Kainate receptors in epilepsy and excitotoxicity.

Kainate (KA), an analog of glutamate, is a potent neurotoxin that has long been known to induce behavioral and electrophysiological seizures as well as neuropathological lesions reminiscent of those found in patients with temporal lobe epilepsy. More than a decade after the initial KA studies, molecular cloning of ionotropic glutamate receptors identified a family of receptors that binds KA with high affinity. The present review explores the links between the epileptogenic and excitotoxic actions of KA and the function of kainate receptors (KARs) in the activity of neuronal networks. We first present evidence that KARs are the main targets of KA to produce the epileptogenic and excitotoxic effects of KA and KA analogs, and we discuss the mechanisms of action of KA. Then the review evaluates the involvement of KARs activated by the endogenous agonist glutamate in the generation and propagation of epileptiform activity. Finally, we report recent findings proposing KARs as targets of antiepileptic drugs and neuroprotective agents.

Kainate receptor-interacting proteins and membrane trafficking.

Kainate receptors are composed of several subunits and splice variants, but the relevance of this diversity is still not well understood. The subunits and splice variants show great divergence in their C-terminal cytoplasmic tail region, which has been identified as a region of interaction with a number of protein partners. Differential trafficking of kainate receptors to neuronal compartments is likely to rely on interactions with distinct subsets of protein partners. This review summarizes our knowledge of the regulation of trafficking of kainate receptors and focuses on the identification and characterization of functions of interacting partners.

Kainate receptor-mediated synaptic currents in cerebellar Golgi cells are not shaped by diffusion of glutamate.

We report the presence of kainate receptors (KARs) in cerebellar Golgi cells of wild-type but not GluR6-deficient mice. Parallel fiber stimulation activates KAR-mediated synaptic currents [KAR-excitatory postsynaptic currents (EPSCs)] of small amplitude. KAR-EPSCs greatly differ from synaptic currents mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors (AMPAR-EPSCs) at the same synapse. KAR-EPSCs display slow rise and decay time and summate in response to a train of stimulations. By using PDA, a low-affinity competitive antagonist and agents that modify the clearance of glutamate, we show that these properties cannot be explained by diffusion of glutamate outside of the synaptic cleft and activation of extrasynaptic KARs. These data suggest that the slow kinetic of KAR-EPSCs is due to intrinsic properties of KARs being localized at postsynaptic sites. The contrasting properties of KAR- and AMPAR-EPSCs in terms of kinetics and summation offer the possibility for a glutamatergic synapse to integrate excitatory inputs over two different time scales.

Functional GluR6 kainate receptors in the striatum: indirect downregulation of synaptic transmission.

Kainate receptors (KARs) are abundantly expressed in the basal ganglia, but their function in synaptic transmission has not been established. In the present study, we show that the GluR6 subunit of KARs is expressed in both substance P- and enkephalin-containing GABAergic projection neurons of the mouse striatum. Using whole-cell voltage-clamp recordings in brain slices, we demonstrate the presence of functional KARs in the dorsal striatum activated by low concentrations of the AMPA/KAR agonist domoate in wild-type but not GluR6-deficient mice. Despite the abundance of KARs, we found no evidence for synaptic activation of these receptors after single or repetitive stimulation of glutamatergic afferents. Domoate induces a transient increase in the frequency of spontaneous IPSCs of small amplitude and a sustained depression of large IPSCs evoked by minimal electrical stimulation within the striatum in wild-type mice but not in GluR6-deficient mice. This depressant effect is inhibited in presence of adenosine A(2A) receptor antagonists, ZM-241385 and SCH-58261. These data strongly suggest that, in striatal neurons, KARs depress GABAergic synaptic transmission indirectly via release of adenosine acting on A(2A) receptors.

Subunit composition of kainate receptors in hippocampal interneurons.

