Project protocol — Contents
Workflow and sampling
Reagents, supplies, and solutions
Time (day) Procedure performed % EtOH % Saccharin Age (wk) Data collected -7 Mice arrived at the animal facility, housed 4 per cage, separated according to sex and strain 0 0 6-8 - -7 to -1 Mice are allowed to acclimate to general housing conditions 0 0 6-8 - -1 to 0 Mice are housed individually with continuous access to 2 bottles (25 mL graduated cylinders with metal sippers) containing tap water and rodent chow distributed near both bottles to avoid food associated tube preferences during acclimation to testing conditions 0 0 7-9 - 1-4 During testing of voluntary ethanol (EtOH) consumption one 25 mL graduated cylinders always contained tap water and the other contained diluted solutions of EtOH 3 0 7-9 - 3 To control for side bias, the positions of the EtOH cylinders on Days 1 and 2 are switched 3 0 7-9 water and 3% EtOH consumption 5-8 Graduated cylinders containing EtOH are replaced and their positions on Days 3 and 4 are switched 6 0 8-9 water and 3% EtOH consumption 7 The positions of the EtOH within a group on Days 5 and 6 are switched 6 0 8-9 water and 6% EtOH consumption 8 Testing cages are changed 6 0 8-9 water and 6% EtOH consumption 9-12 Graduated cylinders containing EtOH are replaced and their positions on Days 7 and 8 are switched 10 0 9-10 water and 6% EtOH consumption 11 The positions of the EtOH cylinders on Days 9 and 10 are switched 10 0 9-10 water and 10% EtOH consumption 13 Graduated cylinders containing EtOH are replaced by 0.2% saccharin solutions and their positions on Days 11through 12 are switched 0 0.2 9-10 water and 0.2% saccharin intakes 14-17 Graduated cylinders containing only saccharin are replaced with solutions containing EtOH and saccharin 3 0.2 10-11 water and 0.2% saccharin intakes 16 The positions of the EtOH and saccharin on Days 13 through 15 are switched and cages are changed 3 0.2 10-11 water and 3% EtOH plus saccharin
18-21 Graduated cylinders containing EtOH and saccharin are replaced and their positions on Days 16 and 17 are switched 6 0.2 11-12 water and 3% EtOH plus saccharin
20 The positions of the EtOH and saccharin on Days 18 through 19 are switched 6 0.2 11-12 water and 6% EtOH plus saccharin
22-25 Graduated cylinders containing EtOH and saccharin are replaced and their positions on Days 20 and 21 are switched 10 0.2 11-12 water and 6% EtOH plus saccharin
24 The positions of the EtOH and saccharin on Days 22 through 23 are switched and cages are changed 10 0.2 11-12 water and 10% EtOH plus saccharin
• 25 mL graduated cylinders fitted with metal sipper tubes
- Ethanol (EtOH) ( 3, 6, and 10% ; v/v solutions with tap water)
- Saccharin (0.2 % solution in tap water)
- 0.2% saccharin + ethanol (EtOH, 3, 6, 10% solutions)
a. Mice 5-6 wks-of-age are acclimated to housing conditions for 3 wks. Initially they are grouped housed at 3-4 mice per cage.
b. Mice are separated and housed individually for 1 day to be acclimated to testing conditions.
c. During the entire day of acclimation the mice are continuously exposed to 2 inverted graduated cylinders with metal sippers and food.
d. EtOH with/out 0.2% saccharin in the same 25 mL graduated cylinders are placed in two empty cages for measuring evaporation and leakage. The average volume depleted from these cylinders is subtracted from individual daily consumption.
e. Mice are first exposed to increasing concentration of EtOH, then 0.2% saccharin in tap water, and then combination of EtOH and 0.2% saccharin.
f. Each novel solution is given for 4 days, and each morning daily fluid consumption (to the nearest 0.2 mL) is recorded from the tap water cylinder and the test cylinder.
g. The cylinder positions are alternated every other day to control for side bias while food is distributed in equal proximity to the cylinders to avoid preference associated with food.
h. With each solution change, fresh tap water is provided and body weight is concurrently obtained.
i. Soiled cages are changed every 8 days.
Data collected by investigator
Average fluid consumption on the 2nd and 4th day of each set of paired solutions per given condition (3%, 6%, 10% EtOH, 0.2% saccharin, EtOH + 0.2% saccharin).
Definitions and calculations
Two-bottle choice test: A component of choice preference test wherein mice are offered two graduated cylinders-one with 3%, 6% or 10% EtOH (with/out saccharin) and the other one with water. Side bias is taken into consideration by alternating the position of the EtOH relative to water cylinders.
Preference ratio = volume of EtOH, EtOH+saccharin, or saccharin consumed divided by total volume of fluid consumed