GluK1-containing Kainate Receptors Research Articles

Delving into the significance of the His289Tyr single-nucleotide polymorphism in the glutamate ionotropic receptor kainate-1 (Grik1) gene of a genetically audiogenic seizure model.

Genetic abnormalities affecting glutamate receptors are central to excitatory overload-driven neuronal mechanisms that culminate in seizures, making them pivotal targets in epilepsy research. Increasingly used to advance this field, the genetically audiogenic seizure hamster from Salamanca (GASH/Sal) exhibits generalized seizures triggered by high-intensity acoustic stimulation and harbors significant genetic variants recently identified through whole-exome sequencing. Here, we addressed the influence of the missense single-nucleotide polymorphism (C9586732T, p.His289Tyr) in the glutamate receptor ionotropic kainate-1 (Grik1) gene and its implications for the GASH/Sal seizure susceptibility. Using a protein 3D structure prediction, we showed a potential effect of this sequence variation, located in the amino-terminal domain, on the stability and/or conformation of the kainate receptor subunit-1 protein (GluK1). We further employed a multi-technique approach, encompassing gene expression analysis (RT-qPCR), Western blotting, and immunohistochemistry in bright-field and confocal fluorescence microscopy, to investigate critical seizure-associated brain regions in GASH/Sal animals under seizure-free conditions compared to matched wild-type controls. We detected disruptions in the transcriptional profile of the Grik1 gene within the audiogenic seizure-associated neuronal network. Alterations in GluK1 protein levels were also observed in various brain structures, accompanied by an unexpected lower molecular weight band in the inferior and superior colliculi. This correlated with substantial disparities in GluK1-immunolabeling distribution across multiple brain regions, including the cerebellum, hippocampus, subdivisions of the inferior and superior colliculi, and the prefrontal cortex. Notably, the diffuse immunolabeling accumulated within perikarya, axonal fibers and terminals, exhibiting a prominent concentration in proximity to the cell nucleus. This suggests potential disturbances in the GluK1-trafficking mechanism, which could subsequently affect glutamate synaptic transmission. Overall, our study sheds light on the genetic underpinnings of seizures and underscores the importance of investigating the molecular mechanisms behind synaptic dysfunction in epileptic neural networks, laying a crucial foundation for future research and therapeutic strategies targeting GluK1-containing kainate receptors.

Presynaptic Kainate Receptors onto Somatostatin Interneurons Are Recruited by Activity throughout Development and Contribute to Cortical Sensory Adaptation.

Somatostatin (SST) interneurons produce delayed inhibition because of the short-term facilitation of their excitatory inputs created by the expression of metabotropic glutamate receptor 7 (mGluR7) and presynaptic GluK2-containing kainate receptors (GluK2-KARs). Using mice of both sexes, we find that as synaptic facilitation at layer (L)2/3 SST cell inputs increases during the first few postnatal weeks, so does GluK2-KAR expression. Removal of sensory input by whisker trimming does not affect mGluR7 but prevents the emergence of presynaptic GluK2-KARs, which can be restored by allowing whisker regrowth or by acute calmodulin activation. Conversely, late trimming or acute inhibition of Ca2+/calmodulin-dependent protein kinase II is sufficient to reduce GluK2-KAR activity. This developmental and activity-dependent regulation also produces a specific reduction of L4 GluK2-KARs that advances in parallel with the maturation of sensory processing in L2/3. Finally, we find that removal of both GluK2-KARs and mGluR7 from the synapse eliminates short-term facilitation and reduces sensory adaptation to repetitive stimuli, first in L4 of somatosensory cortex, then later in development in L2/3. The dynamic regulation of presynaptic GluK2-KARs potentially allows for flexible scaling of late inhibition and sensory adaptation.SIGNIFICANCE STATEMENT Excitatory synapses onto somatostatin (SST) interneurons express presynaptic, calcium-permeable kainate receptors containing the GluK2 subunit (GluK2-KARs), activated by high-frequency activity. In this study we find that their presence on L2/3 SST synapses in the barrel cortex is not based on a hardwired genetic program but instead is regulated by sensory activity, in contrast to that of mGluR7. Thus, in addition to standard synaptic potentiation and depression mechanisms, excitatory synapses onto SST neurons undergo an activity-dependent presynaptic modulation that uses GluK2-KARs. Further, we present evidence that loss of the frequency-dependent synaptic components (both GluK2-KARs and mGluR7 via Elfn1 deletion) contributes to a decrease in the sensory adaptation commonly seen on repetitive stimulus presentation.

