Project protocol — Contents
Workflow, sampling, and experimental treatment
Reagents, supplies, and solutions
Workflow, sampling, and experimental treatment
* The activity data (steps 3 & 6) are collected immediately-to-30 min post injection. However, the data reported are from 0-15 min post-injection because strain effects were strongest during this period.
Step Procedure done Age (wks) Diazepam (mg/kg) Time (min) Equipment Data collected 1 Mice are acclimated 5-6 - - - - 2 Mice are given i.p. injection of treatment vehicle 6 0 - - - 3 Mice are placed in the center of activity monitoring system 6 0 15* Omnitech Activity monitor baseline: total horizontal distance traveled (cm), vertical (rearing) activity (n) 4 Post-activity rectal temperature is obtained 6 0 - Digital thermometer baseline body temperature (°C) 5 Mice are given i.p. injection of benzodiazepine (diazepam) 6 2, 4, 8, or 16 - - - 6 Mice are placed in the center of activity monitoring system 6 2, 4, 8, or 16 15* Omnitech Activity monitor horizontal distance traveled (cm); vertical (rearing) activity (n) 7 Post-activity rectal temperature is obtained 6 2, 4, 8, or 16 <1 Digital thermometer 30 min post-treatment body temperature (°C) 8 Mice are sacrificed and the brains are harvested and stored at -80°C 6 8 or 16 - - - 9 Mice are given 3 test trials before treatment with benzodiazepine (diazepam) and then rested for 1 hr 6 0 >2 Rotarod accelerated at 1 rpm/s (innate rotarod ability is assessed) 10 Mice are given i.p. injection of high-dose of benzodiazepine (diazepam) and then tested on the rotarod 7 20 <2 Rotarod constant speed (5 rpm) latency to fall (s) 11 Mice are sacrificed and the brains are harvested and stored at -80°C 7 20 - - - 12 Treated brains are processed and analyzed 6-7 8, 16, and 20 - gas chromatography concentrations of benzodiazepines (DZ, diazepam) and metabolites (nordazepam and oxazepam) (ng/g)
2 or 4 mg diazepam per kg body weight [mg/kg]
Intermediate doses 8 or 16 mg diazepam per kg body weight [mg/kg] High-dose 20 mg diazepam per kg body weight [mg/kg]
intraperitoneal injection (i.p.)
- Digital thermometer, 0.5 mm diameter x 2.5 cm, Sensortek Thermalert TH-8
- Animal monitoring system, Omnitech Activity Monitoring Chamber
- Rotarod, with constant and accelerating speeds where dowels are suspended 55-60 cm above sawdust bedding
- Gas chromatography
- -80°C and -20°C storage refrigerators
- Plexiglas tube for brief restrain
- Decapitating scissors
- Small rodent dissecting kit
- Benzodiazepine, diazepam
- Treatment vehicle: 1 drop of Tween 20 + 10 mL physiological saline
- Saline (physiological saline): 0.9%, Baxter Healthcare Corp, Deerfield IL, USA
- Isopropyl alcohol: 10% for wipe downs between tests
- Syringes with needles
Acclimation to test conditions
Mice are acclimated for 1 hr before testing begins.
I. Activity and body temperature measurement
a. Each mouse is temporarily restrained using a Plexiglas tube for at most 10 s until baseline rectal (body) temperature is obtained with a pre-lubricated digital thermometer probe.
b. The mouse is then injected i.p. with vehicle or 2, 4, 8, or 16 mg/kg dose of diazepam, and then placed in the center of an animal monitoring system.
c. Activity is automatically measured via infrared beam interruptions every 5 min for a total duration of 30 min.
d. Shortly after 30 min in the activity monitoring chamber, each mouse is again restrained for a final body temperature determination.
e. Following the final body temperature measurement, mice from the 8 and 16 mg/kg dose groups are euthanized and brains dissected and stored at -80°C until assay is performed (see below).
II. Measuring coordination under high-dose diazepam using the rotarod
Because it was not possible to test all strains at once, a 2-block design was adopted. Both C57BL/6J and DBA/2J strains were included in blocks 1 and 2 as internal (seasonal) controls.
a. Initially, mice are tested for their innate ability to perform on a rotarod that is accelerated at a constant rate of 1 rpm/s.
b. After three test trials, performance in Trials 2 and 3 is averaged in revolutions per minute where in the mouse fell from the rotarod is indicative of innate rotarod ability.
c. The mice are rested for 1 hr subsequently after test trials and before coordination under diazepam treatment is measured.
d. After being rested for 1 hr, each mouse is injected i.p. with high-dose of 20 mg/kg of diazepam and placed on a rotarod with a fixed speed of 5 rpm (rotation per min).
e. Testing is terminated when the mouse falls off the rotarod.
f. Shortly after the mouse loses its balance, it is decapitated and the brain is harvested for benzodiazepine determination.
III. Determination of brain benzodiazepines
a. Brain samples are harvested from mice given 8, 16, and 20 mg/kg of diazepam
b. For short-term storage brain samples are kept in -20°C until assay is performed.
c. For long-term storage brain samples are kept in -80°C until assay is performed.
d. Brains samples are processed and then analyzed with gas chromatographic method.
e. Brain concentrations of diazepam, nordazepam, and oxazepam are determined.
f. Only brain concentrations of diazepam and nordazepam are determined in coordination study.
Data collected by investigator
Total horizontal distance traveled, total vertical (rearing) activity, and body (rectal) temperature with baseline/vehicle or 2, 4, 8, or 16 mg/kg of diazepam.
Latency to fall from the rotarod with high-dose 20 mg/kg diazepam.
Brain benzodiazepines concentrations in mice treated with intermediate and high-dose of diazepam.