The synaptic activation of the GluR5 subtype of kainate receptor in area CA3 of the rat hippocampus

Neuropharmucology, Vol. 36, No. 11/12, pp. 1477-1481, 1997 0 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0028-3908/98 $19.00 + 0.00

Pergamon PII: SOO28-3908(!W)OOl58-5

The Synaptic Activation of the GluRS Subtype of Kainate Receptor in Area CA3 of the Rat Hippocampus M. VlGNES,‘* D. BLEAKMAN,’ D. LODGE2 and G. L. COLLINGRIDGE’ ‘Department of Anatomy, University of Bristol, University Walk, Bristol BS8 ITD, U.K. and 2Lilly Research Centre Ltd., Erl Wood Manor, Windlesham, Surrey GU20 6PH, U.K. (Accepted 9 September 1997) Summary-Two new compounds (LY293558 and LY294486), that antagonize homomeric human GluR5 receptors, were examined against responses mediated by kainate receptors in the CA3 region of rat hippocampal slices. Both compounds (applied at a concentration of 10 PM) antagonized reversibly currents induced by 200 nM kainate. They also antagonized reversibly the synaptic activation of kainate receptors, evoked by high-frequency stimulation of mossy fibres, in the presence of NMDA and AMPA receptor antagonists, These results show that GluR5 subunits are likely to contribute to a kainate receptor on CA3 neurones that mediates responses to both kainate and synaptically-released L-glutamate. 0 1998 Elsevier Science Ltd. All rights reserved. Keywords-Kainic

acid, LY293558, LY294486, synaptic transmission,

Native kainate receptors are believed to be assembled, in unknown combinations, from the subunits GluR5, GluR6, GluR7, KA-1 and KA-2 (Bettler and Mulle, 1995). Little is known concerning the function of any of these subunits due to the lack of selective tools. Recently, however, two compounds (LY293558 and LY294486: Fig. 1) have been reported which are selective antagonists for recombinant human GluR5 receptors, when compared with other kainate receptor subunits, expressed in HEK293 cells (Bleakman et al., 1996; Clarke et al., 1997). It has also been reported, recently, that kainate receptors on CA3 neurones in the hippocampus can be activated synaptically by stimulation of the mossy fibre pathway (Vignes and Collingridge, 1997; Castillo et al., 1997). In the present study we have used these subunitselective compounds to determine whether GluR5 subunits are involved in this kainate receptor-mediated synaptic response.

MATERIALS

AND METHODS

Experiments were performed on transverse hippocampal slices (400 pm) obtained from 13-17-day-old Wistar rats using a Vibroslioe. Slices were collected and maintained in medium comprising (n&I) NaCl (124), KC1 (3), NaHC03 (26), NaH2PO4 (1.25), CaC12 (2), MgS04 (l), D-glucose (10) (bubbled with Oz/CO2: 95/ 5%). After at least 1-hr equilibration and recovery time, *To whom correspondence

should be addressed. 1477

hippocampus, mossy fibre.

the slices were transferred to a submerged style recording chamber and perfused with the same medium (at a rate of approximately 2 ml/mm) at room temperature. Wbolecell patch-clamp recordings were obtained from the CA3 region, using the “blind approach’ with glass microelectrodes (5-7 MQ; seal resistance approximately 10 Ga) filled with a solution which comprised (n&I) CsMeSOs (120), NaCl (l), MgC12 (l), BAPTA (lo), N-(2,6dimethyl-phenylcarbamoylmethyl)-trietbylammonium bromide (QX-314) (5), HEPES (5) (adjusted to pH 7.3) and, usually, Mg-ATP (4). Kainate receptor-mediated EPSCs were isolated as described previously (Vignes and Collingridge, 1997) using the following pharmacological cocktail: GYK153655 (LY300168; 50 PM), D-Zamino5-phosphonopentanoate (100 ,uM), L-689,560 (5 ,uM), picrotoxin (50 ,uM), bicuculline (10 PM) and, in some experiments, NBQX (0.5 ,uM). Two successive records per input were averaged to provide 1-min time-points and for presentation of corresponding traces (the time points illustrated being indicated on the graphs by letters). Any apparent differences in stimulus artefacts are due to sampling. Access resistance was monitored continually and neurones discarded if this parameter changed by more than 20%. Compounds were applied by addition to the perfusing medium. 1-(4aminophenyl)-3GYK153655 (LY300168; methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5&2,3_benzodiazepine), LY293558 ((3S, 4aR, 6R, 8aR)-6-[2-( 1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline-3-carboxylic acid) and LY294486 ((3SR, 4aRS, 6SR, 8aRS)-6-((((lH-tetrazol-5-yl)methyl)oxy)methyl)-

M. Vignes et al.

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LY293558

LY294486

Fig. 1. Structures of the GM25 antagonists used in the present study.