Presenilin and APP Regulate Synaptic Kainate Receptors.

Kainate receptors (KARs) form a family of ionotropic glutamate receptors that regulate the activity of neuronal networks by both presynaptic and postsynaptic mechanisms. Their implication in pathologies is well documented for epilepsy. The higher prevalence of epileptic symptoms in Alzheimer's disease (AD) patients questions the role of KARs in AD. Here we investigated whether the synaptic expression and function of KARs was impaired in mouse models of AD. We addressed this question by immunostaining and electrophysiology at synapses between mossy fibers and CA3 pyramidal cells, in which KARs are abundant and play a prominent physiological role. We observed a decrease of the immunostaining for GluK2 in the stratum lucidum in CA3, and of the amplitude and decay time of synaptic currents mediated by GluK2-containing KARs in an amyloid mouse model (APP/PS1) of AD. Interestingly, a similar phenotype was observed in CA3 pyramidal cells in male and female mice with a genetic deletion of either presenilin or APP/APLP2 as well as in organotypic cultures treated with γ-secretase inhibitors. Finally, the GluK2 protein interacts with full-length and C-terminal fragments of APP. Overall, our data suggest that APP stabilizes KARs at synapses, possibly through a transsynaptic mechanism, and this interaction is under the control the γ-secretase proteolytic activity of presenilin.SIGNIFICANCE STATEMENT Synaptic impairment correlates strongly with cognitive deficits in Alzheimer's disease (AD). In this context, many studies have addressed the dysregulation of AMPA and NMDA ionotropic glutamate receptors. Kainate receptors (KARs), which form the third family of iGluRs, represent an underestimated actor in the regulation of neuronal circuits and have not yet been examined in the context of AD. Here we provide evidence that synaptic KARs are markedly impaired in a mouse model of AD. Additional experiments indicate that the γ-secretase activity of presenilin acting on the amyloid precursor protein controls synaptic expression of KAR. This study clearly indicates that KARs should be taken into consideration whenever addressing synaptic dysfunction and related cognitive deficits in the context of AD.

Antagonism of GluK1-containing kainate receptors reduces ethanol consumption by modulating ethanol reward and withdrawal

Alcohol use disorder (AUD) is a neuropsychiatric condition affecting millions of people worldwide. Topiramate (TPM) is an antiepileptic drug that has been shown to reduce ethanol drinking in humans. However, TPM is associated with a variety of adverse effects due to its interaction with many receptor systems and intracellular pathways. GluK1-containing kainate receptors (GluK1*KARs) are non-selectively inhibited by TPM, and genetic association studies suggest that this receptor system could be targeted to reduce drinking in AUD patients. We examined the efficacy of LY466195, a selective inhibitor of GluK1*KAR, in reducing ethanol consumption in the intermittent two-bottle choice paradigm in mice. The effect of LY466195 on various ethanol-related phenotypes was investigated by quantification of alcohol intake, physical signs of withdrawal, conditioned place preference (CPP) and in vivo microdialysis in the nucleus accumbens. Selective GluK1*KAR inhibition reduced ethanol intake and preference in a dose-dependent manner. LY466195 treatment attenuated the physical manifestations of ethanol withdrawal and influenced the rewarding properties of ethanol. Interestingly, LY466195 injection also normalized changes in dopamine levels in response to acute ethanol in ethanol-dependent mice, but had no effect in ethanol-naïve mice, suggesting ethanol state-dependent effects. The data point to GluK1*KARs as an attractive pharmacological target for the treatment of AUD.

