Behavioral assays

BioProtocols Detailed Procedures Behavioral Assays

We only use littermates derived from harem breeding for our testing. No animals are ever singly housed and average cage density is ~2.8 mice/cage. Our behavioral data is derived from at least two separate cohorts of experimental mice with experimental and control mice being tested simultaneously and the experimenters blinded to genotype. All experiments were conducted at ZT6-12 with ZT0=lights on.

Once mice are obtained for a behavioral testing cohort proceed:

Open Field Arena (OFA): 

1. Clean a white Plexiglas OFA (40x40 cm²) prior to use with mild soap, water, and finally 70% ethanol. Stage the testing area (195-lux overhead lighting and 55-dB white noise) prior to bringing mice into the testing room.
2. Move subject mice from their home cage into their individual clean cages containing only bedding. Transfer the mice to a pretesting holding room to acclimate for 60 min before testing.
3. Move one mouse to the testing area and place it in one corner of the OFA, alternating the corner between subjects (ABCD).
4. Allow mouse to explore the arena for 10 min.
5. Data are collected using a video camera (LTC 0335, Bosch) and analyzed using the Ethovision XT video tracking system (Noldus, Wageningen, Netherlands), with the center zone defined as the area 10 cm from the arena walls.
6. Return mouse to its holding cage and clean arena with 70% ethanol in preparation for the next subject.
7. After the last subject is tested, return mice to their home cages and clean arena with mild soap, water, and 70% ethanol.
    

Elevated Plus Maze (EPM): 

1. Clean a white Plexiglas EPM (30-cm arm length) prior to use with mild soap, water, and finally 70% ethanol. Stage the testing area (195-lux overhead lighting and 55-dB white noise) prior to bringing mice into the testing room.
2. Move subject mice singly into a clean cage containing only bedding and transfer them to a pretesting holding room to acclimate for 60 min.
3. Move one mouse to the testing area and deposit mouse at the center of the arena using a bottomless cylinder.
4. Allow mouse to explore the arena for 5 min.
5. Data are collected using a video camera (LTC 0335, Bosch) and analyzed using the Ethovision XT video tracking system (Noldus, Wageningen, Netherlands), with each open and closed arm and the center square where arms intersect defined as testing zones.
6. Return mouse to its holding cage and clean arena with 70% ethanol in preparation for the next subject.
7. After the last subject is tested, return mice to their home cages and clean arena with mild soap, water, and 70% ethanol.
    

Morris Water Maze (MWM): 

Mice are trained over 8 days to locate a hidden escape platform 2-3 cm below the surface of a pool of opaque water using visual cues outside of the pool. After the probe test on day 8, a reversal phase is introduced on days 9-11 in which mice are similarly trained to locate the hidden platform in the opposite quadrant. On days 12-13, mice were tested for visual acuity using a visible escape platform. Data are collected and analyzed using Ethovision XT.
 

Set up prior to start:

1. Set up 24 hours prior to testing to allow water to equilibrate to room temperature.
2. Position the testing tank (112 cm diameter, 61 cm height; www.plastic-mart.com, product # A-ARM-19455) with a different visual cue centered above each quadrant outside of the tank.
3. Place the adjustable escape platform (Med Associates Inc., product # ENV-596M) in the center of one quadrant (quad A) and fill the tank with water until the water level is 2-3 cm above the platform.
4. Add white paint (egg-free, Little Masters Tempera Paint) to water until sufficiently colored white, stirring with a broom handle but avoiding the platform.
5. Set up Ethovision XT video tracking for recording and analyzing data. Make sure the video camera captures the full maze and that subjects are detected properly using a tester mouse unrelated to study with a similar coat. Define the four quadrants, escape platform, and equivalent platform location in the other three quadrants as zones in the maze.
6. Prepare empty holding/drying cages (one per subject) cleaned with 70% ethanol and labeled with subject ID.
    

Everyday:

1. Line the cleaned holding/drying cages with four ‘fluffed’ paper towels.
2. Check that the platform is 2-3 cm below the water line. Add water if necessary. Stir up pool but avoid moving the platform and remove lingering feces with aquarium net.
3. Stage the testing room (55 dB white noise, 550-585 lux overhead lighting) prior to bringing in mice.
4. Put mice in their respective holding/drying cages and allow them to acclimate to the room for 60 min before beginning training. Randomize cage position and mouse testing order every day.
5. Each subject will go through 4 training trials each day. A trial ends either at 60 s or if the mouse successfully locates the escape platform and stays on it for at least 2 s.
6. Upon completion of the day’s testing, return mice to their housing cages. Empty the holding/drying cages and clean with 70% ethanol.
    