1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-c~boxylic acid) were synthesized in house. GYK152466 (l(4-aminophenyl)-4-methy1-7,8-methylenedioxy-SH-2,3benzodiazepine) was obtained from RBI, Poole, UK. Other compounds were obtained from Tocris Cookson, Bristol, UK.

RESULTS LY293558 and LY294486

antagonize

kainute-induced

currents in CA3 neurones

Although it is established that LY293558 and LY294486 antagonize the actions of kainate on HEK293 cells stably expressing human GluRS and on rat dorsal root ganglion (DRG) neurones (Bleakman et al., 1996; Clarke et al., 1997), the effectiveness of these agents on kainate-induced inward currents in neurones in the CNS had not been investigated. We therefore adopted a protocol which we had employed previously to

demonstrate that CNQX antagonizes kainate activation of kainate receptors on hippocampal CA3 neurones (Vignes et al., 1996; Vignes and Collingridge, 1997). Kainate (200 r&I, 30 min) was applied in the presence of a non-competitive AMPA receptor antagonist (100 PM GYK152466 or 50 PM GYKI53655) plus tetrodotoxin (1 PM) and induced an inward current of between 55 and 153 pA (93 + 14 PA; n = 6). At the peak of the response either LY293558 (10 w) or LY294486 (10 PM) was coapplied for 10 min and the percentage reduction of the kainate-induced current calculated for each neurone; these values were 77 f 2% (n = 3) and 65 + 7% (n = 3) respectively. Single examples, which illustrate the reversibility of both antagonists and of kainate are presented in Fig. 2. LY293558 activation The

and LY294486 antagonize of kainate receptors

synaptic

Kainate 200 nh4

I

-40 -

activation

of kainate

the

synaptic

currents

Kainate 200 nM

I

I

-20

LY293558 10 pM

I LY294486 10 pM

-60 -80 -100 -120 -120 -140 -I

I -140 _, 0

-160 c, 10

20

30

Time (min.)

40

50

0

10

20

30

40

50

Time (min.)

Fig. 2. Antagonism of kainate current in CA3 neurones by GluRS antagonists. The graphs plot holding current vs time (V, = - 60 mV) and show the effects of kainate and each antagonist (duration of applications denoted by bars).

was

Synaptic activation of GluR5 kainate receptors

1479

b

LY293558 1 pM

0

10

20

30

40

50

40

50

Time (min.) LY293558 10 pM

0

10

20

30

Time (min.)

Fig. 3. Antagonism of the synaptic activation of k&ate receptors by LY293558. Single examples to illustrate the effects of 1 PM (A) and 10 PM (B) LY293558. Each point plots the amplitude of the average of two successive responses (measured 20 msec following the last stimulus). The traces in (A) are the averages obtained at the times indicated on the graph below (a-c).

obtained, by delivering a brief tetanus (10 shocks at 100 Hz) to the mossy fibre pathway in the presence of 50 PM GYK153655, as dlescribed previously (Vignes and Collingridge, 1997). LY293558 (1 or 10 PM, 10 min) rapidly and reversibly depressed the synaptic response (Fig. 3). The mean depressions were, for 1 PM, 43 f 5% (n = 7) and, for 10 ,ulVl, 73 + 6% (n= 5). Similarly, LY294486 (10 PM, 10 min) rapidly and reversibly depressed the synaptic response (Fig. 4); the mean depression being 75 + 6% (n = 4). The antagonists had no effect on holding current or input resistance per se.