Potential mechanism of GABA secretion in response to the activation of GluK1-containing kainate receptors

Hippocampal GABAergic neurons are subdivided into more than 20 subtypes that are distinguished by features and functions. We have previously described the subpopulation of GABAergic neurons, which can be identified in hippocampal cell culture by the calcium response to the application of domoic acid (DoA), an agonist of kainate receptors (KARs). Here, we investigate the features of DoA-sensitive neurons and their GABA release mechanism in response to KARs activation. We demonstrate that DoA-sensitive GABAergic neurons express GluK1-containing KARs because ATPA, a selective agonist of GluK1-containing receptors, induces the calcium response exclusively in these GABAergic neurons. Our experiments also show that NASPM, previously considered a selective antagonist of calcium-permeable AMPARs, blocks calcium-permeable KARs. We established using NASPM that GluK1-containing receptors of the studied population of GABAergic neurons are calcium-permeable, and their activation is required for GABA release, at least in particular synapses. Notably, GABA release occurs even in the presence of tetrodotoxin, indicating that propagation of the depolarizing stimulus is not required for GABA release in this case. Thus, our data demonstrate that the activation of GluK1-containing calcium-permeable KARs mediates the GABA release by the studied subpopulation of GABAergic neurons.

Kainate Receptors Inhibit Glutamate Release Via Mobilization of Endocannabinoids in Striatal Direct Pathway Spiny Projection Neurons.

Kainate receptors are members of the glutamate receptor family that function by both generating ionotropic currents through an integral ion channel pore and coupling to downstream metabotropic signaling pathways. They are highly expressed in the striatum, yet their roles in regulating striatal synapses are not known. Using mice of both sexes, we demonstrate that GluK2-containing kainate receptors expressed in direct pathway spiny projection neurons (dSPNs) inhibit glutamate release at corticostriatal synapses in the dorsolateral striatum. This inhibition requires postsynaptic kainate-receptor-mediated mobilization of a retrograde endocannabinoid (eCB) signal and activation of presynaptic CB1 receptors. This pathway can be activated during repetitive 25 Hz trains of synaptic stimulation, causing short-term depression of corticostriatal synapses. This is the first study to demonstrate a role for kainate receptors in regulating eCB-mediated plasticity at the corticostriatal synapse and demonstrates an important role for these receptors in regulating basal ganglia circuits.SIGNIFICANCE STATEMENT The GRIK2 gene, encoding the GluK2 subunit of the kainate receptor, has been linked to several neuropsychiatric and neurodevelopmental disorders including obsessive compulsive disorder (OCD). Perseverative behaviors associated with OCD are known to result from pathophysiological changes in the striatum and kainate receptor knock-out mice have striatal-dependent phenotypes. However, the role of kainate receptors in striatal synapses is not known. We demonstrate that GluK2-containing kainate receptors regulate corticostriatal synapses by mobilizing endocannabinoids from direct pathway spiny projection neurons. Synaptic activation of GluK2 receptors during trains of synaptic input causes short-term synaptic depression, demonstrating a novel role for these receptors in regulating striatal circuits.

The auxiliary subunits Neto1 and Neto2 have distinct, subunit-dependent effects at recombinant GluK1- and GluK2-containing kainate receptors

