REVIEW

Kainate receptors

Paulo Pinheiro & Christophe Mulle

Received: 20 April 2006 /Accepted: 31 May 2006 / Published online: 18 July 2006# Springer-Verlag 2006

Abstract Kainate receptors form a family of ionotropicglutamate receptors that appear to play a special role in theregulation of the activity of synaptic networks. This reviewfirst describes briefly the molecular and pharmacologicalproperties of native and recombinant kainate receptors. Itthen attempts to outline the general principles that appear togovern the function of kainate receptors in the activity ofsynaptic networks under physiological conditions. It sub-sequently describes the way that kainate receptors areinvolved in synaptic integration, synaptic plasticity, theregulation of neurotransmitter release and the control ofneuronal excitability, and the manner in which they mightplay an important role in synaptogenesis and synapticmaturation. These functions require the proper subcellularlocalization of kainate receptors in specific functionaldomains of the neuron, necessitating complex cellular andmolecular trafficking events. We show that our comprehen-sion of these mechanisms is just starting to emerge. Finally,this review presents evidence that implicates kainatereceptors in pathophysiological conditions such as epilepsy,excitotoxicity and pain, and that shows that these receptorsrepresent promising therapeutic targets.

Keywords Synapse . Glutamate receptors .

Synaptic plasticity . Trafficking . Excitotoxicity

Genes and properties of recombinant receptors

Discovery, cloning and structure

Kainic acid was first isolated from seaweed more than50 years ago and was known, together with domoic acid, tocause amnesic shellfish poisoning. By the mid-1970s, theexcitatory and neurotoxic actions of kainate were wellestablished and the hypothesis that this compound acted ona specific subset of receptors was formulated (Watkins andEvans 1981). This was supported by the demonstration ofhigh-affinity binding sites for [3H]kainate in the rat brain(London and Coyle 1979) and of distinct depolarizing anddesensitizing responses to kainate in C-fibres of thedorsal root ganglia (DRG; Agrawal and Evans 1986;Huettner 1990).

Subunits of kainate receptors were cloned in the early1990s by screening with low stringency hybridization probesto α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid(AMPA) receptor subunits (Bettler et al. 1990; Egebjerg etal. 1991; Lomeli et al. 1992; Sommer et al. 1992). GluR5,GluR6 and GluR7 subunits form functional homomericreceptor-channels activated by kainate and glutamate whenexpressed in heterologous systems and show an affinity forkainate in the range of 50–100 nM (“low-affinity” subunits;Egebjerg et al. 1991; Sommer et al. 1992; Schiffer et al.1997). KA1 and KA2 subunits show high-affinity [3H]kainate binding, with dissociation constants in the range of5–15 nM (Werner et al. 1991; Herb et al. 1992) but do notform functional homomeric receptor-channels. There is75%–80% homology between GluR5, GluR6 and GluR7,

Cell Tissue Res (2006) 326:457–482DOI 10.1007/s00441-006-0265-6

The work performed in the lab of C. Mulle was supported by grantsfrom the Centre National de la Recherche Scientifique, by the FrenchMinistry of Research and by the EU commission (contracts QLRT-2000-02089 and 2005-511995).

P. Pinheiro :C. Mulle (*)CNRS UMR 5091,Laboratoire “Physiologie Cellulaire de la Synapse”,Bordeaux Neuroscience Institute,University of Bordeaux,33077 Bordeaux Cedex, Francee-mail: mulle@u-bordeaux2.fr

and 68% between KA1 and KA2, whereas the twosubclasses of kainate receptors share just 45% homology.Kainate-receptor subunits display less than 40% homologywith the AMPA receptor subunits GluR1-4 and do not co-assemble with these subunits. KA1 and KA2 can combinewith GluR5, GluR6 and GluR7 to form functional receptorswith modified pharmacological and biophysical properties(Werner et al. 1991; Herb et al. 1992; Sakimura et al. 1992;Howe 1996; Schiffer et al. 1997; Cui and Mayer 1999).GluR5, GluR6 and GluR7 can associate in vitro (Cui andMayer 1999; Paternain et al. 2000) and in vivo (Mulle et al.2000; Christensen et al. 2004; P.S. Pinheiro et al., submitted).

Kainate receptors are tetrameric assemblies of subunitsthat have a structure similar to those of the other ionotropicglutamate receptors (iGluRs; Dingledine et al. 1999). Theyare transmembrane proteins with an extracellularN-terminal domain followed by a first transmembranedomain and a “p-loop” that dips into the lipid bilayer andforms part of the pore. Two other successive transmem-brane domains are connected by an extracellular loop andare followed by an intracellular sequence containing theC-terminus. Recent crystallographic data have revealed thestructural basis for the binding of subtype-specific agonistsof the GluR5 and GluR6 subunits (Mayer 2005; Nanao etal. 2005; Naur et al. 2005). These data help explain themolecular mechanisms underlying the differences in theligand-binding properties between glutamate receptors andwithin the kainate-receptor family. The ligand-bindingcores of GluR5 and GluR6 share many features in commonwith those of GluR2 or NR1. The binding cores arebelieved to assemble as dimers of dimers. The ligand-binding cavity of GluR5 is 40% larger in volume than thatof GluR2, allowing the binding of GluR5-selective ligands.The selectivity of ligands for GluR5 in comparison withGluR6 is achieved by a mixture of the side chains, whichdiffer between GluR5 and GluR6 and lead to stericocclusion (Mayer 2005). These differences may havetherapeutic consequences in allowing the design of selec-tive ligands and allosteric modulators that are missing forthe GluR6 and GluR7 subunits.

Diversity of kainate receptors

Cells from different regions of the brain express distinctsubsets of kainate-receptor subunits (Wisden and Seeburg1993) that display distinct pharmacological and electrophys-iological properties (Wilding and Huettner 2001). Kainatereceptors (Fig. 1) are ligand-gated channels that are perme-able to cations, that are activated by low concentrations ofkainate and that display rapid activation and desensitizationcharacteristics. Kainate receptors share many features incommon with AMPA receptors, although members of theseiGluR families do not cross-assemble. Differences exist

between subtypes of native or recombinant receptors in thepotency of specific agonists for receptor activation ordesensitization, in their sensitivity to antagonists and alloste-ric modulators (see below), in their rectification propertiesand in their Ca2+ permeability (Bettler and Mulle 1995;Huettner 2003; Lerma 2003).

Like those for AMPA receptors, the mRNAs encoding forkainate receptors are subject to post-translational modifica-tions of editing and/or alternative splicing (Bettler and Mulle1995; Lerma 2003; Jaskolski et al. 2005a). All splicevariants of kainate receptors, with the exception of analternative 15-amino-acid exon in the N-terminal domain ofGluR5, differ in their cytoplasmic C-terminal domain. Theinsertion of the 15-amino-acid cassette gives rise to GluR5-1,whereas the originally discovered subunit is termed GluR5-2(Bettler et al. 1990). The GluR5 subunit presents three mainalternative splice variants in the C-terminal domain, viz.GluR5a, GluR5b and GluR5c (Sommer et al. 1992), and anadditional GluR5d only found in humans (Gregor et al.1993; Barbon et al. 2001). Two main splice variants ofGluR6 have been described, GluR6a and GluR6b (Gregor etal. 1993); these are expressed in the same brain regions andco-assemble in native receptors (Coussen et al. 2005). Athird variant, GluR6c, contains an insertion of the 15C-terminal exon and has only been described in humans(Barbon et al. 2001; Jamain et al. 2002). Finally, two splicevariants of GluR7 have been identified, GluR7a andGluR7b, in which almost the entire C-terminus of GluR7ais replaced by an unrelated sequence of 55 amino acids(Schiffer et al. 1997). For all the C-terminal splice variants, ashort stretch of 16 amino acids just after the last transmem-brane domain is conserved between GluR5, GluR6 andGluR7. The physiological properties of the kainate-receptorsubtypes do not appear to be affected by alternative splicingof the C-terminal domains (Schiffer et al. 1997; Jaskolski etal. 2004; Coussen et al. 2005). However, modifications ofC-terminal domains have a strong impact on intracellulartrafficking and regulatory processes of kainate receptors andon their interacting partners (see below; Schiffer et al. 1997;Jaskolski et al. 2004; Coussen et al. 2005). GluR5 andGluR6 can also be edited at a Q/R site in the secondmembrane domain (Fig. 1b) and this determines the extent towhich kainate receptors allow the permeation of Ca2+ ions.In analogy with the GluR2 AMPA receptor, the presence ofan arginine residue results in receptors that have lowpermeability to Ca2+, whereas the presence of a glutamineresidue leads to receptors with higher Ca2+ permeability(Burnashev et al. 1995). As for AMPA receptors, thesedifferent ion permeation properties are correlated with theblockage by intracellular polyamines at positive potentials,leading to inwardly rectifying current-voltage relationshipsfor unedited receptors and linear current-voltage relation-ships for edited receptors (Bowie and Mayer 1995; Bahring

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et al. 1997). In GluR6, in addition to the Q/R site, Ca2+

permeability is also dependent on the edited state of twoother codons in the first transmembrane domain: the I/V(isoleucine/valine) and the Y/C (tyrosine/cysteine) sites(Fig. 1b; Köhler et al. 1993); fully edited GluR6 subunitsare essentially impermeable to Ca2+. Fully unedited receptorsalso exhibit a higher unitary conductance compared withreceptors that include one or more edited subunits (Howe1996; Swanson et al. 1996). The KA1 and KA2 subunits(Fig. 1b) do not undergo any known process of alternativesplicing or RNA editing.

Pharmacology of kainate receptors

In the recent past, the study of kainate-receptor physiologywas hampered by the lack of selective ligands. In particular,many excitatory agonists, including AMPA, kainate anddomoate, are not entirely selective for one class of

receptors. Hence, the more abundant AMPA receptors inmost neurons would systematically obscure the analysis ofa clear kainate-receptor-mediated component. Classicalcompetitive antagonists, such as the quinoxalinedioneCNQX, show little discrimination between either subtypeof receptor. However, the related NBQX displays a morethan 100-fold selectivity for AMPA versus kainate receptors(Mayer et al. 2006) and can be used as a selective AMPAreceptor antagonist in studies of native kainate receptors(Mulle et al. 2000). The study of native kainate receptorshas been further made possible by the development of aseries of 2,3 benzodiazepines, such as GYKI 53655, thatact as AMPA-receptor-selective non-competitive antago-nists (Paternain et al. 1995; Wilding and Huettner 1996),although it should be noted that these compounds have notbeen tested against all kainate-receptor subunits andheteromeric combinations. The potential heterogeneity ofkainate receptors, given the numerous possible modes ofassembly between the various subunits, complicates the

Fig. 1 Kainate-receptorsubunits and splice variants.a The subunit topology isconserved with AMPA andNMDA receptor subunits(ter terminal). b Subunits ofkainate receptors, splice variantsof GluR5, GluR6 and GluR7and editing sites for GluR5 andGluR6

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assessment of pharmacological properties within the family.Nevertheless, a few tools have emerged because of recentefforts with regard to the synthesis and characterization ofnovel compounds (Table 1).

