Chronic Epilepsy and Mossy Fiber Sprouting Following Organophosphate-Induced Status Epilepticus in Rats.

Organophosphate (OP) compounds are highly toxic and include pesticides and chemical warfare nerve agents. OP exposure inhibits the acetylcholinesterase enzyme, causing cholinergic overstimulation that can evolve into status epilepticus (SE) and produce lethality. Furthermore, OP-induced SE survival is associated with mood and memory dysfunction and spontaneous recurrent seizures (SRS). In male Sprague-Dawley rats, we assessed hippocampal pathology and chronic SRS following SE induced by administration of OP agents paraoxon (2 mg/kg, s.c.), diisopropyl fluorophosphate (4 mg/kg, s.c.), or O-isopropyl methylphosphonofluoridate (GB; sarin) (2 mg/kg, s.c.), immediately followed by atropine and 2-PAM. At 1-hour post-OP-induced SE onset, midazolam was administered to control SE. Approximately 6 months after OP-induced SE, SRS were evaluated using video and electroencephalography monitoring. Histopathology was conducted using hematoxylin and eosin (H&E), while silver sulfide (Timm) staining was used to assess mossy fiber sprouting (MFS). Across all the OP agents, over 60% of rats that survived OP-induced SE developed chronic SRS. H&E staining revealed a significant hippocampal neuronal loss, while Timm staining revealed extensive MFS within the inner molecular region of the dentate gyrus. This study demonstrates that OP-induced SE is associated with hippocampal neuronal loss, extensive MFS, and the development of SRS, all hallmarks of chronic epilepsy. SIGNIFICANCE STATEMENT: Models of organophosphate (OP)-induced SE offer a unique resource to identify molecular mechanisms contributing to neuropathology and the development of chronic OP morbidities. These models could allow the screening of targeted therapeutics for efficacious treatment strategies for OP toxicities.

Ketamine Produces Antidepressant Effects by Inhibiting Histone Deacetylases and Upregulating Hippocampal Brain-Derived Neurotrophic Factor Levels in a Diisopropyl Fluorophosphate-Based Rat Model of Gulf War Illness.

Approximately one-third of Gulf War veterans suffer from Gulf War Illness (GWI), which encompasses mood disorders and depressive symptoms. Deployment-related exposure to organophosphate compounds has been associated with GWI development. Epigenetic modifications have been reported in GWI veterans. We previously showed that epigenetic histone dysregulations were associated with decreased brain-derived neurotrophic factor (BDNF) expression in a GWI rat model. GWI has no effective therapies. Ketamine (KET) has recently been approved by the Food and Drug Administration for therapy-resistant depression. Interestingly, BDNF upregulation underlies KET's antidepressant effect in GWI-related depression. Here, we investigated whether KET's effect on histone mechanisms signals BDNF upregulations in GWI. Male Sprague-Dawley rats were injected once daily with diisopropyl fluorophosphate (DFP; 0.5 mg/kg, s.c., 5 days). At 6 months following DFP exposure, KET (10 mg/kg, i.p.) was injected, and brains were dissected 24 hours later. Western blotting was used for protein expression, and epigenetic studies used chromatin immunoprecipitation methods. Dil staining was conducted for assessing dendritic spines. Our results indicated that an antidepressant dose of KET inhibited the upregulation of histone deacetylase (HDAC) enzymes in DFP rats. Furthermore, KET restored acetylated histone occupancy at the Bdnf promoter IV and induced BDNF protein expression in DFP rats. Finally, KET treatment also increased the spine density and altered the spine diversity with increased T-type and decreased S-type spines in DFP rats. Given these findings, we propose that KET's actions involve the inhibition of HDAC expression, upregulation of BDNF, and dendritic modifications that together ameliorates the pathologic synaptic plasticity and exerts an antidepressant effect in DFP rats. SIGNIFICANCE STATEMENT: This study offers evidence supporting the involvement of epigenetic histone pathways in the antidepressant effects of ketamine (KET) in a rat model of Gulf War Illness (GWI)-like depression. This effect is achieved through the modulation of histone acetylation at the Bdnf promoter, resulting in elevated brain-derived neurotrophic factor expression and subsequent dendritic remodeling in the hippocampus. These findings underscore the rationale for considering KET as a potential candidate for clinical trials aimed at managing GWI-related depression.