The kainate-type of ionotropic glutamate receptors are assembled from a combination of five different pore-forming subunits (GluK1–5), which confer distinct functional and pharmacological properties. These receptors are also modulated by co-assembly with the auxiliary subunits Neto1 and Neto2. To determine the impact of variation in subunit composition on the functional interaction between kainate receptors and Neto subunits, the Neto subunits were combined with either GluK1 or GluK2 in HEK-293T cells and responses to glutamate examined through patch-clamp recordings. Co-expression of GluK1 with either Neto1 or Neto2 caused a substantial increase in glutamate sensitivity and a slowing of the onset of desensitization at low agonist concentrations. However, at higher glutamate concentrations the primary effect of Neto2 was to slow the onset of desensitization, while that of Neto1 was to increase recovery from desensitization. In contrast, co-expression of Neto2 with GluK2 homomeric receptors had only modest effects on glutamate sensitivity, but increased the rate of recovery from desensitization as well as slowing its onset at all agonist concentrations. The properties of chimeric Neto1/Neto2 subunits suggested that the extracellular N-terminal region including the two CUB domains was largely responsible for the distinct regulatory effects of Neto1 and Neto2 on the desensitization properties of GluK1 homomeric receptors. These results further demonstrate that the functional effects of Neto subunits depend upon the subunit identity of both the auxiliary and the pore-forming subunits.

Activity-dependent modulation of the axonal conduction of action potentials along rat hippocampal mossy fibers.

The axonal conduction of action potentials in the nervous system is generally considered to be a stable signal for the relaying of information, and its dysfunction is involved in impairment of cognitive function. Recent evidence suggests that the conduction properties and excitability of axons are more variable than traditionally thought. To investigate possible changes in the conduction of action potentials along axons in the central nervous system, we recorded action potentials from granule cells that were evoked and conducted antidromically along unmyelinated mossy fibers in the rat hippocampus. To evaluate changes in axons by eliminating any involvement of changes in the somata, two latency values were obtained by stimulating at two different positions and the latency difference between the action potentials was measured. A conditioning electrical stimulus of 20 pulses at 1 Hz increased the latency difference and this effect, which lasted for approximately 30 s, was inhibited by the application of an α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist or a GluK1-containing kainate receptor antagonist, but not by an AMPA receptor-selective antagonist or an N-methyl-d-aspartate receptor antagonist. These results indicated that axonal conduction in mossy fibers is modulated in an activity-dependent manner through the activation of GluK1-containing kainate receptors. These dynamic changes in axonal conduction may contribute to the physiology and pathophysiology of the brain.

Contribution of Aberrant GluK2-Containing Kainate Receptors to Chronic Seizures in Temporal Lobe Epilepsy

Kainate is a potent neurotoxin known to induce acuteseizures. However, whether kainate receptors (KARs) play any role in the pathophysiology of temporal lobe epilepsy (TLE) is not known. In TLE, recurrent mossy fiber (rMF) axons form abnormal excitatory synapses onto other dentate granule cells that operate via KARs. The present study explores the pathophysiological implications of KARs in generating recurrent seizures in chronic epilepsy. In an invitro model of TLE, seizure-like activity was minimized in mice lacking the GluK2 subunit, which is a main component of aberrant synaptic KARs at rMF synapses. Invivo, the frequency of interictal spikes and ictal discharges was strongly reduced in GluK2(-/-) mice or in the presence of a GluK2/GluK5 receptor antagonist. Our data show that aberrant GluK2-containing KARs play a major role in the chronic seizures that characterize TLE and thus constitute a promising antiepileptic target.

Kainate receptor subunit diversity underlying response diversity in retinal Off bipolar cells

