Time-dependent neuropathology in rats following organophosphate-induced status epilepticus.

Exposure to high levels of a cholinesterase inhibiting organophosphorus (OP) agent often results in seizures that progress to status epilepticus (SE). Survivors of OP-induced SE often display neuropathological consequences the days following SE. In the current study, the temporal profile of neuropathology after SE was investigated in a rat model of diisopropylfluorophosphate (DFP)-induced SE. Adult Sprague-Dawley rats were injected with DFP to induce SE for one hour. Following termination of electrographic SE with urethane (0.8 g/kg, sc), cohorts of rats were euthanized 3, 24 and 48 h later and brain tissue was processed to determine immediate early gene and inflammatory mediator expression as well as blood-brain barrier changes and neurodegeneration. After SE rats displayed a time-dependent upregulation of immediate early genes such as cFos and ΔFosB as well as pro-inflammatory mediators COX-2, IL-1ß and IL-6. The profile of positive cFos staining, but not ΔFosB, coincided temporally with heightened brain activity measured by cortical electroencephalography (EEG). Neurodegeneration in limbic brain regions was absent 3 h after SE, but prominent 24 h later and continued to increase 48 h after SE. Serum albumin was detected in the cortex 3 h after SE suggesting early loss of blood brain barrier integrity. However, the blood-brain barrier appeared repaired 48 h after SE. This study demonstrates that following OP-poisoning in rats, immediate early gene expression in the brain precedes neuroinflammation followed by erosion of the blood-brain barrier and neurodegeneration. The study also demonstrates that seizure activity in brain nuclei coincides with cFos expression. Together, these studies give insight into the temporal molecular changes in the brain following organophosphate-induced status epilepticus.

Pharmacological antagonism of EP2 receptor does not modify basal cardiovascular and respiratory function, blood cell counts, and bone morphology in animal models.

The EP2 receptor has emerged as a therapeutic target with exacerbating role in disease pathology for a variety of peripheral and central nervous system disorders. We and others have recently demonstrated beneficial effects of EP2 antagonists in preclinical models of neuroinflammation and peripheral inflammation. However, it was earlier reported that mice with global EP2 knockout (KO) display adverse phenotypes on fertility and blood pressure. Other studies indicated that EP2 activation with an agonist has a beneficial effect of healing fractured bone in animal models. These results impeded the development of EP2 antagonists, and EP2 antagonism as therapeutic strategy. To determine whether treatment with EP2 antagonist mimics the adverse phenotypes of the EP2 global KO mouse, we tested two EP2 antagonists TG11-77. HCl and TG6-10-1 in mice and rats while they are on normal or high-salt diet, and by two different administration protocols (acute and chronic). There were no adverse effects of the antagonists on systolic and diastolic blood pressure, heart rate, respiratory function in mice and rats regardless of rodents being on a regular or high salt diet. Furthermore, chronic exposure to TG11-77. HCl produced no adverse effects on blood cell counts, bone-volume and bone-mineral density in mice. Our findings argue against adverse effects on cardiovascular and respiratory systems, blood counts and bone structure in healthy rodents from the use of small molecule reversible antagonists for EP2, in contrast to the genetic ablation model. This study paves the way for advancing therapeutic applications of EP2 antagonists against diseases involving EP2 dysfunction.

Sleep Disruption Worsens Seizures: Neuroinflammation as a Potential Mechanistic Link.

Sleep disturbances, such as insomnia, obstructive sleep apnea, and daytime sleepiness, are common in people diagnosed with epilepsy. These disturbances can be attributed to nocturnal seizures, psychosocial factors, and/or the use of anti-epileptic drugs with sleep-modifying side effects. Epilepsy patients with poor sleep quality have intensified seizure frequency and disease progression compared to their well-rested counterparts. A better understanding of the complex relationship between sleep and epilepsy is needed, since approximately 20% of seizures and more than 90% of sudden unexpected deaths in epilepsy occur during sleep. Emerging studies suggest that neuroinflammation, (e.g., the CNS immune response characterized by the change in expression of inflammatory mediators and glial activation) may be a potential link between sleep deprivation and seizures. Here, we review the mechanisms by which sleep deprivation induces neuroinflammation and propose that neuroinflammation synergizes with seizure activity to worsen neurodegeneration in the epileptic brain. Additionally, we highlight the relevance of sleep interventions, often overlooked by physicians, to manage seizures, prevent epilepsy-related mortality, and improve quality of life.