Kainate-receptor agonists

Several kainate-receptor-selective agonists have now beenidentified (Table 1). These include ATPA, (S)-5-iodowillar-

Table 1 Pharmacology of kainate receptors

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diine, SYM2081 and LY339434. ATPA, which is asubstituted analogue of AMPA, and (S)-5-iodowillardiineare potent and selective GluR5 antagonists that display lowaffinity for AMPA and for GluR6- or GluR7-containingreceptors (Clarke et al. 1997; Swanson et al. 1998; Alt et al.2004). AMPA, ATPA and (S)-5-iodowillardiine can alsoactivate GluR6/KA2 heteromeric receptors, despite func-tioning only as partial agonists (Clarke et al. 1997;Swanson et al. 1998; Alt et al. 2004). The gamma-substituted glutamate analogues SYM2081 and LY339434are also more selective for kainate than for AMPAreceptors. Whereas the first displays selectivity for GluR5-and GluR6-containing kainate receptors, the latter seemsmore selective for GluR5 than for GluR6 and GluR7 (Smallet al. 1998; Pedregal et al. 2000; Alt et al. 2004). However,SYM2081 inhibits currents through kainate receptors by aprocess of fast agonist-induced desensitization (Zhou et al.1997) and thus essentially performs as an antagonist ofthese receptors. The naturally occurring marine toxins,dysiherbaine and its natural analogue neodysiherbaine, alsoact as potent kainate-receptor agonists (Sakai et al. 2001;Swanson et al. 2002; Sanders et al. 2005). Dysiherbaine hasallowed the demonstration that the activation of the GluR5subunit within a GluR5/KA2 heteromer suffices to permitthe opening of the receptor channel (Swanson et al. 2002).

Kainate-receptor antagonists

At present, a small number of compounds have beendescribed as selective antagonists of kainate receptors(Table 1); they mainly target GluR5. Compounds of thequinoxalinedione family, including CNQX and NBQX, actas competitive antagonists at native and recombinant kainatereceptors; whereas CNQX exhibits little selectivity forkainate over AMPA receptors, NBQX is far more potent atAMPA receptors (Mayer et al. 2006). Some pyrrolil-quinoxalinedione derivatives have been found to have ahigher affinity for kainate than for AMPA receptors and tobehave as potent anticonvulsants in the kindling model. Thisis particularly true for LU97175, which displays anextremely high selectivity for GluR5 and GluR6 and,especially, for GluR7 (Loscher et al. 1999). Compounds ofa new series of 6-substituted decahydroisoquinolines displaymore selectivity towards kainate receptors and act as potentantagonists at certain subunits. This is the case forLY382884, a compound derived from the non-competitiveAMPA receptor antagonist LY293558, which displaysselectivity towards the GluR5 subunit (Bortolotto et al.1999; Alt et al. 2004), and for LY377770 (O’Neill et al.1998). The willardiine derivative UBP296 has been reportedas the most potent and selective antagonist at GluR5-containing kainate receptors, with activity residing in the Senantiomer, UBP302 (More et al. 2004). More recently,

another series of willardiine derivatives has been synthesizedand tested for antagonist activity. The N3-2-carboxybenzylsubstituted analogue (UBP310) has been found to be apotent and selective antagonist of GluR5-containing kainatereceptors when tested on native rat and human recombinantAMPA and kainate-receptor subtypes (Dolman et al. 2005).NS3763 is the first non-competitive antagonist for kainatereceptors and exhibits selectivity for homomeric GluR5receptors (Christensen et al. 2004). Interestingly, MSVIII-19,a synthetic analogue of the natural agonist dysiherbaine, alsoacts as a potent antagonist of homomeric GluR5 (Sanders etal. 2005). The reason for the wealth of competitive ligandsfor GluR5 as compared with GluR6 probably lies in thepeculiarities of their respective ligand-binding pockets(Mayer 2005; Mayer et al. 2006). The future developmentof selective antagonists (such as non-competitive ligands) forthe major subtype of kainate receptor in the brain, composedof GluR6 and KA2, will be extremely useful. In addition,kainate-receptor function can also be antagonized bylanthanides, particularly lanthanum and gadolinium(Huettner et al. 1998), with a significantly higher potencythan for AMPA receptors. Zinc can also exert an inhibitoryaction on native and recombinant kainate receptors (Huettneret al. 1998; Fukushima et al. 2003). Future work shoulddefine whether zinc inhibition has physiological relevance atmossy fibre synapses, in which zinc is abundant.

Allosteric modulators

Plant lectins, such as concanavalin A, succinyl concanav-alin A, soybean agglutinin and wheat germ agglutinin, havebeen proposed to block the process of desensitization ofkainate receptors, being more effective on homomericassemblies (Partin et al. 1993; Wong and Mayer 1993;Yue et al. 1995). These compounds act via binding to thecarbohydrate side chain of the kainate receptor, N-glyco-sylation of the receptor being essential for the modulationof channel activity (Everts et al. 1997). On GluR6homomeric receptors, concanavalin A exerts its action vianon-specific binding to any of the N-glycosylation sites,even those artificially introduced (Everts et al. 1999), andhas been suggested to “lock” the receptor in the activatablestate, inhibiting the conformational changes required toshift the receptor to the desensitized state (Partin et al.1993; Wong and Mayer 1993; Yue et al. 1995). Analternative mechanism has been proposed whereby Conca-navalin A does not affect the development of or therecovery from desensitization, or the agonist dose-responserelationship (cf. Paternain et al. 1998), but rather shifts thecontribution of different open states of the GluR6 channel(Bowie et al. 2003). Neurons also express endogenouslectins but whether they are involved in regulating kainate-receptor function is not yet known.

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Some cis-unsaturated fatty acids, including arachidonicacid and docosahexanoic acid, which represent majorconstituents of brain membrane phospholipids, can act asblockers of native kainate receptors (Wilding et al. 1998).Membrane fatty acids block GluR6(R) and GluR6(R)/GluR5(R) heteromers but show weak inhibition of GluR5 homo-mers and of unedited kainate receptors (Wilding et al. 2005).The reason that the susceptibility to modulation by fatty acidsstrongly depends on the editing status is not yet understood.

Extracellular anions and cations such as Na+ regulatechannel-gating of native and recombinant kainate receptorsby an allosteric mechanism (Bowie 2002) that is largelydependent on a single residue (M770) in the S2 region ofGluR6 (Paternain et al. 2003) and which appears to bespecific for kainate receptors over AMPA receptors. Changesin pH also affect the function of ionotropic glutamatereceptors. Likewise, protons inhibit most native and recom-binant kainate receptors, except for GluR6/KA1 heteromers(Mott et al. 2003). Inhibition by protons is voltage-independent and relief from this inhibition by extracellularspermine may underlie the potentiation of kainate-receptorcurrents by this polyamine (Mott et al. 2003).

Synaptic function

Low concentrations of kainate reveal the presenceof functional kainate receptors in the brain

Numerous studies have demonstrated the presence offunctional kainate receptors in various neuronal populationsby using kainate receptor agonists, in the presence ofAMPA receptor antagonists when necessary. The presyn-aptic and postsynaptic actions of exogenous kainate-receptor agonists have previously been reviewed (Huettner2003; Lerma 2003). We will comment on these studies onlybriefly because our main purpose is to examine the role ofkainate receptors in synaptic transmission under physiolog-ical conditions, involving activation by the endogenousagonist glutamate. Kainate-receptor agonists can inducepostsynaptic effects characterized by the activation of aninward current or by depolarization of the neuronalmembrane. Inward currents can be observed in CA3pyramidal cells by concentrations of kainate as low as100 nM (Mulle et al. 1998). Since kainate has been widelyused as a neurotoxic agent, it is important to define whetherkainate and AMPA receptors can be activated in differentranges of kainate concentration. The use of mutant mice hashelped define a window of kainate concentrations thatselectively activate kainate receptors. For instance, inhippocampal pyramidal cells, kainate selectively activatesGluR6-containing kainate receptors at concentrations ofless than 1 μM and activates a combination of kainate and

AMPA receptors at higher concentrations (Mulle et al.1998; Bureau et al. 1999). The situation is probably morecomplex in neuronal populations that express a mosaic ofkainate receptors, such as hippocampal interneurons (Mulleet al. 2000; Christensen et al. 2004).

Kainate-receptor agonists induce changes in the proper-ties of γ-aminobutyric acid (GABA)-ergic and glutamatergicsynaptic transmission in electrophysiological experimentsand regulate the release of neurotransmitters from synapto-somal preparations (for reviews, see Huettner 2003; Lerma2003). Because these experiments rely on bath applica-tions of kainate-receptor agonists, it is not always clearwhether kainate exerts a direct effect on kainate receptorslocated on presynaptic terminals or whether GABA orglutamate release is indirectly regulated by alternativemechanisms. One difficulty in interpreting the effects ofkainate-receptor agonists is the frequent presence ofsomatodendritic kainate receptors on presynaptic neurons;this can lead in different ways to synaptic changes. Onesuch example is the kainate-induced activation of CA1interneurons, which leads to substantial GABA release andsubsequent activation of GABAB receptors, depressingsynaptic transmission (Frerking et al. 1999).

Postsynaptic kainate receptors

AMPA and N-methyl-D-aspartate (NMDA) receptors havebeen found to be co-localized in the postsynaptic density ofthe vast majority of glutamatergic synapses in the brain.Both types of iGluRs are activated concomitantly uponrelease of a single quantum of glutamate from thepresynaptic terminal. However, activation of NMDAreceptors only translates into a synaptic current when theMg2+ block is relieved, i.e. at depolarized membranepotentials. Evidence of the existence of postsynaptickainate receptors activated during synaptic transmissionhad long remained elusive. The first demonstration ofpostsynaptic kainate receptors came relatively late fromobservations at synapses between mossy fibres and CA3pyramidal cells in the hippocampus (Castillo et al. 1997;Vignes and Collingridge 1997); in the presence of antag-onists for both AMPA (GYKI 53655) and NMDAreceptors, a slow EPSC was recorded in response to thestimulation of mossy fibres. Because this slow EPSC wasinhibited by CNQX, it was reported to be mediated bykainate receptors. A single stimulation of mossy fibres issufficient to activate synaptic receptors (Castillo et al.1997), although it only yields EPSCs of small amplitude.The analysis of GluR6–/– mice has further demonstratedthat the GYKI-resistant excitatory postsynaptic current(EPSC) is mediated by kainate receptors (Mulle et al.1998). Kainate-receptor-mediated EPSCs have also beenobserved in GABAergic interneurons of the CA1 region

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(Cossart et al. 1998; Frerking et al. 1998), in cerebellarGolgi cells (Bureau et al. 2000), in Purkinje cells (Huang etal. 2004), in both pyramidal cells and interneurons of theneocortex (Kidd and Isaac 1999; Ali 2003; Eder et al. 2003;Wu et al. 2005), in the superficial dorsal horn of the spinalcord (Li et al. 1999), in “Off” bipolar cells of the retina(DeVries and Schwartz 1999) and in the basolateralamygdala (Li and Rogawski 1998).

