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Transcription profiling of two mouse lines displaying different phenotypes on fear conditioning to identify identify differences in gene expression in two key brain regions: amygdala and hippocampus

ABSTRACT: Fear conditioning (FC) is a behavioral paradigm that measures an animalâs ability to learn fear related information. FC is measured by pairing a mild foot-shock with the surroundings in which the shock was received. Upon being placed back in the context, mice exhibit freezing behavior, which is a species-specific response to fear. We have used selective breeding to produce lines of mice with high or low levels of freezing behavior. We are able to identify alleles that govern the genetic variability for FC by using chromosomal markers in these lines. Using microarrays, we will identify differences in gene expression in two key brain regions: amygdala and hippocampus. Gene expression differences and data regarding chromosomal regions involved in the behavior will be compared to identify particular genes that are both differentially expressed and whose expression is governed by alleles that fall into critical chromosomal regions.,We will compare gene expression in the amygdala and hippocampus (brain regions known to be relevant to fear behavior) from the two lines of mice and use Bayesian statistics in an effort to identify gene expression that affects fear behavior.,We hypothesize that selection has acted in part by changing the frequency of alleles that cause differential expression of key genes in the amygdala and hippocampus of our selected lines. Selective breeding changes the frequency of trait relevant (FC) alleles. A relevant allele is expected to increase in one selected line and decrease in the oppositely selected line. Some trait relevant alleles are expected cause changes in the level of expression at particular genes.,Amygdala and hippocampus will be rapidly dissected out of experimentally naÃ¯ve mice from each line. NaÃ¯ve mice will be used for expression studies since the behavior of the mice in the FC test can be reliably anticipated due to their lineage. We have practiced these procedures, and can accurately and reproducibly remove these regions in less than 5 minutes. Different mice will be used to collect each brain region, since the dissection of hippocampus disrupts the removal of amygdala. We will collect enough samples from each region to accommodate a total of 6 microarrays per brain region, per line, thus we will use a total of 24 microarrays. We anticipate that a single brain region will be sufficient to for a microarray. However, we propose to utilize three samples per microarray, because this will reduce variability due to environmental factors and due to slight variability in our dissection procedures. Once this tissue is removed, we will isolate RNA for shipment to the Microarray consortium. We will also collect spleens from each subject as a source of genomic DNA, in order to permit direct comparison of genotype and expression phenotypes. Once we have the results of the microarray analysis, we use WebQTL.org to identify the chromosomal locations of alleles that are know to influence the expression of genes for which we have found differential expression. We will then superimpose this information on trait relevant chromosomal regions identified from our selected lines. This will allow us to rapidly identify genes which may account for genetic variability in FC due to differential expression. Such genes will then be subjected to further study.