Novel therapeutics for treating organophosphate-induced status epilepticus co-morbidities, based on changes in calcium homeostasis.

Organophosphate (OP) chemicals include pesticides such as parathion, and nerve gases such as sarin and soman and are considered major chemical threat agents. Acute OP exposure is associated with a cholinergic crisis and status epilepticus (SE). It is also known that the survivors of OP toxicity exhibit neurobehavioral deficits such as mood changes, depression, and memory impairment, and acquired epilepsy. Our research has focused on addressing the need to develop effective therapeutic agents that could be administered even after prolonged seizures and would prevent or lessen the chronic morbidity associated with OP-SE survival. We have developed rat survival models of OP pesticide metabolite paraoxon (POX) and nerve agent sarin surrogate diisopropyl fluorophosphate (DFP) induced SE that are being used to screen for medical countermeasures against an OP attack. Our research has focused on studying neuronal calcium (Ca2+) homeostatic mechanisms for identifying mechanisms and therapeutics for the expression of neurological morbidities associated with OP-SE survival. We have observed development of a "Ca2+ plateau" characterized by sustained elevations in neuronal Ca2+ levels in OP-SE surviving rats that coincided with the appearance of OP-SE chronic morbidities. These Ca2+ elevations had their origin in Ca2+ release from the intracellular stores such that blockade with antagonists like dantrolene, carisbamate, and levetiracetam lowered OP-SE mediated Ca2+ plateau and afforded significant neuroprotection. Since the Ca2+ plateau lasts for a prolonged period, our studies suggest that blocking it after the control of SE may represent a unique target for development of novel countermeasures to prevent long term Ca2+ mediated OP-SE neuropsychiatric comorbidities such as depression, anxiety, and acquired epilepsy (AE).

Genome-wide analysis of hepatic DNA methylation reveals impact of epigenetic aging on xenobiotic metabolism and transport genes in an aged mouse model.

Hepatic xenobiotic metabolism and transport decline with age, while intact xenobiotic metabolism is associated with longevity. However, few studies have examined the genome-wide impact of epigenetic aging on these processes. We used reduced representation bisulfite sequencing (RRBS) to map DNA methylation changes in liver DNA from mice ages 4 and 24 months. We identified several thousand age-associated differentially methylated sites (a-DMS), many of which overlapped genes encoding Phase I and Phase II drug metabolizing enzymes, in addition to ABC and SLC classes of transporters. Notable genes harboring a-DMS were Cyp1a2, Cyp2d9, and Abcc2 that encode orthologs of the human drug metabolizing enzymes CYP1A2 and CYP2D6, and the multidrug resistance protein 2 (MRP2) transporter. Cyp2d9 hypermethylation with age was significantly associated with reduced gene expression, while Abcc2 expression was unchanged with age. Cyp1a2 lost methylation with age while, counterintuitively, its expression also reduced with age. We hypothesized that age-related dysregulation of the hepatic transcriptional machinery caused down-regulation of genes despite age-related hypomethylation. Bioinformatic analysis of hypomethylated a-DMS in our sample found them to be highly enriched for hepatic nuclear factor 4 alpha (HNF4α) binding sites. HNF4α promotes Cyp1a2 expression and is downregulated with age, which could explain the reduction in Cyp1a2 expression. Overall, our study supports the broad impact of epigenetic aging on xenobiotic metabolism and transport. Future work should evaluate the interplay between hepatic nuclear receptor function and epigenetic aging. These results may have implications for studies of longevity and healthy aging.

Repeated exposure to chlorpyrifos is associated with a dose-dependent chronic neurobehavioral deficit in adult rats.