Interestingly, stimulation of the associational commis-sural fibres fails to elicit a kainate-receptor-mediated EPSCin CA3 pyramidal cells (Castillo et al. 1997). Furthermore,puff application of kainate along the dendritic tree revealsthat postsynaptic kainate receptors are expressed in arestricted manner only in the proximal dendrites facingmossy fibre terminals (in the stratum lucidum; Castillo et al.1997). Glutamatergic synapses in hippocampal CA1 pyra-midal cells (Bureau et al. 1999), medium spiny neurons ofthe dorsal striatum and nucleus accumbens (Chergui et al.2000; Casassus and Mulle 2002) and possibly many otherneuronal populations do not display kainate-receptor-medi-ated EPSCs, although functional postsynaptic kainatereceptors can be activated pharmacologically in these celltypes. Postsynaptic kainate receptors thus show restrictedcellular and subcellular expression (Fig. 2).

The reason that, in most instances, the amplitude ofkainate-receptor-mediated EPSCs is small compared withAMPA-receptor-mediated EPSCs (<10% in response to asingle stimulation) is not clear. This feature, in addition tothe slow rise and decay times, cannot be explained by thediffusion of glutamate out of the synaptic cleft to activateextrasynaptic receptors (Castillo et al. 1997; Vignes andCollingridge 1997; Bureau et al. 2000). A strong argumentagainst such a hypothesis comes from the observation thatkainate receptors take part in miniature EPSCs (attributableto single quantal release events) in CA3 pyramidal cells andin CA1 interneurons (Cossart et al. 2002). To explain theunexpected properties of native kainate-receptor-mediatedEPSCs when compared with the properties of recombinantkainate receptors, an interaction with cytoplasmic proteinssuch as PSD-95 (Garcia et al. 1998) had been proposed tochange the functional properties of kainate receptors.However, reevaluation of these effects has shown only aminor regulation of desensitization by these proteins(Bowie et al. 2003). In CA1 interneurons, despite theirsmall amplitude, the total charge transfer via the twocomponents of the EPSC falls within the same values,because of the slow decay kinetics of kainate-receptor-mediated EPSCs (Frerking et al. 1998). Little has beenundertaken to understand the functional implications of theco-existence of kainate and AMPA receptors (not forgettingNMDA receptors) at certain synapses. The analysis ofFrerking and Ohliger-Frerking (2002) of CA1 interneuronssupports the notion that kainate-receptor-mediated EPSCs

contribute a substantial and prolonged depolarization inconditions of repetitive firing, within a wide range ofphysiological frequencies, whereas AMPA-receptor-medi-ated EPSCs subserve phasic and time-locked excitation.

Regulation of neuronal excitability

Kainate at low nanomolar concentrations (<100 nM)reversibly inhibits the Ca2+-activated K+ current responsiblefor the slow after-hyperpolarizing potential (IsAHP) inhippocampal pyramidal cells (Gho et al. 1986; Melyan etal. 2002; Fisahn et al. 2005; Ruiz et al. 2005). The EC50

value for the inhibition of the IsAHP in CA3 pyramidal cellsby kainate (6 nM; Ruiz et al. 2005) matches well with theaffinity of the so-called high-affinity kainate-binding sites(Monaghan and Cotman 1982) that strongly label the CA3pyramidal cell layer. This value also corresponds to theaffinity of kainate for the recombinant KA1 (Werner et al.1991) and KA2 (Herb et al. 1992) subunits. Accordingly,high-affinity binding sites (J.K. Utvik, T. Giordano, F.Coussen, X. Ottersen and C. Mulle, unpublished) aremarkedly decreased in KA2–/– mice, in parallel with a lossof kainate-induced inhibition of the IsAHP (Ruiz et al. 2005).The high-affinity kainate-binding sites and the inhibition ofthe IsAHP by nanomolar concentrations of kainate are alsolost in GluR6–/– mice (Mulle et al. 1998; Fisahn et al. 2005;Ruiz et al. 2005), although GluR6 does not exhibit high-affinity binding for kainate. This is probably because of anindirect loss of the KA2 protein in the absence of GluR6, asevidenced by a large decrease of KA2 immunoreactivity inCA3 pyramidal cells of GluR6–/– mice (Ruiz et al. 2005)and an overall decrease of KA2 protein in brain extractsfrom these mice (Christensen et al. 2004; Ruiz et al. 2005).Although this has not yet been demonstrated, severalarguments suggest that if KA2 does not take part in aheteromeric receptor with GluR6, the KA2 protein isdirected towards degradation. Of note, no inward current(i.e. direct depolarization) can be observed at concentra-tions of kainate that inhibit the IsAHP, indicating that it doesnot act via an ionotropic action.

Metabotropic actions of kainate, i.e. effects that do notrequire the activation of kainate-receptor-mediated inwardcurrents but that involve indirect pathways such asG-proteins, have been proposed to explain the inhibitionof GABA release in hippocampal cells (Rodriguez-Morenoand Lerma 1998; Cunha et al. 2000; see also below). Thedirect evaluation of these processes at a presynaptic level ischallenging, because of the difficulties in accessing presyn-aptic terminals with patch-clamp electrodes (but see Takagoet al. 2005). In cultured DRG neurons, the activation ofGluR5-containing receptors by kainate elicits an increase inintracellular Ca2+, while inhibiting the activation ofvoltage-dependent Ca2+ channels (Rozas et al. 2003). These

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processes are sensitive to inhibitors of G-proteins andprotein kinase C (PKC) and do not seem to require ionchannel permeation.

The physiological conditions under which the metabo-tropic actions of kainate receptors take place must beconsidered. In CA3 pyramidal cells, postsynaptic kainatereceptors activated by glutamate released from mossy fibresoperate in a bimodal manner, through both an ionotropicaction and the inhibition of the IsAHP (Ruiz et al. 2005).Thus, a short train of stimulation to the mossy fibres notonly directly depolarizes the postsynaptic membrane, butalso increases neuronal excitability. The synaptic inhibitionof the ISAHP is rapidly reversible. Interestingly, theionotropic and metabotropic actions of synaptic kainatereceptors at the mossy fibre synapse can be separated withthe use of mice lacking individual kainate-receptor subunitsand by means of selected kainate-receptor ligands. Forinstance, the metabotropic action is absent in KA2–/– mice,

whereas kainate-receptor-mediated EPSCs are largely pre-served (Ruiz et al. 2005). Stimulation of Schaffer collateralsby a short train at high frequency (100 Hz) induces a long-lasting inhibition of the IsAHP in CA1 pyramidal cells(Melyan et al. 2004). Under these stimulation conditions,no kainate-receptor-mediated inward current is observed,raising questions about the localization and properties ofpostsynaptic kainate receptors in CA1 pyramidal cells.Overall, postsynaptic kainate receptors clearly contribute tosynaptic excitation and to the regulation of neuronalexcitability. The efficiency of kainate receptors in regulat-ing network activity seems to rely on their repetitivesynaptic activation within physiological ranges of firingfrequencies. Thus, kainate receptors appear to be involvedin the temporal integration of excitatory signals.

Some actions of kainate receptors are mediated by non-synaptic glutamate acting on extrasynaptic somatodendritickainate receptors. Exogenous kainate-receptor agonists

Fig. 2 Left Schematic representation of the expression and subcellularlocalization of kainate receptors in the network of hippocampal CA3neurons. Righta Mossy-fibre-mediated EPSCs effected by AMPAreceptors (green) and by kainate receptors (blue; enlarged upperright). b A train of mossy fibre stimuli evokes a compound EPSCmediated by kainate receptors, which is absent in GluR6–/– mice(modified from Mulle et al. 1998). c The role of presynaptic kainate

receptors in frequency facilitation of mossy-fibre-mediated EPSCs.Increasing the stimulation frequency from 0.05 Hz to 0.5 Hz markedlyincreases the amplitude of mossy fibre EPSCs in wild-type but not inGluR6–/– mice. These experiments demonstrate that presynaptickainate receptors are implicated in synaptic facilitation (taken fromContractor et al. 2001)

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trigger firing in GABAergic interneurons and modulateinhibitory synaptic transmission in a number of brainregions including the hippocampus (see (Ben-Ari andCossart 2000; Huettner 2003; Lerma 2003). However, thenature of these somatodendritic extrasynaptic kainatereceptors activated under physiological conditions is notclear. Glutamate released by astrocytes can potentiallyactivate these receptors (Liu et al. 2004); the elevation ofintracellular Ca2+ in astrocytes by Ca2+ uncaging increasesspontaneous inhibitory postsynaptic currents (IPSCs) innearby interneurons by a mechanism that involves theactivation of non-synaptic kainate receptors (Liu et al.2004), probably containing the GluR5 subunit.

Presynaptic kainate receptors

Numerous studies have focused on the actions of exoge-nous kainate-receptor agonists in the regulation of trans-mitter release. This modulation, studied in depth usingsynaptosomal preparations and electrophysiological record-ings, affects both excitatory and inhibitory synaptictransmission (Huettner 2003; Lerma 2003). Exogenouskainate-receptor agonists can either facilitate or inhibitneurotransmitter release, depending on the synapse typeand concentration of the agonist. The mechanisms by whichexogenous kainate-receptor agonists modulate transmitterrelease are unclear because of the difficulty of studyingpresynaptic mechanisms and also, in part, because thepharmacological activation of kainate receptors by bathapplication of exogenous agonists probably affects synaptictransmission both directly and indirectly. In comparison,relatively few reports describe a presynaptic role for kainatereceptors activated by endogenous sources of glutamate.

