Project description:The purpose of this study was to examined the acute actions of the second generation antipsychotic (SGA), olanzapine, on skeletal muscle (gastrocnemius) of Sprague Dawley Rats. SGAs cause metabolic side effects including leading to metabolic inflexibility, hyperglycemia, adiposity and diabetes. These effects are preceded by glucose intolerance and increased FFA flux and metabolism in peripheral tissues. Skeletal muscle is a likely target of glucose intolerance, therefore understanding how olanzapine affects the skeletal muscle transcriptome could elucidate approaches for mitigating these side effects. Male Sprague-Dawley rats freely fed on normal chow with comparable body weights (vehicle: 373±9g, olanzapine: 388±11g, p=0.34) were infused with vehicle or olanzapine for 24h using a dosing regimen leading to mild hyperglycemia (vehicle, 98±2mg/dl; olanzapine 127±4mg/dl, p=0.0023). For the olanzapine group, the venous catheter was attached to a syringe pump (Model NE-300) filled with olanzapine (Dr. Reddy’s Laboratories Ltd, Hyderabad, India) in sterile saline (infusion: 1mg/100g BW loading dose for 0.5h and then 0.04mg/100g/h continuously for 23.5h). Gastrocnemius was then surgically removed under isoflurane anesthesia (carried with 100% O2), and frozen between two aluminum blocks cooled to the temperature of liquid nitrogen and then stored at -80oC until RNA was isolated. With anesthesia gas flow continuing, the animals were euthanized by cutting the diaphram and removing the heart. The mRNA was isolated from from these muscles and used for RNA-Seq followed by alignment of the data with the rat genome assembly 5.0. To determine significant differences in FPKM values between control and olanzapine groups, the DEGexp function of the DEGseq 1.18.0 R package was used with the Likelihood Ratio Test (LRT) and default parameters. In the uploaded excel file, P values with p<0.05 and p<0.001 are shown for each row in different columns indicated by the number 1. The value 0 indicates the row is not significantly different.

Project description:Antipsychotic drugs are classified as typical and atypical based on extrapyramidal effects. However, since the frontal cortex is one of the most important regions for antipsychotic actions, this study attempted to classify antipsychotic drugs based on gene expression in the frontal cortex. Chlorpromazine and thioridazine were selected as typical antipsychotics, and olanzapine and quetiapine as atypical antipsychotics. Since these drugs have similar chemical structures, the effect of the basic structure on gene expression can be eliminated. Cluster analysis of microarray experiments showed thioridazine and olanzapine constituted a robust cluster. K-means clustering separated 4-drug-administered mice into chlorpromazine-quetiapine and thioridazine-olanzapine groups. This classification scheme is different from that which is based on criteria currently used to group the typical and atypical drugs and suggests that antipsychotic drugs can be further separated into multiple groups. Experiment Overall Design: Male 13-week-old ddY mice (Japan SLC Co., Hamamatsu, Japan) weighing 38-43 g were used. Animals were housed with free access to standard food in an air-conditioned room under a constant dark-and-light cycle (light: 7:00 a.m. to 7:00 p.m.) at a temperature of 22 to 24Â°C and 60 to 70% relative humidity. Ether was used for anesthesia during decapitation. All efforts were made to minimize animal suffering and to reduce the number of animals used. The present experiments were carried out after obtaining permission from the Committee of Animal Experimentation of the Graduate School of Medical and Dental Sciences at Kagoshima University. Experiment Overall Design: Doses of 25 mg/kg chlorpromazine HCl (Sigma-Aldrich Co., St. Louis, MO), 25 mg/kg thioridazine HCl (Sigma-Aldrich Co.), 1.25 mg/kg olanzapine (gift from Eli Lilly and Company, Indianapolis, IN) and 18.75 mg/kg quetiapine fumarate (gift from AstraZeneca, Macclesfield, UK) were used in the study. The dosages for these drugs were determined from therapeutically equivalent doses previously reported (Lehman et al, 2003; Woods, 2003). The drugs were dissolved in acetic anhydride and diluted with 0.9% saline, resulting in a final concentration of acetic acid of 0.5%. The drugs were injected intraperitoneally once daily for 28 consecutive days in a volume of 0.1 ml/10 g body weight. Experiment Overall Design: Microarray experiments were performed using an Agilent G4121A Mouse Oligo Microarray Kit (Agilent Technologies, Palo Alto, CA) as per the manufacturerâ??s instructions. The frontal cortex was immediately removed and the RNA was stabilized in RNAlater RNA Stabilization Reagent (Qiagen, Valencia, CA) and stored at -80Â°C until use. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). The RNA was amplified and labeled by the Low RNA Input Fluorescent Linear Amplification Kit (Agilent). To synthesize cDNA, 200 ng total RNA was used. Vehicle-injected controls were labeled by cyanine 3 (PerkinElmer Life Sciences, Inc., Boston, MA) and drug-injected mice were labeled by cyanine 5 (PerkinElmer Life Sciences, Inc.). Hybridizations to the microarray were performed using the In situ Hybridization Kit Plus (Agilent). Doses of 750 ng cyanine 3-labeled cRNA, 750 ng cyanine 5-labeled cRNA, and control targets were mixed and fragmented in the kitâ??s fragmentation buffer, and then hybridized to the microarrays for 17 hours at 60Â°C in a hybridization rotator (Agilent) set to rotate at 4 rpm. Microarrays were washed in 6Ã?SSC with 0.005% Triton X-102 at RT for 10 min, followed by 0.1Ã?SSC with 0.005% Triton X-102 at 4Â°C for 5 min. The slides were dried, and then scanned by the Agilent G2565BA Microarray Scanner System. Data were analyzed using the Agilent Feature Extraction Software version 7.1. A rank consistency filter and LOWESS were used for dye normalization. Control mice and drug-injected mice were processed in parallel. The data discussed in this publication are presented in accordance with the MIAME guidelines (http://www.mged.org/Workgroups/MIAME/miame.html) and were deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/). They can be accessed using the GEO series accession number GSE1501. Experiment Overall Design: Cluster analysis was performed using free software written by M. Eisen (http://rana.lbl.gov/EisenSoftware.htm). The cclust package in the â??Râ?? statistical software system (www.cran.r-project.org) was used to find the most appropriate number of clusters (i.e. â??kâ?? in the k-means clustering).

