Protocol for Forced Swim Test (FST)

• Clean water is used as the swimming medium; published protocols commonly use water deep enough to prevent the animal from touching the bottom, with temperature controlled and kept consistent across animals^{[4][5][6][8][9]}.

• Test compound, vehicle, and a known antidepressant positive control are used only when the experiment is designed to assess antidepressant-like pharmacological effects^{[1][2][3][4][5][7]}.

• Adult mice or rats are used depending on the protocol; strain, sex, genotype, age, and prior stress history should be fixed within an experiment because these factors influence FST behavior^{[3][4][5][10][11]}.

Equipment and instruments

• Use a transparent cylinder or tank, controlled-temperature water source, timer, video camera or behavioral recording system, drying towels or warming recovery area, and scoring software or blinded manual scoring method^[4][5][6][7].

Controls

• Use a vehicle-treated negative control and, for pharmacology studies, a known antidepressant positive control; include locomotor assessment when interpreting reduced immobility, because increased general locomotion can create false-positive anti-immobility effects^{[4][5][7][10]}.

Preparation Steps

• Acclimate animals to the testing room and keep environmental conditions consistent across groups; randomize group order and blind the observer or scorer to treatment whenever possible^[4][5][10].
• Prepare the swim cylinder with water deep enough that animals cannot support themselves by touching the bottom; published rat protocols include a 15-min pretest followed 24 h later by a 5-min test, whereas mouse protocols commonly use a single 6-min session or a related short test format^[3][4][5][6].
• Prepare vehicle, test compound, and positive control according to the experimental design; use identical handling and dosing schedules across groups^{[1][2][3][4][5]}.

Operation Steps

• Place one animal into the water-filled cylinder and begin video recording immediately; avoid testing multiple mice in visually interactive conditions because simultaneous testing without partition can alter immobility time^[4][5][6][9].
• For rats, conduct a pretest lasting 10-15 min when using the traditional or modified rat FST, then perform the main 5-min test 24 h later; this pre-exposure accentuates behavioral differences during the drug test session^[2][5].
• For mice, conduct the test using the published mouse FST format, commonly a 6-min swim session, and quantify immobility during the defined scoring period specified before the experiment^[3][4][6].
• After each test, remove the animal promptly, dry it, keep it warm during recovery, and replace or clean the water as needed to maintain consistent test conditions across animals^[4][5][6]

• Data Acquisition and Analysis
Record immobility time as the primary outcome; immobility is scored when the animal makes only movements necessary to keep the head above water, while active behaviors can be scored as swimming and climbing, especially in modified rat FST protocols^[4][5][7].
• Analyze active behavior categories when mechanism is relevant, because serotonergic antidepressants preferentially increase swimming, whereas noradrenergic antidepressants preferentially increase climbing in the modified rat FST^[7][8].
• Normalize interpretation to control animals tested under identical conditions, report strain, sex, age, water depth, water temperature, cylinder dimensions, session duration, scoring window, and scorer blinding, and include locomotor controls when reduced immobility is interpreted as antidepressant-like activity^{[4][5][10][11]}.
• Use the individual animal as the experimental unit; reported mouse FST literature often used 7-10 animals per group for large known-antidepressant effects, but prospective power analysis may require larger groups when the expected effect size of a new intervention is unknown^[10].

Problem: Immobility differences appear inconsistent between experiments.

Possible Cause: Strain, sex, genotype, prior stress, apparatus dimensions, or testing conditions differ between cohorts.
Literature-supported Solution: Standardize and report animal characteristics and apparatus parameters, and avoid comparing data across protocols with different setup dimensions or scoring rules^{[4][5][9][11]}.

Problem: A treatment appears to reduce immobility but also increases general activity.

Possible Cause: The result may reflect nonspecific locomotor activation rather than antidepressant-like behavior.
Literature-supported Solution: Include locomotor assessment or another control for motor activity before interpreting reduced immobility as antidepressant-like activity^[4][5][10].

Problem: Mouse FST results change when two animals are tested at the same time.

Possible Cause: Visual/social exposure during simultaneous testing can alter immobility.
Literature-supported Solution: Test animals individually or use conditions that prevent visual interaction, and report the testing arrangement^[9].

Problem: Manual scoring gives variable immobility values.

Possible Cause: Immobility thresholds and scoring criteria can bias quantification.
Literature-supported Solution: Use predefined blinded scoring criteria, video-based analysis, or validated sampling approaches for immobility and active behaviors^[5][7][12].