Presynaptic autoreceptors

The most compelling evidence for a physiological functionof presynaptic kainate receptors is found at synapsesformed onto CA3 pyramidal cells by mossy fibresoriginating from dentate granule cells (Mf-CA3 synapses).Mf-CA3 synapses exhibit characteristic features of short-term plasticity (Salin et al. 1996) in the form of a prominentfacilitation of mossy fibre EPSCs in response to a paired-pulse protocol, to repetitive stimulation in the low frequen-cy range (0.1–3 Hz) or to short trains of higher frequencies(20–100 Hz). Several studies have concurred in implicatingpresynaptic kainate autoreceptors in these processes. Whenmonitoring mossy fibre NMDA receptor EPSCs (recordedin the presence of 20 μM GYKI 53655), CNQX used as akainate-receptor antagonist does not directly affect synaptictransmission in response to a single stimulus but partiallyinhibits frequency facilitation (Schmitz et al. 2001). Asimilar reduction in frequency-dependent facilitation has

been observed by using LY382884 (10 μM), a kainate-receptor antagonist (Lauri et al. 2001b). These data indicatethat synaptically released glutamate can activate a presyn-aptic facilitatory autoreceptor. The analysis of mice withdisrupted kainate-receptor subunit genes has confirmed therole of kainate receptors as facilitatory autoreceptors.Paired-pulse facilitation and frequency facilitation at lowand high stimulation rates are markedly impaired inGluR6–/– mice but not in GluR5–/– or KA2–/– mice(Contractor et al. 2001, 2003). The lack of changes inGluR5–/– mice is at odds with the reported effects ofLY382884 used as a selective antagonist for GluR5-containing kainate receptors. However, it matches with thefinding that the expression of GluR5 mRNA is notdetectable in dentate gyrus granule cells (Wisden andSeeburg 1993; Bureau et al. 1999). In addition, the abilityof LY382884 to block synaptic facilitation has beendisputed (Breustedt and Schmitz 2004). The electrophysi-ological analysis of GluR7–/– mice has provided a new turnin the study of presynaptic kainate receptors. GluR7, whichis expressed at substantial levels in the dentate gyrus(Wisden and Seeburg 1993; Bureau et al. 1999), is aputative presynaptic kainate autoreceptor. Indeed, short-term synaptic plasticity is markedly impaired at thehippocampal mossy fibre synapse of GluR7–/– mice (P.S.Pinheiro et al., submitted; Pinheiro et al. 2005b), as hasbeen observed in GluR6–/– mice. Philanthotoxin, a blockerof Ca2+-permeable AMPA/kainate receptors, decreases themagnitude of synaptic facilitation in wild-type mice but notin GluR7–/– mice. Together with the results from immuno-precipitation experiments, these data suggest that presyn-aptic autoreceptors are composed of GluR6 and GluR7(Fig. 3).

What is the mechanism of action of facilitatory presyn-aptic kainate autoreceptors? Importantly, the synapticactivation of presynaptic kainate receptors takes placewithin less than 10 ms, since inhibition by kainate-receptorantagonists is observed for the second EPSC in highfrequency trains (100 Hz; Schmitz et al. 2001). In keepingwith these data, paired-pulse facilitation is impaired inGluR6–/– and GluR7–/– mice at short interstimulus intervals(<20 ms; Contractor et al. 2001; P.S. Pinheiro et al.,submitted; Pinheiro et al. 2005b). The rapid action ofpresynaptic kainate receptors makes it unlikely that aG-protein-coupled process is involved. Accordingly, cal-phostin C, which inhibits the metabotropic action of kainatein mediating the inhibition of GABA release (Rodriguez-Moreno and Lerma 1998), does not influence synapticfacilitation (Lauri et al. 2001c). Moreover, short-termplasticity is not affected by the low-affinity competitiveantagonist kynurenate, at a concentration (1 mM) thatblocks more than 80% of glutamatergic synaptic transmis-sion (see Bortolotto et al. 1999). This tends to rule out the

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hypothesis of a network effect. Instead, the fast actionfavours a presynaptic mechanism relying directly on theactivation of the receptor channel, causing depolarization ofthe nerve terminal membrane and/or direct Ca2+ influx.Imaging of the presynaptic Ca2+ and membrane voltage ofthe mossy fibre nerve terminals during short-term synapticplasticity (Kamiya et al. 2002) suggests that activation ofpresynaptic kainate receptors leads to depolarization of thenerve terminal, which could thereby augment the actionpotential-dependent activation of voltage-gated Ca2+ chan-nels. At the calyx of Held, a mild depolarization of thepresynaptic membrane potential elicits small Ca2+ eleva-tions that are sufficient to strongly enhance the probabilityof release (Awatramani et al. 2005; Alle and Geiger 2006).Moreover, a small depolarization attributable to the activa-tion of presynaptic kainate receptors might be amplified bythe activation of voltage-gated Na+ channels, which areabundant in mossy fibre terminals (Engel and Jonas 2005).

In addition to a direct depolarization, the finding that theaction of presynaptic kainate receptors can be blocked byphilanthotoxin raises the possibility that direct Ca2+ influxthrough the receptor channel (Lauri et al. 2003; Pinheiro etal. 2005b) can participate in synaptic facilitation. Finally,evidence suggests that Ca2+ release from intracellular storescontributes to the actions of presynaptic kainate receptors atMf-CA3 synapses (Lauri et al. 2003; but see Breustedt andSchmitz 2004). In summary, at the mossy fibre synapse,presynaptic kainate autoreceptors are probably composed ofGluR6 and GluR7 and might be located in the presynapticactive zone close to glutamate release sites. Their activa-tion triggers either depolarization of the nerve terminalor direct Ca2+ influx that may lead to Ca2+ release fromintracellular stores. This remains, up to now, the bestexample for a clearly defined autoreceptor function ofkainate receptors (Fig. 3). Presynaptic kainate receptorsare involved in long-term potentiation (LTP; Henze et al.2000), a form of plasticity that is induced and expressedpresynaptically in mossy fibres (Bortolotto et al. 1999;Contractor et al. 2001; Lauri et al. 2001a; Schmitz et al.2003). Presynaptic kainate receptors are not essential formossy fibre LTP but strongly influence this form ofsynaptic plasticity by changing the induction threshold,possibly by their direct facilitatory effects on synaptictransmission (Schmitz et al. 2003).

Presynaptic kainate receptors are also involved inregulating thalamocortical synaptic depression at earlystages of postnatal development. Synaptic depression ofthalamocortical transmission during trains of stimulation isinhibited by LY382884 (10 μM), but only at the highestfrequencies tested (50 and 100 Hz; Kidd et al. 2002). Theseare frequencies corresponding to those observed duringwhisker activation in vivo. Finally, in the cerebellum, lowfrequency stimulation (5 stimuli, 10 Hz) of parallel fibresfacilitates glutamatergic synaptic transmission onto stellatecells through the activation of presynaptic kainate receptors,whereas high frequency stimulation inhibits synaptictransmission (5 stimuli, 100 Hz; Delaney and Jahr 2002).Interestingly, the situation is different at synapses betweenparallel fibres and Purkinje cells in which facilitation isobserved at all stimulation frequencies. Here, the effects ofkainate receptors activated by endogenous glutamate arealso likely to be mediated by an ionotropic action (Delaneyand Jahr 2002). The difference in the sensitivity of the twoparallel fibre synapses has not been explained and mightrely on subtle differences in receptor composition (granulecells express GluR6 and KA2), density or precise location.

Presynaptic heteroreceptors

Apart from autoreceptors, there is evidence for the presenceof presynaptic kainate receptors acting as heteroreceptors.

Fig. 3 Schematic and hypothetical representation of the mechanismsof action of presynaptic and postsynaptic kainate receptors (PKCprotein kinase C, VSCC voltage-gated Ca2+ channels). This represen-tation takes the mossy fibre synapse as an example. Presynaptickainate receptors acting as autoreceptors are probably localized closeto the synaptic release site where they function as fast facilitatoryreceptors, either by a direct influx of Ca2+ through the kainate-receptorchannel or through depolarization of the presynaptic membrane.Heterosynaptic kainate receptors are thought to be localized at somedistance from the neurotransmitter release site, are activated by thediffusion of glutamate from its source and act with a slower timecourse to facilitate or depress synaptic transmission. Activation ofpostsynaptic kainate receptors (1) generates an EPSC with integrativeproperties that leads to the cumulative depolarization of the postsyn-aptic membrane and (2) induces a down-regulation of the slow IAHP,leading to an increase in neuronal excitability. These various actionsare proposed to arise from kainate receptors with distinct subunitcompositions

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Heterosynaptic regulation of synaptic transmission by theactivation of kainate receptors occurs both at glutamatergicand GABAergic synapses. At mossy fibre synapses,synaptic release of glutamate by brief stimulus trains athigh frequency (200 Hz) causes the heterosynaptic activa-tion of kainate receptors, leading either to synapticinhibition or facilitation (Schmitz et al. 2000, 2001).Heterosynaptic regulation shifts from facilitation to inhibi-tion when increasing the duration of the conditioningtetanus (Schmitz et al. 2001). The mechanism of kainate-receptor-mediated heterosynaptic regulation is not clear andrequires strong stimulation conditions. It appears to dependon receptors composed of GluR6 and KA2 (Contractor etal. 2003), unlike presynaptic autoreceptors, which seem todepend on GluR6 and GluR7, but not KA2. Althoughpresynaptic kainate receptors are proposed to be theeffectors of this regulation, the precise location of thekainate receptors that are activated under these experimen-tal conditions is unclear. Because a general increase inglutamate concentration in the extracellular medium isnecessary, these heterosynaptic kainate receptors might bepositioned at some distance from glutamate release sites, atvariance with presynaptic autoreceptors.

GABA release is also regulated by kainate-receptoragonists (Huettner 1990; Lerma 2003), although it remainscontroversial whether all these effects depend on receptorsthat are specifically localized on GABAergic terminals oron somatodendritic receptors. A few studies have describedthe physiological conditions under which these (hetero-synaptic) kainate receptors are activated. In hippocampalCA1 pyramidal cells, the synaptic release of glutamate by aconditioning train to the Schaffer collaterals inhibits evokedIPSCs by a kainate-receptor-dependent mechanism (Min etal. 1999). A similar conditioning train also enhances axonalexcitability of GABAergic interneurons, an effect suggestedto be attributable to a direct kainate-receptor-dependentdepolarization of axons (Semyanov and Kullmann 2001).Finally, in an elegant study, Ali and collaborators (2001)have recorded unitary IPSCs elicited by fast-spikinginterneurons onto layer V pyramidal neurons by using dualrecording techniques and have provided evidence for theinhibition of evoked GABA release by exogenous kainate-receptor agonists, an effect that is mimicked by apronounced depolarization of the postsynaptic cell (for1.5 seconds). This has been interpreted as being aconsequence of glutamate release from the somatodendriticcompartment of the pyramidal cell and subsequent activa-tion of presynaptic kainate receptors.

