Project protocol — Contents
Workflow, sampling, and experimental treatment
Equipment for all procedures
Definitions and calculations
Equipment Age (wk)
1 Chronic fluoxetine treatment (21 days) - 5-8 - 2 Mice undergo behavioral testing (tail suspension and open field tests); blood collected for drug and metabolite concentration see Wiltshire2 9-10 see Wiltshire2 3 Cortex harvested dissecting kit 10-11 - 4 Neurobiochemical analytes quantified parallelized reverse ELISA, protein array 10-11 quantification of analytes from control and fluoxetine-treated mice
- Dissecting kit: decapitator
- -80°C Freezer
- Parallelized reverse ELISA methodology (Zeptosens â a division of Bayer (Schweiz) AG, Witterswil, Switzerland
- QIAgen TissueLyser II (QIAgen, Valencia, CA)
- Tumbling shaker
- ZeptoREADER (Zeptosens â a division of Bayer (Schweiz) AG, Witterswil, Switzerland)
- Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Inc. Redwood, CA)
- Fluoxetine HCl (Spectrum Chemicals, Gardena, CA)
- CLB1 buffer with zirconium oxide beads
- Coomassie Protein Plus Assay (Pierce Biotechnology, Rockford, IL)
- 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. Behavioral testing (see Wiltshire2)
III. Brain tissue collection
a. Mice are sacrificed by cervical dislocation and decapitation by fully train personnel between 0900h and 1300h upon completion of the study.
b. Trunk blood is quickly collected and allowed to clot on ice (see Wiltshire2 for assessing fluoxetine and norfluoxetine serum levels).
c. Cortex samples are taken, snap frozen on dry ice, and stored at -80°C until analysis.
IV. Neurobiochemical analyte quantification and data normalization for Reference Fluorescence Intensity (RFI)
a. Neurobiochemical markers are quantified using a parallelized reverse ELISA methodology.
b. Cortex tissues are homogenized in CLB1 buffer with zirconium oxide beads for 30 s using QIAgen TissueLyser II.
c. Semi-solid brain tissues are mixed gently on a tumbling shaker for 30 min at room temperature.
d. Following centrifugation (2 min at 10,000 x g), supernatants are transferred and stored at -80°C until further analysis.
e. Total protein concentrations for each sample are determined using a modified Bradford assay (Coomassie Protein Plus Assay).
f. All samples are adjusted with CLB1 buffer to obtain a uniform concentration of 2 mg/mL.
g. Immediately after dilution, each protein sample is spotted twice at concentrations of 0.05, 0.10, 0.15, and 0.2 mg/mL.
h. Reference signals are obtained from simultaneous spotting of assay buffer and labeled antibodies on the array chip.
i. Following overnight incubation with primary antibodies, arrays are washed and incubated with fluorescence-labeled anti-species secondary antibody for 2.5 h in the dark.
j. Arrays are imaged simultaneously using a ZeptoREADER at excitation/emission wavelengths of 635/670 nm with exposure times of 0.3, 1, 5, and 10 s.
k. All images are analyzed using ZeptoVIEW 3.0 (version 184.108.40.206) software.
l. Background intensities are determined by taking the mean signals of four additional spots equidistant from the sample spot.
m. Net fluorescence intensities (NFI) are calculated by subtracting background signal from each sample spot signaling.
n. Following NFI determination, each NFI value is normalized to the mean intensity of the reference spots.
o. Using least-squares method, eight normalized NFI values obtained from each sample are fitted linearly against tissue lysate protein concentrations. The extrapolated signal intensity that corresponds to the mid-point of the concentration range (0.125 mg/mL) is defined as the reference fluorescence intensity (RFI).
Submitting investigator's notes: "Levels of thirty-six neurobiochemical markers proposed to be involved in depression and anxiety were measured. Dopamine transporter (DAT), interleukin-6 (IL-6), FK506-binding protein 51 (FKBP51), glucocorticoid receptor (NR3C1), and neuron specific family gene member 2 (NSG2) were excluded from the analysis due to weak intensities, irregular staining, or non-linear dose-response signals..."
Data collected by investigator
Tail Suspension Test: Wiltshire2
Open Field Test: Wiltshire2
Drug and metabolite concentration: Wiltshire2
Neurobiochemical analyte quantification: reference fluorescence intensity (RFI)
MPD computed values
Change index = log ratio of fluoxetine-treated protein levels to control protein levels
Fluoxetine (also known by the trade names Prozac, Sarafem, Fontex) is a selective serotonin reuptake inhibitor (SSRI), commonly used as an antidepressant.
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.