition curves, and ECjO is the half-maximal concentration previously calculated for functional kainate receptors in hippocampal neurons (22 pM; Lerma et al., 1993). Then, eK50 is the value that would be obtained if an antagonist were to inhibit functional kainate receptors with the described Ki. For CNQX, considering a Ki of 0.75 pM (Johansen et al., 1993; Table l), the value of eK50 is equivalent to 10.9 pM. This value is not vastly different from the ICS0 of 6.1 pM we actually obtained. Similar calculations may be performed for NS 102; taking the Ki for the low-affinity kainate site to be 0.63 pM (Table l), the e K S o value is 3.5 pM, which again is not far from the 2.2 pM that we obtained from dose-inhibition curves of native receptors. These results therefore support the idea that low-affinity kainate sites in the brain correspond to functional kainate-selective receptors. It is true, however, that ICsO values obtained in our preparations are slightly smaller than the calculated eZCso values. This may be accounted for by the fact that, in our experiments, agonist and antagonist were not allowed to reach equilibrium before desensitization took place, since antagonist dissociation is a limiting step in governing the binding of the agonist to the receptor. Consequently, high-affinity competitive antagonists would influence transient responses more than expected from equilibrium affinity constants. In fact, competitive antagonists behave as if they are non-competitive in that the degree of inhibition becomes independent of agonist concentration (Pennefather and Quastel, 1981). Actually, for one of the antagonists studied, NS394, the degrees of inhibition produced by 0.1 pM at 300 pM and 1 mM kainate were similar (48.9 ? 5.4 and 40.8 2 9.2 respectively).

The e K 5 , for NS394 (assuming a Ki of 0.5 pM; Table I ) was 7.5 pM, which differs from the IC50 obtained from the kainate dose- inhibition curve (0.13 pM). There is a factor that may contribute to this difference. As the dissociation rate of NS394 is very slow (we have estimated a k,ff value of l/s; not shown) during the -20 ms that the peak kainate response lasts, dissociation of NS394 should be -5%. If there is essentially no dissociation of the antagonist, then the IC50 should approach the equilibrium inhibitory constant for the antagonist (Colquhoun et al., 1992). However, the Ki was 0.5 pM in experiments in which [3H]kainate bound to the low-affinity site, performed in cortical membranes. This value is larger than, but not very different from, our IC50 value. Still, this result supports the suggestion of identity between low-affinity kainate binding sites and functional kainate receptors. Moreover, this reasoning indicates that ICS0 values obtained in non-equilibrium conditions (underestimated with respect to those calculated at equilibrium) may be more meaning- ful than equilibrium data in considering antagonism of synaptic transmission, for example, where equilibration with the transmitter is never completed.

We have demonstrated recently that the GluR6 subunit is involved in the formation of functional kainate receptors in hippocampal cells in culture (Ruano et al., 1995). Accordingly, our results strongly support the idea that the presence of the GluR6 subunit reflects low- affinity binding sites in the brain (Verdoorn et al., 1994). However, EC50 values for recombinant GluR6 and native kainate receptors differ a lot (1 versus 22 pM) (Egebjerg et al., 1991; Lerma et al., 1993), suggesting that native kainate receptors may include other (regulatory?) protein(s) in addition to the GluR6 subunit, as previously suggested (Ruano et al., 1995).

Finally, our data show that the kainate receptors of cultured hippocampal cells lack the benzothiadiazide site(s) known to be present at AMPA receptors. A similar result has been found for kainate receptors expressed by dorsal root ganglion cells (Wong and Mayer, 1993; Wilding and Huettner, 1995). glial progenitor cells and

recombinant GluR6 receptors (Partin et al., 1993; Patneau et al., 1994). This makes selective antagonism of AMPA receptors by drugs acting on that site possible. Availability of these compounds may be of great help in elucidating a function for kainate receptors, which so far remains an enigma. In fact, the lack of compounds able to separate AMPA from kainate receptors has been one important reason why the demonstration of functional receptors in central neurons has proved to be very challenging both in situ and in vitro.

Most cells in culture express kainate receptors, as seen by both electrophysiological recordings (Fig. 1) and immunocytochemistry (Fig. 2), a result which resembles what has been found in the brain at the levels of mRNA and protein (Huntley et al., 1993; Wisden and Seeburg, 1993; Bahn et al., 1994; Petralia et al., 1994). Independently of the age in culture, 79 I 3.8% of the recorded cells presented functional kainate receptors, and 78.8 2 4.7% were strongly immunol- abelled with the GluR5/6/7 antibody. However, there was a slight tendency to detect fewer positive cells in electrophysiological record- ings as the cultures aged. This might reflect either some difficulty in electrical access to the receptor-generated signal as neurons mature (e.g. functional channels may be targeted to dendrites) or insurmount- able masking of tiny kainate receptor-mediated currents by the rapid expression of AMPA receptors. All these data suggest that the expression of kainate receptors does not undergo significant variation during development in vitro, and that clear electrophysiological responses may rather depend on cell size and/or cell geometry.

Acknowledgements We thank Dr J. Drejer (NeuroSearch, Glostrup, Denmark) for providing us with NS102, Dr M. Casaiia (Lilly Espaiia, Madrid, Spain) for a sample of cyclothiazide, and Drs A. Fernis and M. Nieto-Sampedro from Instituto Cajal for allowing us to use their fluorescence microscopy facilities. English corrections by M. Sefton are appreciated. This work was supported in part by grants to J. L. from the Direccidn General de Investigacibn Cientifica y Tkcnica (PB93/0150), Fondo de Investigaciones Sanitarias (95/0869) and the Biotech Program of the European Community (BI02-CT930243). A. V. P. holds a fellowship from Glaxo.

Abbreviations AMPA a-hydroxy-5-methyl-4-isoxazolepropionic acid CNQX 6-cyano-nitroquinoxaline-2,3-dione NS102 5-nitro-6,7,8,9-tetrahydrobenzo(G)indole-2,3,-dione-3-oxime NS394 8-methyl-5-(4-nitrophenyl)-6,7,8,9-tetrahydro- 1 H-pyrrolo[3.2-

h]isoquinoline-2,3-dione-3-oxime

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Note added in proof While this paper was in the press, Wilding and Huettner (1996) reported a study determining the relative potency of several competitive antagonists, including NS102 and CNQX, at AMPA receptors expressed by cultured rat cortical neurons, and at kainate receptors in freshly dissociated rat dorsal root ganglia cells. Their results show that most competitive antagonists display weak selectivity between AMPA and kainate receptors, emphasizing the broad overlap in potency of competitive antagonists at peripheral kainate receptors and CNS AMPA receptors.