Project description:In order to understand how biochemical and genetic differences correlate with treatment response, we measured depressive-like behavior, gene expression and the levels of thirty-six neurobiochemical analytes across a panel of genetically-diverse mouse inbred lines after chronic treatment with vehicle or fluoxetine. Neurobiochemical markers were chosen based on their putative molecular function within pathways proposed to underlie depression, which include neuronal transmission, HPA-axis regulation, and neuroimmune processes. The goal of this study is to establish genetic and biochemical biomarkers that can predict treatment response and to propose a molecular pathway that is critical in mediating anti-depressant response. Thirty mouse inbred strains (129S1/SvImJ, A/J, AKR/J, BALB/cJ, BTBRT T(+) tf/J, BUB/BnJ, C3H/HeJ, C57BL/6J, C57BLKS/J, C57BR/cdJ, C58/J, CBA/J, CE/J, DBA/2J, FVB/NJ, I/LnJ, LG/J, LP/J, MA/MyJ, MRL/MpJ, NOD/LtJ, NOR/LtJ, NZB/BlNJ, NZW/LacJ, P/J, PL/J, RIIIS/J, SJL/J, SM/J, and SWR/J) aged 5-6 weeks old were obtained from The Jackson Laboratory (Bar Harbor, ME). Male mice were kept in polycarbonate cages on a 12-hour light/dark cycle (lights on at 0700h) with access to food and water ad libitum. Following one week of habituation, mice were randomized to either control or treatment group (n=12 male mice per strain per treatment group). Mean water intake for each strain was determined previously by measuring daily water consumption for three weeks (n=12 mice per strain). This information, along with average weight measurements for each strain, was used to determine the amount of fluoxetine required to provide a daily oral dose of 0 or 18 mg/kg per mouse. After chronic administration of 0 or 18 mg/kg of fluoxetine for 21 days, mice aged 9-10 weeks were tested in a tail-suspension and open field apparatus. Upon completion of the study, mice were sacrificed by cervical dislocation and decapitation between 0900h and 1300h. Trunk blood was quickly collected and allowed to clot on ice. Following centrifugation, serum samples were collected and stored at -20C for determination of fluoxetine and norfluoxetine levels using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Micro-dissections of individual regions were performed on serial coronal brain sections (approximately 10 µm) that were placed on a cold metal block. Cortex was taken from the same section for each animal and immediately snaps frozen on dry ice. Samples were stored at -80C until analysis.

Project description:Both the mechanism of action and the factors determining the behavioral response to antidepressants are unknown. It has been shown that antidepressant treatment promotes the proliferation and survival of hippocampal neurons via enhanced serotonergic signaling, but it is still unclear whether hippocampal neurogenesis is responsible for the behavioral response to antidepressants. Furthermore, a large subpopulation of patients fails to respond to antidepressant treatment due to presumed underlying genetic factors. In the present study, we have used the phenotypic and genotypic variability of inbred mouse strains to show that there is a genetic component to both the behavioral and neurogenic effects of chronic fluoxetine treatment, and that this antidepressant induces an increase in hippocampal cell proliferation only in the strains that also show a positive behavioral response to treatment. The behavioral and neurogenic responses are associated with an upregulation of genes known to promote neuronal proliferation and survival. These results suggest that inherent genetic predisposition to increased serotonin-induced neurogenesis is a determinant of antidepressant efficacy. Experiment Overall Design: Male DBA/2J, age 3-5 weeks, were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained on a 12:12 light:dark cycle with lights on at 0700 hours. Following at least one week of acclimation, mice were provided with either untreated or fluoxetine-treated drinking water, available ad libitum. Fluoxetine-HCl was obtained from Spectrum Chemicals (Gardena, CA). Differences in daily water consumption were previously determined by measuring water intake for three weeks from at least 12 mice (6.4 mL (+/- .58 mL) per day). We also determined the average weight at 10 weeks of age (25.5 g +/- 1.4 g), and used this information to dilute fluoxetine in the water in order to provide a daily dosage of 0 or 18 mg/kg/mouse. Experiment Overall Design: Mice were 8-12 weeks of age at the time of behavioral testing, which was performed during the light phase between 1300h and 1600h. Behavioral despair was measured using a mouse Tail Suspension apparatus from Med Associates (Georgia, VT). Experiment Overall Design: Individual bilateral hippocampii from 21-day-treatment 0 and 18 mg/kg/day DBA/2J mice were homogenized in 500 Âµl TRIzol using a QIAgen Tissuelyser (15 minutes at 30 Hz, QIAgen, Chatsworth, CA). RNA was extracted by phenol-chloroform phase separation and further processed using the RNAeasy miniprep kit (QIAgen). For each treatment group of 12 animals, separate RNA pools were made using hippocampal RNA from 6 mice per pool. These biological replicates were processed separately through the entirety of the microarray procedure. Five Âµg of total RNA from each pool was used as a template to synthesize complementary DNA (cDNA) and biotinylated cRNA (Enzo kit, Affymetrix, Santa Clara, CA) using standard Affymetrix protocols. cRNA was hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Array. Data were analyzed using ArrayAssist software (Stratagene, La Jolla, CA) and normalized with the GCRMA algorithm. A probeset had to obtain a minimal intensity value 100 to be considered as detecting expression above background. Therefore, any probeset that failed to meet this cutoff was removed from the analysis. Ratios of intensity differences were generated and genes showing at least a two-fold difference between experimental conditions were identified for further analysis.

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Mus musculus

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