Kainate receptor activation affects GABAergic inhibition in the hippocampus by mechanisms that are thought to involve the GluR5 subunit. We report that disruption of the GluR5 subunit gene does not cause the loss of functional KARs in CA1 interneurons, nor does it prevent kainate-induced inhibition of evoked GABAergic synaptic transmission onto CA1 pyramidal cells. However, KAR function is abolished in mice lacking both GluR5 and GluR6 subunits, indicating that KARs in CA1 stratum radiatum interneurons are heteromeric receptors composed of both subunits. In addition, we show the presence of presynaptic KARs comprising the GluR6 but not the GluR5 subunit that modulate synaptic transmission between inhibitory interneurons. The existence of two separate populations of KARs in hippocampal interneurons adds to the complexity of KAR localization and function.

Kainate receptor-mediated responses in the CA1 field of wild-type and GluR6-deficient mice.

Kainate receptors are abundantly expressed in the hippocampus. Mice with disruption of kainate receptor subunits allow the genetic dissection of the role of each kainate receptor subunits in the synaptic physiology of the hippocampus, as well as in excitotoxic processes. We have compared the action of domoate and kainate on CA1 pyramidal neurons in slices from wild-type and GluR6-/- mice. The difference in the amplitude of inward currents evoked by domoate and kainate between wild-type and GluR6-/- mice demonstrates the presence of functional kainate receptors in CA1 pyramidal neurons. Block of domoate-activated inward currents by the AMPA receptor antagonists 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo(F)quinoxaline (1 microM) and 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine) (GYKI 53655) (50 microM) is complete in GluR6-/- mice but only partial in wild-type mice. In the presence of GYKI 53655, kainate receptor activation dramatically increases the frequency of spontaneous IPSCs in CA1 pyramidal cells from wild-type, as well as GluR6-/-, mice. This results from the kainate receptor-mediated activation of a sustained inward current and an increased action potential firing in afferent GABAergic interneurons of the CA1 field. These effects are observed in wild-type, as well as GluR6-/-, mice. Kainate receptors also decrease the amplitude of evoked IPSCs in CA1 pyramidal cells by increasing synaptic failures in wild-type and GluR6-/- mice. These results indicate that in CA1 pyramidal cells, distinct subtypes of kainate receptors mediate several functionally antagonistic effects.

Altered synaptic physiology and reduced susceptibility to kainate-induced seizures in GluR6-deficient mice.

L-glutamate, the neurotransmitter of the majority of excitatory synapses in the brain, acts on three classes of ionotropic receptors: NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptors. Little is known about the physiological role of kainate receptors because in many experimental situations it is not possible to distinguish them from AMPA receptors. Mice with disrupted kainate receptor genes enable the study of the specific role of kainate receptors in synaptic transmission as well as in the neurotoxic effects of kainate. We have now generated mutant mice lacking the kainate-receptor subunit GluR6. The hippocampal neurons in the CA3 region of these mutant mice are much less sensitive to kainate. In addition, a postsynaptic kainate current evoked in CA3 neurons by a train of stimulation of the mossy fibre system is absent in the mutant. We find that GluR6-deficient mice are less susceptible to systemic administration of kainate, as judged by onset of seizures and by the activation of immediate early genes in the hippocampus. Our results indicate that kainate receptors containing the GluR6 subunit are important in synaptic transmission as well as in the epileptogenic effects of kainate.

Potentiation of GABAergic synaptic transmission by AMPA receptors in mouse cerebellar stellate cells: changes during development.

