Protocol to induce memory generalization and stress-like behaviors without memory strengthening using contextual fear conditioning in mice

Summary

Over-generalization of negative memory is common in psychiatric disorders, including depression and anxiety. Here, we present a protocol to dissociate memory generalization from memory strengthening and induce lasting stress-related behavioral alterations using contextual fear conditioning in adult mice. We describe the steps for repeated conditioning in identical or distinct contexts, followed by memory testing and behavioral assays of stress-related phenotypes. This protocol enables the investigation of the behavioral and biological mechanisms underlying pathological memory generalization and its consequences.

For complete details on the use and execution of this protocol, please refer to Cheng et al.¹

Graphical abstract

Highlights

• •Protocol for double-dissociating fear memory generalization from memory strengthening
• •Instructions for inducing lasting stress-related behaviors after acute training
• •Guidance on evaluating memory, motivation loss, social deficits, and anhedonia in mice
• •Steps for linking negative memory over-generalization to stress-like behaviors

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Over-generalization of negative memory is common in psychiatric disorders, including depression and anxiety. Here, we present a protocol to dissociate memory generalization from memory strengthening and induce lasting stress-related behavioral alterations using contextual fear conditioning in adult mice. We describe the steps for repeated conditioning in identical or distinct contexts, followed by memory testing and behavioral assays of stress-related phenotypes. This protocol enables the investigation of the behavioral and biological mechanisms underlying pathological memory generalization and its consequences.

Before you begin

Innovation

This protocol presents a reproducible approach to induce and assess negative memory generalization in mice using a modified contextual fear conditioning (CFC) paradigm.¹ A key advance of this design is its ability to double-dissociate memory generalization from memory strengthening. In contrast to conventional paradigms, in which high-intensity foot shocks often elicit both stronger and more generalized fear,^2,3,4 or paradigms that require extended incubation periods to reveal generalized memory over time,^5,6 our protocol enables efficient induction of robust memory generalization within a defined experimental framework.

A further advance is that the same paradigm that induces memory generalization also produces long-lasting psychological distress-related behaviors, creating an integrated platform to examine how memories of acute aversive experiences give rise to broader behavioral outcomes. In contrast to standard models based on chronic stress, this approach reveals that two training sessions in different, but not identical, contexts are sufficient to induce enduring stress-related phenotypes. Importantly, this contextual differentiation relies on distinct multisensory environmental configurations, including unique chamber geometry, illumination, tactile features, and olfactory cues provided by the cleaning agents used for each context. This feature makes the assay particularly useful not only for studying how the neural substrates underlying negative memory generalization contribute to behavioral abnormalities, but also for preclinical screening and evaluating intervention strategies aimed at reducing memory overgeneralization and its associated psychological stress phenotypes.

This workflow allows the combined evaluation of behavioral, physiological, circuit, and molecular outcomes. It is compatible with other analyses, including circuit tracing and functional manipulation, in vivo miniscope imaging in freely moving mice to identify neural activity patterns, and circuit-specific transcriptomic sequencing to resolve the molecular signatures underlying memory-associated psychological distress.

Institutional permissions

All experimental procedures should be performed in compliance with relevant institutional, national and international guidelines for the care and use of laboratory animals. The study protocol described here was approved by the Laboratory Animal Welfare and Ethics Committee of Sun Yat-sen University (Reference No. SYSU-IACUC-2026-000957).

Animal acclimatization and handling

Timing: 1 to 2 weeks

• 1.Within each experimental batch, use age-matched animals across all groups, preferably littermates when possible.

Note: We used C57BL/6J mice aged 8–12 weeks at the start of the experiments.

• 2.House animals in groups of 4 to 5 in a standard specific pathogen-free (SPF) facility at 23 ± 2°C and 50∼60% humidity on a 12-h light/dark cycle, with water and food available ad libitum.

Note: For example, light on at 8 am; light off at 8 pm.

CRITICAL: Both male and female mice are compatible with this protocol. For males, continuous group housing from 5 to 6 weeks of age is essential to avoid aggression caused by introducing unfamiliar males. In contrast, grouping unfamiliar females does not typically present such challenges.

