Project description:Neurodegenerative brain disorders become more common in the aged. Most of these disorders are associated with or caused by selective death of certain neuronal subpopulations. The mechanisms underlying the differential vulnerability of certain neuronal populations are still largely unexplored and few neuroprotective treatments are available to date. Elucidation of these mechanisms may lead to a greater understanding of the pathogenesis and treatment of neurodegenerative diseases. Moreover, preconditioning by a short seizure confers neuroprotection following a subsequent prolonged seizure. Our goal is to identify pathways that confer vulnerability and resistance to neurotoxic conditions by comparing the basal and preconditioned gene expression profiles of three differentially vulnerable hippocampal neuron populations. Hippocampal CA1 and CA3 pyramidal neurons are highly susceptible to seizures and ischemia, whereas dentate gyrus granule cells are relatively resistant. A brief preconditioning seizure confers protection to the pyramidal cells. We will first determine gene expression profiles of untreated rat CA1 and CA3 pyramidal cells, and dentate granule cells, using laser capture microscopy to obtain region-specific neuronal mRNA. We will then determine the effect of a brief preconditioning seizure, which is neuroprotective in CA1 and CA3, on these expression profiles. We hypothesize that common molecular mechanisms exist in neurons that determine their susceptibility to seizure-induced injury. Intrinsic differences in gene expression exist between hippocampal glutamatergic CA1 and CA3 pyramidal neurons, on the one hand, and dentate granule cells on the other, which contribute to the greater susceptibility of pyramidal neurons to degeneration in experimental stroke and epilepsy. We specifically hypothesize that differences in basal energy metabolism genes may confer differential susceptibility to neurodegeneration produced by seizures and ischemia. Anesthetized animals will be sacrificed by decapitation, and frozen 10 micron sections will be lightly stained with cresyl violet to identify cell layers in the hippocampus. Approximately 1000 neurons from each of the three cell layers will be isolated by LCM. Poly-A RNA will be amplified using a modified Eberwine protocol. The quality of our aRNA will be evaluated by quantitative RT-PCR of GluR6 and KA2 mRNA levels before we send the samples to the Center for labeling and hybridization to Affymetrix rat 230A arrays. We will provide a one-round amplification cDNA product to the center for labeling and hybridization. This protocol is identical to a previously approved study by Jim Greene in our laboratory.

Project description:Neither the molecular basis of the pathologic tendency of neuronal circuits to generate spontaneous seizures (epileptogenicity) nor anti-epileptogenic mechanisms that maintain a seizure-free state are well understood. Here, we performed transcriptomic analysis in the intrahippocampal kainate model of temporal lobe epilepsy in rats using both Agilent and Codelink microarray platforms to characterize the epileptic processes. The experimental design allowed subtraction of the confounding effects of the lesion, identification of expression changes associated with epileptogenicity, and genes upregulated by seizures with potential homeostatic anti-epileptogenic effects. Using differential expression analysis, we identified several hundred expression changes in chronic epilepsy, including candidate genes associated with epileptogenicity such as Bdnf and Kcnj13. To analyze these data from a systems perspective, we applied weighted gene co-expression network analysis (WGCNA) to identify groups of co-expressed genes (modules) and their central (hub) genes. One such module contained genes upregulated in the epileptogenic region, including multiple epileptogenicity candidate genes, and was found to be involved the protection of glial cells against oxidative stress, implicating glial oxidative stress in epileptogenicity. Another distinct module corresponded to the effects of chronic seizures and represented changes in neuronal synaptic vesicle trafficking. We found that the network structure and connectivity of one hub gene, Sv2a, showed significant changes between normal and epileptogenic tissue, becoming more highly connected in epileptic brain. Since Sv2a is a target of the antiepileptic levetiracetam, this module may be important in controlling seizure activity. Bioinformatic analysis of this module also revealed a potential mechanism for the observed transcriptional changes via generation of longer alternatively polyadenlyated transcripts through the upregulation of the RNA binding protein HuD. In summary, combining conventional statistical methods and network analysis allowed us to interpret the differentially regulated genes from a systems perspective, yielding new insight into several biological pathways underlying homeostatic anti-epileptogenic effects and epileptogenicity. Four condition experiment with five samples per condition. The samples include right dentate gyrus from animals with seizures, left dentate gyrus from animals with seizures, right dentate gyrus from animals without seizures, and left dentate gyrus from animals without seizures. A dye swap was included.