Organophosphate (OP) chemicals include commonly used pesticides and chemical warfare agents, and mechanistically they are potent inhibitors of the cholinesterase (ChE) enzyme. Epidemiological studies report long-term neuropsychiatric issues, including depression and cognitive impairments in OP-exposed individuals. Chlorpyrifos (CPF) is one of the most widely used pesticides worldwide. Multiple laboratory studies have reported on either the long-term behavioral effect of an acute high-dose CPF (30-250 mg/kg) or studied sub-chronic behavioral effects, particularly the motor and cognitive effects of repeated low-dose CPF. However, studies are lacking on chronic mood and depression-related morbidities following repeated CPF doses that would mimic occupationally relevant OP exposures during adulthood. In this study, adult male rats were injected with CPF (1, 3, 5, or 10 mg/kg/d, s.c.) for 21 consecutive days. Dependent on the CPF dose, ChE activity was inhibited approximately 60-80% in the blood and about 20-50% in the hippocampus at 2-days after the end of CPF exposures. Following a 12-week washout period, a complete recovery of ChE activity was noted. However, CPF-treated rats exhibited a dose-dependent increase in signs related to anhedonia (sucrose preference test), anxiety (open-field and elevated plus-maze), and despair (forced swim test) at this stage. To the best of our knowledge, this could be the first laboratory study that demonstrates a cause-effect relationship between occupational-like CPF exposures in adult rats and the development of long-term depression-related outcomes and could provide an experimental system to study molecular mechanisms underlying environmental OP exposures and the elevated risk for chronic behavioral deficits.

A review of pre-clinical models for Gulf War Illness.

Gulf War Illness (GWI) is a chronic multisymptomatic disorder that afflicts over 1/3rd of the 1991 GW veterans. It spans multiple bodily systems and presents itself as a syndrome exhibiting diverse symptoms including fatigue, depression, mood, and memory and concentration deficits, musculoskeletal pain and gastrointestinal distress in GW veterans. The etiology of GWI is complex and many factors, including chemical, physiological, and environmental stressors present in the GW arena, have been implicated for its development. It has been over 30 years since the end of the GW but, GWI has been persistent in suffering veterans who are also dealing with paucity of effective treatments. The multifactorial aspect of GWI along with genetic heterogeneity and lack of available data surrounding war-time exposures have proved to be challenging in developing pre-clinical models of GWI. Despite this, over a dozen GWI animal models exist in the literature. In this article, following a brief discussion of GW history, GWI definitions, and probable causes for its pathogenesis, we will expand upon various experimental models used in GWI laboratory research. These animal models will be discussed in the context of their attempts at mimicking GW-related exposures with regards to the variations in chemical combinations, doses, and frequency of exposures. We will discuss their advantages and limitations in modeling GWI followed by a discussion of behavioral and molecular findings in these models. The mechanistic data obtained from these preclinical studies have offered multiple molecular pathways including chronic inflammation, mitochondrial dysfunction, oxidative stress, lipid disturbances, calcium homeostatic alterations, changes in gut microbiota, and epigenetic modifications, amongst others for explaining GWI development and its persistence. Finally, these findings have also informed us on novel druggable targets in GWI. While, it has been difficult to conceive a single pre-clinical model that could express all the GWI signs and exhibit biological complexity reflective of the clinical presentation in GWI, animal models have been critical for identifying molecular underpinnings of GWI and evaluating treatment strategies for GWI.

Epigenetic histone acetylation and Bdnf dysregulation in the hippocampus of rats exposed to repeated, low-dose diisopropylfluorophosphate.

AIMS: Deployment-related exposures to organophosphate (OP) compounds are implicated for Gulf War Illness (GWI) development in First GW veterans. However, reasons for the persistence of GWI are not fully understood. Epigenetic modifications to chromatin are regulatory mechanisms that can adaptively or maladaptively respond to external stimuli. These include DNA methylation and histone acetylation. DNA methylation changes have been reported in GWI but the role of histone acetylation in GWI has been less explored, despite its importance as an epigenetic mechanism for neurological disorders. MAIN METHODS: Male Sprague-Dawley rats were exposed to OP diisopropyl fluorophosphate (DFP, 0.5 mg/kg s.c., 5-d) and 6-m later brains were dissected for hippocampus. Western blotting, activity assays and chromatin immunoprecipitation (ChIP) were utilized for epigenetic analyses. Behavior was assessed using the Forced Swim Test (FST) and the Elevated Plus Maze (EPM). KEY FINDINGS: We observed a significant upregulation in HDAC1 protein along with a significant increase in HDAC enzyme activity in the hippocampus of DFP rats. A locus-specific ChIP study revealed decreases in H3K9ac at the brain derived neurotrophic factor (Bdnf) promoter IV coupled with a significant decrease in BDNF protein in DFP rat hippocampus. Treatment with HDAC inhibitor valproic acid reduced HDAC activity and decreased the FST immobility time in DFP rats. SIGNIFICANCE: Our research suggests that epigenetic alterations to histone acetylation pathways and decreased BDNF expression could represent novel mechanisms for GWI symptomatology and may provide new targets for developing effective drugs for GWI treatment.