• 3.Allow animals to acclimate to the facility for at least 1 week after transportation before starting any experimental procedures.
• 4.Gently handle mice 2 to 3 min per day for 4 consecutive days before training. This procedure habituates mice to human touch and scent, thereby reducing baseline stress and improving behavioral stability during subsequent assays.

Note: Use the same handler and perform handling at a relatively consistent time of day to maintain baseline stability.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Experimental models: Organisms/strains
Mouse: C57BL/6J mice (wild-type, male and female, 8–12 weeks old)	Institute of Experimental Animals of Sun Yat-sen University	RRID:IMSR_JAX:000664
Software and algorithms
EthoVision XT	Noldus	RRID:SCR_000441
Prism 9	GraphPad	RRID:SCR_002798
Other
Shock delivery apparatus	Med Associates	N/A
Shock delivery apparatus	Shanghai Jiliang Software Technology Co., Ltd.	N/A
DigBeh system	Shanghai Jiliang Software Technology Co., Ltd.	N/A

Materials and equipment

Memory training and testing contexts

• •CFC and testing are performed in three environmental configurations.
• • •
    Context A: Place a rectangular chamber (30 cm × 25 cm × 20 cm) lined with white wallpaper in a sound-attenuating cabinet under 90 lux white illumination. The floor consists of stainless-steel rods connected to a foot-shock delivery apparatus (Med Associates, St. Albans, Vermont, USA). Clean the chamber with 4% acetic acid between animals. Position a camera directly in front of the chamber (Figure 1A).
  • •
    Context B: Place an acrylic rectangular chamber (25 cm × 25 cm × 45 cm) with striped walls in a different sound-attenuating cabinet under 90 lux red illumination. The floor consists of stainless-steel rods connected to a foot-shock delivery apparatus (Shanghai Jiliang Software Technology Co., Ltd., China). Clean the chamber with 75% ethanol between animals. Mount a camera above the chamber (Figure 1B).
  • •
    Context C: Place an acrylic triangular chamber (28 cm × 20 cm × 20 cm) with clear walls in a sound-attenuating cabinet matched to those used for other contexts, under 60 lux red illumination that provides an additional visual cue for contextual discrimination. The floor consists of a solid acrylic sheet covered with smooth white wallpaper. Clean the chamber with 2% ammonia solution between animals. Mount a camera above the chamber (Figure 1C).
• •Commercial fear-conditioning systems from different vendors are acceptable, provided that chamber dimensions, floor structure, illumination, odor cues, and shock-delivery parameters are kept consistent within each context across experiments.
• •Prepare all cleaning solutions fresh each week and use them within 1 week of preparation to maintain consistent olfactory cues across experimental cohorts. After cleaning, allow each chamber to air-dry before introducing the next subject, while retaining a faint residual odor as part of the contextual cue.

Figure 1.

Environmental configurations used for CFC and memory tests

(A–C) Top: Representative photographs showing the front view of Context A (A), Context B (B) and Context C (C).

Bottom: Corresponding camera views showing a mouse in each context during training or testing.

Step-by-step method details

CFC

Timing: 2 days

In this protocol, each training session consists of a 3 min habituation period in Context A or B, followed by three 2 s foot-shocks (0.75 mA, alternating current) delivered at 60 s intervals. Mice remain in the chamber for 1 additional min after the final shock before removal, as previously described.⁷

Note: Although rodents are nocturnal, previous studies have shown that, under the experimental conditions examined, mice acquire fear conditioning and exhibit stronger memory retrieval during the light phase, whereas training or testing during the dark phase can be associated with impaired fear memory performance.^8,9 Therefore, we perform all behavioral procedures during the light cycle. Conducting all behavioral procedures within a consistent time window is important for minimizing time-of-day-related variability.

• 1.
  Group allocation: Before the first training session, randomly assign mice to one of the following experimental groups according to the training schedule:

  • a.
    “1×Tr” group: mice receive a single CFC session in either Context A or B, with context assignment counterbalanced across animals.
  • b.
    “2×Tr same” group: mice receive two CFC sessions in the same context (A+A or B+B), spaced 24 h apart, with context assignment counterbalanced across animals.
  • c.
    “2×Tr altered” group: mice receive two CFC sessions in different contexts (A+B or B+A), spaced 24 h apart, with training order counterbalanced across animals.

Note: Mark each mouse uniquely to ensure unambiguous individual identification throughout the experiment.