Postsynaptic kainate receptors mediate excitatory synaptic transmission over a broad range of temporal frequencies. In heterologous systems, the temporal responses of kainate receptors vary when different channel-forming and auxiliary subunits are co-expressed but how this variability relates to the temporal differences at central synapses is incompletely understood. The mammalian cone photoreceptor synapse provides advantages for comparing the different temporal signalling roles of kainate receptors, as cones release glutamate over a range of temporal frequencies, and three functionally distinct Off bipolar cell types receive cone signals at synapses that contain either AMPA or kainate receptors, all with different temporal properties. A disadvantage is that the different receptor subunits are not identified. We used in situ hybridization, immunocytochemistry, and pharmacology to identify the kainate receptor and auxiliary subunits in ground squirrel (Ictidomys tridecimlineatus) cb1a/b, cb2, and cb3a/b Off bipolar cell types. As expected, the types showed distinct subunit expression patterns. Kainate receptors mediated ∼80% of the synaptic response in cb3a/b cells and were heteromers of GluK1 and GluK5. Cb3a/b cells contained message for GluK1 and GluK5, and also GluK3 and the auxiliary subunit Neto1. The synaptic responses in cb1a/b cells were mediated by GluK1-containing kainate receptors that behaved differently from the receptors expressed by cb3a/b cells. AMPA receptors mediated the entire synaptic response in cb2 cells and the remaining synaptic response in cb3a/b cells. We conclude that GluK1 is the predominant kainate receptor subunit in cb1 and cb3 Off bipolar cells. Different temporal response properties may result from selective association with GluK3, GluK5, or Neto1.

Electrophysiological Effects of Kainic Acid on Vasopressin-Enhanced Green Fluorescent Protein and Oxytocin-Monomeric Red Fluorescent Protein 1 Neurones Isolated from the Supraoptic Nucleus in Transgenic Rats

The supraoptic nucleus (SON) contains two types of magnocellular neurosecretory cells: arginine vasopressin (AVP)-producing and oxytocin (OXT)-producing cells. We recently generated and characterised two transgenic rat lines: one expressing an AVP-enhanced green fluorescent protein (eGFP) and the other expressing an OXT-monomeric red fluorescent protein 1 (mRFP1). These transgenic rats enable the visualisation of AVP or OXT neurones in the SON. In the present study, we compared the electrophysiological responses of AVP-eGFP and OXT-mRFP1 neurones to glutamic acid in SON primary cultures. Glutamate mediates fast synaptic transmission through three classes of ionotrophic receptors: the NMDA, AMPA and kainate receptors. We investigated the contributions of the three classes of ionotrophic receptors in glutamate-induced currents. Three different antagonists were used, each predominantly selective for one of the classes of ionotrophic receptor. Next, we focused on the kainate receptors (KARs). We examined the electrophysiological effects of kainic acid (KA) on AVP-eGFP and OXT-mRFP1 neurones. In current clamp mode, KA induced depolarisation and increased firing rates. These KA-induced responses were inhibited by the non-NMDA ionotrophic receptor antagonist 6-cyano-7-nitroquinoxaline-2,3(1H4H)-dione in both AVP-eGFP and OXT-mRFP1 neurones. In voltage clamp mode, the application of KA evoked inward currents in a dose-dependent manner. The KA-induced currents were significantly larger in OXT-mRFP1 neurones than in AVP-eGFP neurones. This significant difference in KA-induced currents was abolished by the GluK1-containing KAR antagonist UBP302. At high concentrations (250-500 μm), the specific GluK1-containing KAR agonist (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) induced significantly larger currents in OXT-mRFP1 neurones than in AVP-eGFP neurones. Furthermore, the difference between the AVP-eGFP and OXT-mRFP1 neurones in the ATPA currents was approximately equal to the difference in the KA currents. These findings suggest that the GluK1-containing KARs may be more highly expressed in OXT neurones than in AVP neurones. These results may provide new insight into the physiology and synaptic plasticity of SON neurones.

Neuroprotection of GluK1 kainate receptor agonist ATPA against ischemic neuronal injury through inhibiting GluK2 kainate receptor–JNK3 pathway via GABAA receptors

It is well known that GluK2-containing kainate receptors play essential roles in seizure and cerebral ischemia-induced neuronal death, while GluK1-containing kainate receptors could increase tonic inhibition of post-synaptic pyramidal neurons. This research investigated whether GluK1 could inhibit activation of c-Jun N-terminal kinase 3 (JNK3) signaling pathway mediated by the GluK2 in cerebral ischemia–reperfusion. The results show that GluK1 activation by (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) at 1nmol per rat could inhibit the assembly of GluK2·Postsynaptic density 95·mixed lineage kinase 3 signaling module, activation of JNK3 and its downstream signal molecules. However, the inhibition of ATPA could be prevented by GluK1 antagonist NS3763, GluK1 antisense, and GABAA receptor antagonist bicuculline. In addition, ATPA played a neuroprotective role against cerebral ischemia. In sum, the findings indicate that activation of GluK1 by ATPA at specific dosages may promote GABA release, which then suppresses post-synaptic GluK2–JNK3 signaling-mediated cerebral ischemic injury via GABAAR.

