Protocol for Sucrose Preference Test (SPT)

• Sucrose is used to prepare the sweet solution; published SPT protocols commonly use approximately 1-2% sucrose solution, although some studies used other concentrations^[1][2][3][8].

• Drinking water is used as the comparison fluid in the two-bottle choice test^[1][2][3].

• Vehicle or untreated control condition is used as the negative control in pharmacological or stress-model experiments^[2][3][4].

• Antidepressant treatment may be used as a positive control when testing reversibility of stress-induced sucrose preference deficits^[2][4][8].

• Mice or rats are used depending on the model; species, strain, sex, body weight, stress history, housing condition, and feeding status should be reported because they affect sucrose intake and preference^{[1][3][5][6][7][9]}.

Equipment and instruments

• Use two drinking bottles per cage or animal, bottle holders that allow simultaneous access, a balance or graduated measurement method to quantify fluid intake, cages suitable for individual measurement when individual intake is required, and data sheets or software for calculating preference^[1][3].

Controls

• Use water-only or vehicle-treated controls, non-stressed controls for stress models, and antidepressant-treated positive controls when testing rescue of stress-induced anhedonia-like behavior^[2][4][8].

Preparation Steps

• Before testing, habituate animals to the two-bottle setup and sucrose solution when required by the selected protocol, because novelty, side bias, and learning can influence measured preference^[1][3][7].
• Prepare two bottles for each test unit: one containing sucrose solution and one containing water; record starting bottle weights or volumes before placement^[1][3].
• Avoid long food or water deprivation unless it is the specific variable under study, because deprivation can confound sucrose preference by adding hunger and thirst drives^[5][6].
• Randomize or alternate bottle positions across the test to reduce side preference bias^[1][3].

Operation Steps

• Place the sucrose bottle and water bottle on the cage simultaneously and allow the animal free access for the predefined test period; published protocols include short tests and overnight or multi-day testing, so the selected duration should be reported explicitly and kept identical across groups^[1][2][3][5].
• At the end of the test, remove bottles and measure remaining sucrose solution and water by weight or volume to calculate intake^[1][3].
• Measure total fluid intake as well as sucrose intake, because reduced sucrose preference may reflect altered total drinking rather than reduced reward sensitivity^[1][5][6].
• Record body weight when interpreting sucrose intake, because body weight and metabolic status can influence consumption^[5][6][9]

• Data Acquisition and Analysis
Calculate sucrose preference as: sucrose intake / (sucrose intake + water intake) × 100%^[1][3].
• Analyze sucrose preference together with sucrose intake, water intake, total fluid intake, body weight, stress condition, sex, and treatment group^[5][6][7][9].
• Use the individual animal as the biological replicate when intake is measured individually; if animals are group-housed during testing, the cage rather than the individual animal is the measured unit unless individual drinking is tracked^[1][3].
• Interpret reduced sucrose preference cautiously, because recent studies show that SPT performance can reflect non-hedonic processes such as metabolic drive, learning, motivation, choice behavior, or altered fluid intake^[5][6][7].

Problem: Sucrose preference decreases after fasting or water restriction.

Possible Cause: Hunger and thirst drives can confound reward-related interpretation.
Literature-supported Solution: Avoid long deprivation unless experimentally required, and report feeding and water-access conditions clearly^[5][6].

Problem: Sucrose preference changes but total fluid intake also changes.

Possible Cause: The phenotype may reflect altered drinking, metabolic state, or body weight rather than anhedonia.
Literature-supported Solution: Report sucrose intake, water intake, total intake, and body weight, not preference percentage alone^[5][6][9].

Problem: Animals consistently drink from one side.

Possible Cause: Side bias can distort two-bottle preference results.
Literature-supported Solution: Alternate or randomize bottle positions during testing^[1][3].

Problem: SPT results do not align with other depression-like behavioral outcomes.

Possible Cause: SPT reflects multiple behavioral components, including reward sensitivity, learning, motivation, and choice.
Literature-supported Solution: Interpret SPT alongside complementary behavioral assays rather than as a standalone measure of depression-like behavior^[6][7].