In acute slices of the spinal cord, endogenous glutamatereleased upon stimulation of primary afferent fibres with aconditioning train of stimuli (50 Hz, 20 pulses) inhibitsevoked IPSCs via the activation of kainate receptors(Kerchner et al. 2001a); this suppression of inhibition is

prevented by GABAB receptor antagonists (Kerchner et al.2001a). Because glutamate-containing sensory fibres comeinto close proximity with the GABA/glycine terminals oflocal interneurons, presynaptic kainate receptors have beensuggested to enhance GABA/glycine release via an iono-tropic action, leading to a negative feedback pathway onglutamatergic synapses mediated by GABAB autoreceptors(Kerchner et al. 2001a). In such a scheme, presynaptickainate receptors exert a facilitatory action on GABArelease. More direct evidence for a facilitatory action ofpresynaptic kainate receptors on GABAergic terminals hasbeen provided by recordings from cell pairs in the CA1region of the hippocampus. Although kainate-receptoragonists induce biphasic changes in unitary IPSCs in CA1pyramidal cells, depending on the concentration of theagonist, endogenous glutamate only exerts a facilitatoryaction on GABAergic transmission (Jiang et al. 2001)under basal conditions or in response to conditioning trainsto glutamatergic afferents in the stratum radiatum (40stimuli at 100 Hz). Interestingly, CNQX, but not GYKI53655, decreases GABAergic connections with high releaseprobability, indicating that ambient glutamate tonicallyfacilitates synaptic transmission for this group of connec-tions (Jiang et al. 2001). In contrast to a previous study(Min et al. 1999), high frequency trains to the Schaffercollaterals have been shown to increase both evoked unitaryIPSCs and spontaneous IPSCs (Jiang et al. 2001). Thekainate-receptor-mediated potentiation of inhibitory trans-mission may function as a negative feedback control inlocal circuits, although this has not been directly tested. Themechanisms underlying the facilitatory actions of presyn-aptic kainate receptors on GABAergic terminals and theprecise localization of kainate receptors in relation toGABA release sites have not yet been identified. Thegeneral assumption is that the facilitatory actions of pre-synaptic ionotropic receptors require a slight depolarizationof the presynaptic membrane (Engelman and MacDermott2004), possibly leading to large changes in synaptic release(Awatramani et al. 2005). Higher concentrations of theagonist might depress synaptic transmission as a conse-quence of a larger depolarization and inactivation ofpresynaptic voltage-dependent ion channels or via anindirect mechanism, the possibility of which must alwaysbe considered. The release of glutamate from varioussources (e.g. astrocytes; Liu et al. 2004) can also activateextrasynaptic somatodendritic kainate receptors on inter-neurons, leading to increased spontaneous synaptic activity.In this case, the level of synaptic transmission can bealtered through a mechanism independent of presynaptickainate receptors. Compelling data support a role forG-protein-dependent mechanisms in the regulation ofsynaptic transmission by exogenous kainate-receptor ago-nists (for reviews, see Huettner 2003; Lerma 2003).

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However, evidence for these processes in the conditions ofactivation of kainate receptors by endogenous agonists isscarce. One such example of this concerns the regulation ofhippocampal synaptic circuits in the newborn rat. Duringthe first postnatal week, endogenous glutamate tonicallyactivates kainate receptors at CA3 glutamatergic synapsesto inhibit glutamate release via a G-protein and PKC-dependent mechanism (Lauri et al. 2005).

Much remains to be learned regarding the mechanismsof regulation of synaptic transmission by presynaptickainate receptors. The mossy fibre synapse onto CA3pyramidal cells is certainly a promising model synapse,given the possibility of directly recording from thepresynaptic terminals (Bischofberger et al. 2002).

Trafficking of kainate receptors

Subcellular localization

Functional studies strongly suggest that kainate receptorsare localized at diverse subcellular compartments thatdictate the functions that they subserve. Kainate receptorsare targeted to various subcellular domains, with segregatedpopulations of receptors within the same neuron possiblydiffering in their subunit composition and properties. Thehippocampus is a good example of the complexity of thetargeting of kainate receptors within neurons. CA3 pyrami-dal cells express GluR6, KA1 and KA2 subunits of kainatereceptors (Wisden and Seeburg 1993; Bureau et al. 1999)and these cells receive input from different pathways. Atthe synapses formed with mossy fibre inputs, kainate-receptor-mediated EPSCs can be recorded when blockingthe AMPA-receptor-mediated component (Castillo et al.1997; Vignes and Collingridge 1997). However, at the moredistal synapses formed with the associational/commissuralpathway, no EPSCs mediated by these receptors have beendetected and local application of kainate reveals functionalreceptors only in the stratum lucidum, where mossy fibrescontact pyramidal cells (Castillo et al. 1997). The kainatereceptors involved in mossy fibre EPSCs are composed ofat least GluR6 and KA2 (Mulle et al. 1998; Contractor et al.2003) and the development of the presynaptic mossy fibrepathway may potentially play a role in the recruitment ofpostsynaptic kainate receptors to such sites (Marchal andMulle 2004). On the other hand, granule cells in the dentategyrus express GluR6, GluR7, KA1 and KA2 subunits ofkainate receptors. These cells do not display kainate-receptor-mediated EPSCs, although they can potentiallyform recurrent mossy fibre synapses with kainate-receptor-mediated EPSCs in epileptic rats (Epsztein et al. 2005).Instead, in dentate granule cells, kainate receptors probablycomprising GluR6 and GluR7 (Contractor et al. 2001;

Pinheiro 2005b) are targeted to the presynaptic compart-ment where they function as autoreceptors at the synapsesformed with CA3 pyramidal cells.

The subcellular localization of kainate receptors in thesevarious neuronal domains is mainly inferred from electro-physiological studies. Immunohistological evaluation of thedistribution of the kainate-receptor subunits GluR6, KA1and KA2 has confirmed the preferential localization ofthese subunits in the stratum lucidum, the region of mossyfibre synaptic contacts (Petralia et al. 1994; Darstein et al.2003; Ruiz et al. 2005). Immunogold studies with subunit-specific antibodies for KA1 and KA2 receptor subunitshave provided evidence for a preferential localization ofKA1 in presynaptic terminals, with a subset of goldparticles being found near active zones, and a predomi-nantly postsynaptic localization for KA2 (Petralia et al.1994; Darstein et al. 2003). The localization of kainatereceptors to presynaptic compartments has also beendescribed in the rat spinal cord (Hwang et al. 2001).Finally, biochemical enrichment procedures have shownthat KA2 and GluR6 (and/or GluR7) are concentrated infractions corresponding to presynaptic membranes(Pinheiro et al. 2005a). The mechanisms involved in thispolarized targeting of kainate receptors are not yetunderstood. Targeting of kainate receptors to pre- versuspostsynaptic sites is not clearly related to specific subunitsor splice variants but may involve the differential associ-ation of the various subunits and/or splice variants todifferent sets of interacting proteins (Jaskolski et al. 2005a).

Trafficking

Our understanding of the intracellular and membranetrafficking of kainate receptors lags well behind that ofAMPA and NMDA receptors. Studies with recombinantreceptors in cell lines and cultured neurons have recentlydefined a number of rules for the trafficking of kainatereceptors to the plasma membrane. The relative level ofsurface expression of kainate receptors depends on subunitsand alternative splicing of their C-terminal domain and onsubunit composition of heteromeric receptors. Some sub-unit splice variants are endowed with a forward traffickingmotif, whereas others are strongly retained in the endoplas-mic reticulum (ER) by intracellular retention signals(Fig. 4).

KA2 was initially thought to require the presence ofother subunits to form receptor complexes but a recentstudy has shown that KA2 can form homomeric assembliesin heterologous cells (Ren et al. 2003a), although it isheavily retained in the ER in the absence of GluR5, GluR6or GluR7 (Gallyas et al. 2003; Hayes et al. 2003; Ren et al.2003a). This subunit possesses an ER retention motif in itsC-terminus consisting of a stretch of five positively charged

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arginines and also contains a di-leucine endocytic motif thatmay mediate its rapid clathrin-dependent endocytosis andlow steady-state plasma membrane expression. The ERretention/retrieval signal in KA2 is sterically shieldedduring heteromeric assembly, allowing the delivery offunctional heteromeric receptors to the plasma membrane(Ren et al. 2003a). The polyarginine ER retention motiffunctions as an interaction domain for coatomer proteincomplex I (COPI), a complex that plays a central role in the

retrograde trafficking from the Golgi to the ER ofmisfolded or unassembled proteins (Vivithanaporn et al.2006). The assembly of KA2 with GluR6 markedly reducesinteraction between KA2 and COPI.

GluR5, GluR6 and GluR7 can form functional homo-meric receptor channels and have multiple isoforms derivedfrom alternative splicing and RNA editing. These isoformsdisplay different patterns of expression at the plasmamembrane strongly depending on the alternative splicing

Fig. 4 Trafficking of kainatereceptors. a Exit from the endo-plasmic reticulum and targetingof kainate receptors to the plas-ma membrane depends on thecomposition in terms of subunitsand splice variants. b Update ofthe known interacting proteinsof kainate-receptor subunits

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at their C-terminus (Ren et al. 2003b; Jaskolski et al. 2004).By analogy with KA2, the GluR5c splice variant isessentially retained in the ER because of the presence ofan RXR retention motif, similar to that described forNMDA receptors (Standley et al. 2000) and potassiumchannels (Zerangue et al. 1999). GluR7b also contains anRXR motif present in its amino acid sequence but this doesnot act as an ER retention signal and may play a role duringreceptor biogenesis and assembly (Jaskolski et al. 2005b).

GluR6a and GluR7a are expressed at high levels in theplasma membrane and can promote the surface expressionof other subunits that contain ER retention motifs (Jaskolskiet al. 2004; Yan et al. 2004; Jaskolski et al. 2005b), becauseof the existence in their C-terminal portion of a forwardtrafficking motif. This stretch of positively charged aminoacids (CQRRLKHK) is crucial for ER exit but, whenmutated, the receptors can nevertheless reach the plasmamembrane if associated with other receptors containing theintact sequence. The molecular mechanisms by which theCQRRLKHK motif acts as a forward trafficking signal arenot known. The other subunit splice variants studied(GluR5a, GluR5b, GluR6b, GluR7b) are present at lowlevels at the plasma membrane when expressed ashomomers. Other positively charged residues that areconserved between GluR5b and GluR5c are also importantfor their exit from the ER (Ren et al. 2003b). Oligomeri-zation of kainate-receptor subunits plays a major role intheir surface expression as demonstrated, for instance, inthe case of GluR6a/GluR6b heteromers, which form nativekainate receptors (Coussen et al. 2005).

In conclusion, cis-acting regulatory elements encodedwithin the C-terminal domain of kainate-receptor subunitsaffect the trafficking and surface expression of thesereceptors. These processes are not regulated by C-terminalPDZ-binding domains of kainate receptors (Ren et al. 2003a;Jaskolski et al. 2004; Yan et al. 2004), contrary to findingsfrom NMDA receptor subunits. In addition, recent studieshave demonstrated that the N-terminal domain of kainatereceptors also plays an important role in their trafficking andsurface expression (Fleck et al. 2003; Mah et al. 2005;Valluru et al. 2005). The formation of an intact glutamate-binding site has been suggested to act as a quality controlmeasure for the forward trafficking of intracellular kainatereceptors (Mah et al. 2005; Valluru et al. 2005).