1. The effects of low concentrations of domoate, an agonist at both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and kainate receptors (AMPARs and KARs, respectively), were investigated in stellate cells in slices of mouse cerebellum at two developmental stages (postnatal day (PN) 11-13 and PN21-25). 2. Low concentrations of domoate enhanced the frequency of miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin (TTX) at PN11-13 but not at PN21-25. 3. The effects of low concentrations of domoate on synaptic activity were probably mediated by the activation of AMPARs and not KARs, since they were blocked by GYKI 53655 (LY300168), a selective AMPAR antagonist. 4. Domoate increased mIPSC frequency in part by activation of presynaptic voltage-dependent Ca2+ channels since potentiation was reduced by 60 % in the presence of Cd2+. AMPARs in stellate cells were found to be permeable to Ca2+. The residual potentiation in the presence of Cd2+ could thus be due to a direct entry of Ca2+ through AMPAR channels. 5. In the presence of TTX, potentiation of synaptic activity by focal application of domoate was not restricted to the region of the cell body, but was observed within distances of 120 micro(m). These experiments also revealed a strong spatial correlation between the location of the presynaptic effects of domoate and the activation of postsynaptic AMPARs. 6. Our data show a developmentally regulated presynaptic potentiation of synaptic transmission between cerebellar interneurones mediated by AMPARs. We discuss the possibility that the developmental switch could be due to a shift in the localization of AMPARs from the axonal to the somato-dendritic compartment.

Spatial distribution of kainate receptor subunit mRNA in the mouse basal ganglia and ventral mesencephalon.

In an attempt to gain knowledge of the possible functions of kainate receptors, we have used in situ hybridization to examine the regional and cellular expression patterns of glutamate receptor subunits GluR5-7, KA1 and KA2 in the adult mouse basal ganglia, known to play a pivotal role in the translation of motivation into actions. Kainate receptor subunits were found to be differentially expressed in the circuitry forming the basal ganglia. They differ from each other in expression levels and their spatial localization. GluR6 appeared as the key subunit for the descending gamma-aminobutyric acid (GABA)ergic-glutamatergic pathways, with highest message levels in the caudate putamen, globus pallidus and subthalamic nucleus as well as in the nucleus accumbens and olfactory tubercle. GluR7 exhibited highest expression in the ascending nigrostriatal and mesolimbic dopaminergic neurons. GluR5 had a restricted distribution pattern, with high expression in the ventral pallidum, the islands of Calleja and pars compacta of the substantia nigra. KA2 was usually coexpressed with GluR6, although with a generally lower level of expression. Finally, KA1 mRNA was barely detectable in these neuronal circuits. These data suggest that kainate receptors in general may be involved in the functions associated with the basal ganglia, with a key role in the control of the central dopaminergic transmission. Thus, they might be implicated in the neurodegenerative and psychic disorders associated with an impairment of the basal ganglia.

Evidence for "preterminal" nicotinic receptors on GABAergic axons in the rat interpeduncular nucleus.

Presynaptic nicotinic ACh receptors (nAChRs) are abundant in the nervous system, where they are thought to regulate the release of various neurotransmitters. Whole-cell recordings performed on rat interpeduncular nucleus neurons using the thin-slice technique showed that nicotine dramatically increased the frequency of postsynaptic GABAergic currents. This effect was observed at low micromolar concentration of agonist; it was mimicked by cytisine, dimethylphenylpiperazinium, and ACh in the presence of eserine. It was blocked by hexamethonium, dihydro-beta-erythroidine, and mecamylamine. The presynaptic action was suppressed in the presence of TTX. A comparable effect of nicotine was found using a preparation of acutely isolated neurons that had retained synaptic terminals attached to their cell body as evidenced by immunoreactivity to synaptophysin and presence of spontaneous GABAergic and glutamatergic synaptic activity. Nicotinic agonists increased the frequency of GABAergic postsynaptic currents, an effect blocked by curare and mecamylamine. This action was also suppressed in the presence of TTX. These data suggest the presence of nAChRs at a preterminal level on axons of intrinsic GABAergic neurons. We propose that, in contrast to presynaptic nAChRs, activation of these "preterminal" nAChRs can trigger a spike discharge and thus have a generalized action on the GABAergic afferent.

Photoactivatable agonist of the nicotinic acetylcholine receptor: potential probe to characterize the structural transitions of the acetylcholine binding site in different states of the receptor.