Project description:Antipsychotic drugs are commonly used to treat psychosis, mood disorders, and anxiety. While there is indirect evidence that some component of the antipsychotic effect of these drugs may involve modulation of dopamine transmission, their mechanism of action is poorly understood. We hypothesized that antipsychotic drugs mediate their effects via epigenetic modulation. Here we tested the effect of an antipsychotic, olanzapine, on the methylation status of genes following chronic treatment. These effects have been revealed through significantly increased (p<0.01) DNA methylation of genes involved in dopaminergic and non-dopaminergic pathways including the glutamatergic, GABAergic, cholinergic, neuregulin and ErbB signaling pathways. The affected genes included GLS in hippocampus, NR1 in cerebellum and GLUD1 and NR2B in liver. Further, from a set of genes in the 22q11.2 micro-deletions that has been previously implicated in psychosis, 22 genes showed increased methylation following olanzapine treatment. Ingenuity Pathway Analysis (IPA) revealed that chronic olanzapine treatment significantly affected several important pathways such as CREB and CDK5 signaling (p=1.4E-05). Also, DNA replication, recombination and repair, cellular movement and cell cycle have been identified as the top networks affected by olanzapine. The results suggest that these downstream effects, aside from D2 blockade, may play a critical role in the biological actions of antipsychotics. These include altered expressions of relevant genes involved in GABAergic, glutamatergic, cholinergic, neuregulin and ErbB signaling pathways. Epigenetic mechanisms involving changes in DNA methylation could, therefore, explain the delay and individualized non-specificity of biological effects of olanzapine. The results also suggest that DNA methylation may play a role in the process of therapeutic efficacy of olanzapine by altering the transcriptome via tissue-specific methylation of genes involved in schizophrenia signaling pathways. comparison of olanzapine treated rats vs. control rats for genome-wide DNA methylation changes

Project description:Antipsychotic drugs are commonly used to treat psychosis, mood disorders, and anxiety. While there is indirect evidence that some component of the antipsychotic effect of these drugs may involve modulation of dopamine transmission, their mechanism of action is poorly understood. We hypothesized that antipsychotic drugs mediate their effects via epigenetic modulation. Here we tested the effect of an antipsychotic, olanzapine, on the methylation status of genes following chronic treatment. These effects have been revealed through significantly increased (p<0.01) DNA methylation of genes involved in dopaminergic and non-dopaminergic pathways including the glutamatergic, GABAergic, cholinergic, neuregulin and ErbB signaling pathways. The affected genes included GLS in hippocampus, NR1 in cerebellum and GLUD1 and NR2B in liver. Further, from a set of genes in the 22q11.2 micro-deletions that has been previously implicated in psychosis, 22 genes showed increased methylation following olanzapine treatment. Ingenuity Pathway Analysis (IPA) revealed that chronic olanzapine treatment significantly affected several important pathways such as CREB and CDK5 signaling (p=1.4E-05). Also, DNA replication, recombination and repair, cellular movement and cell cycle have been identified as the top networks affected by olanzapine. The results suggest that these downstream effects, aside from D2 blockade, may play a critical role in the biological actions of antipsychotics. These include altered expressions of relevant genes involved in GABAergic, glutamatergic, cholinergic, neuregulin and ErbB signaling pathways. Epigenetic mechanisms involving changes in DNA methylation could, therefore, explain the delay and individualized non-specificity of biological effects of olanzapine. The results also suggest that DNA methylation may play a role in the process of therapeutic efficacy of olanzapine by altering the transcriptome via tissue-specific methylation of genes involved in schizophrenia signaling pathways.

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Olanzapine vs. Saline rat pfc

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