Project description:Organophosphorus nerve agents irreversibly inhibit acetylcholinesterase, causing a toxic buildup of acetylcholine at muscarinic and nicotinic receptors. Current medical countermeasures to nerve agent intoxication increase survival if administered within a short period of time following exposure but may not fully prevent neurological damage. Therefore, there is a need to discover drug treatments that are effective when administered after the onset of seizures and secondary responses that lead to brain injury. Methods To determine potential therapeutic targets for such treatments, we analyzed gene expression changes in the rat piriform cortex following sarin (O-isopropyl methylphosphonofluoridate) exposure. Male Sprague-Dawley rats were challenged with 1.0 x LD50 sarin and subsequently treated with atropine sulfate, 2-pyridine aldoxime methylchloride (2-PAM), and the anticonvulsant diazepam. Control animals received an equivalent volume of vehicle and drug treatments. The piriform cortex, a brain region particularly sensitive to neural damage from sarin-induced seizures, was extracted at 0.25, 1, 3, 6, and 24 h after seizure onset, and total RNA was processed for microarray analysis. Principal component analysis identified sarin-induced seizure occurrence and time point following seizure onset as major sources of variability within the dataset. Based on these variables, the dataset was filtered and analysis of variance was used to determine genes significantly changed in seizing animals at each time point. The calculated p-value and geometric fold change for each probeset identifier were subsequently used for gene ontology analysis to identify canonical pathways, biological functions, and networks of genes significantly affected by sarin-induced seizure over the 24-h time course. Results A multitude of biological functions and pathways were identified as being significantly altered following sarin-induced seizure. Inflammatory response and signaling pathways associated with inflammation were among the most significantly altered across the five time points examined. Conclusions This analysis of gene expression changes in the rat brain following sarin-induced seizure and the molecular pathways involved in sarin-induced neurodegeneration will allow a better identification of potential therapeutic targets for the development of effective neuroprotectants to treat nerve agent exposure. Three animals were used for each experimental group (naM-CM-/ve, sarin-exposed non-seizure, non-seizure control [saline], sarin-exposed seizure, and seizure control [saline]) at each time point (0.25, 1, 3, 6, 24 h), with the exception of 1 h seizure control, 3 h sarin-exposed seizure, 24 h sarin-exposed non-seizure, and 24 h sarin-exposed seizure (n=4).

Project description:Early childhood convulsions have been correlated with hippocampal neuron loss in patients with intractable temporal lobe epilepsy. Using a "two-hit" rat seizure model, we have shown that animals subjected to kainate (KA)- or hypoxia-induced seizures during early postnatal period showed no cell death, yet sustained more extensive neuronal death after second seizures in adulthood. An early life seizure, without causing overt cellular injury, predisposes the brain to the damaging effect of seizures in later life. Cellular and molecular changes that accompany early seizures and that lead to subsequent epileptogenesis and increased susceptibility to seizure-induced neuronal injury, however, remain poorly understood. We propose to investigate age-specific, time-dependent changes in gene expression that may underlie this priming effect of early-life seizures. We will determine the sequence of gene expression pattern in the hippocampus at various times following KA induced seizures at postnatal day (P) 15. Previous studies have shown that AMPA receptor subtype of glutamate receptors play a crucial role in the age-specific vulnerability and in the long-term epileptogenic effects of perinatal hypoxia seizures. We found that AMPA receptor antagonists block the increased susceptibility caused by early life seizures to later seizures and seizure-induced brain damage. We hypothesize that an alteration of AMPA receptor composition is one of many changes caused by early-life seizures that leads to an increase in Ca2+ permeability, which then results in cascade of downstream events and modifies array of gene expression that promote epileptogenesis and susceptibility to neuronal death in later life. We will examine three time points: 1hr, 72 hr, and 15 days following systemic KA-induced seizures at P15 as we have previously observed structural changes within the hippocampus at these time points. Within an hour of KA seizures, a marked swelling of dendrites, disassembly of dendritic microtubules and glycogen depletion are observed by electron microscopy. Within 5 days, basal dendrites of CA3 hippocampal pyramidal neurons show abnormal spine morphology and decreased branching pattern. 15 days after the seizures, aberrant growth of mossy fibers in the CA3 stratum oriens is observed in animals exposed to KA. Ten hippocampi will be pooled from five animals treated with KA (3mg/kg i.p.) and from five littermate controls injected with PBS. Animals will be decapitated and hippocampi will be rapidly dissected from the brain, flash frozen in liquid nitrogen, and stored at -80C until extraction of total RNA, which will be sent to the center. We will provide 4 tissue samples-2 controls and 2 KA, each a pool of five animals - for each time points. Mixing tissues from multiple rats will normalize single nucleotide polymorphisms and tissue heterogeneity.

