Project protocol — ContentsWorkflow and sampling
Workflow, sampling, and experimental treatment
Equipment for all procedures
Step Procedure performed Equipment Duration (min) Age (wk) Data collected 1 Water consumption and body weight are assessed daily for 3 wks. balance scale - 5-8 daily body weight and water consumption for dose determination 2 Chronic fluoxetine treatment (21 days) - - 5-8 - 3 Mice are assessed for the tail suspension test Tail suspension apparatus 7 9-10 percent immobility 4 Body weights are obtained from each mouse after tail suspension test balance scale - 9-10 body weight 5 Exploratory behavior is assessed in an open field arena Open field arena 10 10-11 percent center occupancy, total activity 6 Blood is collected for serum drug and metabolite concentration LC-MS/MS - 10-11 serum fluoxetine and norfluoxetine 7 Cortex harvested dissecting kit 10-11 - 8 Neurobiochemical analytes quantified parallelized reverse ELISA, protein array 10-11 quantification of analytes (see Wiltshire3)
• Mouse Tail Suspension setup (PHM-300 TST Cubicle, Med Associates, St. Albans, VT)) see Figure 1 below
• Open field arena: The open field is a 27.3 cm x 27.3 cm arena with a white Plexiglas floor and clear Plexiglas walls (MED-OFA-MS; Med Associates, St. Albans, VT, USA), which is surrounded by infrared detection beams on the x-, y- and z-axes that track the animals' position and activity over the course of the experiment (see Figure 2 below).
• Sciex 4000 LC/MS/MS (Sciex Inc., Concord ON) equipped with a Waters YMC Cyano HPLC column (2.0 x 23 mm) (Milford, MA).
• Freezer at -80°C
• Dissecting kit: decapitator
Figure 1. Schematic layout of the tail suspension test (not drawn to scale).
Figure 2. Schematic layout of the open field arena (not drawn to scale).
- Fluoxetine HCl (Spectrum Chemicals, Gardena, CA)
- Light bleach solution (0.1%)
- Paper towels
I. Pre-treatment essentials
a. Water consumption and body weight are initially determined daily for 3 wks.
b. To obtain a daily oral dose of 0 or 18 mg/kg per mouse of fluoxetine, mean water intake and body weight measurements are taken. (There is no evidence that fluoxetine provided in drinking water alters water consumption.)
c. A chronic fluoxetine regimen of 18 mg/kg for 21 days is implemented, based on a previously published dose-response study of fluoxetine administration in drinking water (Miller et al., 2008).
d. An equal number of control and treated mice in each cohort are subjected to the 3-wk regimen, and each strain is represented within a test group for which the order of mice are randomized and counterbalanced.
II. Tail Suspension Test (TST)
a. After chronic administration of 0 or 18 mg/kg of fluoxetine for 21 days, mice 9-10 wks of age are tested in a tail-suspension apparatus between 1300h and 1600h.
b. Behavioral despair is then evaluated by measuring percent time spent immobile while the mouse is suspended by its tail.
c. Immobility is recorded for 7 min in 60 sec blocks using the following parameters: threshold = 3, gain = 8, and resolution = 200ms.
d. Percent time spent immobile is calculated for the last 5 min of the test allowing a period of habituation and apparent increased activity to the novel environment in the first 2 min.
e. Mice that climbed up their tail during testing are excluded from analysis.
III. Open Field Test (OFT)
a. Behavior in the open field is recorded for all mice between 1300h and 1600h using an open field apparatus 1 wk following TST.
b. Vertical and horizontal movements are automatically recorded by infrared beams located horizontally and vertically above the arena.
c. Anxiety-like behavior is measured as percent time spent in the center of the open field. Following the administration of anxiolytic drugs like fluoxetine, increase in exploratory behavior at the center of the open field is predicted based on previous observations.
d. Activity in the open field is recorded for a total of 10 min.
IV. Brain tissue and blood collection
a. Mice are sacrificed by cervical dislocation and decapitation by fully train personnel between 0900h and 1300h.
b. Trunk blood is then quickly collected and allowed to clot on ice.
c. Serum samples are collected following blood centrifugation and then stored at -20°C for later analysis.
d. Serial coronal brain sections (approximately 10 µm) placed on a cold metal block are subjected to micro-dissections of individual regions of interest.
e. Cortex is taken from the same section for each mouse and immediately frozen on dry ice.
f. Brain samples are stored at -80Â°C until further analysis (see Wiltshire3).
V. Serum fluoxetine/norfluoxetine quantification
a. A mixture of 10 µL of serum sample and 150 µL of acetonitrile solvent are spiked with 5 µg/mL of each internal standard (fluoxetineD6 and norfluoxetineD6).
b. Serum samples are filtered with 0.45 Âµm filter plate, and 50 µL of the filtrate is diluted with 0.1% formic acid and injected into a Sciex 4000 LC MS/MS equipped with a high performance liquid chromatography (HPLC) column (2.0 x 23 mm).
c. A mobile phase consisting of water/acetonitrile/formic acid (75:25:0.1) is used for running the HPLC.
d. A standard curve with concentrations from 20 to10,000 ng/mL is conducted for extrapolating the amounts of fluoxetine and norfluoxetine in each sample.
e. Peak areas are detected for ions with the following mass to charge (m/z) ratios: m/z 310 → (fluoxetine), m/z 296 → 134 (norfluoxetine), m/z 316 → 44 (fluoxetineD6), and m/z 302 → 140 (norfluoxetineD6).
f. Extrapolated information is then used to quantify serum fluoxetine and norfluoxetine levels.
IV. Neurobiochemical analyte quantification and data normalization for Reference Fluorescence Intensity (RFI) See Wiltshire3
Definitions and calculations
Fluoxetine (also known by the trade names Prozac, Sarafem, Fontex) is a selective serotonin reuptake inhibitor (SSRI) that is commonly used as an antidepressant.
OFT center avoidance (thigmotaxis) = 100 â percent center occupancy
Data collected by investigator
Tail Suspension Test: duration of immobility
Open Field Test: total distance traveled and percent center occupancy
Drug and metabolite concentration: serum fluoxetine and norfluoxetine
Neurobiochemical analyte quantification: Wiltshire3
MPD computed values
OFT center avoidance (thigmotaxis)
Bailey JS, Grabowski-Boase L, Steffy BM, Wiltshire T, Churchill GA, Tarantino LM. Identification of quantitative trait loci for locomotor activation and anxiety using closely related inbred strains. Genes Brain Behav. 2008 Oct;7(7):761-9.
Eisener-Dorman AF, Grabowski-Boase L, Steffy BM, Wiltshire T, Tarantino LM. Quantitative trait locus and haplotype mapping in closely related inbred strains identifies a locus for open field behavior. Mamm Genome. 2010 Jun;21(5-6):231-46. Epub 2010 May 15.
Segall SK, Nackley AG, Diatchenko L, Lariviere WR, Lu X, Marron JS, Grabowski-Boase L, Walker JR, Slade G, Gauthier J, Bailey JS, Steffy BM, Maynard TM, Tarantino LM, Wiltshire T. Comt1 genotype and expression predicts anxiety and nociceptive sensitivity in inbred strains of mice. Genes Brain Behav. 2010 Nov;9(8):933-46. doi: 10.1111/j.1601-183X.2010.00633.x.