Project description:Epilepsy is a chronic disorder characterized by recurring seizures, and results from excessive, synchronized discharge in populations of cells. Epilepsy affects 1% of the population and results from a variety of CNS insults. Traumatic physical brain injury causes 4% of cases. Posttraumatic epilepsy (PTE) is characterized by a delay of weeks to years between the trauma and the first seizures. Proper prophylactic intervention during this incubation time might prevent PTE. Many PTE patients are resistant to anticonvulsant medications and suffer from negative side effects. Better knowledge of the cell biology underlying injury-induced epileptogenesis should lead to better therapeutic strategies for interrupting the events occurring between the injury and the development of epilepsy. The aim of my experiments is to identify cellular factors underlying PTE using an in vitro model system to study two key aspects of brain injury in isolation: deafferentation and axonal transection. My aim is to perform a non-biased comparison of the expression of genes in area CA1, and separately in area CA3, in unlesioned cultures and in cultures lesioned 1, 3 and 7 days earlier. Changes in gene expression due to neuronal denervation will be apparent in area CA1, whereas changes in expression due to axonal transection will be apparent in area CA3. In performing these experiments, I hope to gain insight into the molecular underpinnings of the electrophysiological and immunocytochemical effects that we have already detected, as well as to generate new hypotheses that we can test further using our own expertise and techniques. We use cultured slices of rat hippocampus to study the effects of brain injury. Our model is to lesion the Schaffer collateral projection pathway between areas CA1 and CA3, producing a selective injury of the axons of CA3 cells and selective denervation of CA1 cells. Our preliminary electrophysiological and immunocytochemical data indicate that axonal sprouting is triggered in area CA3 and that the dendritic excitability is increased in CA1 pyramidal cells. Both effects occur only after about 7 days, suggesting they are mediated by changes in gene expression. My hypothesis is that the expression of numerous genes changes after Schaffer collateral transection. I predict that neurotrophins and their receptors change in area CA3 to trigger axonal sprouting, along with genes for proteins mediated axonal extension, and that changes in the expression of genes for glutamate receptors and voltage-dependent ion channels occur in area CA1 to account for their increased excitability. Hippocampal slice cultures prepared from male Sprague-Dawley rats at PN day 6. Slices will be maintained in vitro for 14 days in serum-containing medium. Cultures will then be examined and unhealthy cultures discarded. Remaining sister cultures will be divided into two groups: cultures to be lesioned and cultures remaining unlesioned. Lesioned cultures will have a sterile transection of the Schaffer collateral pathway using a razor blade shard. All cultures then returned to the incubator. At various times , cultures will be removed from the incubator. Individual cultures will be soaked in RNAlater buffer(Ambion), and a razor blade shard will be used to subdissect the CA3 and CA1 segments. Segments are sucked up with a sterile pipette-tip containing RNAlater buffer and transferred into a sterile tube. Total RNA will be prepared by homogenization in TRIzol reagent (GibcoBRL) and precipitation in isopropyl alcohol. Rneasy Kit (Qiagen) will further isolate RNA. Spectrographic analysis of RNA prepared indicates 260nm:280nm ratios above 1.6, and insignificant amounts of either degraded RNA or DNA contaminants detected on a 2% denaturing gel. We have found that it is necessary to pool 15 CA3 or CA1 segments to obtain 5+ ug of total RNA. Total RNA samples will be sent to the consortium for additional quality control, labeling, hybridizations using the Affymetrix Neuro U34 chip, scanning, and preliminary analyses. The gene array data will then be compared in several pairwise manners (lesion vs. age matched control and region vs. region, for each time point), as well as time sequence comparisons for gene expression in each region.