Dantrolene inhibits the calcium plateau and prevents the development of spontaneous recurrent epileptiform discharges following in vitro status epilepticus.

Status epilepticus is a clinical emergency that can lead to the development of acquired epilepsy following neuronal injury. Understanding the pathophysiological changes that occur between the injury itself and the expression of epilepsy is important in the development of new therapeutics to prevent epileptogenesis. Currently, no anti-epileptogenic agents exist; thus, the ability to treat an individual immediately after status epilepticus to prevent the ultimate development of epilepsy remains an important clinical challenge. In the Sprague-Dawley rat pilocarpine model of status epilepticus-induced acquired epilepsy, intracellular calcium has been shown to increase in hippocampal neurons during status epilepticus and remain elevated well past the duration of the injury in those animals that develop epilepsy. This study aimed to determine if such changes in calcium dynamics exist in the hippocampal culture model of status epilepticus-induced acquired epilepsy and, if so, to study whether manipulating the calcium plateau after status epilepticus would prevent epileptogenesis. The in vitro status epilepticus model resembled the in vivo model in terms of elevations in neuronal calcium concentrations that were maintained well past the duration of the injury. When used following in vitro status epilepticus, dantrolene, a ryanodine receptor inhibitor, but not the N-methyl-D-aspartic acid channel blocker MK-801 inhibited the elevations in intracellular calcium, decreased neuronal death and prevented the expression of spontaneous recurrent epileptiform discharges, the in vitro correlate of epilepsy. These findings offer potential for a novel treatment to prevent the development of epileptiform discharges following brain injuries.

Calcium Hypothesis of Gulf War Illness: Role of Calcium Ions in Neurological Morbidities in a DFP-Based Rat Model for Gulf War Illness.

Gulf War Illness (GWI) refers to a multi-system disorder that afflicts approximately 30% of First Gulf War (GW) veterans. Amongst the symptoms exhibited, mood and memory impairment are commonly reported by GW veterans. Exposure to organophosphate (OP) compounds which target the cholinergic system is considered a leading cause for GWI symptoms. It is hypothesized that chronic OP-based war-time stimulation of cholinergic signaling led to recruitment of excitatory glutamatergic signaling and other downstream signaling cascades leading to neuronal injury, neuroinflammation, generation of reactive oxygen species, oxidative stress, and mitochondrial damage within the central nervous system. These findings have been observed in both experimental models and GWI veterans. In this context the role of calcium (Ca2+) signaling in GWI has come to the forefront. Here we present our Ca2+ hypothesis of GWI that suggests sustained neuronal Ca2+ elevations serve as a molecular trigger for pathological synaptic plasticity that has allowed for the persistence of GWI symptoms. Subsequently we discuss that therapeutic targeting of Ca2+ homeostatic mechanisms provides novel targets for effective treatment of GWI-related neurological signs in our rodent model.

Assessment of Ketamine and Its Enantiomers in an Organophosphate-Based Rat Model for Features of Gulf War Illness.

Approximately 33% of U.S. soldiers from the first Gulf War suffer from a multi-system disorder known as the Gulf War Illness (GWI). GW veterans suffer from a cluster of symptoms that prominently include fatigue and can include mood-related symptoms. Compared to traditional antidepressants, ketamine (KET) produces a fast-onset and long-lasting antidepressant response, but assessments of KET for GWI-related depression are lacking. The etiology of GWI is multi-factorial and exposure to organophosphates (OP) during deployment is one of the factors underlying GWI development. Here, male Sprague-Dawley rats were repeatedly exposed to an OP DFP and three months later these rats, when assessed on a battery of rodent behavioral assays, displayed signs consistent with aspects of GWI characteristics. When treated with a sub-anesthetic dose of KET (3, 5, or 10 mg/kg, i.p.), DFP-treated rats exhibited a significant improvement in immobility time, open-arm exploration, and sucrose consumption as early as 1 h and much of these effects persisted at 24-h post-KET injection. KET's stereoisomers, R-KET and S-KET, also exhibited such effects in DFP rats, with R-KET being the more potent isomer. Our studies provide a starting point for further assessment of KET for GWI depression.