Impact of Stress on Epilepsy: Focus on Neuroinflammation-A Mini Review.

Epilepsy, one of the most common neurological disorders worldwide, is characterized by recurrent seizures and subsequent brain damage. Despite strong evidence supporting a deleterious impact on seizure occurrence and outcome severity, stress is an overlooked component in people with epilepsy. With regard to stressor duration and timing, acute stress can be protective in epileptogenesis, while chronic stress often promotes seizure occurrence in epilepsy patients. Preclinical research suggests that chronic stress promotes neuroinflammation and leads to a depressive state. Depression is the most common psychiatric comorbidity in people with epilepsy, resulting in a poor quality of life. Here, we summarize studies investigating acute and chronic stress as a seizure trigger and an important factor that worsens epilepsy outcomes and psychiatric comorbidities. Mechanistic insight into the impact of stress on epilepsy may create a window of opportunity for future interventions targeting neuroinflammation-related disorders.

A Novel Second-Generation EP2 Receptor Antagonist Reduces Neuroinflammation and Gliosis After Status Epilepticus in Rats.

Prostaglandin-E2 (PGE2), an important mediator of inflammation, achieves its functions via four different G protein-coupled receptors (EP1, EP2, EP3, and EP4). We previously demonstrated that the EP2 receptor plays a proinflammatory and neurodegenerative role after status epilepticus (SE). We recently developed TG8-260 as a second-generation highly potent and selective EP2 antagonist. Here, we investigate whether TG8-260 is anti-inflammatory and combats neuropathology caused by pilocarpine-induced SE in rats. Adult male Sprague-Dawley rats were injected subcutaneously with pilocarpine (380-400 mg/kg) to induce SE. Following 60 min of SE, the rats were administered three doses of TG8-260 or vehicle and were allowed to recover. Neurodegeneration, neuroinflammation, gliosis, and blood-brain barrier (BBB) integrity were examined 4 days after SE. The results confirmed that pilocarpine-induced SE results in hippocampal neurodegeneration and a robust inflammatory response that persists days after SE. Furthermore, inhibition of the EP2 receptor by TG8-260 administered beginning 2 h after SE significantly reduced hippocampal neuroinflammation and gliosis but, in distinction to the earlier generation EP2 antagonist, did not mitigate neuronal injury or BBB breakdown. Thus, attenuation of neuroinflammation and gliosis is a common feature of EP2 inhibition following SE.

Comparison of neuropathology in rats following status epilepticus induced by diisopropylfluorophosphate and soman.

The increasing number of cases involving the use of nerve agents as deadly weapons has spurred investigation into the molecular mechanisms underlying nerve agent-induced pathology. The highly toxic nature of nerve agents restrict their use in academic research laboratories. Less toxic organophosphorus (OP) based agents including diisopropylfluorophosphate (DFP) are used as surrogates in academic research laboratories to mimic nerve agent poisoning. However, neuropathology resulting from DFP-induced status epilepticus (SE) has not been compared directly to neuropathology observed following nerve agent poisoning in the same study. Here, the hypothesis that neuropathology measured four days after SE is the same for rats exposed to DFP and soman was tested. Adult Sprague-Dawley rats were injected with soman or DFP to induce SE. Cortical electroencephalography (EEG) was recorded prior to and during soman-induced SE. EEG power analysis of rats administered soman revealed prolonged electrographic SE similar to that of rats that endure uninterrupted SE following injection of DFP. Rats that experienced soman-induced SE displayed less hippocampal neuroinflammation and gliosis compared to rats administered DFP. Seizure-induced weight change, blood-brain barrier (BBB) leakiness and neurodegeneration in most seizure sensitive limbic brain regions were similar for rats that endured SE following soman or DFP. The amalgamated pathology score calculated by combining pathological measures (weight loss, hippocampal neuroinflammation, gliosis, BBB integrity and neurodegeneration) was similar in rats administered the OP agents. These findings support use of the rat DFP model of SE as a suitable surrogate for investigating some, but not all delayed consequences produced by nerve agents.