The surface expression of glutamate receptors isdynamically regulated. GluR6-containing receptors aresubject to rapid endocytosis followed by sorting to eitherrecycling or degradation pathways, depending on theendocytic stimulus. In cultured hippocampal neurons,whereas exogenous kainate causes a PKC-dependentinternalization of kainate receptors targeted to lysosomesfor degradation, NMDA triggers a Ca2+-, PKA- and PKC-dependent endocytosis of kainate receptors to early endo-

somes for recycling to the plasma membrane (Martin andHenley 2004).

Protein interactions

Many kainate-receptor-interacting proteins have been iden-tified in recent studies. Some of these proteins have beenimplicated in the trafficking, synaptic localization andmodulation of the properties of kainate receptors. Likeother glutamate receptors, kainate receptors contain, at theirC-terminus, a PDZ-binding motif that can bind to proto-typic PDZ-domain-containing proteins such as PSD-95,SAP97 and SAP102. In heterologous systems, the bindingof GluR6a to PSD-95 causes the clustering of kainatereceptors (Garcia et al. 1998; Mehta et al. 2001; Coussen etal. 2002). Interestingly, the moderate specificity in theassociation of kainate receptors with this family of proteinsmay dictate the subcellular localization of the receptors;both SAP90 and SAP102 can associate with KA2 andGluR6 but the presynaptically localized SAP97 has beenfound only to associate with GluR6 (Garcia et al. 1998).The PDZ-domain-containing proteins PSD-95, PICK1,GRIP and syntenin also associate with several subunits ofkainate receptors (Hirbec et al. 2003) but none of them isspecific for this class of receptors. These proteins differen-tially regulate the function and synaptic stability of kainateand AMPA receptors. However, PSD-95, PICK1 and GRIPdo not seem to play any role in the exit from the ER ofeither GluR5 or GluR6 (Ren et al. 2003b; Jaskolski et al.2004). The association between GluR6a and PSD-95 mightalso play an important role in triggering excitotoxicity, byinitiating the formation of a complex with MLK2/3 proteinseventually leading to the activation of c-Jun N-terminalkinase (JNK; Savinainen et al. 2001).

Kainate receptors can also bind to β-catenin and proteinsof the cadherin/catenin complex through an indirectinteraction with the extreme C-terminus of GluR6 (Coussenet al. 2002). Activation of the cadherin/catenin complex, inconditions mimicking the establishment of cell-cell contactsin heterologous cells, triggers the recruitment of GluR6a.Interestingly, overexpression of PSD-95 appears to disruptthe interaction between GluR6a and the cadherin/catenincomplex. This sequence of events suggests that interactionswith cadherin/catenin complexes play an initial role inrecruiting and stabilizing kainate receptors at the synapticmembrane during synapse formation (Coussen et al. 2002).

Although the two splice variants GluR6a and GluR6b donot significantly differ in their functional properties, they canco-assemble into the same heteromeric complex in native andrecombinant receptors, bringing into close proximity differentsets of interacting cytosolic proteins (Coussen et al. 2005).The set of proteins identified by a proteomic approach asinteracting with GluR6a/GluR6b includes dynamin-1, NSF,

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dynamitin and 14-3-3 protein, which are possibly involvedin the assembly and trafficking of membrane receptors, andalso spectrin and profillin II, which may participate incytoskeletal organization. GluR6b also interacts with a groupof proteins including calcineurin, calmodulin, VILIP-1 andVILIP-3, which are involved in the regulation of receptorsand ion channels by Ca2+. Given the apparent complexity ofthe processes of kainate-receptor trafficking and polarizedtargeting in neurons, they are probably governed by intricateprotein interactions with as yet unknown partners.

Plasticity of kainate-receptor function

Mechanisms of synaptic plasticity at the postsynaptic levelinvolve either modifications of the functional properties ofneurotransmitter receptors or changes in their number andsubcellular localization. The functional properties of re-combinant kainate receptors can be modified by directcAMP-dependent protein kinase phosphorylation of theC-terminal domain (Raymond et al. 1993; Wang et al.1993; Traynelis and Wahl 1997). The probability of channelopening of GluR6a receptors expressed in HEK-293 cells isincreased by PKA and decreased by calcineurin (Traynelisand Wahl 1997). In cultured hippocampal neurons, aconditioning activation of NMDA receptors down-regulatesnative kainate receptors in a rapid and reversible manner(Ghetti and Heinemann 2000). This depression depends onCa2+ influx through the NMDA-receptor channel andactivation of calcineurin (Ghetti and Heinemann 2000). Italso requires the heteromeric assembly of GluR6a andGluR6b and the association of calcineurin with theC-terminal domain of GluR6b in the receptor complex(Coussen et al. 2005). PKCα can phosphorylate theC-terminal domain of GluR5b (and possibly equivalentresidues in GluR6a) under the control of PICK1, a kainate-receptor-interacting protein (Hirbec et al. 2003). Disruptionof either PICK1 or GRIP binding (possibly with GluR6/KA2) by the infusion of competitive peptides in CA3pyramidal cells causes a loss in functional synaptic kainatereceptors (Hirbec et al. 2003). In perirhinal cortex neurons,the activation of PKC by the stimulation of mGluR5enhances kainate-receptor-mediated Ca2+ responses andkainate-receptor-mediated EPSCs (Cho et al. 2003). Inter-estingly, this regulation appears to be specific for mGluR5over mGluR1 and the tonic activation of mGluR5 inneurons of the perirhinal cortex regulates synaptic kainatereceptors (Park et al. 2006). In these neurons, a train of 200stimuli at 5 Hz results in long-term depression of kainate-receptor-mediated EPSCs via a mechanism involvingmGluR5, PKC and PICK1 PDZ-domain interactions (Parket al. 2006). The precise way that the interaction of kainatereceptors with these PDZ-domain proteins affects synaptic

function or synaptic localization is still under investigation.In all cases, an understanding of the mechanisms by whichkainate-receptor function is regulated during synapticplasticity lags far behind similar studies for AMPA andNMDA receptors.

Kainate receptors in the development of neuronalcircuits

Several lines of evidence suggest a role for kainatereceptors in neuronal development. mRNAs for kainate-receptor subunits have been detected in the brain at earlydevelopmental stages (Bettler et al. 1990; Bahn et al. 1994),although this does not appear as a specific feature ofkainate receptors amongst other ionotropic glutamatereceptors. In contrast to the other subunits, the GluR5subunit undergoes the clearest qualitative changes, forexample in the developing sensory cortex (Bahn et al.1994), and might thus play a special role in developmentalplasticity. The editing status of GluR5 and GluR6 alsochanges during the critical period of late embryonic andearly postnatal ages (Bernard et al. 1999). In rat DRGneurons, the developmental changes in the editing ofGluR5, which is mostly unedited at embryonic day 17and partially edited in the adult, is reflected by a rapidswitch of kainate receptors from high to low Ca2+

permeability (Lee et al. 2001).Spontaneous network activity is an inherent property of

the developing hippocampus (Ben-Ari et al. 1989) and isthought to be instrumental in the maturation of synapticnetwork activity in this structure. Activation of kainatereceptors comprising the GluR5 subunit by ambientglutamate in the neonatal hippocampus increases thisbursting activity (Lauri et al. 2005). Although the mecha-nisms generating neonatal bursts are complex, presynaptickainate receptors have been proposed to down-regulateglutamatergic inputs onto interneurons, thereby reducingthe synchronization of interneuronal activity and impedingburst generation. The consequences of ablating the GluR5gene on mouse cerebral development have not yet beendirectly addressed. Stages of maturation of hippocampal orneocortical circuitry will probably be affected or delayed bythe lack of GluR5-containing kainate receptors, althoughcompensatory mechanisms might exist such that only subtledifferences occur at the adult stage. Whereas this needs tobe examined in detail, preliminary evidence suggests thatthe postnatal development of network activity is altered inGluR5–/– mice (Lauri et al. 2005). In addition, micedeficient for both GluR5 and GluR6 subunits displayimpaired mossy fibre synaptic transmission not only ofkainate-receptor-mediated EPSCs, but also of AMPA-receptor-mediated synaptic transmission (Marchal and

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Mulle 2004). Whether this is attributable to the lack ofGluR5, of GluR6 or of both subunits remains to be tested.

In layer IV of the somatosensory cortex, developingthalamocortical synapses express postsynaptic kainatereceptors (Kidd and Isaac 1999). The thalamocorticalkainate-receptor-mediated EPSCs can readily be separatedfrom the AMPA-receptor-EPSCs on the basis of their slowkinetics and insensitivity to GYKI 53655 (Kidd and Isaac1999). During the first week of life, the kainate-receptor-mediated component decreases. Interestingly, an LTP-induction protocol causes a rapid reduction in thekainate-receptor-mediated component, in parallel with anincrease of the AMPA-receptor-mediated component. Adetailed and comparative study of the two componentsindicates that two types of thalamocortical synapses existin the developing barrel cortex, viz. kainate synapses andAMPA synapses (Kidd and Isaac 1999; Bannister et al.2005), and that the developmental change might beattributable to a decrease of kainate synapses rather thanto changes in the properties of postsynaptic receptors(Bannister et al. 2005). At the same stages of corticaldevelopment in the rat (postnatal days P3-P6), presynaptickainate receptors (autoreceptors) mediate a form of short-term depression at thalamocortical synapses in response tobrief trains of stimulation at high frequency (100 Hz; Kiddet al. 2002). At the end of the first postnatal week, thismechanism is no longer evident, possibly allowing thetransfer of high-frequency sensory-evoked activity fromthe thalamus to the cortex (Kidd et al. 2002).

Unlike at thalamocortical synapses, kainate-receptor-mediated synaptic transmission at the mossy fibre synapseonto CA3 pyramidal cells is positively regulated during thefirst 2 weeks of postnatal development in the mouse(Marchal and Mulle 2004) and can only be observed atP6. Interestingly, between P6 and P9, the emergence of apostsynaptic kainate receptor component of synaptic trans-mission tightly coincides with the emergence of AMPAreceptor-mediated EPSCs of large amplitude and with thefeatures of a mature synapse (Marchal and Mulle 2004).Thus, the emergence of a kainate receptor-mediated com-ponent is an index of maturity of mossy fibre synapses.Together with the finding that smaller AMPA-receptor-mediated EPSCs are observed in double-mutant mice(GluR5–/–xGluR6–/–), this suggests that kainate receptorsplay a role in the maturation of these synapses (Marchaland Mulle 2004). In addition, the activation of kainatereceptors induces a bi-directional regulation of the motilityof mossy fibre filopodia in the developing hippocampus(Tashiro et al. 2003). Axonal filopodia of mossy fibres,imaged over time with two-photon microscopy, are highlymotile at early developmental stages. Filopodial motility isdifferentially regulated by kainate receptors and by electri-cal stimulation of the slices according to the developmental

stage. In young slices (13–15 DIV), activation of kainatereceptors up-regulates motility, possibly facilitating theexploration of the environment by the filopodia (Tashiroet al. 2003). At later stages (20–22 DIV), down-regulationof filopodia motility by kainate receptors may help instabilizing presynaptic terminals (Tashiro et al. 2003). Themechanisms underlying the bi-directional control of axonalfilopodia motility remain unclear and whether the kainatereceptors involved in this process are localized on presyn-aptic terminals is also not known. Since the reduction ofmotility is blocked by tetrodotoxin (Tashiro et al. 2003), theoverall activation of presynaptic neurons by kainatereceptors might be involved in this process, whereas theincrease in motility might depend on more local activationof kainate receptors at the tip of the axon.