• 2.Acclimation: Transfer mice to the experimental room at least 1 h before each training session.

Note: Transport from the vivarium to the behavioral suite can induce acute stress. Adequate acclimation is essential to minimize behavioral variability associated with elevated baseline stress.

• 3.Equipment setup: Ensure that the shock delivery apparatus is clean and that all instruments are properly connected and powered on.

Note: Mice are highly sensitive to olfactory cues. Thoroughly clean and fully dry the chamber before each trial to prevent residual odors or pheromones from the previous animal from affecting behavioral outcomes.

• 4.Shock intensity calibration: Set the shock intensity to 0.75 mA and verify the output current across the floor rods using an appropriate current meter.
• 5.
  The first training session (Day 1, ∼10 min per mouse):

  • a.
    Clean Context A with 4% acetic acid or Context B with 75% ethanol.

    Note: This step serves both as sanitation and as part of the contextual odor cue.

  • b.
    Transfer a mouse from its home cage into the training chamber.
  • c.
    Initiate the pre-programmed CFC protocol and record video. Troubleshooting 1.
  • d.
    At the end of the session, remove the mouse and gently place it into a clean holding cage.
  • e.
    Repeat steps a-d for the remaining animals.
  • f.
    Return mice to their home cage after all cage-mates have completed training.

    Note: Naïve mice receive no CFC or contextual exposure. Do not house naïve mice together with experimentally trained mice, as exposure to odors or pheromones from trained animals may affect the state of naïve cage-mates.

• 6.
  The second training session (Day 2, ∼10 min per mouse):

  • a.
    24 h after the first session, perform the second training session.
  • b.
    Train mice according to their assigned schedules, following the same procedure described in Step 5. Troubleshooting 1.

CRITICAL: Counterbalance context assignments to avoid chamber bias. For the “1×Tr” group, distribute training evenly across Days 1 and 2, with similar numbers of mice conditioned in Context A and Context B on each day. For the “2×Tr same” group, balance animals between A+A and B+B schedules. For the “2×Tr altered” group, balance animals between the A+B and B+A schedules. This protocol has been validated using a 24-h interval between the two training sessions. The effects of shorter or longer intersession intervals on memory strength, memory generalization, and subsequent stress-related behaviors have not been evaluated.

Memory tests

Timing: 2 days for recent memory tests; 16 days in total for remote memory tests, including a 14-day retention interval followed by 2 days of behavioral testing.

Assess memory strength and memory generalization by re-exposing mice to the conditioned context (A or B) or a novel context (C), respectively, for 3 min. Recent memory tests begin 1 day after the final training session, whereas remote memory tests begin 14 days after the final training session.

• 7.Acclimation: Transport mice to the behavioral testing room and allow them to acclimate to the environment for at least 1 h before each test.
• 8.
  Recent memory strength test (S test, performed on Day 3 or Day 4 in a counterbalanced order, ∼5 min per mouse):

  • a.
    Clean the chambers using the same procedures described for training in Step 5a.
  • b.
    Place each mouse into one of its conditioned contexts for 3 min to assess memory strength and record video. Troubleshooting 2.
  • c.
    At the end of the session, transfer the mouse to a clean holding cage to prevent interaction with untested cage-mates.
  • d.
    Repeat steps a-c for the remaining mice.
  • e.
    Return mice to their home cage after all cage-mates have completed testing.
• 9.
  Recent memory generalization test (G test, performed on Day 4 or Day 3 in a counterbalanced order, ∼5 min per mouse):

  • a.
    Clean Context C with 2% ammonia solution.

    Note: This step serves both as sanitation and as part of the contextual odor cue.

  • b.
    Place each mouse into Context C for 3 min to assess memory generalization and record video. Troubleshooting 2.
  • c.
    Follow the same procedure described in Step 8c-e.

    Note: Context C presents distinct visual and olfactory cues to assess memory over-generalization. It retains limited similarities to Context A and B, rather than being completely distinct, thereby modeling partial contextual overlap for valid generalization testing.