Intramuscular atenolol and levetiracetam reduce mortality in a rat model of paraoxon-induced status epilepticus.

Organophosphorus (OP) compounds are chemical threat agents and are irreversible inhibitors of the enzyme acetylcholinesterase that lead to a hypercholinergic response that could include status epilepticus (SE). SE particularly targets the heart and brain and despite existing therapies, it is still associated with significant mortality and morbidity. Here, we investigated the effect of intramuscular (i.m.) adjunct therapy consisting of atenolol (AT) and levetiracetam (LV) when administered after paraoxon (POX)-induced SE. The combination therapy was administered twice daily for 2, 7, or 14 days. POX exposure in rats produced rapid SE onset that was treated with atropine, pralidoxime chloride, and midazolam. Here, AT + LV therapy produced significant reductions in POX SE mortality assessed at 30 days post-SE. AT + LV therapy exhibited muscle pathology inflammation scores that were not significantly different from saline-treated controls. Pharmacokinetic analyses revealed that the i.m. route achieved faster and stabler plasma therapeutic levels for both AT and LV under OP SE conditions compared with oral administrations. Our data provide evidence of the safety and efficacy of i.m. AT + LV therapy for reducing mortality following POX SE.

Neuronal-Specific Inhibition of Endoplasmic Reticulum Mg2+/Ca2+ ATPase Ca2+ Uptake in a Mixed Primary Hippocampal Culture Model of Status Epilepticus.

Loss of intracellular calcium homeostasis is an established mechanism associated with neuronal dysfunction and status epilepticus. Sequestration of free cytosolic calcium into endoplasmic reticulum by Mg2+/Ca2+ adenosinetriphosphatase (ATPase) is critical for maintenance of intracellular calcium homeostasis. Exposing hippocampal cultures to low-magnesium media is a well-accepted in vitro model of status epilepticus. Using this model, it was shown that endoplasmic reticulum Ca2+ uptake was significantly inhibited in homogenates from cultures demonstrating electrophysiological seizure phenotypes. Calcium uptake was mainly neuronal. However, glial Ca2+ uptake was also significantly inhibited. Viability of neurons exposed to low magnesium was similar to neurons exposed to control solutions. Finally, it was demonstrated that Ca2+ uptake inhibition and intracellular free Ca2+ levels increased in parallel with increasing incubation in low magnesium. The results suggest that inhibition of Mg2+/Ca2+ ATPase-mediated endoplasmic reticulum Ca2+ sequestration contributes to loss of intracellular Ca2+ homeostasis associated with status epilepticus. This study describes for the first time inhibition of endoplasmic reticulum Mg2+/Ca2+ ATPase in a mixed primary hippocampal model of status epilepticus. In combination with animal models of status epilepticus, the cell culture model provides a powerful tool to further elucidate mechanisms that result in inhibition of Mg2+/Ca2+ ATPase and downstream consequences of decreased enzyme activity.

Molecular mechanisms for the antidepressant-like effects of a low-dose ketamine treatment in a DFP-based rat model for Gulf War Illness.

Exposure to organophosphates (OP) during the First Gulf War is among one of the factors for Gulf War Illness (GWI) development in veterans and it has been challenging to treat GWI symptoms with existing therapies. Ketamine produces a rapid-onset and sustained antidepressant response, but there is no evidence whether ketamine treatment is effective for GWI depression. Repeated, low-dose exposure to diisopropyl fluorophosphate (DFP) mimic Gulf War related OP exposures and produces a chronic depressive state in rats. In this study, DFP-exposed rats treated with ketamine (10 mg/kg, i.p.) exhibited antidepressant-like effect on the Forced Swim Test at 1-h. This effect persisted at 24-h post ketamine, a time-point by which it is eliminated from the brain suggesting involvement of mechanisms that affect long-term synaptic plasticity. Western blot analysis showed significantly lower Brain-Derived Neurotrophic Factor (BDNF) levels in DFP rat brains. Ketamine produced a nonsignificant increase in BDNF expression at 1-h but produced a larger, significant (2.2-fold) increase at 24-h in DFP rats. We previously reported chronic hippocampal calcium elevations ([Ca2+]i) in DFP rats. Ketamine-treated DFP rats exhibited significantly lower [Ca2+]i at 1-h but not at 24-h. Interestingly, treatment with ANA-12, a TrkB-BDNF receptor antagonist, in DFP rats blunted ketamine's antidepressant-like effect at 24-h but not at 1-h. These experiments suggest that in a rat model of DFP-induced depression, inhibition of the NMDAR-Ca2+ contributes to the rapid-onset antidepressant effects of ketamine while the antidepressant actions that persisted at 24-h post ketamine administration involve upregulation of BDNF signaling.