Pathophysiological roles of kainate receptors

Epilepsy

Classical studies have shown that systemic or intracerebralinjections of kainate in rodents cause seizures and epilep-tiform discharges in the hippocampus that propagate toother limbic structures. This has been the most investigatedanimal model of temporal lobe epilepsy. Seminal studies onthis kainate model date back long before the molecularidentification of kainate receptors (for a review, see Ben-Ariand Cossart 2000). However, the finding that submicromo-lar concentrations of kainate generate epileptogenic effectsin slice preparations in the CA3 region, a region enrichedwith high-affinity kainate-binding sites, was highly sugges-tive that kainate receptors (and not AMPA receptors, whichare also activated by kainate) were primarily responsible forthe epileptogenic action of kainate (Ben-Ari and Cossart2000). In the absence of selective ligands for kainatereceptors, the direct demonstration of their involvementcame from the use of GluR6–/– mice, which proved to beresistant to epileptic seizures induced by kainate injection(Mulle et al. 1998). In these mice, high-affinity kainatebinding sites in the hippocampus and activation of currentsby submicromolar concentrations of kainate were abolished(Mulle et al. 1998). The lack of GluR6 prevents bothkainate-induced gamma oscillations and epileptiform burstsin slice preparations (Fisahn et al. 2004). Conversely, over-expression of fully edited GluR6a in the hippocampus byviral delivery rapidly leads to seizures and spontaneousbursting in vitro (Telfeian et al. 2000). Finally, miceengineered to express unedited GluR6 have been found tobe more vulnerable to kainate-induced seizures, eitherbecause of enhanced GluR6-mediated currents or becauseof increased Ca2+ permeability (Vissel et al. 2001). Thehigh levels of kainate-receptor expression in CA3 pyrami-

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dal cells (Wisden and Seeburg 1993; Bureau et al. 1999)and their recurrent connectivity render this region especial-ly sensitive to the epileptogenic effects of kainate. Howev-er, the susceptibility of mice mutant for GluR6 to othermodels of seizure induction (i.e. pilocarpine-induced,picrotoxin-induced, kindling) has not yet been analysedand the outcome might be markedly different from thekainate injection model.

The implication of the GluR5 subunit in epilepsy is morecontroversial. GluR5 is widely accepted as being expressedin GABAergic interneurons of the hippocampus (Wisdenand Seeburg 1993; Bureau et al. 1999) and probablyparticipates in the formation of various subtypes of kainatereceptors, together with GluR6 or KA2 (Mulle et al. 2000;Christensen et al. 2004). Kainate or ATPA, a GluR5-selective agonist, depolarize interneurons and triggersustained spike discharge (Cossart et al. 1998; Frerking etal. 1998; Bureau et al. 1999), which, in turn, leads toincreased spontaneous IPSCs recorded in pyramidal cells ofCA1. On the other hand, GluR5-containing receptors areunlikely to be present in CA3 pyramidal cells (or CA1pyramidal cells; Contractor et al. 2000; Mulle et al. 2000).Because of the dramatic increase of inhibitory drive onpyramidal cells, selective GluR5 agonists have the potentialto increase general inhibition of the hippocampal network.Consistent with this hypothesis, ATPA decreases thedischarge of pyramidal cells and prevents the propagationof seizure activity triggered by bicuculline in an in vitromodel of the neonatal hippocampus (Khalilov et al. 2002).Conversely, the lack of GluR5 leads to higher susceptibilityof the hippocampal network to the epileptogenic effects ofkainate (Fisahn et al. 2004). The paradoxical idea thatGluR5 agonists can act as anti-epileptic agents has yet to beconfirmed in other models of epilepsy, especially in theadult animal. In contrast to this notion, strong inhibition ofevoked IPSCs has also been observed in response tokainate; this has been suggested to underlie its epilepto-genic effects (Rodriguez-Moreno et al. 1997), although,when recording spontaneous activity in the hippocampus,the net effect of kainate is clearly a large increase in IPSCfrequency. In agreement with an epileptogenic action ofGluR5 agonists, intravenous injections of ATPA induceclonic seizures in mice (Kaminski et al. 2004). In addition,topiramate, an antiepileptic drug proposed to antagonizeGluR5-mediated synaptic currents in the amygdala (Gryderand Rogawski 2003), selectively protects against clonicseizures induced by ATPA and, to a lesser extent, bykainate (Kaminski et al. 2004). The implication of GluR5 ingenerating seizures induced by ATPA should be confirmed,since ATPA is also a weak agonist of AMPA receptors andof GluR6/KA2 heteromers, the main kainate-receptorsubtype in the brain. Because of the variety of possibletargets for topiramate, the issue of its selectivity for GluR5

during epileptogenesis also deserves further attention,for instance regarding whether it affects other kainate-receptor subtypes. In hippocampal slices, selectiveantagonists of GluR5-containing receptors (LY377770,LY382884) prevent the development of epileptiformactivity evoked by the muscarinic agonist pilocarpine(Smolders et al. 2002). In vivo, these antagonists preventseizures induced in mice by corneal stimulation (Smolderset al. 2002). However, LY377770 (or LY382884) does notprevent picrotoxin-induced seizures and epileptiformbursts (Smolders et al. 2002). In future studies, the effectsof both agonists and antagonists of GluR5 should becompared in similar models of temporal lobe epilepsy andthe specificity of the targeted receptors must be establishedgiven the large combinatorial possibilities for heteromeri-zation of kainate receptors.

A related problem is the way in which the statusepilepticus changes the expression and functional propertiesof kainate receptors. In the rat hippocampus followingkainate-induced seizures (Bernard et al. 1999) and inhuman patients (Grigorenko et al. 1998; Kortenbruck etal. 2001), the level of editing of both GluR5 and GluR6 issignificantly altered. The increase in editing in neocorticaltissues has been proposed to serve as an adaptative reactionto prevent excessive Ca2+ influx during epileptic seizures.In parallel, the levels of kainate-receptor mRNAs inpatients with temporal lobe epilepsy are altered in acomplex manner, depending on the cell type (Mathernet al. 1998). The immediate early gene c-fos, which isinduced by activity, regulates the expression of GluR6 inmice with kainate-induced seizures (Zhang et al. 2002). Inboth human patients and animal models of temporal lobeepilepsy, the axons of dentate granule cells sprout togenerate recurrent mossy fibres, leading to enhancedexcitation that can facilitate seizure generation (Nadler2003). An elegant study has recently demonstratedthat recurrent mossy fibres in pilocarpine-treated ratsestablish aberrant kainate-receptor-operated synapsesonto granule cells (Epsztein et al. 2005). Kainate receptorsthus contribute to the enhanced synchronized networkactivity in the dentate gyrus of epileptic rats (Epsztein etal. 2005).

Excitotoxicity

Administration of kainate is known to cause an increase inintracellular Ca2+ and to trigger Ca2+-dependent pathwaysthat lead to neuronal apoptosis in many regions of the brain.Systemic injection of kainate into rodents also results in theactivation of glial cells and inflammatory responsestypically found in neurodegenerative diseases (Sun et al.2006). Regions that are particularly sensitive to kainateexcitotoxicity, such as the striatum or the CA3 pyramidal

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cell region of the hippocampus (Nadler 1978), are regionswith high expression levels of the kainate-receptor genesGluR6 and KA2 (Wisden and Seeburg 1993; Bureau et al.1999). Although receptors with high affinity for kainate areprobably involved in this process, there have beenrelatively few direct demonstrations that kainate receptorsare indeed involved. Of these, immunocytochemical andmorphological examinations of the CA3 pyramidal cellregion in response to kainate injections have clearlyidentified kainate receptors comprising the GluR6 subunitas key receptors in triggering excitotoxicity (Mulle et al.1998). Kainate-induced toxicity in hippocampal slicecultures (with 5 μM kainate) is restricted to the CA3 region(Best et al. 1996), is prevented in cultures from GluR6–/–

mice (C. Brana, L.E. Sundstrom and C. Mulle, unpub-lished) and can be attenuated by preconditioning thecultures with a prior exposure to a sub-lethal concentrationof kainate in the presence of the AMPA receptor antagonistGYKI 53655 (Nvue et al. 2004). Oligodendrocytes are alsovulnerable to excitotoxic insults mediated by Ca2+ influxthrough kainate receptors, although this is also the case forAMPA receptors (Sanchez-Gomez and Matute 1999). Inaddition, a brief period of activation of kainate receptorsincreases the death of oligodendrocytes caused by comple-ment attack (Alberdi et al. 2006). The signaling mechanismslinking kainate-receptor activation to cell death appear toinvolve the activation of JNK3 and the anchoring of mixed-lineage kinase to the receptor complex (Savinainen et al.2001). Interestingly, as for NMDA receptors (Aarts et al.2002), PSD-95 plays a critical role in GluR6-mediatedJNK activation (Savinainen et al. 2001). In the rat,cerebral ischaemia followed by reperfusion enhances theassembly of the GluR6/PSD-95/MLK2/3 signaling moduleand JNK activation (Pei et al. 2006) suggesting thatimpairment of the association between GluR6 and PSD-95might be neuroprotective. In support of this hypothesis,peptides comprising the 9 C-terminus amino-acids ofGluR6 fused to the Tat peptide exhibit a protective effectagainst neuronal death when administered by cerebro-ventricular injection to a rat 40 min before ischaemia(Pei et al. 2006).

Two independent groups have demonstrated that part ofthe variance in the age of onset of Huntington’s disease, aneurodegenerative condition that particularly affects thestriatum with its high expression of GluR6, could beattributable to a polymorphism in the 3′-untranslated regionof GluR6 (Rubinsztein et al. 1997; MacDonald et al. 1999).GluR6–/– mice do not display obvious motor impairmentsin basal conditions but they die earlier and display earliermotor symptoms after injection of 3-nitropropionic acid,suggesting a complex role of GluR6 during the neuro-degeneration process in this model of Huntington’s disease(Diguet et al. 2004).