Urethane attenuates early neuropathology of diisopropylfluorophosphate-induced status epilepticus in rats.

Seizures can be evident within minutes of exposure to an organophosphorus (OP) agent and often progress to status epilepticus (SE) resulting in a high mortality if left untreated. Effective medical countermeasures are necessary to sustain patients suffering from OP poisoning and to mitigate the ensuing brain injury. Here, the hypothesis was tested that a single subanesthetic dose of urethane prevents neuropathology measured 24 h following diisopropylfluorophosphate (DFP)-induced SE. Adult Sprague-Dawley rats were injected with DFP to induce SE. During SE rats displayed increased neuronal activity in the hippocampus and an upregulation of immediate early genes as well as pro-inflammatory mediators. In additional experiments rats were administered diazepam (10 mg/kg, ip) or urethane (0.8 g/kg, sc) 1 h after DFP-induced SE and compared to rats that experienced uninterrupted SE. Cortical electroencephalography (EEG) and power analysis strengthen the conclusion that urethane effectively terminates SE and prevents the overnight return of seizure activity. Neurodegeneration in limbic brain regions and the seizure-induced upregulation of key inflammatory mediators present 24 h after DFP-induced SE were strongly attenuated by administration of urethane. A trivial explanation for these beneficial effects, that urethane simply reactivates acetylcholinesterase, has been ruled out. These findings indicate that, by contrast to rats administered diazepam or rats that experience uninterrupted SE, the early neuropathology after SE is prevented by subanesthetic urethane, which terminates rather than interrupts, SE.

Potent, Selective, Water Soluble, Brain-Permeable EP2 Receptor Antagonist for Use in Central Nervous System Disease Models.

Activation of prostanoid EP2 receptor exacerbates neuroinflammatory and neurodegenerative pathology in central nervous system diseases such as epilepsy, Alzheimer's disease, and cerebral aneurysms. A selective and brain-permeable EP2 antagonist will be useful to attenuate the inflammatory consequences of EP2 activation and to reduce the severity of these chronic diseases. We recently developed a brain-permeable EP2 antagonist 1 (TG6-10-1), which displayed anti-inflammatory and neuroprotective actions in rodent models of status epilepticus. However, this compound exhibited moderate selectivity to EP2, a short plasma half-life in rodents (1.7 h) and low aqueous solubility (27 µM), limiting its use in animal models of chronic disease. With lead-optimization studies, we have developed several novel EP2 antagonists with improved water solubility, brain penetration, high EP2 potency, and selectivity. These novel inhibitors suppress inflammatory gene expression induced by EP2 receptor activation in a microglial cell line, reinforcing the use of EP2 antagonists as anti-inflammatory agents.

A rat model of organophosphate-induced status epilepticus and the beneficial effects of EP2 receptor inhibition.

This review describes an adult rat model of status epilepticus (SE) induced by diisopropyl fluorophosphate (DFP), and the beneficial outcomes of transient inhibition of the prostaglandin-E2 receptor EP2 with a small molecule antagonist, delayed by 2-4 h after SE onset. Administration of six doses of the selective EP2 antagonist TG6-10-1 over a 2-3 day period accelerates functional recovery, attenuates hippocampal neurodegeneration, neuroinflammation, gliosis and blood-brain barrier leakage, and prevents long-term cognitive deficits without blocking SE itself or altering acute seizure characteristics. This work has provided important information regarding organophosphate-induced seizure related pathologies in adults and revealed the effectiveness of delayed EP2 inhibition to combat these pathologies.

Novel Microglia Cell Line Expressing the Human EP2 Receptor.