    Note: We define freezing as the absence of any visible movement except that required for respiration. We quantify the percentage of freezing time during each 3 min test offline, either automatically using EthoVision XT software or by an examiner blinded to group assignment. We calculate the generalization index (G index) as follows:

    $$Gindex=\frac{thepercentageoffreezingtimeintheGtest}{thepercentageoffreezingtimeintheStest}$$
• 10.Remote memory tests: Repeat the S test and G test on Day 15 to 16 (2 weeks after training) to assess the persistence of memory strength and generalization. Troubleshooting 2.

CRITICAL: Counterbalance the order of the S test and G test across cohorts.

Stress-related behavioral tests

Timing: 7 days for recent tests; 21 days in total for remote tests, including a 14-day retention interval followed by 7 days of behavioral testing.

In this section, we perform a battery of behavioral assays to evaluate stress-related phenotypes associated with memory strengthening or memory generalization. These assays include the novelty-suppressed feeding test (NSFT) for motivational conflict, the splash test (ST) for self-care deficits, the tail suspension test (TST) for immobility behavior, the three-chamber social interaction test (SIT) for social withdrawal, and the sucrose preference test (SPT) for anhedonia.

CRITICAL: The overall behavioral sequence can introduce cumulative stress. Perform less stressful assays before more stressful ones to minimize carryover effects. Use gentle handling and conduct all tests during a fixed time window of the light cycle. Keep housing conditions stable throughout the experiment unless a specific assay requires otherwise.

• 11.Acclimation: Transport mice to the behavioral testing room and allow them to acclimate to the environment for at least 1 h before each test.
• 12.
  NSFT (Day 5 or 17, 1 day after completion of recent or remote memory tests, ∼15 min per mouse):

  • a.
    Food-deprive mice for 24 h before the NSFT by removing all chow from the food hopper and inspecting the bedding to ensure that no residual food pellets remain in the home cage.
  • b.
    Clean the test apparatus with 75% ethanol.

    Note: The apparatus consists of an open-field arena (40 cm × 40 cm × 40 cm) under 80–100 lux red illumination, placed in a sound-attenuating cabinet (Figure 2A).

  • c.
    Place two pieces of palatable chow on a filter paper in the center of the arena.
  • d.
    Introduce the mouse into one corner of the arena, facing the food.

    • i.
      Allow the mouse to explore freely until it takes the first bite.

      Note: Terminate the test if no feeding occurs within 10 min.

    • ii.
      Record behavior using the DigBeh system. Troubleshooting 3.
  • e.
    Follow the same procedure described in Step 8c-e.

    Note: A researcher blinded to experimental group assignment analyzes the video offline and records the latency to the first bite as the primary outcome measure.

• 13.
  ST (Day 6 or 18, ∼10 min per mouse):

  • a.
    Clean the testing apparatus thoroughly with 75% ethanol.

    Note: The apparatus consists of a standard-size mouse cage under 80-100 lux red illumination in a sound-attenuating cabinet (Figure 2B).

  • b.
    Transfer the mouse to the test cage and allow it to habituate for 1 min.
  • c.
    Gently remove the mouse from the test cage and hold it by hand.
  • d.
    Spray the dorsal fur of the test mouse 3 times with 10% sucrose solution, then gently return it to the test cage and record behavior for 5 min. Troubleshooting 4.

    Note: Prepare the 10% sucrose solution fresh before use or store it at 4°C for no longer than 1 week.

  • e.
    Follow the same procedure described in Step 8c-e.

    Note: A researcher blinded to group assignment analyzes the video offline and records the total grooming time. Grooming behavior is defined as organized self-cleaning movements, including paw licking, facial wiping, head washing, licking or stroking of the torso, and fur combing with the forepaws.

• 14.
  SIT (Day 7 or 19, ∼25 min per mouse):

  • a.
    Clean the testing apparatus thoroughly with 75% ethanol.

    Note: The apparatus consists of a three-chamber arena (60 cm x 40 cm x 20 cm) under 80-100 lux white illumination in a sound-attenuating cabinet (Figure 2C).

  • b.
    Place two empty perforated plastic cups with barred side openings at opposite diagonal corners of the arena.

    Note: The cups should prevent direct physical contact while allowing visual, olfactory, and limited tactile investigation.

  • c.
    Transfer the test mouse to the arena and allow it to explore freely for 10 min.
  • d.
    Remove the test mouse from the arena. Place an unfamiliar conspecific mouse into one cup and a toy mouse into the other cup.

