This is the first ever mouse judgment bias task to be validated as sensitive to changes in affective state, in other words, mood or emotion. It can be used to investigate the effects of any variable on mouse affect. This protocol uses an ethological design, making it easy for mice to learn, and it's also welfare friendly, since the task avoids the use of punishment or negative reinforcement.
This task can assess the impact of husbandry or experimental procedures on mouse affective states, making it valuable across diverse fields, like animal welfare research or modeling human affective disorders. Assuming researchers are already comfortable with non-aversive handling methods, our main advice is to use pilots before training to identify appropriate odor mixtures, in other words, the ones that mice treat as ambiguous. This behavioral test is a scent-based go-go digging task in which a mouse must dig for high-or low-value food rewards.
The experimental apparatus comprises a rectangular arena with two arms. One arm is always centered and marked with either the positive or negative discriminative stimulus odor, and the other arm is always unscented. Digging pots include an accessible and an inaccessible compartment separated by a perforated plastic barrier.
Both pots in the apparatus always contain the high-and low-value food, but treats are made accessible depending on the arm and trial type. During positive training trials, the mouse can choose to explore and dig in the scented arm, which contains a pot marked with a positive discriminative stimulus odor and a buried high-value food reward. The mouse can also choose to dig in the unscented arm, which is marked with distilled water and contains a low-value food reward.
During negative training trials, The mouse can again choose between the scented arm, which now contains a pot marked with the negative discriminative stimulus odor and no accessible food reward, or the unscented arm, which is again marked with distilled water and contains a low-value food reward. Then, mice are tested in positive and negative un-reinforced trials, where no food rewards are available, and those who have learned the task will undergo an un-reinforced ambiguous trial. In the ambiguous trial, the scented arm contains a pot marked with a mixture of the positive and negative discriminative stimulus odor cues, making the ambiguous odor, while the unscented arm is marked with only distilled water, as always.
Mice reveal their judgment biases in this ambiguous trial. For experimental preparation, using a one-milliliter syringe or micropipette, add mint and vanilla extract into labeled centrifuge tubes. Dilute the extracts 1:4 with distilled water and mix by inverting several times.
Make these daily and repeatedly invert before use to ensure that the mixtures are fresh and consistent. For the ambiguous odor mixture, after the mint and vanilla essences have been diluted, add equal volumes to a centrifuge tube to make a 1:1 mixture of the diluted essences. Next, during the reinforced trials, for the positive discriminative stimulus odor pot, place one piece of chow in the inaccessible compartment and bury one piece of banana chip in the accessible area of the pot.
For the negative discriminative stimulus odor pot, place one piece of chow and one piece of banana chip in the inaccessible compartment. Then, fill the pot with three centimeters of corn cob bedding. No food rewards will be available in the accessible area of this pot.
For unscented pots, place one piece of banana chip in the inaccessible compartment and bury one piece of chow in the accessible area of the pot. During all un-reinforced trial tests, place one piece of chow and one piece of banana chip in the inaccessible compartment. Then, fill the pot with three centimeters of corn cob bedding.
No food rewards will be available in the accessible area of this pot. After filling the pots, use a syringe or micropipette to draw 100 microliters of the appropriate odor mixture or distilled water and inject it directly on top of the corn cob in a circular motion. Next, to prepare scent dispensers, place one cotton pad piece in the base of the tissue cassette.
Then, using a one-milliliter syringe or micropipette, draw 100 microliters of the appropriate odor mixture or distilled water and inject it onto the cotton piece. Cover the tissue cassette with its lid to enclose the scented cotton and create a scent dispenser. Place the digging pots at the end of the arms of the arena and the scent dispensers at the beginning of each arm.
Insert the removable door immediately before the cassette slots to block entry to the arena arms and create the start compartment. Perform all training and testing sessions under red light during the dark and active phase of the mouse. Fast the mouse for one hour in their cage before each training or testing session by removing food from the hopper.
On day one of digging training, place the food rewards on top of the three-centimeter corn cob bedding instead of burying them underneath. Progressively bury the rewards deeper under the corn cob over the following four days until they're located at the bottom of the three-centimeter bedding by day five. To start training trials, move the mouse from the transport cage to the start compartment of the arena.
Remove the start door, immediately lower the plexiglass lid over the arena, and start the five-minute trial timer. Then, record latency to dig as the time at which the first occurrence of digging is observed and latency to eat as the time at which the first occurrence of eating is observed. For discrimination training, conduct one positive reinforced trial followed by one negative reinforced trial on days one to five.
On days 6 to 10, pseudo-randomize the order of trials so that each mouse undergoes two positive and two negative trials per day. In the testing phase, conduct one positive or negative un-reinforced test trial for each mouse daily until the learning criteria are met. Each day, perform one positive and one negative reinforced trial in a randomized order before the video-recorded test trial, and another positive and negative reinforced trial after the test trial.
Ensure that the positive and negative test trials are conducted in alternating order across days. To perform a video-recorded un-reinforced test trial, move the mouse from the transport cage to the start compartment of the arena. Then, set up a video camera on a tripod so that both pots at the end of the arms are in view and start recording.
Ensure to record the cue card with the mouse blind code and the trial type during video scoring. Remove the start door, immediately lower the plexiglass lid over the arena, and start the two-minute trial timer. After testing, return the mouse to its transport cage.
Score the positive and negative test trial videos on the day of testing to assess whether the mice have met the learning criteria. Ensure same day video scoring, since animals who meet learning criteria will undergo ambiguous tests the following day. Using event recording software or a stopwatch, let the researcher who is blind to treatment record each mouse's latency to dig and digging duration in each pot during the first minute of positive and negative test trials.
Record latency to dig as the time at which the first occurrence of digging is observed, and digging duration as the total time a mouse spends digging. Compare digging duration in the scented pot between positive and negative trials to determine whether mice can discriminate the task. Learning criteria are met if digging duration in the positive discriminative stimulus odor pot is at least double that for the negative discriminative stimulus odor pot during the first minute of testing.
For mice that have met the learning criteria, test responses to the ambiguous odor mixture. Perform one positive and one negative reinforced trial in a randomized order. Then, perform one video-recorded test trial using the ambiguous odor mixture.
For data analysis, plot the least-squared means of latency in each trial type to confirm that the ambiguous cue presented was interpreted as intermediate. While assessing judgment bias in both the one and two minutes of testing, the trial type by positive discriminative stimulus-odor interaction was significant. This reveals that mice with mint as their positive discriminative stimulus unexpectedly interpreted the ambiguous odor mixtures as positive, while mice with vanilla as their positive discriminative stimulus treated the same ambiguous odor mixtures as intermediate.
This finding indicated that only mice with vanilla as their positive discriminative stimulus met the technical requirement for treating the scent mixture as intermediate between the positive and negative discriminative stimulus. Thus, mice with mint as their positive discriminative stimulus were excluded from subsequent judgment bias analyses. Within mice with vanilla as their positive discriminative stimulus, housing influenced the digging latencies, with conventionally housed animals being slower to dig in ambiguous trials than environmentally enriched mice, but not for positive or negative trials.
These pessimistic interpretations of ambiguous cues by conventionally housed mice reflect negative judgment biases indicative of negative affect. While performing this protocol, it's important that technical criterion are met. The mice should meet learning criterion showing they're able to discriminate between cues, and interpret the ambiguous cue as intermediate.
The development of this new judgment bias task opens the door to test important hypotheses about mouse affective states with both animal welfare and clinical implications.