The nicotinic acetylcholine receptor exhibits at least four different affinity states for agonists such as acetylcholine. In order to identify the structural changes occurring at or near the agonist binding site during the allosteric transitions, three photoactivatable compounds designed to display agonist activity were synthesized. Inhibition constants of these compounds for the cholinergic and the noncompetitive blocker binding sites were determined for the resting and the desensitized states of the receptor. Among these probes, two ligands, AC5 and AC7, displayed a high affinity for the agonist binding site and were poorly recognized by the binding site for noncompetitive blockers. Electrophysiological experiments revealed that these ligands behaved as agonists at low concentrations. We used these two compounds in photolabeling experiments and observed that they were able to inactivate the agonist binding site. Up to 50% of these sites were irreversibly inhibited, depending on the ligand, the irradiation conditions, and the selected receptor state. The compound with the most interesting properties (high affinity and selectivity for the acetylcholine binding site, as well as agonist activity and high photolabeling yield) is AC5, a structural analogue of the fluorescent agonist dansyl-C6-choline, which has been previously used to characterize the different states of the nicotinic receptor. After radioactive synthesis, [3H]AC5 was shown to label all four receptor subunits, in a protectable manner. This radioligand, thus, appears suitable for investigation of the dynamics of allosteric transitions occurring at the activated acetylcholine binding site.

Potentiation of nicotinic receptor response by external calcium in rat central neurons.

Nicotinic acetylcholine receptor (nAChR) responses of rat medial habenular neurons are potentiated up to 3.5-fold by increasing the concentration of external Ca2+ in the millimolar range. This effect, independent of voltage, is probably due to the binding of Ca2+ to an external site. External Ca2+ decreases nAChR single-channel conductance at negative but not positive potentials, and it markedly enhances the frequency of opening of acetylcholine activated channels. The potentiating effect of Ca2+ is mimicked by Ba2+ and Sr2+, but barely by Mg2+. These data support the existence of positively acting allosteric sites for Ca2+, distinct from those involved in the decrease of single-channel conductance. A model in which external Ca2+ changes the properties of activation of the nAChR appears consistent with these data.

Unconventional pharmacology of a neuronal nicotinic receptor mutated in the channel domain.

The putative channel-forming MII domains of the nicotinic, gamma-aminobutyric acid type A, and glycine receptors contain a highly conserved leucine residue. Mutation of this hydrophobic amino acid in the neuronal nicotinic receptor alpha 7 (Leu-247), reconstituted in Xenopus oocytes, modifies the ionic response to acetylcholine and alters desensitization. Furthermore, the Leu----Thr (L247T) mutant has two conducting states (46 pS and 80 pS), in contrast with the wild-type (WT) receptor, which has only one (45 pS). We now show that this mutant possesses a rather paradoxical pharmacology: antagonists of the WT receptor such as dihydro-beta-erythroidin, hexamethonium, or (+)-tubocurarine elicit ionic currents when applied to the L247T alpha 7 mutant and these responses are blocked by alpha-bungarotoxin. Furthermore, prolonged application of acetylcholine causes desensitization in the WT but leads to a potentiation of the responses to acetylcholine or dihydro-beta-erythroidin in the mutant. These data are consistent with a scheme in which mutation of Leu-247 renders a desensitized state in the WT channel a conducting state. They also strengthen the proposal that, in the WT, some competitive antagonists may stabilize desensitized states. Finally, these observations may shed light on properties of other ion channels, in particular the glutamate receptors, which display multiple conductance levels associated with various pharmacological agents.

Calcium influx through nicotinic receptor in rat central neurons: its relevance to cellular regulation.