DI!SCUSSION The principal finding of the present study is that two GluR5 antagonists inhibit the activation of kainate

receptors on CA3 neurones by both exogenously applied kainate and synaptically-released glutamate. This provides the first evidence that GluR5 subunits contribute to synaptic transmission. Activation

of postsynaptic

kuinate receptors

It has been shown previously that kainate is a potent excitant of CA3 neurones, inducing large depolarizations at nanomolar concentrations (Robinson and Deadwyler, 1981; Westbrook and Lothman, 1983). These concentrations of kainate are likely to be subthreshold for activating AMPA receptors under normal conditions (Bettler and Mulle, 1995). In the present study, any activation of AMPA receptors, by nanomolar concentrations of kainate, was completely excluded by the use of the potent and selective non-competitive AMPA receptor

M. Vignes et al.

1480 b

25 pA

500 ms LY294486 10 pM

0

1 0

I

I

I

I

I

I

I

I

10

20

30

40

50

60

70

80

Time (min.)

Fig. 4. Antagonism of the synaptic activation of kainate receptors by LY294486. Data presented as in Fig. 3.

antagonists (GYK152466 or GYK153655) (Tarnawa et al., 1990; Palmer and Lodge, 1993; Donevan and Rogawski, 1993; Zorumski et al., 1993) which have been shown to substantially antagonize AMPA but to spare kainate receptor-mediated responses in CA3 neurones (Vignes et al., 1996; Vignes and Collingridge, 1997; Castillo et al., 1997) and elsewhere (Patemain et al., 1995; Renard et al., 1995; Wilding and Huettner, 1995; Wilding and Huettner, 1997; Bleakman et al., 1996; Chittajallu et al., 1996).

The synaptic activation of kainute receptors Although it has been known for circa 20 years that kainate receptors can be distinguished from AMPA receptors (Davies et al., 1979; McLennan and Lodge, 1979), the lack of selective kainate receptor agonists and antagonists has greatly hindered the identification of the roles of kainate receptors in the vertebrate CNS. By the use of GYKI53655 to eliminate activation of AMPA receptors, it has been shown that kainate receptors can be activated synaptically by high-frequency stimulation of the mossy fibre pathway. This conclusion was based mainly on the use of CNQX, which is a weak kainate receptor antagonist (Vignes and Collingridge, 1997; Castillo et aZ., 1997). The present observation that LY293558 and LY294486 antagonize the synaptic response, evoked by high-frequency stimulation of mossy

fibres in the presence of GYKI53655, over the same concentration range as responses to kainate, confirms the involvement of kainate receptors in this synaptic response. A role for GZuR5 in postsynaptic excitation The finding that the postsynaptic kainate receptor on CA3 neurones is sensitive to GluR5 antagonists may seem surprising based on the predominant distribution of GluR6, KA-1 and KA-2 mRNA in the hippocampus (Wisden and Seeburg, 1993) and the finding that cultured embryonic hippocampal neurones express predominantly GluR6 mRNA and respond to kainate, in the presence of GYKI53655, in a manner indicative of activation of homomeric GluR6 (Ruano et al., 1995). However, if only homomeric GluR6 receptors were present on CA3 neurones, then little or no steady-state currents to kainate would be observed, due to pronounced desensitization. Recently, it has been shown that hippocampal neurones cultured from 2-5-day-old rats respond very differently to kainate; a large steady-state current which exhibited an approximately linear Z-V relationship was observed (Wilding and Huettner, 1997). It was suggested that this may be due to the developmental increase in GluR5 expression, which has been observed in vivo (Bahn et al., 1994). The present results are in full accord with this hypothesis, as is the approximately linear Z-V relation-

Synaptic activation of GluRS kainate receptors

ship of the kainate receptor-mediated EPSC (Vignes and Collingridge, 1997; Castillo et al., 1997). Concluding remarks Although it has been known for a considerable time that kainate receptors constitute a distinct pharmacological entity from AMPA or NMDA receptors (Davies et al., 1979; McLennan and Lodge, 1979; Watkins and Evans, 1981), their functions are largely unknown (Feldmeyer and Cull-Candy, 1994). The recent availability of antagonists for the GluRS subunit of the kainate receptor family (Bettler et al., 1990) is enabling the roles of kainate receptors composed of, or containing, this subunit to be determined. Further studies will be required to establish which other subunits also contribute to the kainate receptor which i.s highly enriched in the mossy fibre termination zone of area CA3 (Monaghan et al., 1985) and is activated during high-frequency synaptic transmission in the mossy fibre pathway (Vignes and Collingridge, 1997; Castillo et al., 1997). Acknowledgements-Supported by the MRC. The authors wish to thank Paul Omstein for the synthesis of LY293558 and LY294486 and Marvin Hansen for the synthesis of LY300168 (GYKI53655).

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