Project description:Neither the molecular basis of the pathologic tendency of neuronal circuits to generate spontaneous seizures (epileptogenicity) nor anti-epileptogenic mechanisms that maintain a seizure-free state are well understood. Here, we performed transcriptomic analysis in the intrahippocampal kainate model of temporal lobe epilepsy in rats using both Agilent and Codelink microarray platforms to characterize the epileptic processes. The experimental design allowed subtraction of the confounding effects of the lesion, identification of expression changes associated with epileptogenicity, and genes upregulated by seizures with potential homeostatic anti-epileptogenic effects. Using differential expression analysis, we identified several hundred expression changes in chronic epilepsy, including candidate genes associated with epileptogenicity such as Bdnf and Kcnj13. To analyze these data from a systems perspective, we applied weighted gene co-expression network analysis (WGCNA) to identify groups of co-expressed genes (modules) and their central (hub) genes. One such module contained genes upregulated in the epileptogenic region, including multiple epileptogenicity candidate genes, and was found to be involved the protection of glial cells against oxidative stress, implicating glial oxidative stress in epileptogenicity. Another distinct module corresponded to the effects of chronic seizures and represented changes in neuronal synaptic vesicle trafficking. We found that the network structure and connectivity of one hub gene, Sv2a, showed significant changes between normal and epileptogenic tissue, becoming more highly connected in epileptic brain. Since Sv2a is a target of the antiepileptic levetiracetam, this module may be important in controlling seizure activity. Bioinformatic analysis of this module also revealed a potential mechanism for the observed transcriptional changes via generation of longer alternatively polyadenlyated transcripts through the upregulation of the RNA binding protein HuD. In summary, combining conventional statistical methods and network analysis allowed us to interpret the differentially regulated genes from a systems perspective, yielding new insight into several biological pathways underlying homeostatic anti-epileptogenic effects and epileptogenicity. Four condition experiment with five samples per condition. The samples include right dentate gyrus from animals with seizures, left dentate gyrus from animals with seizures, right dentate gyrus from animals without seizures, and left dentate gyrus from animals without seizures.

Project description:Neurodegenerative brain disorders become more common in the aged. Most of these disorders are associated with or caused by selective death of certain neuronal subpopulations. The mechanisms underlying the differential vulnerability of certain neuronal populations are still largely unexplored and few neuroprotective treatments are available to date. Elucidation of these mechanisms may lead to a greater understanding of the pathogenesis and treatment of neurodegenerative diseases. Moreover, preconditioning by a short seizure confers neuroprotection following a subsequent prolonged seizure. Our goal is to identify pathways that confer vulnerability and resistance to neurotoxic conditions by comparing the basal and preconditioned gene expression profiles of three differentially vulnerable hippocampal neuron populations. Hippocampal CA1 and CA3 pyramidal neurons are highly susceptible to seizures and ischemia, whereas dentate gyrus granule cells are relatively resistant. A brief preconditioning seizure confers protection to the pyramidal cells. We will first determine gene expression profiles of untreated rat CA1 and CA3 pyramidal cells, and dentate granule cells, using laser capture microscopy to obtain region-specific neuronal mRNA. We will then determine the effect of a brief preconditioning seizure, which is neuroprotective in CA1 and CA3, on these expression profiles. We hypothesize that common molecular mechanisms exist in neurons that determine their susceptibility to seizure-induced injury. Intrinsic differences in gene expression exist between hippocampal glutamatergic CA1 and CA3 pyramidal neurons, on the one hand, and dentate granule cells on the other, which contribute to the greater susceptibility of pyramidal neurons to degeneration in experimental stroke and epilepsy. We specifically hypothesize that differences in basal energy metabolism genes may confer differential susceptibility to neurodegeneration produced by seizures and ischemia. Anesthetized animals will be sacrificed by decapitation, and frozen 10 micron sections will be lightly stained with cresyl violet to identify cell layers in the hippocampus. Approximately 1000 neurons from each of the three cell layers will be isolated by LCM. Poly-A RNA will be amplified using a modified Eberwine protocol. The quality of our aRNA will be evaluated by quantitative RT-PCR of GluR6 and KA2 mRNA levels before we send the samples to the Center for labeling and hybridization to Affymetrix rat 230A arrays. We will provide a one-round amplification cDNA product to the center for labeling and hybridization. This protocol is identical to a previously approved study by Jim Greene in our laboratory. Keywords: other

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Differential Circadian Time Modulates of Dentate Gyrus and CA1 Excitation-Inhibition Balance and Seizure Threshold.

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