ORGANISM(S): Mus musculus

SUBMITTER: Elizabeth Salomon

PROVIDER: E-GEOD-4035 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Fear conditioning (FC) is a behavioral paradigm that measures an animal's ability to learn fear related information. FC is measured by pairing a mild foot-shock with the surroundings in which the shock was recieved. Upon being placed back in the context, mice exhibit freezing behavior, which is a species-specific response to fear. We have previously used selective breeding to produce lines of mice with high or low levels of freezing behavior. This experiment is a replication of a previous experiment that produced lines of mice with high or low levels of freezing behavior. These lines derive from different progenitor mouse strains. We are able to identify alleles that govern the genetic variability for FC by using chromosomal markers in these selected lines. Using microarrays, we will identify differences in gene expression in two key brain regions: amygdala and hippocampus. Gene expression differences and data regarding chromosomal regions involved in the behavior will be compared to identify particular genes that are both differentially expressed and whose expression is governed by alleles that fall into critical chromosomal regions.,We will compare gene expresion in the amygdala and hippocampus (brain regions known to be relevant to fear behavior) from the these two lines of mice and to the those in the previous experiment. Bayesian statistics will be used in an effort to identify gene expression that affects fear behavior.,We hypothesize that selection has acted in part by changing the frequency of alleles that cause differential expression of key genes in the amygdala and hippocampus of our selected lines. Slective breeding changes the frequency of trait relevand (FC) alleles. A relevant allele is expected to increas in one selected line and decrease in the oppositely selected line. Some trait relevant alleles are expected to cause changes in the level of expression at particular genes.,Amygdala and hippocampus will be rapidly dissected out of experimentally naÃ¯ve mice from each line. NaÃ¯ve mice will be used for expression studies since the behavior of the mice in the FC test can be reliably anticipated due to their lineage. We have practiced these procedures, and can accurately and reproducibly remove these regions in less than 5 minutes. Different mice will be used to collect each brain region, since the dissection of hippocampus disrupts the removal of amygdala. We will collect enough samples from each region to accommodate a total of 6 microarrays per brain region, per line, thus we will use a total of 24 microarrays. We anticipate that a single brain region will be sufficient to for a microarray. However, we propose to utilize three samples per microarray, because this will reduce variability due to environmental factors and due to slight variability in our dissection procedures. Once this tissue is removed, we will isolate RNA for shipment to the Microarray consortium. We will also collect spleens from each subject as a source of genomic DNA, in order to permit direct comparison of genotype and expression phenotypes. Once we have the results of the microarray analysis, we use WebQTL.org to identify the chromosomal locations of alleles that are know to influence the expression of genes for which we have found differential expression. We will then superimpose this information on trait relevant chromosomal regions identified from our selected lines. This will allow us to rapidly identify genes which may account for genetic variability in FC due to differential expression. Such genes will then be subjected to further study.

Project description:Prolonged stress has adverse consequences for neurons in the hippocampus (HIPP). The amygdala (AMY) is a brain region that is similar to HIPP across many measures, suggesting that chronic stress might modulate AMY and HIPP function in similar ways. However, studies addressing this issue have produced surprising results. For example, a regimen of chronic stress shown to produce atrophy in HIPP neurons caused dendritic branching in AMY neurons. These and other data have revealed that excessive stress induces fundamentally opposing processes in the AMY and HIPP, such that AMY function is facilitated by levels of stress that produce deleterious effects in HIPP. The mechanisms that contribute to the opposing responses of these brain regions to chronic stress are unknown. Such knowledge may suggest novel directions for pharmacological interventions to protect the HIPP from stress-mediated damage.,We will determine gene expression patterns in the hippocampus and amygdala under basal and stressful conditions, with a particular interest in those genes that are differentially regulated across the two regions. ,We hypothesize that genes that are oppositely regulated in the amygdala and hippocampus after stress mediate the opposing functional consequences of chronic stress in these two brain regions. Moreover, genes which are differentially expressed in these regions under basal conditions may underlie the distinct responses of these regions to stress.,Rats will receive either 30sec of handling (Control group; n=15) or 3hr of immobilization stress (Stress group; n=15) each day for 14 consecutive days. This stress paradigm has previously been shown to produce opposing effects on dendritic morphology and region-dependent behaviors for the hippocampus versus amygdala. Twenty-four hours after the last stress or handling session, the rats will be overanaesthetized, and the brain will be removed and placed in ice-cold buffer. Tissue will be rapidly dissected from the basolateral complex of the amygdala and the CA3 region of the hippocampus of both hemispheres, flash frozen in liquid nitrogen, and stored at -80C until further processing. The tissue from each brain region will be pooled across groups, yielding four samples (Control-Hippocampus, Control-Amygdala, Stress-Hippocampus, and Stress-Amygdala). Total RNA will be extracted using standard methods, and sent to the centers. Each sample will be run in triplicate, utilizing a total of 12 Affymetrix Rat Genome U34A gene chips. A triplicate array assay of pooled samples has previously been shown to both substantially increase the sensitivity of detecting genes of interest and reduce the variability associated with individual microarrays. In addition, the large number of rats used in each group will reduce the variability in gene expression associated with individual responses to chronic stress, as well as variability due to slight anatomical differences in the tissue extracted from each rat.

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Transcription profiling of two mouse lines displaying different phenotypes on fear conditioning to identify identify differences in gene expression in two key brain regions: amygdala and hippocampus

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