Enhanced Membrane Incorporation of H289Y Mutant GluK1 Receptors from the Audiogenic Seizure-Prone GASH/Sal Model: Functional and Morphological Impacts on Xenopus Oocytes.

Epilepsy is a neurological disorder characterized by abnormal neuronal excitability, with glutamate playing a key role as the predominant excitatory neurotransmitter involved in seizures. Animal models of epilepsy are crucial in advancing epilepsy research by faithfully replicating the diverse symptoms of this disorder. In particular, the GASH/Sal (genetically audiogenic seizure-prone hamster from Salamanca) model exhibits seizures resembling human generalized tonic-clonic convulsions. A single nucleotide polymorphism (SNP; C9586732T, p.His289Tyr) in the Grik1 gene (which encodes the kainate receptor GluK1) has been previously identified in this strain. The H289Y mutation affects the amino-terminal domain of GluK1, which is related to the subunit assembly and trafficking. We used confocal microscopy in Xenopus oocytes to investigate how the H289Y mutation, compared to the wild type (WT), affects the expression and cell-surface trafficking of GluK1 receptors. Additionally, we employed the two-electrode voltage-clamp technique to examine the functional effects of the H289Y mutation. Our results indicate that this mutation increases the expression and incorporation of GluK1 receptors into an oocyte's membrane, enhancing kainate-evoked currents, without affecting their functional properties. Although further research is needed to fully understand the molecular mechanisms responsible for this epilepsy, the H289Y mutation in GluK1 may be part of the molecular basis underlying the seizure-prone circuitry in the GASH/Sal model.

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.

mRNA editing of kainate receptor subunits: what do we know so far?

Kainate receptors (KARs) are considered one of the key modulators of synaptic activity in the mammalian central nervous system. These receptors were discovered more than 30 years ago, but their role in brain functioning remains unclear due to some peculiarities. One such feature of these receptors is the editing of pre-mRNAs encoding GluK1 and GluK2 subunits. Despite the long history of studying this phenomenon, numerous questions remain unanswered. This review summarizes the current data about the mechanism and role of pre-mRNA editing of KAR subunits in the mammalian brain and proposes a perspective of future investigations.

Structural basis for positive allosteric modulation of AMPA and kainate receptors.

This paper summarizes the present knowledge on how positive allosteric modulators (PAMs) interact with the ligand-binding domain (LBD) of AMPA and kainate receptors, based on structure determinations. AMPA and kainate receptors belong to the family of ionotropic glutamate receptors that are responsible for mediating the majority of fast excitatory neurotransmission. These receptors have been related to brain disorders, e.g. Alzheimer's disease and attention deficit hyperactivity disorder. PAMs are small molecules that potentiate AMPA and kainate receptor currents by interfering with receptor desensitization. Therefore, PAMs are considered to be of interest for the development of pharmacological tools. Whereas PAMs for AMPA receptors have been known for several years, only recently have PAMs for kainate receptors been reported. Today, >80 structures are available for AMPA receptors with PAMs. These PAMs bind at the interface between two LBD subunits in the vicinity of residue 775, which is important for functional differences between flip and flop isoforms of AMPA receptors. PAMs can be divided into five classes based on their binding mode. The most potent PAM reported to date belongs to class 3, which comprises dimerized PAMs. Three structures of the kainate receptor GluK1 were determined with PAMs belonging to class 2. One PAM enhances kainate receptor currents 5- to 59-fold but shows 100-fold lower potency compared to AMPA receptors. Selective PAMs for kainate receptors will be of great use as pharmacological tools for functional investigations in vivo and might potentially prove useful as drugs in controlling the activity of neuronal networks.

Properties of GABAergic Neurons Containing Calcium-Permeable Kainate and AMPA-Receptors.