Kainate Receptor-Induced Retrograde Inhibition of Glutamatergic Transmission in Vasopressin Neurons

Presynaptic kainate receptors (KARs) exert a modulatory action on transmitter release. We here report that applications of agonists of GluK1-containing KARs in the rat supraoptic nucleus has an opposite action on glutamatergic transmission according to the phenotype of the postsynaptic neuron. Whereas glutamate release was facilitated in oxytocin (OT) neurons, it was inhibited in vasopressin (VP) cells. Interestingly, an antagonist of GluK1-containing KARs caused an inhibition of glutamate release in both OT and VP neurons, revealing the existence of tonically activated presynaptic KARs that are positively coupled to transmitter release. We thus postulated that the inhibition of glutamate release observed with exogenous applications of GluK1 agonists on VP neurons could be indirect. In agreement with this hypothesis, we first showed that functional GluK1-containing KARs were present postsynaptically on VP neurons but not on OT cells. We next showed that the inhibitory effect induced by exogenous GluK1 receptor agonist was compromised when BAPTA was added in the recording pipette to buffer intracellular Ca2+ and block the release of a putative retrograde messenger. Under these conditions, GluK1-containing KAR agonist facilitates glutamatergic transmission in VP neurons in a manner similar to that observed for OT neurons and that resulted from the activation of presynaptic GluK1 receptors. GluK1-mediated inhibition of glutamate release in VP neurons was also blocked by a κ-opioid receptor antagonist. These findings suggest that activation of postsynaptic GluK1-containing KARs on VP neurons leads to the release of dynorphin, which in turn acts on presynaptic κ-opioid receptors to inhibit glutamate release.

Piperazine-2,3-dicarboxylic Acid Derivatives as Dual Antagonists of NMDA and GluK1-Containing Kainate Receptors

Competitive N-methyl-d-aspartate receptor (NMDAR) antagonists bind to the GluN2 subunit, of which there are four types (GluN2A-D). We report that some N(1)-substituted derivatives of cis-piperazine-2,3-dicarboxylic acid display improved relative affinity for GluN2C and GluN2D versus GluN2A and GluN2B. These derivatives also display subtype selectivity among the more distantly related kainate receptor family. Compounds 18i and (-)-4 were the most potent kainate receptor antagonists, and 18i was selective for GluK1 versus GluK2, GluK3 and AMPA receptors. Modeling studies revealed structural features required for activity at GluK1 subunits and suggested that S674 was vital for antagonist activity. Consistent with this hypothesis, replacing the equivalent residue in GluK3 (alanine) with a serine imparts 18i antagonist activity. Antagonists with dual GluN2D and GluK1 antagonist activity may have beneficial effects in various neurological disorders. Consistent with this idea, antagonist 18i (30 mg/kg ip) showed antinociceptive effects in an animal model of mild nerve injury.