Targeting Intracellular Calcium Stores Alleviates Neurological Morbidities in a DFP-Based Rat Model of Gulf War Illness.

Gulf War Illness (GWI) is a chronic multi-symptom disorder afflicting the veterans of the First Gulf War, and includes neurological symptoms characterized by depression and memory deficits. Chronic exposure to organophosphates (OPs) is considered a leading cause for GWI, yet its pathobiology is not fully understood. We recently observed chronic elevations in neuronal Ca2+ levels ([Ca2+]i) in an OP-diisopropyl fluorophosphate (DFP)-based rat model for GWI. This study was aimed at identifying mechanisms underlying elevated [Ca2+]i in this DFP model and investigating whether their therapeutic targeting could improve GWI-like neurological morbidities. Male Sprague-Dawley rats (9 weeks) were exposed to DFP (0.5 mg/kg, s.c., 1×-daily for 5 days) and at 3 months postDFP exposure, behavior was assessed and rats were euthanized for protein estimations and ratiometric Fura-2 [Ca2+]i estimations in acutely dissociated hippocampal neurons. In DFP rats, a sustained elevation in intracellular Ca2+ levels occurred, and pharmacological blockade of Ca2+-induced Ca2+-release mechanisms significantly lowered elevated [Ca2+]i in DFP neurons. Significant reductions in the protein levels of the ryanodine receptor (RyR) stabilizing protein Calstabin2 were also noted. Such a posttranslational modification would render RyR "leaky" resulting in sustained DFP [Ca2+]i elevations. Antagonism of RyR with levetiracetam significantly lower elevated [Ca2+]i in DFP neurons and improved GWI-like behavioral symptoms. Since Ca2+ is a major second messenger molecule, such chronic increases in its levels could underlie pathological synaptic plasticity that expresses itself as GWI morbidities. Our studies show that treatment with drugs targeted at blocking intracellular Ca2+ release could be effective therapies for GWI neurological morbidities.

Traumatic brain injury causes a long-lasting calcium (Ca2+)-plateau of elevated intracellular Ca levels and altered Ca2+ homeostatic mechanisms in hippocampal neurons surviving brain injury.

Traumatic brain injury (TBI) survivors often suffer chronically from significant morbidity associated with cognitive deficits, behavioral difficulties and a post-traumatic syndrome and thus it is important to understand the pathophysiology of these long-term plasticity changes after TBI. Calcium (Ca2+) has been implicated in the pathophysiology of TBI-induced neuronal death and other forms of brain injury including stroke and status epilepticus. However, the potential role of long-term changes in neuronal Ca2+ dynamics after TBI has not been evaluated. In the present study, we measured basal free intracellular Ca2+ concentration ([Ca2+](i)) in acutely isolated CA3 hippocampal neurons from Sprague-Dawley rats at 1, 7 and 30 days after moderate central fluid percussion injury. Basal [Ca2+](i) was significantly elevated when measured 1 and 7 days post-TBI without evidence of neuronal death. Basal [Ca2+](i) returned to normal when measured 30 days post-TBI. In contrast, abnormalities in Ca2+ homeostasis were found for as long as 30 days after TBI. Studies evaluating the mechanisms underlying the altered Ca2+ homeostasis in TBI neurons indicated that necrotic or apoptotic cell death and abnormalities in Ca2+ influx and efflux mechanisms could not account for these changes and suggested that long-term changes in Ca2+ buffering or Ca2+ sequestration/release mechanisms underlie these changes in Ca2+ homeostasis after TBI. Further elucidation of the mechanisms of altered Ca2+ homeostasis in traumatized, surviving neurons in TBI may offer novel therapeutic interventions that may contribute to the treatment and relief of some of the morbidity associated with TBI.