Calcium-permeable kainate and AMPA receptors (CP-KARs and CP-AMPARs), as well as NMDARs, play a pivotal role in plasticity and in regulating neurotransmitter release. Here we visualized in the mature hippocampal neuroglial cultures the neurons expressing CP-AMPARs and CP-KARs. These neurons were visualized by a characteristic fast sustained [Ca2+]i increase in response to the agonist of these receptors, domoic acid (DoA), and a selective agonist of GluK1-containing KARs, ATPA. Neurons from both subpopulations are GABAergic. The subpopulation of neurons expressing CP-AMPARs includes a larger percentage of calbindin-positive neurons (39.4 ± 6.0%) than the subpopulation of neurons expressing CP-KARs (14.2 ± 7.5% of CB+ neurons). In addition, we have shown for the first time that NH4Cl-induced depolarization faster induces an [Ca2+]i elevation in GABAergic neurons expressing CP-KARs and CP-AMPARs than in most glutamatergic neurons. CP-AMPARs antagonist, NASPM, increased the amplitude of the DoA-induced Ca2+ response in GABAergic neurons expressing CP-KARs, indicating that neurons expressing CP-AMPARs innervate GABAergic neurons expressing CP-KARs. We assume that CP-KARs in inhibitory neurons are involved in the mechanism of outstripping GABA release upon hyperexcitation.

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.

Kainate receptor modulation of glutamatergic synaptic transmission in the CA2 region of the hippocampus.

Kainate (KA) receptors (KARs) are important modulators of synaptic transmission. We studied here the role of KARs on glutamatergic synaptic transmission in the CA2 region of the hippocampus where the actions of these receptors are unknown. We observed that KA depresses glutamatergic synaptic transmission at Schaffer collateral-CA2 synapses; an effect that was antagonized by NBQX (a KA/AMPA receptors antagonist) under condition where AMPA receptors were previously blocked. The study of paired-pulse facilitation ratio, miniature responses, and fluctuation analysis indicated a presynaptic locus of action for KAR. Additionally, we determined the action mechanism for this depression of glutamate release mediated by the activation of KARs. We found that inhibition of protein kinase A suppressed the effect of KAR activation on evoked excitatory post-synaptic current, an effect that was not suppressed by protein kinase C inhibitors. Furthermore, in the presence of Pertussis toxin, the depression of glutamate release mediated by KAR activation was not present, invoking the participation of a Gi/o protein in this modulation. Finally, the KAR-mediated depression of glutamate release was not suppressed by treatments that affect calcium entry trough voltage-dependent calcium channels or calcium release from intracellular stores. We conclude that KARs present at these synapses mediate a depression of glutamate release through a mechanism that involves the activation of G protein and protein kinase A.

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.

Molecular Correlates of Topiramate and GRIK1 rs2832407 Genotype in Pluripotent Stem Cell-Derived Neural Cultures.

BACKGROUND: There is growing evidence that the anticonvulsant topiramate is efficacious in reducing alcohol consumption. Further, an intronic single nucleotide polymorphism (rs2832407, C A) in the GRIK1 gene, which encodes the GluK1 subunit of the excitatory kainate receptor, predicted topiramate's effectiveness in reducing heavy drinking in a clinical trial. The molecular correlates of GRIK1 genotype that may relate to topiramate's ability to reduce drinking remain unknown. METHODS: We differentiated induced pluripotent stem cells (iPSCs) characterized by GRIK1 rs2832407 genotype from 8 A/A and 8 C/C donors into forebrain-lineage neural cultures. Our differentiation protocol yielded mixed neural cultures enriched for glutamatergic neurons. Basal mRNA expression of the GRIK1 locus was examined via quantitative polymerase chain reaction (qPCR). The effects of acute topiramate exposure on excitatory spontaneous synaptic activity were examined via whole-cell patch-clamp electrophysiology. Results were compared and contrasted between iPSC donor genotypes. RESULTS: Although characterization of the GRIK1 locus revealed no effect of rs2832407 genotype on GRIK1 isoform mRNA expression, a significant difference was observed on GRIK1 antisense-2 expression, which was greater in C/C neural cultures. Differential effects of acute exposure to 5 µM topiramate were observed on spontaneous synaptic activity in A/A versus C/C neurons, with a smaller reduction in excitatory event frequency observed in C/C donor neurons. CONCLUSIONS: This work highlights the use of iPSC technologies to study pharmacogenetic treatment effects in psychiatric disorders and furthers our understanding of the molecular effects of topiramate exposure in human neural cells.

