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Transcriptional responses of the nerve agent-sensitive brain regions amygdala, hippocampus, piriform cortex, septum, and thalamus following exposure to the organophosphonate anticholinesterase sarin.

ABSTRACT: BACKGROUND:Although the acute toxicity of organophosphorus nerve agents is known to result from acetylcholinesterase inhibition, the molecular mechanisms involved in the development of neuropathology following nerve agent-induced seizure are not well understood. To help determine these pathways, we previously used microarray analysis to identify gene expression changes in the rat piriform cortex, a region of the rat brain sensitive to nerve agent exposure, over a 24-h time period following sarin-induced seizure. We found significant differences in gene expression profiles and identified secondary responses that potentially lead to brain injury and cell death. To advance our understanding of the molecular mechanisms involved in sarin-induced toxicity, we analyzed gene expression changes in four other areas of the rat brain known to be affected by nerve agent-induced seizure (amygdala, hippocampus, septum, and thalamus). METHODS:We compared the transcriptional response of these four brain regions to sarin-induced seizure with the response previously characterized in the piriform cortex. In this study, rats were challenged with 1.0 × LD?? sarin and subsequently treated with atropine sulfate, 2-pyridine aldoxime methylchloride, and diazepam. The four brain regions were collected at 0.25, 1, 3, 6, and 24 h after seizure onset, and total RNA was processed for microarray analysis. RESULTS:Principal component analysis identified brain region and time following seizure onset as major sources of variability within the dataset. Analysis of variance identified genes significantly changed following sarin-induced seizure, and gene ontology analysis identified biological pathways, functions, and networks of genes significantly affected by sarin-induced seizure over the 24-h time course. Many of the molecular functions and pathways identified as being most significant across all of the brain regions were indicative of an inflammatory response. There were also a number of molecular responses that were unique for each brain region, with the thalamus having the most distinct response to nerve agent-induced seizure. CONCLUSIONS:Identifying the molecular mechanisms involved in sarin-induced neurotoxicity in these sensitive brain regions will facilitate the development of novel therapeutics that can potentially provide broad-spectrum protection in five areas of the central nervous system known to be damaged by nerve agent-induced seizure.

SUBMITTER: Spradling KD

PROVIDER: S-EPMC3180277 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Transcriptional responses of the nerve agent-sensitive brain regions amygdala, hippocampus, piriform cortex, septum, and thalamus following exposure to the organophosphonate anticholinesterase sarin.

Spradling Kimberly D KD Lumley Lucille A LA Robison Christopher L CL Meyerhoff James L JL Dillman James F JF

Journal of neuroinflammation 20110721

<h4>Background</h4>Although the acute toxicity of organophosphorus nerve agents is known to result from acetylcholinesterase inhibition, the molecular mechanisms involved in the development of neuropathology following nerve agent-induced seizure are not well understood. To help determine these pathways, we previously used microarray analysis to identify gene expression changes in the rat piriform cortex, a region of the rat brain sensitive to nerve agent exposure, over a 24-h time period followi ...[more]

PMID: 21777430

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Project description:BackgroundOrganophosphorus 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.MethodsTo determine potential therapeutic targets for such treatments, we analyzed gene expression changes in the rat piriform cortex following sarin (O-isopropyl methylphosphonofluoridate)-induced seizure. Male Sprague-Dawley rats were challenged with 1 × 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.ResultsA 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.ConclusionsThis analysis of gene expression changes in the rat brain following sarin-induced seizure and the molecular pathways involved in sarin-induced neurodegeneration will facilitate the identification of potential therapeutic targets for the development of effective neuroprotectants to treat nerve agent exposure.

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.

Project description:The goal was to test 14 nerve agent model compounds of soman, sarin, tabun, and cyclohexyl methylphosphonofluoridate (GF) for their suitability as substitutes for true nerve agents. We wanted to know whether the model compounds would form the identical covalent adduct with human butyrylcholinesterase that is produced by reaction with true nerve agents. Nerve agent model compounds containing thiocholine or thiomethyl in place of fluorine or cyanide were synthesized as Sp and Rp stereoisomers. Purified human butyrylcholinesterase was treated with a 45-fold molar excess of nerve agent analogue at pH 7.4 for 17 h at 21 degrees C. The protein was denatured by boiling and was digested with trypsin. Aged and nonaged active site peptide adducts were quantified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of the tryptic digest mixture. The active site peptides were isolated by HPLC and analyzed by MALDI-TOF-TOF mass spectrometry. Serine 198 of butyrylcholinesterase was covalently modified by all 14 compounds. Thiocholine was the leaving group in all compounds that had thiocholine in place of fluorine or cyanide. Thiomethyl was the leaving group in the GF thiomethyl compounds. However, sarin thiomethyl compounds released either thiomethyl or isopropyl, while soman thiomethyl compounds released either thiomethyl or pinacolyl. Thiocholine compounds reacted more rapidly with butyrylcholinesterase than thiomethyl compounds. Labeling with the model compounds resulted in aged adducts that had lost the O-alkyl group (O-ethyl for tabun, O-cyclohexyl for GF, isopropyl for sarin, and pinacolyl for soman) in addition to the thiocholine or thiomethyl group. The nerve agent model compounds containing thiocholine and the GF thiomethyl analogue were found to be suitable substitutes for true soman, sarin, tabun, and GF in terms of the adduct that they produced with human butyrylcholinesterase. However, the soman and sarin thiomethyl compounds yielded two types of adducts, one of which was thiomethyl phosphonate, a modification not found after treatment with authentic soman and sarin.