Project description:Epilepsy is a chronic disorder characterized by recurring seizures, and results from excessive, synchronized discharge in populations of cells. Epilepsy affects 1% of the population and results from a variety of CNS insults. Traumatic physical brain injury causes 4% of cases. Posttraumatic epilepsy (PTE) is characterized by a delay of weeks to years between the trauma and the first seizures. Proper prophylactic intervention during this incubation time might prevent PTE. Many PTE patients are resistant to anticonvulsant medications and suffer from negative side effects. Better knowledge of the cell biology underlying injury-induced epileptogenesis should lead to better therapeutic strategies for interrupting the events occurring between the injury and the development of epilepsy. The aim of my experiments is to identify cellular factors underlying PTE using an in vitro model system to study two key aspects of brain injury in isolation: deafferentation and axonal transection. My aim is to perform a non-biased comparison of the expression of genes in area CA1, and separately in area CA3, in unlesioned cultures and in cultures lesioned 1, 3 and 7 days earlier. Changes in gene expression due to neuronal denervation will be apparent in area CA1, whereas changes in expression due to axonal transection will be apparent in area CA3. In performing these experiments, I hope to gain insight into the molecular underpinnings of the electrophysiological and immunocytochemical effects that we have already detected, as well as to generate new hypotheses that we can test further using our own expertise and techniques. We use cultured slices of rat hippocampus to study the effects of brain injury. Our model is to lesion the Schaffer collateral projection pathway between areas CA1 and CA3, producing a selective injury of the axons of CA3 cells and selective denervation of CA1 cells. Our preliminary electrophysiological and immunocytochemical data indicate that axonal sprouting is triggered in area CA3 and that the dendritic excitability is increased in CA1 pyramidal cells. Both effects occur only after about 7 days, suggesting they are mediated by changes in gene expression. My hypothesis is that the expression of numerous genes changes after Schaffer collateral transection. I predict that neurotrophins and their receptors change in area CA3 to trigger axonal sprouting, along with genes for proteins mediated axonal extension, and that changes in the expression of genes for glutamate receptors and voltage-dependent ion channels occur in area CA1 to account for their increased excitability. Hippocampal slice cultures prepared from male Sprague-Dawley rats at PN day 6. Slices will be maintained in vitro for 14 days in serum-containing medium. Cultures will then be examined and unhealthy cultures discarded. Remaining sister cultures will be divided into two groups: cultures to be lesioned and cultures remaining unlesioned. Lesioned cultures will have a sterile transection of the Schaffer collateral pathway using a razor blade shard. All cultures then returned to the incubator. At various times , cultures will be removed from the incubator. Individual cultures will be soaked in RNAlater buffer(Ambion), and a razor blade shard will be used to subdissect the CA3 and CA1 segments. Segments are sucked up with a sterile pipette-tip containing RNAlater buffer and transferred into a sterile tube. Total RNA will be prepared by homogenization in TRIzol reagent (GibcoBRL) and precipitation in isopropyl alcohol. Rneasy Kit (Qiagen) will further isolate RNA. Spectrographic analysis of RNA prepared indicates 260nm:280nm ratios above 1.6, and insignificant amounts of either degraded RNA or DNA contaminants detected on a 2% denaturing gel. We have found that it is necessary to pool 15 CA3 or CA1 segments to obtain 5+ ug of total RNA. Total RNA samples will be sent to the consortium for additional quality control, labeling, hybridizations using the Affymetrix Neuro U34 chip, scanning, and preliminary analyses. The gene array data will then be compared in several pairwise manners (lesion vs. age matched control and region vs. region, for each time point), as well as time sequence comparisons for gene expression in each region. Keywords: time-course

			Action	DRS
	20140709_001_SWATH_6CA1.wiff	Wiff
	20140709_001_SWATH_6CA1.wiff.scan	Wiff
	20140709_002_SWATH_29_CA1.wiff	Wiff
	20140709_002_SWATH_29_CA1.wiff.scan	Wiff
	20140709_003_SWATH_42_CA1.wiff	Wiff

Dataset Information

Subregion-specific proteomic signature in the hippocampus for memory formation in adult mice

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Publications

Subregion-Specific Proteomic Signature in the Hippocampus for Recognition Processes in Adult Mice.

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