Targeting the glutamatergic system to counteract organophosphate poisoning: A novel therapeutic strategy.

One of the devastating effects of acute exposure to organophosphates, like nerve agents, is the induction of severe and prolonged status epilepticus (SE), which can cause death, or brain damage if death is prevented. Seizures after exposure are initiated by muscarinic receptor hyperstimulation-after inhibition of acetylcholinesterase by the organophosphorus agent and subsequent elevation of acetylcholine-but they are reinforced and sustained by glutamatergic hyperexcitation, which is the primary cause of brain damage. Diazepam is the FDA-approved anticonvulsant for the treatment of nerve agent-induced SE, and its replacement by midazolam is currently under consideration. However, clinical data derived from the treatment of SE of any etiology, as well as studies on the control of nerve agent-induced SE in animal models, have indicated that diazepam and midazolam control seizures only temporarily, their antiseizure efficacy is reduced as the latency of treatment from the onset of SE increases, and their neuroprotective efficacy is limited or absent. Here, we review data on the discovery of a novel anticonvulsant and neuroprotectant, LY293558, an AMPA/GluK1 receptor antagonist. Treatment of soman-exposed immature, young-adult, and aged rats with LY293558, terminates SE with limited recurrence of seizures, significantly protects from brain damage, and prevents long-term behavioral deficits, even when LY293558 is administered 1 h post-exposure. More beneficial effects and complete neuroprotection is obtained when LY293558 administration is combined with caramiphen, which antagonizes NMDA receptors. Further efficacy studies may bring the LY293558 + caramiphen combination therapy on the pathway to approval for human use.

Reduced synaptic function of Kainate receptors in the insular cortex of Fmr1 Knock-out mice.

Fragile X syndrome is caused by the loss of fragile X mental retardation protein (FMRP). Kainate receptor (KAR) is a subfamily of ionotropic glutamate receptors (iGluR) that acts mainly as a neuromodulator of synaptic transmission and neuronal excitability. However, little is known about the changes of synaptic KAR in the cortical area of Fmr1 KO mice. In this study, we performed whole-cell patch-clamp recordings from layer II/III pyramidal neurons in the insular cortex of Fmr1 KO mice. We found that KARs mediated currents were reduced in Fmr1 KO mice. KARs were mainly located in the synaptosomal fraction of the insular cortex. The abundance of KAR subunit GluK1 and GluK2/3 in the synaptosome was reduced in Fmr1 KO mice, whereas the total expressions of these KARs subunits were not changed. Finally, lack of FMRP impairs subsequent internalization of surface GluK2 after KAR activation, while having no effect on the surface GluK2 expression. Our studies provide evidence indicating that loss of FMRP leads to the abnormal function and localization of KARs. This finding implies a new molecular mechanism for Fragile X syndrome.

Full Protection Against Soman-Induced Seizures and Brain Damage by LY293558 and Caramiphen Combination Treatment in Adult Rats.