Pain

Small diameter DRG neurons, including cells that conveynociceptive information into the spinal cord, are known toexpress functional kainate receptors (Huettner 1990; Lee etal. 2001). Early studies demonstrated depolarization ofC-fibres and blockade of action potentials (Agrawal andEvans 1986) in dorsal root fibres by exposure to kainate.Kainate receptors contribute to synaptic transmission in theperiphery and spinal cord and are located on the centralendings of DRG neurons, where they regulate glutamater-gic spinal sensory transmission (Kerchner et al. 2001b,2002; Youn and Randic 2004). In addition, postsynaptickainate receptors contribute to EPSCs evoked by high-threshold dorsal root fibre stimulation (Li et al. 1999).Finally, presynaptic kainate receptors are expressed onspinal cord inhibitory interneurons (Kerchner et al. 2001a).The involvement of kainate receptors at many stages ofsynaptic transmission of noxious stimuli offers promisingclinical perspectives. Several studies have provided evi-dence for anti-nociceptive effects of compounds directed atthe GluR5 subunit (Procter et al. 1998; Simmons et al.1998; Dominguez et al. 2005), the key receptor expressedin DRG neurons (Mulle et al. 2000; Kerchner et al. 2002).In keeping with these pharmacological data, noxiousresponses to capsaicin and formalin are strongly reducedin GluR5–/– but not GluR6–/– mice (Ko et al. 2005). Despitethis, no behavioural change has been observed in models ofmechanical allodynia (Ko et al. 2005).

Human molecular genetic studies

In humans, the gene coding for GluR5 (GRIK1) is dividedinto 18 exons and covers 402 kb in region 21q22.1 (Barbonand Barlati 2000), a region triplicated in Down syndrome.The gene coding for GluR6 (GRIK2) is split into 17 exonsand spans a region of about 670 kb on chromosome 6,position q21 (Barbon et al. 2001; Jamain et al. 2002). Agrowing body of literature implicates glutamatergic dys-function as a possible substrate for the pathophysiology ofschizophrenia (Table 2), with a strong focus on abnormal-ities in NMDA-receptor-mediated neurotransmission(Moghaddam 2003). GluR7 (GRIK3) has been proposedto be linked to schizophrenia in two genetic studies, oneassociating the disease with a GluR7 polymorphism(ser310ala; Begni et al. 2002) and the other reportingaberration in GluR7 DNA copy-number, among a numberof other important proteins (Wilson et al. 2006). However,none of the kainate-receptor genes have been reported to belinked to schizophrenia in the Japanese population (Shibataet al. 2001, 2002, 2006), indicating that the link betweenGluR7 and schizophrenia needs to be further explored. Inschizophrenic patients, maternal transmission disequilib-

474 Cell Tissue Res (2006) 326:457–482

rium of GRIK2 (GluR6) also suggests a role for this gene asa susceptibility gene for schizophrenia (Bah et al. 2004).

Two independent studies performed in families withautistic children show a significant linkage and associationbetween GRIK2 and autism (Table 2), with an excess ofmaternal transmission of GRIK2 haplotypes (Jamain et al.2002; Shuang et al. 2004), as in schizophrenia. Only one ofthe nucleotide variations identified leads to a modificationof the protein sequence, at amino acid M867I, in theC-terminal domain of GluR6a. An evaluation of theconsequences of this mutation for synaptic trafficking orfunction of GluR6-containing receptors will be interesting.GluR6–/– mice have not yet been tested in behavioural tasksrelevant to the autism phenotype.

Epilepsy is present in an estimated 30% of the autisticpopulation. However, no genetic link has been foundbetween GRIK2 and familial forms of epilepsy (Sander etal. 1995), although GluR6-containing receptors are thoughtto be key players in models of temporal lobe epilepsy (seeabove). Juvenile absence epilepsy is a common subtype ofidiopathic generalized epilepsies for which hereditaryfactors play a major role (Table 2). An allelic associationof a GRIK1 (GluR5) polymorphism (a nine-copy tetranu-cleotide repeat) with the disease has been observed in 20families of patients (Sander et al. 1997), although thistetranuclotide repeat is localized in an intron, leading to noobvious functional mutation (Izzi et al. 2002).

Perspectives

Kainate receptors are closely related to AMPA receptors. Incontrast to AMPA receptors, which play a major role inglutamatergic synaptic transmission in the vast majority ofexcitatory synapses in the brain, the roles of kainatereceptors in the activity of synaptic networks and in brainfunction are still a matter of uncertainty. Kainate receptors

are now thought to be key modulators of network activitythrough a variety of distinct mechanisms that include: (1)the depolarization of the neuronal membrane by activationof an inward current, (2) the regulation of neuronalexcitability through the modulation of ionic conductanceand (3) the regulation of excitatory and inhibitory neuro-transmitter release. At variance with AMPA receptors,which exert a phasic action through fast activation bysynaptically released glutamate, kainate receptors appear toexert both phasic and tonic actions at presynaptic andpostsynaptic sites. Future experiments will need to addressthe way in which this variety of effects is integrated todefine and to regulate specific brain functions. A parameterthat might clearly specify the difference between AMPAand kainate receptors is temporal integration, as previouslyproposed (Frerking and Ohliger-Frerking 2002), becausethe activation of kainate receptors seems to be prominentupon repetitive synaptic stimulation or tonic activation.These hypotheses have to be extended not simply to asynaptic locus, but to the overall activity of networks underphysiological conditions of kainate-receptor activation.

Current approaches to test the importance of kainatereceptors in defined brain functions include the analysis ofmutant mice for kainate-receptor subunits. Invalidation ofany of the kainate-receptor subunits in the mouse does notlead to any overt phenotype. However, these mice displaysubtle phenotypes that need to be better explored. Forinstance, in GluR6–/– mice, locomotor activity appearsnormal under basal conditions but is largely decreased inresponse to novelty and during high locomotion phases ofnictemeral activity (X. Cador and C. Mulle, unpublished).In addition, GluR6–/– mice do not show clear impairmentsin the basic configuration of the Morris water maze butdisplay strongly reduced fear conditioning (Ko et al. 2005).Given the high expression of kainate receptors at the mossyfibre synapses onto CA3 pyramidal cells, behavioural teststhat engage this synaptic input should be used (Nakazawa

Table 2 Kainate receptors and human genetics of disease (SNP single-nucleotide polymorphism)

Receptor Gene Locus Associated disease Alteration References

GluR5 GRIK1 21q22.1 (11q11) Juvenile absenceepilepsy

Tetranucleotide polymorphismin non-coding region

Sander et al. 1997

GluR6 GRIK2 6q16.3-q21 (20q13) Schizophrenia Maternal transmissiondisequilibrium

Bah et al. 2004

Early-onsetHuntington’s disease

Polymorphism in the3′-untranslated region

MacDonald et al. 1999Rubinsztein et al. 1997

Autism Maternal transmission disequilibriumof Met867Ile polymorphism

Jamain et al. 2002

Transmission disequilibriumof SNPs rs2227281and rs2227283

Shuang et al. 2004

GluR7 GRIK3 1p34-p33 (5q36) Schizophrenia Ser310Ala polymorphism Begni et al. 2002Change in DNA copy number Wilson et al. 2006

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et al. 2002). Because of possible compensatory mecha-nisms, new mouse models with conditional gene ablationshould also be developed. An understanding of the role ofkainate receptors in higher brain functions is also importantfor the emerging involvement of these receptors in complexcognitive diseases such as autism or schizophrenia.

The relative lack of specific compounds for subtypes ofkainate receptors continues to hamper physiological andpsychopharmacological studies. GluR5 has proven to be aprominent target for the development of competitiveligands, probably because of a favourable structure of thebinding site (Mayer et al. 2006). These tools have played animportant role in implicating GluR5 receptors in severalphysiological and pathophysiological conditions. By con-trast, this underscores the lack of convenient pharmacolog-ical tools for the most abundant kainate-receptor subtype inthe brain, namely GluR6/KA2 heteromers.

Much remains to be understood with respect to themechanisms of action of native kainate receptors. The ques-tion of the slow kinetics and small amplitude of the EPSCsmediated by kainate receptors is unresolved and does notdepend in a simple manner on an interaction with anyidentified cytosolic protein or the localization at somedistance from glutamate release sites. The mode of action ofpresynaptic kainate receptors has been intensely debated butthe difficulty of recording electrophysiological responsesmediated by kainate receptors from presynaptic terminalshas limited the evaluation of such mechanisms. Bothionotropic and metabotropic mechanisms are possible; thismight depend on subtype and precise localization with respectto synaptic release sites. “Non-canonical” signalling bykainate receptors (Rozas et al. 2003; Melyan et al. 2002),eagerly awaits a molecular explanation of the way that thesereceptors are coupled to the activation of a G-protein-dependent mechanism in the absence of any detectableionotropic action. Extrasynaptic kainate receptors activatedby ambient glutamate either at pre- or postsynaptic sitesprobably play a prominent role in the tonic regulation ofneuronal excitability or synaptic release properties. Recentstudies showing that stimulation of astrocytes plays a role inthe tonic activation of somatodendritic kainate receptors (Liuet al. 2004) should be extended in order to examine thecellular and pathophysiological conditions in which extra-synaptic glutamate engages kainate receptors.

The disparate actions of kainate receptors are supportedby the variety shown in their subcellular localization. Ourknowledge of the mechanisms of the polarized traffickingand stabilization of kainate receptors in discrete neuronaldomains is far less advanced than for those of AMPA andNMDA receptors. Various approaches have identifiedsubsets of proteins interacting with the C-terminal domainof kainate-receptor subunits for which a role in assembly,trafficking and function of kainate receptors has not yet

been unraveled. More protein interactors will probably beidentified in the near future and might help in identifyingmolecular determinants for the polarized trafficking andanchoring of kainate receptors in discrete functionaldomains. This will require the use of techniques amenableto the study of these mechanisms in an in vivo situation.Indeed, the role of the cellular environment in defining theprecise localization and density of kainate receptors isprobably crucial. In this regard, the finding that exuberantmossy fibres that sprout in epileptic rats create recurrentsynaptic contacts activating postsynaptic kainate receptorsis intriguing (Epsztein et al. 2005). These data point to amaster role of the presynaptic afferent in recruiting kainatereceptors at the synapse.

As is evident from this short and incomplete survey ofproblems to be solved in the field of kainate receptors, thereis an urgent need to combine a wide array of methodolog-ical approaches, from the structural study of potentialligand-binding sites to behavioural analyses. Because theyplay a prominent role in the regulation of synapticnetworks, kainate receptors will continue to emerge ashighly promising therapeutic targets for the amelioration ofhigher cognitive disorders and for the treatment ofexcitotoxic insults and pain conditions.

Acknowledgements We are grateful to David Perrais and NeilDavies for careful reading of the manuscript.

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