    CRITICAL: The unfamiliar stimulus mouse should be age-, weight-, and sex-matched to the test mouse.

  • e.
    Gently return the test mouse to the center of the arena and allow it to explore freely for 10 min. Record behavior throughout the session. Troubleshooting 5.
  • f.
    Follow the same procedure described in Step 8c-e.

    Note: A researcher blinded to group assignment analyzes the video offline and quantifies the time spent investigating the unfamiliar mouse and the toy mouse. Investigation is typically defined as directing the nose toward the cup at close proximity (∼1 cm).¹⁰ We calculate the social preference differential index (DI) as follows:

    $$DI=\frac{{Time}_{mouse}-{Time}_{toy}}{{Time}_{mouse}+{Time}_{toy}}$$
• 15.
  TST (Day 8 or 20, ∼10 min per mouse):

  • a.
    The TST apparatus consists of a three-sided rectangular enclosure under 100-120 lux white illumination in a quiet testing room. A horizontal metal bar is fixed 50 cm above the floor for mouse suspension (Figure 2D).
  • b.
    Clean the apparatus and surrounding area with 75% ethanol.
  • c.
    Pass the distal tail through a 1 mL pipette tip with its narrow end removed, and secure the tail to the suspension bar with adhesive tape, positioning the mouse with its head ∼35 cm above the floor.

    Note: The pipette tip with its narrow end removed serves as a climbing deterrent and prevents mice from grasping their tails and climbing upward.

  • d.
    Suspend the mouse and record behavior for 6 min.
  • e.
    Follow the same procedure described in Step 8c-e.

    Note: We define immobility as the cessation of struggling in all four limbs and the absence of substantial body movement. We measure immobility time during the final 4 min offline using EthoVision XT software or by a researcher blinded to group assignment.

• 16.
  SPT (Days 8–11 or Days 20–23, ∼30 min per day)

  • a.
    After completion of the TST, house mice individually and maintain single housing throughout the SPT procedure.
  • b.
    Allow mice to acclimate for 1 day with access to two identical bottles filled with water. Record the initial weight of each water bottle before placing them on the cages.

    Note: Mice have ad libitum access to standard chow (Figure 2E).

    CRITICAL: Ensure that all bottles are leak-proof before the experiment. Pre-examine each bottle to prevent unintended fluid loss that could confound intake measurements.

  • c.
    Twenty-four hours later, weigh both water bottles again.
  • d.
    Replace one water bottle with 0.8% sucrose solution while keeping the other bottle filled with plain water. Record the initial weight of both bottles. Troubleshooting 6.
  • e.
    Twenty-four hours after sucrose exposure begins, weigh both bottles to quantify fluid intake. Afterwards, switch the positions of the sucrose and water bottles to minimize side bias.
  • f.
    Twenty-four hours after switching bottle positions, weigh both bottles again to quantify fluid intake. Calculate the sucrose preference differential index (DI) using the following formula:

    $$DI=\frac{sucroseintake-waterintake}{sucroseintake+waterintake}$$
  • g.
    After completion of intake measurements, return mice to group housing.

Figure 2.

Experimental setups for stress-related behavioral tests

(A) Camera view of the open-field arena used for the novelty-suppressed feeding test (NSFT).

(B) Camera view of the mouse cage used for the splash test (ST).

(C) Camera view of the three-chamber arena used for the social interaction test (SIT).

(D) Photograph of the suspension bracket used for the tail suspension test (TST).

(E) Photograph of the home cage and water bottles used for the sucrose preference test (SPT).

Expected outcomes

Using this protocol, mice subjected to repeated CFC are expected to exhibit distinct behavioral profiles associated with memory strengthening versus memory generalization. Compared with the “1×Tr” group, mice in the “2×Tr same” group are expected to show stronger, context-specific fear memory, reflected by high freezing levels in the trained context and limited freezing in the novel context. In contrast, mice in the “2×Tr altered” group are expected to display enhanced memory generalization, as indicated by increased freezing in the novel context and an elevated G index, while maintaining freezing levels in the trained context comparable to those of the “1×Tr” group (Figures 3A and 3B).

Figure 3.