Acute exposure to nerve agents induces status epilepticus (SE), which causes brain damage or death. LY293558, an antagonist of AMPA and GluK1 kainate receptors is a very effective anticonvulsant and neuroprotectant against soman; however, some neuronal damage is still present after treatment of soman-exposed rats with LY293558. Here, we have tested whether combining LY293558 with an NMDA receptor antagonist can eliminate the residual damage. For this purpose, we chose caramiphen (CRM), an antimuscarinic compound with NMDA receptor antagonistic properties. Adult male rats were exposed to 1.2 × LD50 soman, and at 20 min after soman exposure, were injected with atropine + HI-6, or atropine + HI-6 + LY293558 (15 mg/kg), or atropine + HI-6 + LY293558 + CRM (50 mg/kg). We found that (1) the LY293558 + CRM treatment terminated SE significantly faster than LY293558 alone; (2) after cessation of the initial SE, seizures did not return in the LY293558 + CRM-treated group, during 72 h of monitoring; (3) power spectrum analysis of continuous EEG recordings for 7 days post-exposure showed increased delta and decreased gamma power that lasted beyond 24 h post-exposure only in the rats who did not receive anticonvulsant treatment; (4) spontaneous recurrent seizures appeared on day 7 only in the group that did not receive anticonvulsant treatment; (5) significant neuroprotection was achieved by LY293558 administration, while the rats who received LY293558 + CRM displayed no neurodegeneration; (6) body weight loss and recovery in the LY293558 + CRM-treated rats did not differ from those in control rats who were not exposed to soman. The data show that treatment with LY293558 + CRM provides full antiseizure and neuroprotective efficacy against soman.

Susceptibility to Soman Toxicity and Efficacy of LY293558 Against Soman-Induced Seizures and Neuropathology in 10-Month-Old Male Rats.

Acute nerve agent exposure causes prolonged status epilepticus (SE), leading to death or long-term brain damage. We have previously demonstrated that LY293558, an AMPA/GluK1 kainate receptor antagonist, terminates SE induced by the nerve agent soman and protects from long-term brain damage, in immature rats and young-adult rats, even if administered with a relatively long latency from the time of exposure. However, susceptibility to the lethal consequences of SE increases with age, and mortality by SE induced by soman is substantially greater in older animals. Therefore, in the present study, we compared the susceptibility to soman toxicity of 10-month-old male rats with that of young-adult male rats (42 to 50 days old) and examined the protective efficacy of LY293558 in the older group. A lower percentage of the 10-month-old rats developed SE after injection of 1.2 × LD50 soman, compared to the young adults, the latency to seizure onset was longer in the older rats, and seizure intensity did not differ between the two age groups. However, mortality rate in the older rats who developed SE was higher than in the young adults. Acetylcholinesterase activity in the amygdala, hippocampus, and piriform cortex did not differ between the two age groups. Administration of LY293558 at 20 or 60 min post-exposure suppressed SE, increased 24-h survival rate, decreased the long-term risk of death, reduced neuronal degeneration in the amygdala, hippocampus, piriform, and entorhinal cortices, and facilitated recovery from body weight loss. Thus, LY293558 is an effective countermeasure against soman toxicity also in older animals.

Phosphorylation of the kainate receptor (KAR) auxiliary subunit Neto2 at serine 409 regulates synaptic targeting of the KAR subunit GluK1.

Synaptic strength at excitatory synapses is determined by the presence of glutamate receptors (i.e. AMPA, NMDA, and kainate receptors) at the synapse. Synaptic strength is modulated by multiple factors including assembly of different receptor subunits, interaction with auxiliary subunits, and post-translational modifications of either the receptors or their auxiliary subunits. Using mass spectrometry, we found that the intracellular region of neuropilin and tolloid-like proteins (Neto) 1 and Neto2, the auxiliary subunits of kainate receptor (KARs), are phosphorylated by multiple kinases in vitro Specifically, Neto2 was phosphorylated at serine 409 (Ser-409) by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA) both in vitro and in heterologous cells. Interestingly, we observed a substantial increase in Neto2 Ser-409 phosphorylation in the presence of CaMKII, and this phosphorylation was reduced in the presence of the KAR subunit GluK1 or GluK2. We also found endogenous phosphorylation of Neto2 at Ser-409 in the brain. Moreover, Neto2 Ser-409 phosphorylation inhibited synaptic targeting of GluK1 because, unlike WT Neto2 and the phosphodeficient mutant Neto2 S409A, the Neto2 S409D phosphomimetic mutant impeded GluK1 trafficking to synapses. These results support a molecular mechanism by which Neto2 phosphorylation at Ser-409 helps restrict GluK1 targeting to the synapse.

