Project protocol — Contents
Workflow, sampling, and experimental treatment
Reagents, supplies, and solutions
Procedure: Measurement of behavioral despair or stress reactivity using the tail suspension (TST) paradigm
Procedure: Measuring exploratory activity using the Open field test (OFT)
Procedure: Measuring anxiety or behavioral conflict using the light-dark box test (LDT)
Day Procedure done Treatment Trial Time (min) Data collected 1 mice are weighed none bw1 (baseline) 1 mice are subjected to baseline tail suspension test (TST) none 1 6 baseline duration of immobility 3 mice are subjected to TST for the second time i.p. saline or imipramine 30mg/kg 1 6 duration of immobility under saline or imipramine 3 mice are weighed none bw2 following TST under saline or imipramine 10 mice are tested on the open field (OFT) none 3 5 baseline distance traveled, vertical movements, center time 10 mice are tested on the open field (OFT) i.p. saline or imipramine 30mg/kg 3 5 distance traveled, vertical movements, center time 11 mice are tested on the light-dark box (LDT) none 1 10 baseline number of transitions 11 mice are tested on the light-dark box (LDT) i.p. saline or imipramine 30mg/kg 1 10 number of transitions from light to dark compartment
An automated TST device (Med Associates Inc.) is used to measure the duration (s) of immobility in the TST. The strain gauge detected movements of the mouse and the electrical signals are transmitted to a central unit. A computer automatically calculated the total duration of immobility, which is the time the force transducer detected the mouse's movements- is below a pre-set threshold criterion (i.e. immobile, not struggling) during a 6-min TST. The most discriminating settings for detecting immobility are determined empirically. The device is configured with the following settings: time constant = 0.25, gain = 4, threshold 1 = 3, and resolution = 200 ms. Whenever the mouse's movements are lower than our threshold 1 (<3) for 200 ms, the duration of immobility accumulated.
The automated open field apparatus (Opto-Varimex 0043- 501L; Columbus Instruments, Columbus, OH) consisted of a square field (42 cm x 42 cm x 30.5 cm) with a photocell sensor system consisting of an array of 15 photo beams per side used to measure distance traveled (cm), vertical movements (rearing), and center time. The vertical sensors are positioned 8 cm above the cage floor. Illumination is adjusted to give 730 lux at the cage floor.
The light–dark apparatus consisted of a polypropylene rat cage (44 cm x 21 cm x 21 cm) unequally divided (2/3 and 1/3) into two chambers by a wall with a small vestibule. The larger chamber is open, transparent, and brightly illuminated by two 20-W fluorescent lights (1388 lux at cage floor), while the smaller chamber is closed, painted black, and dark. The vestibule contained four photocells that detected the transitions from light to dark compartment, which is then recorded during a 10-min session by a computer.
- Imipramine HCl (30 mg/kg; RBI/Sigma, Natick, MA) is dissolved in pyrogen-free saline (0.9% NaCl) solution and prepared on the day of testing. Imipramine or saline solution is administered, i.p., 30 min prior to tail suspension testing, in a volume of 0.1 ml/10 g body weight.
- Scotch tape use to suspend mice via their tail (Scotch Super Strength mailing tape Cat. #341, 3M Corp, St. Paul, MN)
- 70% alcohol
- Paper towel
Acclimation to test conditions
All animals are brought to the testing room at least 1h before the beginning of each behavioral test, and remained in the same room throughout the test.
Notes common to all tests
After each trial and/or session, the apparatus is cleaned to remove potential olfactory cues.
a. Initially mice are suspended via their tail using Scotch tape.
b. The tail is secured to an aluminum bar connected to a strain gauge.
c. While the mice struggle and try to escape, their movements are transmitted to a computer processor.
d. The duration of immobility or secession of struggling is automatically calculated during the 6min TST test.
e. The TST baseline or basal immobility is the total time (s) during which the mice are immobile on the first session.
f. After obtaining baseline TST measurements, the randomly chosen treatment groups are given i.p. injections of either saline or imipramine 30mg/kg dose before being subjected to a second session of TST.
g. Tail suspension test imipramine immobility is the duration of immobility in the second session following i.p. injection of imipramine 30mg/kg or saline.
h. The mice are given 1wk of rest before being subjected the open field test (OFT).
a. Baseline measurements are first obtained in the initial session with each mouse gently placed at the front left corner (nose to the corner) of the apparatus at the beginning of each trial.
b. There are three consecutive trials, each trial consisted of 5min interval for a total 15min per session.
c. The horizontal ambulatory (activities) and vertical movements of the mouse are automatically detected by built-in photocell sensor system consisting of an array of strategically placed photobeams.
d. Likewise, center time duration and "thigmotaxis" or tendency to move away from the center are measured and recorded by the same multi-zone motion monitor software. The average of only the 2nd and 3rd trials is used for the center time value.
e. Baseline number of fecal boli is counted after the initial 15min session.
f. After obtaining baseline values for distance traveled, vertical movements or rearings, center time duration, the mice are treated with i.p. injection of imipramine at 30mg/kg dose.
g. Imipramine treated mice are then subjected to a second session of OFT testing consisting of 3 trials of 5min intervals.
h. Open field behavioral performance of individual mouse under imipramine are automatically detected and recorded as above.
i. At the end of the treatment (second) session, the number of fecal boli is again counted and recorded.
a. The following day after the open field test, the mice are subjected to a conflict test where each mouse is initially placed in the bright side of a 2-compartment light-dark box.
b. Since mice naturally prefer to be in the dark, their exploratory tendencies conflict with their avoidance of bright light. Hence, the light-dark box is used to measure this state of conflict or anxiety.
c. Baseline performance in the light-dark box test is obtained and recorded automatically during the 10min session.
d. Following baseline testing on the light-dark box, the mice are treated with i.p. injection of imipramine at 30mg/kg dose, and then tested again on the light-dark box for the second time.
f. A computer also recorded the latencies for the first transition to the dark compartment during baseline session as well as with imipramine session.
• TST baseline or basal immobility, TST imipramine and saline immobility, body weight before testing and after testing and treatment with imipramine or saline, change in immobility with imipramine relative to saline.
• Baseline values for distance traveled, vertical movements, center time, and number of fecal boli in the open field test (OFT); values for distance traveled, vertical movements, center time, and number of fecal boli in the open field test (OFT) with imipramine treatment.
• The number of transitions, from light-dark compartment under baseline conditions or with imipramine treatment, during the light-dark box test.
Definitions & formulas
TST baseline or basal immobility durimmob_preRx or durimmob_pre_saline (TSTBAS) is the total time (s) during which the mouse is immobile on the first session.
TST imipramine/saline immobility, durimmob_postRx/durimmob_post_saline, (TSTIMI/ TSTSAL) is the duration of immobility in the second session following the injection of imipramine/saline.
TST percentage imipramine immobility, immobpct_Rx, (TSTIMI%) in the inbred strains is calculated as follows:
(Group mean TST imipramine immobility − Group mean TST saline immobility time) ÷ Group mean TST saline immobility time) X 100 = TSTIMI%
Individual mouse immobility percentage = ((individual immobility with imipramine - group average immobility with saline) ÷ group average immobility with saline) x 100
Change in TST immobility with imipramine (ΔTST) is as follows:
ΔTST = durimmob_preRx - durimmob_postRx
Change in tail suspension induced hyperthermia- TSIH (ΔT) is calculated by subtracting imipramine TSIH from baseline TSIH.