Training:

1. Following acclimation, on day 1 only, hold each mouse by the tail on the platform for 15 s. Ensure that all four paws are flat on the platform (but if the mouse keeps kicking off the platform, front two paws are sufficient). After all subjects are shown the platform location, proceed to training trial 1. For all other days, begin with trial 1 following the acclimation period.
2. Create a random order of quadrant entry for the 4 trials each day (CADB, DCBA, etc.); this is the order in which mice are placed in the maze. Randomize the order every day. On the first two days of training, do not use the target quadrant (where the escape platform is located) as the entry point for the first trial.
3. To start a trial, begin recording and drop the mouse by its tail into the pool from 3 cm above the water near the wall of the tank at the quadrant midpoint, with the mouse facing the wall.
4. If the mouse finds the platform and stays on for 2 s minimum, retrieve and return it immediately to its holding cage to dry off. If the mouse leaps off after 2 s (before retrieval), hold it on the platform for 10 s before returning it to its holding cage. If the mouse does not locate the platform after 60 s, retrieve and hold the mouse on the platform for 10 s before returning it to its cage.
5. If the mouse floats for >2 s during the trial, prod it at a 45-degree angle near its mid to lower back.
6. Test/train the next subject. After all subjects have completed trial 1, proceed to trial 2 and repeat the procedure until trial 4 is done. Ensure at least 15-30 min have elapsed since the mouse’s last trial before its next trial begins.
7. Repeat for eight consecutive days of training. After day 7, analyze escape latency data to determine if mice for a group have met training criteria of 50% of day 1 latency or <12 s to reach the platform. If a group meet one of these criteria, continue with training followed by a probe test on day 8. If a group fails to reach criterion, add additional training days until either criteria is met before performing the probe test. 
    

Probe test:

1. On day 8 (or later when a training criterion is reached) after all subjects have completed training trial 4, remove the platform from the maze and allow water to settle.
2. After 30-60 min has passed since the subject completed training trial 4, drop the mouse (facing and near the tank wall) into quad C, the quadrant opposite of the target quadrant where the escape platform had been located for training.
3. Each subject is given 60 s to swim in the maze and behavior is recorded before it is returned to its holding cage.
4. Analyze time spent swimming in each quadrant, the number of platform location crossings, and swim distance.

Reversal platform training:

1. After the probe test, move the escape platform to the respective location in quad C (opposite of the target quadrant).
2. Beginning the day after the probe test, train mice to find the new platform location using the same process as above (Training #2-6) over three consecutive days minimum.
3. After the third day, analyze data to determine if latency to find platform is 50% less than on the first day of reversal training. If mice do not meet this criterion, proceed with 2 more days of reversal training.
    

Visible platform training:

1. After successful reversal training, proceed with visible platform training for the next two days.
2. Move escape platform to the respective location in an adjacent quadrant (quad B) and add a ‘flag’ (plastic-coated thick electrical wire twisted into the appropriate shape) that sticks out of the water above the hidden platform (10 cm above).
3. Train mice to find this platform location using the same process as above (Training #2-6). If the mouse climbs on the flag, this counts as a successful trial to find the platform, so retrieve and return mouse immediately to its holding cage (do not hold on the platform).
    

Fear conditioning

This protocol applies to using Coulbourn Instruments isolation cubicles and shock equipment with the Actimetrics FreezeFrame software to measure freezing behavior:

ISOLATION CUBICLE, WIDE (ID: 30" W X 17.75" D X 18.5" H)

FREEZEFRAME 4 SOFTWARE (SHOCK CONTROL EXPANSION KIT, LIGHT/TONE CONTROL EXPANSION KIT, FAN/HOUSE LIGHT CONTROL KIT, AUDIO EXPANSION KIT)

OPTIONAL IR ILLUMINATOR - MOUSE

FAN MODULE - MOUSE

A12-33 -> PROG. TONE/NOISE GENERATOR 110V

H13-15 -> PRECISION ANIMAL SHOCKER 110V

SONY STR-DH130: https://www.sony.com/electronics/av-receivers/str-dh130

SPEAKER MODULE - MOUSE
 

Everyday:

1. Turn on equipment and open appropriate program.
2. Stage the testing room (55-dB white noise, 550-585 lux overhead lighting) prior to bringing in mice.
3. Move subject mice from their home cage into their designated individual holding cages containing only bedding and labeled with subject ID. Transfer the mice to a pretesting holding room to acclimate for 60 min before beginning training or testing.
4. Set up fear conditioning chambers as described below for each phase.
5. Clean chambers between subjects as described below for each phase.
6. After all subjects have been trained or tested, return mice to their home cages. Reuse the same holding cages until the end of the study.
7. Clean fear conditioning chambers: rinse drop pans thoroughly with water followed with 70% ethanol; clean shock/floor grids with water followed by 70% ethanol; and clean test cages with 70% ethanol. Leave chambers open to air out.
8. Turn off all equipment.
    