The behavioral results after different patterns of CFC

(A and B), Quantification of the percentage of time spent in freezing during S test, G test and the G index in recent memory test (A) and remote memory tests (B).

G index: % freezing during G test divided by % freezing during S test. n = 9–10 per group.

(C and H) Quantification of the latency to eat in NSFT.

(D and I) Quantification of grooming duration in ST. (E, J) Quantification of time exploring the non-social target (NS) and the social mouse (S) during SIT, as well as the differential index (DI) during SIT. DI: time exploring the stranger mouse minus the time exploring the toy mouse as a fraction of the total exploration time.

(F and K) Quantification of the immobile time during TS test. (G, L) Quantification of sucrose preference (DI) during SPT.

(C–G) tests starting 1 day after CFC. (H–L) tests starting 2 w after CFC. (C–F, H−K) n = 10–12 per group.

(G and L) n = 4–8 per group. S test, fear memory strength test; G test, fear generalization test; NSFT, novelty-suppressed feeding test; ST, splash test; SIT, social interaction test; TST, tail suspension test; SPT, sucrose preference test. Naïve: control mice without training; 1×Tr: a single CFC session; 2×Tr same: two CFC sessions in the same context; 2×Tr altered: two CFC sessions in two different contexts.

Data are presented as mean ± s.e.m. and analyzed by one-way ANOVA followed by Tukey’s post hoc test or two-way ANOVA followed by the Sidak’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Data presented in Figure 3 are reprinted with permission from Cheng et al.^1.

In stress-related behavioral assays, mice with generalized memory in the “2×Tr altered” group are expected to exhibit increased feeding latency in the NSFT, decreased grooming duration in the ST, increased immobility in the TST, reduced social preference in the SIT, and reduced sucrose preference in the SPT (Figures 3C–3G). These behavioral alterations can persist for at least 2 weeks after the final conditioning session (Figures 3H-3L), supporting the use of this paradigm for studying sustained stress-related behavioral changes following acute aversive experiences.

Behavioral outcomes are expected to be reproducible across cohorts when context assignment, handling, and testing conditions are carefully controlled.

Limitations

While this protocol provides a reproducible behavioral framework to dissociate memory generalization from memory strengthening and to examine subsequent stress-related behavioral alterations, several limitations should be considered when applying this paradigm.

First, the performance of the protocol is highly sensitive to the degree of environmental contrast between the training contexts. Successful dissociation between the “2×Tr same” and “2×Tr altered” conditions requires carefully controlled differences in sensory features, such as visual cues, illumination, chamber geometry, and odor. If the contrast between Context A and Context B is too weak, the “2×Tr altered” condition may be less effective in inducing memory generalization and subsequent stress-related behavioral changes. Researchers should therefore calibrate these parameters according to their specific laboratory environment.

Second, the protocol has so far been optimized in a single inbred strain, which may limit generalizability across genetic backgrounds. Future studies should evaluate its robustness in additional inbred strains or outbred mice.

Finally, although this protocol identifies a unique link between memory generalization and stress-related phenotypes, it has not yet been validated with pharmacological interventions. Whether clinically relevant treatments, such as selective serotonin reuptake inhibitors or ketamine, can reverse memory generalization and the associated stress-related phenotypes induced by the “2×Tr altered” condition remains to be determined.

Troubleshooting

Problem 1

No obvious behavioral response to foot shock during conditioning (related to Steps 5 and 6).

This problem can arise for several reasons. First, the electrical circuit may be compromised. Grid floors contaminated with feces or urine can increase electrical resistance, and moisture on the grid can cause partial short-circuiting, thereby reducing the effective current delivered to the animal. Second, a small number of mice may reduce effective shock exposure by contacting only a single grid bar during shock presentation.

Potential solution

• •Calibrate the shock intensity using an ammeter before each experiment and confirm that all electrical connections are secure.
• •Thoroughly clean and dry the grid floor between subjects to maintain stable conductivity.

Problem 2

Mice show little or no freezing during memory tests (related to Steps 8-10).

Stress can exert intensity-dependent effects on fear learning and memory. Although moderate stress may facilitate fear conditioning, excessive environmental stress may impair the formation or retrieval of the context-foot-shock association or interfere with the stable expression of freezing during testing. Unintended cues present during training or testing, such as noise or other disturbances, may also interfere with the association between the context and foot shock, thereby reducing subsequent freezing behavior.