Carisbamate prevents the development and expression of spontaneous recurrent epileptiform discharges and is neuroprotective in cultured hippocampal neurons.

PURPOSE: Although great advances have been made in the development of treatments for epilepsy, acquired epilepsy following brain injury still comprises approximately 50% of all the cases of epilepsy. Thus, development of drugs that would prevent or decrease the onset of epilepsy following brain injury represents an important area of research. METHODS: Here, we investigated effects of carisbamate (RWJ 333369) on the development and expression of spontaneous recurrent epileptiform discharges (SREDs) and its neuroprotective potential in cultured hippocampal neurons. This model utilizes 3 h of low Mg(2+) treatment to mimic status epilepticus (SE-like) injury in vitro. Following the injury, networks of neurons manifest synchronized SREDs for their life in culture. Neuronal cultures were treated with carisbamate (200 microM) for 12 h immediately after the SE-like injury. The drug was then removed and neurons were patch clamped 24 h following drug washout. RESULTS: Treatment with carisbamate after neuronal injury prevented the development and expression of epileptiform discharges. In the few neurons that displayed SREDs following carisbamate treatment, there was a significant reduction in SRED frequency and duration. In contrast, phenytoin and phenobarbital, when used in place of carisbamate, did not prevent the development and expression of SREDs. Carisbamate was also effective in preventing neuronal death when administered after SE-like injury. CONCLUSIONS: Carisbamate prevents the development and generation of epileptiform discharges and is neuroprotective when administered following SE-like injury in vitro and may offer a novel treatment to prevent the development of epileptiform discharges following brain injuries.

Time course and mechanism of hippocampal neuronal death in an in vitro model of status epilepticus: role of NMDA receptor activation and NMDA dependent calcium entry.

The hippocampus is especially vulnerable to seizure-induced damage and excitotoxic neuronal injury. This study examined the time course of neuronal death in relationship to seizure duration and the pharmacological mechanisms underlying seizure-induced cell death using low magnesium (Mg2+) induced continuous high frequency epileptiform discharges (in vitro status epilepticus) in hippocampal neuronal cultures. Neuronal death was assessed using cell morphology and fluorescein diacetate-propidium iodide staining. Effects of low Mg2+ and various receptor antagonists on spike frequency were assessed using patch clamp electrophysiology. We observed a linear and time-dependent increase in neuronal death with increasing durations of status epilepticus. This cell death was dependent upon extracellular calcium (Ca2+) that entered primarily through the N-methyl-d-aspartate (NMDA) glutamate receptor channel subtype. Neuronal death was significantly decreased by co-incubation with the NMDA receptor antagonists and was also inhibited by reduction of extracellular (Ca2+) during status epilepticus. In contrast, neuronal death from in vitro status epilepticus was not significantly prevented by inhibition of other glutamate receptor subtypes or voltage-gated Ca2+ channels. Interestingly this NMDA-Ca2+ dependent neuronal death was much more gradual in onset compared to cell death from excitotoxic glutamate exposure. The results provide evidence that in vitro status epilepticus results in increased activation of the NMDA-Ca2+ transduction pathway leading to neuronal death in a time-dependent fashion. The results also indicate that there is a significant window of opportunity during the initial time of continuous seizure activity to be able to intervene, protect neurons and decrease the high morbidity and mortality associated with status epilepticus.

Levetiracetam inhibits both ryanodine and IP3 receptor activated calcium induced calcium release in hippocampal neurons in culture.

Epilepsy affects approximately 1% of the population worldwide, and there is a pressing need to develop new anti-epileptic drugs (AEDs) and understand their mechanisms of action. Levetiracetam (LEV) is a novel AED and despite its increasingly widespread clinical use, its mechanism of action is as yet undetermined. Intracellular calcium ([Ca2+]i) regulation by both inositol 1,4,5-triphosphate receptors (IP3R) and ryanodine receptors (RyR) has been implicated in epileptogenesis and the maintenance of epilepsy. To this end, we investigated the effect of LEV on RyR and IP3R activated calcium-induced calcium release (CICR) in hippocampal neuronal cultures. RyR-mediated CICR was stimulated using the well-characterized RyR activator, caffeine. Caffeine (10mM) caused a significant increase in [Ca2+]i in hippocampal neurons. Treatment with LEV (33 microM) prior to stimulation of RyR-mediated CICR by caffeine led to a 61% decrease in the caffeine induced peak height of [Ca2+]i when compared to the control. Bradykinin stimulates IP3R-activated CICR-to test the effect of LEV on IP3R-mediated CICR, bradykinin (1 microM) was used to stimulate cells pre-treated with LEV (100 microM). The data showed that LEV caused a 74% decrease in IP3R-mediated CICR compared to the control. In previous studies we have shown that altered Ca2+ homeostatic mechanisms play a role in seizure activity and the development of spontaneous recurrent epileptiform discharges (SREDs). Elevations in [Ca2+]i mediated by CICR systems have been associated with neurotoxicity, changes in neuronal plasticity, and the development of AE. Thus, the ability of LEV to modulate the two major CICR systems demonstrates an important molecular effect of this agent on a major second messenger system in neurons.