Recently, EP2 signaling pathways were shown to regulate the classical activation and death of microglia in rat primary microglial culture. The study of microglial cells has been challenging because they are time-consuming to isolate in culture, they are demanding in their growth requirements, and they have a limited lifespan. To circumvent these difficulties, we created a murine BV2 microglial cell line stably expressing human EP2 receptors (BV2-hEP2) and further explored EP2 modulation of microglial functions. The BV2-hEP2 cells displayed cAMP elevation when exposed to the selective EP2 receptor agonists (ONO-AE1-259-1 and CP544326), and this response was competitively inhibited by TG4-155, a selective EP2 antagonist (Schild KB = 2.6 nM). By contrast, untransfected BV2 cells were unresponsive to selective EP2 agonists. Similar to the case of rat primary microglia, BV2-hEP2 microglia treated with lipopolysaccharide (LPS) (100 ng/mL) displayed rapid and robust induction of the inflammatory mediators COX-2, IL-1ß, TNFα, and IL-6. EP2 activation depressed TNFα induction but exacerbated that of the other inflammatory mediators. Like primary microglia, classically activated BV2 microglia phagocytose fluorescent-labeled latex microspheres. The presence of EP2, but not its activation by agonists, in BV2-hEP2 microglia reduced phagocytosis and proliferation by 65% and 32%, respectively, compared to BV2 microglia. Thus, BV2-hEP2 is the first microglial cell line that retains the EP2 modulation of immune regulation and phagocytic ability of native microglia. Suppression of phagocytosis by the EP2 protein appears unrelated to classical EP2 signaling pathways, which has implications for therapeutic development of EP2 antagonists.

5xFAD Mice Display Sex-Dependent Inflammatory Gene Induction During the Prodromal Stage of Alzheimer's Disease.

Alzheimer's disease (AD) pathology consists of extracellular deposits of amyloid-ß peptides (Aß) and intracellular neurofibrillary tangles. These pathological alterations are accompanied by a neuroinflammatory response consisting of increased expression of inflammatory mediators. An anti-inflammatory strategy designed to prevent or delay the development of AD would benefit from knowing when neuroinflammation appears in the transgenic models during prodromal disease stages relative to Aß pathology. We investigated the expression patterns of inflammatory mediators in the brain of 5xFAD mice in comparison to development of Aß deposition. Expression changes in inflammatory mediators and glial markers are more robust in female mice starting at three months of age, in contrast to males in which there is no clear trend through five months. Female and male 5xFAD mice also displayed an age-dependent increase in cortical Aß deposition congruent with neuroinflammation. Thus, in the 5xFAD mouse model of AD, administration of an anti-inflammatory agent would be most efficacious when administered before three months of age.

The COX-2/prostanoid signaling cascades in seizure disorders.

Introduction：A robust neuroinflammatory response is a prevalent feature of multiple neurological disorders, including epilepsy and acute status epilepticus. One component of this neuroinflammatory reaction is the induction of cyclooxygenase-2 (COX-2), synthesis of several prostaglandins and endocannabinoid metabolites, and subsequent activation of prostaglandin and related receptors. Neuroinflammation mediated by COX-2 and its downstream effectors has received considerable attention as a potential target class to ameliorate the deleterious consequences of neurological injury. Areas covered: Here we describe the roles of COX-2 as a major inflammatory mediator. In addition, we discuss the receptors for prostanoids PGE2, prostaglandin D2, and PGF2α as potential therapeutic targets for inflammation-driven diseases. The consequences of prostanoid receptor activation after seizure activity are discussed with an emphasis on the utilization of small molecules to modulate prostanoid receptor activity. Expert opinion: Limited clinical trial experience is supportive but not definitive for a role of the COX signaling cascade in epileptogenesis. The cardiotoxicity associated with chronic coxib use, and the expectation that COX-2 inhibition will influence the levels of endocannabinoids, leukotrienes, and lipoxins as well as the prostaglandins and their endocannabinoid metabolite analogs, is shifting attention toward downstream synthases and receptors that mediate inflammation in the brain.

Beneficial Outcome of Urethane Treatment Following Status Epilepticus in a Rat Organophosphorus Toxicity Model.