NETO1 Guides Development of Glutamatergic Connectivity in the Hippocampus by Regulating Axonal Kainate Receptors.

Kainate-type glutamate receptors (KARs) are highly expressed in the developing brain, where they are tonically activated to modulate synaptic transmission, network excitability and synaptogenesis. NETO proteins are auxiliary subunits that regulate biophysical properties of KARs; however, their functions in the immature brain are not known. Here, we show that NETO1 guides the development of the rodent hippocampal CA3-CA1 circuitry via regulating axonal KARs. NETO deficiency reduced axonal targeting of most KAR subunits in hippocampal neurons in a subtype independent manner. As an interesting exception, axonal delivery of GluK1c was strongly and selectively impaired in the Neto1-/-, but not Neto2-/-, neurons. Correspondingly, the presynaptic GluK1 KAR activity that tonically inhibits glutamate release at immature CA3-CA1 synapses was completely lost in the absence of NETO1 but not NETO2. The deficit in axonal KARs at Neto1-/- neurons resulted in impaired synaptogenesis and perturbed synchronization of CA3 and CA1 neuronal populations during development in vitro. Both these Neto1-/- phenotypes were fully rescued by overexpression of GluK1c, emphasizing the role of NETO1/KAR complex in development of efferent connectivity. Together, our data uncover a novel role for NETO1 in regulation of axonal KARs and identify its physiological significance in development of the CA3-CA1 circuit.

Design, synthesis and structure-activity relationships of novel phenylalanine-based amino acids as kainate receptors ligands.

A new series of carboxyaryl-substituted phenylalanines was designed, synthesized and pharmacologically characterized in vitro at native rat ionotropic glutamate receptors as well as at cloned homomeric kainate receptors GluK1-GluK3. Among them, six compounds bound to GluK1 receptor subtypes with reasonable affinity (Ki values in the range of 4.9-7.5µM). A structure-activity relationship (SAR) for the obtained series, focused mainly on the pharmacological effect of structural modifications in the 4- and 5-position of the phenylalanine ring, was established. To illustrate the results, molecular docking of the synthesized series to the X-ray structure of GluK1 ligand binding core was performed. The influence of individual substituents at the phenylalanine ring for both the affinity and selectivity at AMPA, GluK1 and GluK3 receptors was analyzed, giving directions for future studies.

A proteomic analysis reveals the interaction of GluK1 ionotropic kainate receptor subunits with Go proteins.

Kainate receptors (KARs) are found ubiquitously in the CNS and are present presynaptically and postsynaptically regulating synaptic transmission and excitability. Functional studies have proven that KARs act as ion channels as well as potentially activating G-proteins, thus indicating the existance of a dual signaling system for KARs. Nevertheless, it is not clear how these ion channels activate G-proteins and which of the KAR subunits is involved. Here we performed a proteomic analysis to define proteins that interact with the C-terminal domain of GluK1 and we identified a variety of proteins with many different functions, including a Go α subunit. These interactions were verified through distinct in vitro and in vivo assays, and the activation of the Go protein by GluK1 was validated in bioluminescence resonance energy transfer experiments, while the specificity of this association was confirmed in GluK1-deficient mice. These data reveal components of the KAR interactome, and they show that GluK1 and Go proteins are natural partners, accounting for the metabotropic effects of KARs.

High-Throughput Screen of GluK1 Receptor Identifies Selective Inhibitors with a Variety of Kinetic Profiles Using Fluorescence and Electrophysiology Assays.