Alterations in neuronal calcium levels are associated with cognitive deficits after traumatic brain injury.

Traumatic brain injury (TBI) survivors often suffer from a post-traumatic syndrome with deficits in learning and memory. Calcium (Ca(2+)) has been implicated in the pathophysiology of TBI-induced neuronal death. However, the role of long-term changes in neuronal Ca(2+) function in surviving neurons and the potential impact on TBI-induced cognitive impairments are less understood. Here we evaluated neuronal death and basal free intracellular Ca(2+) ([Ca(2+)](i)) in acutely isolated rat CA3 hippocampal neurons using the Ca(2+) indicator, Fura-2, at seven and thirty days after moderate central fluid percussion injury. In moderate TBI, cognitive deficits as evaluated by the Morris Water Maze (MWM), occur after injury but resolve after several weeks. Using MWM paradigm we compared alterations in [Ca(2+)](i) and cognitive deficits. Moderate TBI did not cause significant hippocampal neuronal death. However, basal [Ca(2+)](i) was significantly elevated when measured seven days post-TBI. At the same time, these animals exhibited significant cognitive impairment (F(2,25)=3.43, p<0.05). When measured 30 days post-TBI, both basal [Ca(2+)](i) and cognitive functions had returned to normal. Pretreatment with MK-801 blocked this elevation in [Ca(2+)](i) and also prevented MWM deficits. These studies provide evidence for a link between elevated [Ca(2+)](i) and altered cognition. Since no significant neuronal death was observed, the alterations in Ca(2+) homeostasis in the traumatized, but surviving neurons may play a role in the pathophysiology of cognitive deficits that manifest in the acute setting after TBI and represent a novel target for therapeutic intervention following TBI.

The novel antiepileptic drug carisbamate (RWJ 333369) is effective in inhibiting spontaneous recurrent seizure discharges and blocking sustained repetitive firing in cultured hippocampal neurons.

This study was initiated to investigate effects of the novel neuromodulator carisbamate (RWJ 333369) in the hippocampal neuronal culture model of status epilepticus and spontaneous epileptiform discharges. Whole-cell current clamp techniques were used to determine the effects of carisbamate on spontaneous recurrent epileptiform discharges (SREDs, in vitro epilepsy), depolarization-induced sustained repetitive firing (SRF) and low Mg(2+)-induced continuous high frequency spiking (in vitro status epilepticus). This in vitro model is an important tool to study the effects of anticonvulsant drugs (AEDs) on SREDs that occur for the life of the neurons in culture. Carisbamate dose dependently blocked the expression and reoccurrence of SREDs. The ED(50) value for its antiepileptic effect was 58.75+/-2.43 microM. Inhibition of SRF is considered a common attribute of many AEDs. Carisbamate (100 microM) significantly decreased SRF in hippocampal neurons. All these effects of carisbamate were reversed during a 5 to 30 min drug washout period. When exposed to low Mg(2+) medium cultured hippocampal neurons exhibit high frequency spiking. This form of in vitro status epilepticus is not effectively blocked by conventional AEDs that are known to be effective in treating status epilepticus in humans. Carisbamate, like phenytoin and phenobarbital, had little or no effect on low Mg(2+)-induced continuous high frequency spiking. These results characterize the effects of carisbamate in the hippocampal neuronal culture model of epileptiform discharges and suggest that the ability of carisbamate to inhibit depolarization-induced SRF may account in part for some of it's anticonvulsant effect.