The efficacy of benzodiazepines to terminate electrographic status epilepticus (SE) declines the longer a patient is in SE. Therefore, alternative methods for ensuring complete block of SE and refractory SE are necessary. We compared the ability of diazepam and a subanesthetic dose of urethane to terminate prolonged SE and mitigate subsequent pathologies. Adult Sprague Dawley rats were injected with diisopropylfluorophosphate (DFP) to induce SE. Rats were administered diazepam (10 mg/kg, ip) or urethane (0.8 g/kg, s.c.) 1 h after DFP-induced SE and compared to rats that experienced uninterrupted SE. Large-amplitude and high-frequency spikes induced by DFP administration were quenched for at least 46 h in rats administered urethane 1 h after SE onset as demonstrated by cortical electroencephalography (EEG). By contrast, diazepam interrupted SE but seizures with high power in the 20- to 70-Hz band returned 6-10 h later. Urethane was more effective than diazepam at reducing hippocampal neurodegeneration, brain inflammation, gliosis and weight loss as measured on day 4 after SE. Furthermore, rats administered urethane displayed a 73% reduction in the incidence of spontaneous recurrent seizures after four to eight weeks and a 90% reduction in frequency of seizures in epileptic rats. By contrast, behavioral changes in the light/dark box, open field and a novel object recognition task were not improved by urethane. These findings indicate that in typical rodent SE models, it is the return of SE overnight, and not the initially intense 1-2 h of SE experience, that is largely responsible for neurodegeneration, accompanying inflammation, and the subsequent development of epilepsy.

Ethylatropine Bromide as a Peripherally Restricted Muscarinic Antagonist.

Quaternary ammonium analogues of atropine that are unable to cross the blood-brain barrier are used to alleviate peripheral muscarinic toxicity in animal models of epilepsy produced by systemic administration of pilocarpine or diisopropylfluorophosphate (DFP). Currently, methylatropine is the most popular and potent of these quaternary derivatives; however, it is expensive and produced in limited quantity. Here, we propose the use of ethylatropine bromide as an alternative to methylatropine. The synthesis of ethylatropine bromide is simple, inexpensive and has low environmental impact. We demonstrate the efficacy of ethylatropine bromide to antagonize the carbachol induced rise in intracellular calcium in a calcium mobilization assay, and its ability to prevent pilocarpine-induced total fluid secretions in mice without blocking pilocarpine-induced seizures. The ease of synthesis, cost effectiveness, and efficacy makes ethylatropine bromide a desirable alternative to methylatropine as a peripherally restricted acetylcholine receptor antagonist.

Inhibition of the prostaglandin E2 receptor EP2 prevents status epilepticus-induced deficits in the novel object recognition task in rats.

Survivors of exposure to an organophosphorus nerve agent may develop a number of complications including long-term cognitive deficits (Miyaki et al., 2005; Nishiwaki et al., 2001). We recently demonstrated that inhibition of the prostaglandin E2 receptor, EP2, attenuates neuroinflammation and neurodegeneration caused by status epilepticus (SE) induced by the soman analog, diisopropylfluorophosphate (DFP), which manifest within hours to days of the initial insult. Here, we tested the hypothesis that DFP exposure leads to a loss of cognitive function in rats that is blocked by early, transient EP2 inhibition. Adult male Sprague-Dawley rats were administered vehicle or the competitive EP2 antagonist, TG6-10-1, (ip) at various times relative to DFP-induced SE. DFP administration resulted in prolonged seizure activity as demonstrated by cortical electroencephalography (EEG). A single intraperitoneal injection of TG6-10-1 or vehicle 1 h prior to DFP did not alter the development of seizures, the latency to SE or the duration of SE. Rats administered six injections of TG6-10-1 starting 90 min after the onset of DFP-induced SE could discriminate between a novel and familiar object 6-12 weeks after SE, unlike vehicle treated rats which showed no preference for the novel object. By contrast, behavioral changes in the light-dark box and open field assays were not affected by TG6-10-1. Delayed mortality after DFP was also unaffected by TG6-10-1. Thus, selective inhibition of the EP2 receptor may prevent SE-induced memory impairment in rats caused by exposure to a high dose of DFP.