GluK1, a kainate subtype of ionotropic glutamate receptors, exhibits an expression pattern in the CNS consistent with involvement in pain processing and migraine. Antagonists of GluK1 have been shown to reduce pain signaling in the spinal cord and trigeminal nerve, and are predicted to provide pain and migraine relief. We developed an ultra-high-throughput small-molecule screen to identify antagonists of GluK1. Using the calcium indicator dye fluo-4, a multimillion-member small-molecule library was screened in 1536-well plate format on the FLIPR (Fluorescent Imaging Plate Reader) Tetra against cells expressing a calcium-permeable GluK1. Following confirmation in the primary assay and subsequent counter-screen against the endogenous Par-1 receptor, 6100 compounds were selected for dose titration to assess potency and selectivity. Final triage of 1000 compounds demonstrating dose-dependent inhibition with IC50 values of less than 12 µM was performed in an automated whole-cell patch clamp electrophysiology assay. Although a weak correlation between electrophysiologically active and calcium-imaging active compounds was observed, the identification of electrophysiologically active compounds with a range of kinetic profiles revealed a broad spectrum of mechanisms of action.

A rat model of nerve agent exposure applicable to the pediatric population: The anticonvulsant efficacies of atropine and GluK1 antagonists.

Inhibition of acetylcholinesterase (AChE) after nerve agent exposure induces status epilepticus (SE), which causes brain damage or death. The development of countermeasures appropriate for the pediatric population requires testing of anticonvulsant treatments in immature animals. In the present study, exposure of 21-day-old (P21) rats to different doses of soman, followed by probit analysis, produced an LD50 of 62µg/kg. The onset of behaviorally-observed SE was accompanied by a dramatic decrease in brain AChE activity; rats who did not develop SE had significantly less reduction of AChE activity in the basolateral amygdala than rats who developed SE. Atropine sulfate (ATS) at 2mg/kg, administered 20 min after soman exposure (1.2×LD50), terminated seizures. ATS at 0.5mg/kg, given along with an oxime within 1 min after exposure, allowed testing of anticonvulsants at delayed time-points. The AMPA/GluK1 receptor antagonist LY293558, or the specific GluK1 antagonist UBP302, administered 1h post-exposure, terminated SE. There were no degenerating neurons in soman-exposed P21 rats, but both the amygdala and the hippocampus were smaller than in control rats at 30 and 90days post-exposure; this pathology was not present in rats treated with LY293558. Behavioral deficits present at 30 days post-exposure, were also prevented by LY293558 treatment. Thus, in immature animals, a single injection of atropine is sufficient to halt nerve agent-induced seizures, if administered timely. Testing anticonvulsants at delayed time-points requires early administration of ATS at a low dose, sufficient to counteract only peripheral toxicity. LY293558 administered 1h post-exposure, prevents brain pathology and behavioral deficits.

Neto auxiliary proteins control both the trafficking and biophysical properties of the kainate receptor GluK1.

Kainate receptors (KARs) are a subfamily of glutamate receptors mediating excitatory synaptic transmission and Neto proteins are recently identified auxiliary subunits for KARs. However, the roles of Neto proteins in the synaptic trafficking of KAR GluK1 are poorly understood. Here, using the hippocampal CA1 pyramidal neuron as a null background system we find that surface expression of GluK1 receptor itself is very limited and is not targeted to excitatory synapses. Both Neto1 and Neto2 profoundly increase GluK1 surface expression and also drive GluK1 to synapses. However, the regulation GluK1 synaptic targeting by Neto proteins is independent of their role in promoting surface trafficking. Interestingly, GluK1 is excluded from synapses expressing AMPA receptors and is selectively incorporated into silent synapses. Neto2, but not Neto1, slows GluK1 deactivation, whereas Neto1 speeds GluK1 desensitization and Neto2 slows desensitization. These results establish critical roles for Neto auxiliary subunits controlling KARs properties and synaptic incorporation.