Pre- and post-synaptic functions of kainate receptors at glutamate and GABA synapses in the rat entorhinal cortex

Oscillatory network activity in cortical areas is seen as vital to physiological processes of cognition, learning, and memory, and fundamental to disorders such as epilepsy. Increasing attention is being paid to the role of kainate receptors (KAr) in the generation of network oscillations and synchrony. The entorhinal cortex (EC) plays a key role in learning and memory, and is a major site of dysfunction in temporal lobe epilepsy. KAr have been implicated in oscillogenesis in the EC, but limited information is available concerning the physiological roles of KAr in synaptic transmission in this area. Here, we make a detailed analysis of KAr function in Layer III of the EC, a site known to be highly susceptible to oscillogenesis, using whole-cell patch clamp recording of evoked and spontaneous synaptic currents in rat brain slices. We demonstrate that KAr containing the GluK1-subunit act as facilitatory autoreceptors at glutamatergic synapses on pyramidal neurones in Layer III. In addition, GluK1-containing KAr mediate an excitatory drive at glutamatergic synapses on GABAergic interneurones. In contrast, a different KAr, which is likely to contain the GluK2-subunit mediates a slow postsynaptic excitation at glutamatergic synapses on principal neurones, and may also act as a heteroreceptor, facilitating GABA release at inhibitory terminals on principal neurones. Reducing [Mg(2+) ](o) , which we have previously shown can generate KAr-dependent slow network oscillations in Layer III, enhances both glutamate and GABA release. Both effects are partly sustained by increased activation of GluK1-containing KAr. Increased activation of the GluK1-containing autoreceptor also results in an enhancement of the postsynaptic response mediated by GluK2-containing receptors. Finally, spontaneous release of both transmitters shows a rhythmic periodicity in low-Mg, and, again, this is dependent on GluK1-containing KAr. The results show that KAr contribute a facilitatory function at multiple levels in the networks of the EC, and provide a basis for dissecting the role of these receptors in oscillogenesis in this area.

Energy deprivation transiently enhances rhythmic inhibitory events in the CA3 hippocampal network in vitro

Oxygen glucose deprivation (OGD) leads to rapid suppression of synaptic transmission. Here we describe an emergence of rhythmic activity at 8 to 20 Hz in the CA3 subfield of hippocampal slice cultures occurring for a few minutes prior to the OGD-induced cessation of evoked responses. These oscillations, dominated by inhibitory events, represent network activity, as they were abolished by tetrodotoxin. They were also completely blocked by the GABAergic antagonist picrotoxin, and strongly reduced by the glutamatergic antagonist NBQX. Applying CPP to block NMDA receptors had no effect and neither did UBP302, an antagonist of GluK1-containing kainate receptors. The gap junction blocker mefloquine disrupted rhythmicity. Simultaneous whole-cell voltage-clamp recordings from neighboring or distant CA3 pyramidal cells revealed strong cross-correlation of the incoming rhythmic activity. Interneurons in the CA3 area received similar correlated activity. Interestingly, oscillations were much less frequently observed in the CA1 area. These data, together with the observation that the recorded activity consists primarily of inhibitory events, suggest that CA3 interneurons are important for generating these oscillations. This transient increase in inhibitory network activity during OGD may represent a mechanism contributing to the lower vulnerability to ischemic insults of the CA3 area as compared to the CA1 area.

Glia-Dependent Switch of Kainate Receptor Presynaptic Action

Presynaptic kainate receptors (KARs) exert a modulatory action on transmitter release. This effect can be switched from facilitation to inhibition by an increased concentration of KAR agonists. We here report that activation of presynaptic GluK1-containing KARs facilitates GABA release on oxytocin and vasopressin neurons in the supraoptic nucleus of the hypothalamus. Increase in ambient levels of glutamate associated with the physiological reduction of astrocytic coverage of oxytocin neurons in lactating rats switches this KAR-mediated facilitation to inhibition of GABAergic transmission. This effect was reproduced in both oxytocin and vasopressin neurons of virgin rats when glutamate transporters were blocked pharmacologically, thereby establishing that enhanced levels of extracellular glutamate induce the switch in KAR-mediated action. The facilitation of GABA release was inhibited with philanthotoxin, a Ca(2+)-permeable KAR antagonist, suggesting that this effect was associated with an ionotropic mode of action. Conversely, KAR-mediated inhibition was compromised in the presence of U73122, a phospholipase C inhibitor, in agreement with the involvement of a metabotropic pathway. We thus reveal that physiological astrocytic plasticity modifies the mode of action of presynaptic KARs, thereby inversing their coupling with GABA release.