Potential solution

• •Extend the days of handling before training to reduce baseline anxiety in the animals and improve behavioral stability.
• •Avoid unintended external cues during training and testing, such as auditory disturbances (e.g., phone alerts or loud conversation), strong odors (e.g., perfume), or environmental disturbances (e.g., nearby construction).
• •If such disturbances occur, discard the affected trials. Resume the experiment only after the environment has stabilized, or repeat the experiment using a new cohort of animals.

Problem 3

Mice show unusually short or long feeding latencies in the NSFT (related to Step 12).

This is often caused by a poorly controlled hunger state at the time of testing. Extremely short or long latencies may indicate inappropriate fasting durations.

Potential solution

• •Standardize the duration of food deprivation (e.g., 18–24 h).
• •Replace the bedding at the start of food deprivation to minimize access to residual chow or feces.
• •Use bedding material that is less likely to be ingested during fasting, such as aspen shavings instead of corn cob bedding.

Problem 4

Mice show little, no, or highly variable grooming behavior during the ST (related to Step 13).

This may result from acute stress or high baseline anxiety of the animals, both of which can alter grooming performance and increase variability. In addition, old or improperly prepared sucrose solution may reduce the salience of the grooming stimulus.

Potential solution

• •Allow sufficient habituation to the test room and test cage before sucrose spraying to minimize acute stress responses.
• •Place a thin layer of bedding on the floor of the test cage to reduce anxiety associated with a novel environment.
• •Prepare fresh 10% sucrose solution before testing to ensure consistent stimulus effectiveness.

Problem 5

Mice climb onto the tops of the cups or the walls during the SIT (related to Step 14).

This may result from insufficient habituation to the testing chamber. It may also reflect elevated anxiety in the subject mouse.

Potential solution

• •Extend the habituation period in the chamber to ensure that the subject mouse is fully acclimated before the unfamiliar conspecific is introduced.
• •Increase the height of the social cups. Use cups made of smoother materials to reduce grip, or place a bottle on top of each cup to prevent climbing.

Problem 6

Mice show abnormally low or high sucrose preference during the SPT (related to Step 16).

In naïve mice, the sucrose preference differential index should typically range from 0.7 to 0.85. Values outside this range may suggest that the sucrose concentration is not optimal for the experimental setting. Baseline preference may vary with housing conditions and with the brand or purity of the sucrose used.

Potential solution

• •Before the formal experiment, perform a preliminary concentration-gradient test in a small cohort of naïve mice, using 1% sucrose as the starting concentration.
• •Adjust the sucrose concentration within a range of 0.5% to 2%. Reduce the concentration if naïve mice show excessively high preference, and increase it if preference is too low.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Xiaojing Ye (yexiaoj8@mail.sysu.edu.cn).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Xin Cheng (chengx265@mail.sysu.edu.cn).

Materials availability

This study did not generate new materials.

Data and code availability

• •All data reported in this paper will be shared by the lead contact upon reasonable request.
• •This paper does not generate new code.

Acknowledgments

This work is supported by grants from the Brain Science and Brain-like Intelligence Technology, National Science and Technology Major Project (no. 2021ZD0202000 to X.Y.); the National Natural Science Foundation of China (no. 32271068 to X.Y. and no. 81972967 to W.-J.L.); the Guangdong Basic and Applied Basic Research Foundation (no. 2024A1515011474 to X.Y. and no. 2025A1515011369 to W.-J.L.); the Science and Technology Planning Project of Guangdong Province (no. 2023B1212060013 and no. 2020B1212030004 to W.-J.L. and no. 2023B1212060018 to X.Y. and W.J.L.); and the China Postdoctoral Science Foundation (2025M772577 to X.C.). We thank the Animal Facility and the Core Facility of Medical Science (Guangzhou Campus), Sun Yat-sen University for their help in equipment use and data analysis. We thank all the lab members for discussion and technical assistance of this project.

Author contributions

X.Y. and W.-J.L. designed the research project. X.C., Y.Z., and Y.H. carried out the experiments and analyzed the data. X.C. wrote the manuscript. X.Y. and W.-J.L. reviewed and edited the manuscript.

Declaration of interests

The authors declare no competing interests.