The Ca2+ permeability of a nicotinic acetylcholine receptor (nAChR) in the rat CNS was determined using both current and fluorescence measurements on medial habenula neurons. The elementary slope conductance of the nAChR channel was 11 pS in pure external Ca2+ (100 mM) and 42 pS in standard solution. Ca2+ influx through nAChRs resulted in the rise of cytosolic Ca2+ concentration ([Ca2+]i) to the micromolar range. This increase was maximal under voltage conditions (below -50 mV) in which Ca2+ influx through voltage-activated channels was minimal. Ca2+ influx through nAChRs directly activated a Ca(2+)-dependent Cl- conductance. In addition, it caused a decrease in the GABAA response that outlasted the rise in [Ca2+]i. These results underscore the physiological significance of Ca2+ influx through nAChR channel in the CNS.

Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor.

A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor.

Functional adult acetylcholine receptor develops independently of motor innervation in Sol 8 mouse muscle cell line.

We have defined culture conditions, using a feeder layer of cells from the embryonic mesenchymal cell line, 10T1/2 and a serum-free medium, which allow cells from the mouse myogenic cell line Sol 8 to form contracting myotubes for two weeks. Under these culture conditions, Sol 8 myotubes undergo a maturation process characterized by a sequential expression of two phenotypes. An early phenotype is typified by the expression of the nicotinic acetylcholine receptor (AChR) gamma-subunit transcripts and the presence of low conductance ACh-activated channels, typical of embryonic AChR. A late phenotype is characterized by the expression of AChR epsilon-subunit transcripts, the decreased accumulation of gamma-subunit transcripts and the appearance of high conductance ACh-activated channels, typical of adult AChR. These results indicate that the expression of functional adult type AChR does not require the presence of the motor nerve and therefore represents an intrinsic feature of the Sol 8 muscle cells. Chronic exposure of the cells to the voltage-sensitive Na+ channel blocking agent tetrodotoxin does not affect the appearance of the AChR epsilon-subunit transcripts but prevents the reduction of the steady-state level of the AChR gamma-subunit transcripts and yields a reduced proportion of the adult type channels. Thus, activity seems to facilitate the switch from the embryonic to the adult phenotype of the AChR protein. The Sol 8 cell system might be useful to analyse further the genetic and epigenetic regulation of muscle fibre maturation in mammals.

Existence of different subtypes of nicotinic acetylcholine receptors in the rat habenulo-interpeduncular system.

Neuronal nicotinic ACh receptors (nAChRs) are present in the rat medial habenula (MHB) and interpeduncular nucleus (IPN), two brain regions connected through the fasciculus retroflexus (FR). The goal of the present study was to compare the electrophysiological and pharmacological characteristics of nAChRs located at pre- and postsynaptic sites within the MHB-IPN system. nAChRs located on the soma of IPN neurons were studied using patch-clamp techniques and a preparation of acutely isolated neurons. Whole-cell currents evoked by Ach and nicotine showed an intense rectification at positive membrane potentials. nAChR channels were relatively nonselective for cations, had a unitary conductance of 35 pS, and were activated by several nicotinic agonists with the following rank order: cytisine greater than ACh greater than nicotine greater than dimethylphenylpiperazinium (DMPP). They were blocked by mecamylamine, hexamethonium, curare, and dihydro-beta-erythroidine (DHBE), but were insensitive to alpha-bungarotoxin and neuronal bungarotoxin. In contrast, nAChRs recorded on the soma of MHB neurons under equivalent experimental conditions exhibited different characteristics for single-channel conductance and agonist and antagonist sensitivity. The pharmacological properties of presynaptic nAChRs in the IPN were analyzed in a rat brain slice preparation. Stimulation of the FR produced a presynaptic afferent volley recorded in the rostral subnucleus of the IPN. Nicotinic agonists decreased the amplitude of the afferent volley with different efficacies: nicotine greater than cytisine greater than ACh greater than DMPP. The action of nicotine was insensitive to alpha-bungarotoxin and to neuronal bungarotoxin, but was blocked by mecamylamine, hexamethonium, curare, and DHBE, with IC50 values different from those reported for IPN postsynaptic nAChRs. This study thus demonstrates the functional diversity of nAChRs in the rat CNS.