Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Ovariectomy and sham surgeries
• Procedure: Open field test
• Procedure: Forced swim test
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Mice anesthetized and ovariectomy or sham surgery performed	Surgery equipment	-
2	Mice allowed to recover for 17 days	-	-
3	Open field test	Open field arena	Locomotor activity, exploratory and anxiety-like behavior
4	Forced swim test	Cylinder filled halfway with water	Depression-like behavior

Equipment and supplies

• Surgical tools
  
• Sutures (Med Rep Express, Prescott AZ)
• Open field arena and software (VersaMax Animal Activity Monitoring System, AccuScan Instruments Inc., Columbus OH)
  • 42 x 42 x 30 cm arena consisting of a white Plexiglas floor and clear Plexiglas walls
  • 16 photocells on each side of the arena, dividing it into 256 equal squares that allow for tracking of horizontal and vertical activity
  • In a sound-attenuating chamber
• Forced swim test apparatus (Noldus, Wageningen, The Netherlands)
  • 25-cm high glass-polycarbonate cylinder 17 cm in diameter, filled halfway with water maintained at room temperature
• Noldus Ethovision 7.0 software (Noldus, Wageningen, The Netherlands)

Reagents and solutions

• Tribromoethanol (Sigma-Aldrich, St. Louis MO)
• Buprenorphine HCl (Patterson Veterinary, Devens MA)

Procedure: Ovariectomy and sham surgeries

1. Mice are anesthesized with a 1.2% solution of tribromoethanol administered by intraperitoneal injection at a dose of 0.2 mL/10g body weight.
2. A dorsal midline incision is made in the skin caudal to the posterior border of the ribs.
3. An incision is made in the muscle, and the fat pad located just beneath the muscles is pulled out to expose the ovary.
4. The fallopian tube is clamped off, the ovary removed and muscle and skin are sutured.
5. This procedure is performed bilaterally.
6. The procedure for sham surgery is identical except for clamping and tying off fallopian tubes and removal of ovaries.
7. Buprenorphine HCl is administered subcutaneously at a dose of 0.05 mg/kg body weight after surgery as needed during the recovery period.
8. Mice are allowed to recover for 17 days.

Procedure: Open field test

1. Mice are placed in the open field arena individually for a 10-min test.
2. Behavioral measures are recorded and analyzed in five 2-min bins using the VersaMax activity monitor and analyzer software.

Procedure: Forced swim test

1. Mice are placed in the apparatus for a 6-min test.
2. Test sessions are video recorded and analyzed using Noldus Ethovision software.
3. Immobility is defined as no movements other than those required to stay afloat.
4. NOTE: C58/J mice were not tested due to an inability to remain above water.

Data collected by investigator

• Open field test
  
  • distance traveled
  • movement time
  • center distance
  • margin distance (thigmotaxis)
  • vertical movement
  • percent time spent in the center of the arena
• Forced swim test
  • immobility