Pathological alterations in GABAergic interneurons and reduced tonic inhibition in the basolateral amygdala during epileptogenesis

An acute brain insult such as traumatic head/brain injury, stroke, or an episode of status epilepticus can trigger epileptogenesis, which, after a latent, seizure-free period, leads to epilepsy. The discovery of effective pharmacological interventions that can prevent the development of epilepsy requires knowledge of the alterations that occur during epileptogenesis in brain regions that play a central role in the induction and expression of epilepsy. In the present study, we investigated pathological alterations in GABAergic interneurons in the rat basolateral amygdala (BLA), and the functional impact of these alterations on inhibitory synaptic transmission, on days 7 to 10 after status epilepticus induced by kainic acid. Using design-based stereology combined with glutamic acid decarboxylase (GAD) 67 immunohistochemistry, we found a more extensive loss of GABAergic interneurons compared to the loss of principal cells. Fluoro-Jade C staining showed that neuronal degeneration was still ongoing. These alterations were accompanied by an increase in the levels of GAD and the α1 subunit of the GABAA receptor, and a reduction in the GluK1 (previously known as GluR5) subunit, as determined by Western blots. Whole-cell recordings from BLA pyramidal neurons showed a significant reduction in the frequency and amplitude of action potential–dependent spontaneous inhibitory postsynaptic currents (IPSCs), a reduced frequency but not amplitude of miniature IPSCs, and impairment in the modulation of IPSCs via GluK1-containing kainate receptors (GluK1Rs). Thus, in the BLA, GABAergic interneurons are more vulnerable to seizure-induced damage than principal cells. Surviving interneurons increase their expression of GAD and the α1 GABAA receptor subunit, but this does not compensate for the interneuronal loss; the result is a dramatic reduction of tonic inhibition in the BLA circuitry. As activation of GluK1Rs by ambient levels of glutamate facilitates GABA release, the reduced level and function of these receptors may contribute to the reduction of tonic inhibitory activity. These alterations at a relatively early stage of epileptogenesis may facilitate the progress towards the development of epilepsy.

ACET is a highly potent and specific kainate receptor antagonist: Characterisation and effects on hippocampal mossy fibre function

Kainate receptors (KARs) are involved in both NMDA receptor-independent long-term potentiation (LTP) and synaptic facilitation at mossy fibre synapses in the CA3 region of the hippocampus. However, the identity of the KAR subtypes involved remains controversial. Here we used a highly potent and selective GluK1 (formerly GluR5) antagonist (ACET) to elucidate roles of GluK1-containing KARs in these synaptic processes. We confirmed that ACET is an extremely potent GluK1 antagonist, with a Kb value of 1.4±0.2nM. In contrast, ACET was ineffective at GluK2 (formerly GluR6) receptors at all concentrations tested (up to 100μM) and had no effect at GluK3 (formerly GluR7) when tested at 1μM. The X-ray crystal structure of ACET bound to the ligand binding core of GluK1 was similar to the UBP310–GluK1 complex. In the CA1 region of hippocampal slices, ACET was effective at blocking the depression of both fEPSPs and monosynaptically evoked GABAergic transmission induced by ATPA, a GluK1 selective agonist. In the CA3 region of the hippocampus, ACET blocked the induction of NMDA receptor-independent mossy fibre LTP. To directly investigate the role of pre-synaptic GluK1-containing KARs we combined patch-clamp electrophysiology and 2-photon microscopy to image Ca2+ dynamics in individual giant mossy fibre boutons. ACET consistently reduced short-term facilitation of pre-synaptic calcium transients induced by 5 action potentials evoked at 20–25Hz. Taken together our data provide further evidence for a physiological role of GluK1-containing KARs in synaptic facilitation and LTP induction at mossy fibre–CA3 synapses.