Project description:Vagal Nerve Stimulation (VNS) Therapy® is a United States Food and Drug Administration approved neurotherapeutic for medically refractory partial epilepsy and treatment-resistant depression. The molecular mechanisms underlying its beneficial effects are unclear. We hypothesized that one mechanism involves neuronal activity-dependent modifications of central nervous system excitatory synapses. To begin to test this hypothesis, we asked whether VNS modifies the activity of neurons in amygdala and hippocampus. Neuronal recordings from adult, freely moving rats revealed that activity in both amygdala and hippocampus was modified by VNS immediately after its application, and changes were detected following 1 week of stimulation. To investigate whether VNS modifies the proteome of excitatory synapses, we established a label-free, quantitative liquid chromatography-tandem mass spectrometry workflow that enables global analysis of the constituents of the postsynaptic density (PSD) proteome. PSD proteins were biochemically purified from amygdala/piriform cortex of VNS- or dummy-treated rats following 1-week stimulation, and individual PSD protein levels were quantified by liquid chromatography-tandem mass spectrometry analysis. We identified 1899 unique peptides corresponding to 425 proteins in PSD fractions, of which expression levels of 22 proteins were differentially regulated by VNS with changes greater than 150%. Changes in a subset of these proteins, including significantly increased expression of neurexin-1α, cadherin 13 and voltage-dependent calcium channel α2δ1, the primary target of the antiepileptic drug gabapentin, and decreased expression of voltage-dependent calcium channel γ3, were confirmed by western blot analysis of PSD samples. These results demonstrate that VNS modulates excitatory synapses through regulating a subset of the PSD proteome. Our study reveals molecular targets of VNS and point to possible mechanisms underlying its beneficial effects, including activity-dependent formation of excitatory synapses.

Project description:Altered hippocampal morphology and reduced volumes have been found in children born preterm compared to full-term. Stress inhibits neurogenesis in the hippocampus, and neonatal stress/noxious stimulation in rodent pups are associated with long-term alterations in hippocampal volumes. We have previously shown reduced cortical thickness and cerebellar volumes in relation to more exposure to pain-related stress of neonatal invasive procedures in children born very preterm. We have reported targeted gene-by-pain environment interactions that contribute to long-term brain development and outcomes in this population. We now aim to determine whether exposure to pain-related stress (adjusted for clinical factors and genotype) differentially impacts regional structures within the limbic system and thalamus, and investigate relationships with outcomes in very preterm children. Our study included 57 children born very preterm (<32 weeks GA) followed longitudinally from birth who underwent 3-D T1 MRI neuroimaging at ∼8 years. Hippocampal subfields and white matter tracts, thalamus and amygdala were automatically segmented using the MAGeT Brain algorithm. The relationship between those subcortical brain volumes (adjusted for total brain volume) and neonatal invasive procedures, gestational age (GA), illness severity, postnatal infection, days of mechanical ventilation, number of surgeries, morphine exposure, and genotype (COMT, SLC6A4, and BDNF) was examined using constrained principal component analysis. We found that neonatal clinical factors and genotypes accounted for 46% of the overall variance in volumes of hippocampal subregions, tracts, basal ganglia, thalamus and amygdala. After controlling for clinical risk factors and total brain volume, greater neonatal invasive procedures was associated with lower volumes in the amygdala and thalamus (p = 0.0001) and an interaction with COMT genotype predicted smaller hippocampal subregional volume (p = 0.0001). More surgeries, days of ventilation, and lower GA were also related to smaller volumes in various subcortical regions (p < 0.002). These reduced volumes were in turn differentially related to poorer cognitive, visual-motor and behavioral outcomes. Our findings highlight the complexity that interplays when examining how exposure to early-life stress may impact brain development both at the structural and functional level, and provide new insight on possible novel avenues of research to discover brain-protective treatments to improve the care of children born preterm.

Dataset Information

Transcriptional responses of the nerve agent-sensitive brain regions amygdala, hippocampus, piriform cortex, septum, and thalamus following exposure to the organophosphonate anticholinesterase sarin.

Publications

Transcriptional responses of the nerve agent-sensitive brain regions amygdala, hippocampus, piriform cortex, septum, and thalamus following exposure to the organophosphonate anticholinesterase sarin.

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