Inhibition of the prostaglandin EP2 receptor is neuroprotective and accelerates functional recovery in a rat model of organophosphorus induced status epilepticus.

Exposure to high levels of organophosphorus compounds (OP) can induce status epilepticus (SE) in humans and rodents via acute cholinergic toxicity, leading to neurodegeneration and brain inflammation. Currently there is no treatment to combat the neuropathologies associated with OP exposure. We recently demonstrated that inhibition of the EP2 receptor for PGE2 reduces neuronal injury in mice following pilocarpine-induced SE. Here, we investigated the therapeutic effects of an EP2 inhibitor (TG6-10-1) in a rat model of SE using diisopropyl fluorophosphate (DFP). We tested the hypothesis that EP2 receptor inhibition initiated well after the onset of DFP-induced SE reduces the associated neuropathologies. Adult male Sprague-Dawley rats were injected with pyridostigmine bromide (0.1 mg/kg, sc) and atropine methylbromide (20 mg/kg, sc) followed by DFP (9.5 mg/kg, ip) to induce SE. DFP administration resulted in prolonged upregulation of COX-2. The rats were administered TG6-10-1 or vehicle (ip) at various time points relative to DFP exposure. Treatment with TG6-10-1 or vehicle did not alter the observed behavioral seizures, however six doses of TG6-10-1 starting 80-150 min after the onset of DFP-induced SE significantly reduced neurodegeneration in the hippocampus, blunted the inflammatory cytokine burst, reduced microglial activation and decreased weight loss in the days after status epilepticus. By contrast, astrogliosis was unaffected by EP2 inhibition 4 d after DFP. Transient treatments with the EP2 antagonist 1 h before DFP, or beginning 4 h after DFP, were ineffective. Delayed mortality, which was low (10%) after DFP, was unaffected by TG6-10-1. Thus, selective inhibition of the EP2 receptor within a time window that coincides with the induction of cyclooxygenase-2 by DFP is neuroprotective and accelerates functional recovery of rats.

The prostaglandin EP1 receptor potentiates kainate receptor activation via a protein kinase C pathway and exacerbates status epilepticus.

Prostaglandin E2 (PGE2) regulates membrane excitability, synaptic transmission, plasticity, and neuronal survival. The consequences of PGE2 release following seizures has been the subject of much study. Here we demonstrate that the prostaglandin E2 receptor 1 (EP1, or Ptger1) modulates native kainate receptors, a family of ionotropic glutamate receptors widely expressed throughout the central nervous system. Global ablation of the EP1 gene in mice (EP1-KO) had no effect on seizure threshold after kainate injection but reduced the likelihood to enter status epilepticus. EP1-KO mice that did experience typical status epilepticus had reduced hippocampal neurodegeneration and a blunted inflammatory response. Further studies with native prostanoid and kainate receptors in cultured cortical neurons, as well as with recombinant prostanoid and kainate receptors expressed in Xenopus oocytes, demonstrated that EP1 receptor activation potentiates heteromeric but not homomeric kainate receptors via a second messenger cascade involving phospholipase C, calcium and protein kinase C. Three critical GluK5 C-terminal serines underlie the potentiation of the GluK2/GluK5 receptor by EP1 activation. Taken together, these results indicate that EP1 receptor activation during seizures, through a protein kinase C pathway, increases the probability of kainic acid induced status epilepticus, and independently promotes hippocampal neurodegeneration and a broad inflammatory response.

Cyclooxygenase-2 in epilepsy.