Project description:Early childhood convulsions have been correlated with hippocampal neuron loss in patients with intractable temporal lobe epilepsy. Using a "two-hit" rat seizure model, we have shown that animals subjected to kainate (KA)- or hypoxia-induced seizures during early postnatal period showed no cell death, yet sustained more extensive neuronal death after second seizures in adulthood. An early life seizure, without causing overt cellular injury, predisposes the brain to the damaging effect of seizures in later life. Cellular and molecular changes that accompany early seizures and that lead to subsequent epileptogenesis and increased susceptibility to seizure-induced neuronal injury, however, remain poorly understood. We propose to investigate age-specific, time-dependent changes in gene expression that may underlie this priming effect of early-life seizures. We will determine the sequence of gene expression pattern in the hippocampus at various times following KA induced seizures at postnatal day (P) 15. Previous studies have shown that AMPA receptor subtype of glutamate receptors play a crucial role in the age-specific vulnerability and in the long-term epileptogenic effects of perinatal hypoxia seizures. We found that AMPA receptor antagonists block the increased susceptibility caused by early life seizures to later seizures and seizure-induced brain damage. We hypothesize that an alteration of AMPA receptor composition is one of many changes caused by early-life seizures that leads to an increase in Ca2+ permeability, which then results in cascade of downstream events and modifies array of gene expression that promote epileptogenesis and susceptibility to neuronal death in later life. We will examine three time points: 1hr, 72 hr, and 15 days following systemic KA-induced seizures at P15 as we have previously observed structural changes within the hippocampus at these time points. Within an hour of KA seizures, a marked swelling of dendrites, disassembly of dendritic microtubules and glycogen depletion are observed by electron microscopy. Within 5 days, basal dendrites of CA3 hippocampal pyramidal neurons show abnormal spine morphology and decreased branching pattern. 15 days after the seizures, aberrant growth of mossy fibers in the CA3 stratum oriens is observed in animals exposed to KA. Ten hippocampi will be pooled from five animals treated with KA (3mg/kg i.p.) and from five littermate controls injected with PBS. Animals will be decapitated and hippocampi will be rapidly dissected from the brain, flash frozen in liquid nitrogen, and stored at -80C until extraction of total RNA, which will be sent to the center. We will provide 4 tissue samples-2 controls and 2 KA, each a pool of five animals - for each time points. Mixing tissues from multiple rats will normalize single nucleotide polymorphisms and tissue heterogeneity. Keywords: time-course

Project description:Nicotine, acting through the neuronal nicotinic acetylcholine receptors (nAChR), can induce seizures in mice. We aimed to study brain transcriptional response to seizure and to identify genes whose expression is altered after nicotine-induced seizures. Whole brains of untreated mice were compared to brains one hour after seizure activity, using Affymetrix U74Av2 microaarays. Experimental groups included wild-type mice and both nicotine-induced seizures sensitive and resistant nAChR mutant mice. Each genotype group received different nicotine doses to generate seizures. This approach allowed the identification of significantly changed genes whose expression was dependent on seizure activity, nicotine administration or both, but not on the type of nAChR subunit mutation or the amount of nicotine injected. Significant expression changes were detected in 62 genes (p < 0.05, FDR correction). Among them, GO functional annotation analysis determined that the most significantly over-represented categories were of genes encoding MAP kinase phosphatases, regulators of transcription and nucleosome assembly proteins. In-silico bioinformatic analysis of the promoter regions of the 62 changed genes detected the significant enrichments of 16 transcription regulatory elements (TREs), creating a network of transcriptional regulatory responses to seizures. The TREs for ATF and SRF were most significantly enriched, supporting their association with seizure activity. Our data suggest that nicotine-induced seizure in mice is a useful model to study seizure activity and its global brain transcriptional response. The differentially expressed genes detected here can help understand the molecular mechanisms underlying seizures in animal models, and may also serve as candidate genes to study epilepsy in humans. Experiment Overall Design: Whole brain expression profiles were determined in two experimental groups of mice, sixteen mice that were not treated with nicotine and twelve mice one hour after experiencing nicotine-induced seizure. The untreated group included six wild-type mice, five alpha7+/T and five beta4-/- mice. The group of mice that underwent nicotine-induced seizures included three wild-types, five alpha7+/T and four beta4-/- mice. Different doses of nicotine were injected intraperitoneally (i.p.) to each genotype group of mice in order to achieve a similar seizure score in all three genotypes.

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Transcriptomic Analysis of Rat Brain Following Exposure to the Organophosphonate Anticholinesterase Sarin

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