Epilepsy is one of the more prevalent neurologic disorders in the world, affecting approximately 50 million people of different ages and backgrounds. Epileptic seizures propagating through both lobes of the forebrain can have permanent debilitating effects on a patient's cognitive and somatosensory brain functions. Epilepsy, defined by the sporadic occurrence of spontaneous recurrent seizures (SRS), is often accompanied by inflammation of the brain. Pronounced increases in the expression of key inflammatory mediators (e.g., interleukin -1ß [IL-1ß], tumor necrosis factor alpha [TNFα], cyclooxygenase-2 [COX-2], and C-X-C motif chemokine 10 [CXCL10]) after seizures may cause secondary damage in the brain and increase the likelihood of repetitive seizures. The COX-2 enzyme is induced rapidly during seizures. The increased level of COX-2 in specific areas of the epileptic brain can help to identify regions of seizure-induced brain inflammation. A good deal of effort has been expended to determine whether COX-2 inhibition might be neuroprotective and represent an adjunct therapeutic strategy along with antiepileptic drugs used to treat epilepsy. However, the effectiveness of COX-2 inhibitors on epilepsy animal models appears to depend on the timing of administration. With all of the effort placed on making use of COX-2 inhibitors as therapeutic agents for the treatment of epilepsy, inflammation, and neurodegenerative diseases there has yet to be a selective and potent COX-2 inhibitor that has shown a clear therapeutic outcome with acceptable side effects.

Ionotropic glutamate receptors: regulation by G-protein-coupled receptors.

The function of many ion channels is under dynamic control by coincident activation of G-protein-coupled receptors (GPCRs), particularly those coupled to the Gαs and Gαq family members. Such regulation is typically dependent on the subunit composition of the ionotropic receptor or channel as well as the GPCR subtype and the cell-specific panoply of signaling pathways available. Because GPCRs and ion channels are so highly represented among targets of U.S. Food and Drug Administration-approved drugs, functional cross-talk between these drug target classes is likely to underlie many therapeutic and adverse effects of marketed drugs. GPCRs engage a myriad of signaling pathways that involve protein kinases A and C (PKC) and, through PKC and interaction with ß-arrestin, Src kinase, and hence the mitogen-activated-protein-kinase cascades. We focus here on the control of ionotropic glutamate receptor function by GPCR signaling because this form of regulation can influence the strength of synaptic plasticity. The amino acid residues phosphorylated by specific kinases have been securely identified in many ionotropic glutamate (iGlu) receptor subunits, but which of these sites are GPCR targets is less well known even when the kinase has been identified. N-methyl-d-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and heteromeric kainate receptors are all downstream targets of GPCR signaling pathways. The details of GPCR-iGlu receptor cross-talk should inform a better understanding of how synaptic transmission is regulated and lead to new therapeutic strategies for neuropsychiatric disorders.

Activation of group I metabotropic glutamate receptors potentiates heteromeric kainate receptors.

Kainate receptors (KARs), a family of ionotropic glutamate receptors, are widely expressed in the central nervous system and are critically involved in synaptic transmission. KAR activation is influenced by metabotropic glutamate receptor (mGlu) signaling, but the underlying mechanisms are not understood. We undertook studies to examine how mGlu modulation affects activation of KARs. Confocal immunohistochemistry of rat hippocampus and cultured rat cortex revealed colocalization of the high-affinity KAR subunits with group I mGlu receptors. In hippocampal and cortical cultures, the calcium signal caused by activation of native KARs was potentiated by activation of group I mGlu receptors. In Xenopus laevis oocytes, activation of group I mGlu receptors potentiated heteromeric but not homomeric KAR-mediated currents, with no change in agonist potency. The potentiation of heteromeric KARs by mGlu1 activation was attenuated by GDPßS, blocked by an inhibitor of phospholipase C or the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), prolonged by the phosphatase inhibitor okadaic acid, but unaffected by the tyrosine kinase inhibitor lavendustin A. Protein kinase C (PKC) inhibition reduced the potentiation by mGlu1 of GluK2/GluK5, and conversely, direct activation of PKC by phorbol 12-myristate,13-acetate potentiated GluK2/GluK5. Using site-directed mutagenesis, we identified three serines (Ser833, Ser836, and Ser840) within the membrane proximal region of the GluK5 C-terminal domain that, in combination, are required for mGlu1-mediated potentiation of KARs. Together, these data suggest that phosphorylation of key residues in the C-terminal domain changes the overall charge of this domain, resulting in potentiated agonist responses.