Day 1: Training

Mice are trained with two pairings of a tone (CS, 30 s, 85-dB white noise) and foot-shock (US, 2 s, 0.5 mA) presentation in a 5-min session.

1. Set stereo volume to 60 (Sony Amplifier); use Sd/cb (85-dB white noise).
2. Prior to start, set up the shock grid and drop pan in each fear conditioning chamber.
3. Check that shock grids are functional by placing a wet sponge on each grid while connected to the shock distributor cable and testing each “Animal Shocker” box to make sure that current is set at 0.5 mA.
4. Open the program for 2 CS-US training.
5. Manually adjust the camera in each chamber for optimal video brightness and resolution to track mice. Use tester mice unrelated to the study with a similar coat to subject mice and adjust cameras accordingly. Note camera settings.
6. Clean the test cage, shock grid, and drop pan in each chamber with 70% ethanol. 
7. Set up the fear conditioning chambers for training (Context A):
   • House light on
   • White light beneath the house light is screwed in but not turned on
   • Clean each test cage, shock grid, and drop pan with 70% ethanol
   • Add peppermint odor (Dr. Bronner’s soap) to each drop pan (enough to coat the surface in a thin layer) and place beneath shock grid
8. Train the first set of mice by placing one in each chamber and running the program for 2 CS-US training:
   • 0-120 s, baseline period with no tone or shock (pre-CS)
   • 120-150 s, tone on (CS1)
   • 148-150 s, shock on (US1)
   • 150-210 s, no tone or shock
   • 210-240 s, tone on (CS2) 
   • 238-240 s, shock on (US2)
   • 240-300 s, no tone or shock
9. Retrieve mice after the 5-min training session and return to their holding cages.
10. Prepare chambers for the next set of mice: rinse drop pans thoroughly with water before cleaning with 70% ethanol and adding peppermint odor; clean shock grids with water followed by 70% ethanol; and clean test cages with 70% ethanol. 
11. Analyze percent time spent freezing prior to the first CS presentation (pre-CS) to determine baseline freezing and then percent time freezing during the 30 s after each CS presentation to assess learning.
     

Day 2: Contextual and Cued Long-Term Memory (LTM) testing

In contextual LTM testing, mice are re-exposed ~24 h later to the training context from Day 1 and behavior is recorded for 5 min. In cued LTM testing, mice are re-exposed ~24 h later to the CS in a novel context using two 30-s CS presentations given 1 min apart in a 5-min session. 

1. Plan out mouse test order to counterbalance testing contextual or cued LTM first, with one hour between the two tests for any given set of mice.
2. Prior to start, set up the fear conditioning chambers as though for cued testing: add white acrylic floor over shock grid, plastic inserts with different display patterns or textures over test cage walls, and open the cued testing program.
3. Manually adjust the camera in each chamber for optimal video brightness and resolution to track mice in the cued testing environment. Use tester mice unrelated to the study with a similar coat to the subject mice and adjust cameras accordingly. Note camera settings. Taking note of these camera settings and those from Day 1 will enable alternating between cued and contextual testing, respectively, more easily.
4. Set stereo volume to 60 (Sony Amplifier); use Sd/cb (85-dB white noise).
5. Following acclimation, proceed with contextual or cued testing as described below.
6. When switching between contextual and cued tests, allow 15-20 minutes so the previous odor can air out and the new odor can permeate the chambers before testing.

Contextual testing:

1. Set up fear conditioning chambers with Context A.
2. Adjust camera settings for appropriate video brightness and resolution.
3. Place mice in the chambers and record their behavior using the contextual testing program:
   • 0-300 s, no tone or shock
4. Retrieve mice when the program finishes and return them to their holding cages.
5. Clean and prepare chambers for contextual or cued LTM testing of the next set of mice.
6. Contextual memory is measured as percent time spent freezing during the 5-min test.
    

Cued testing: 

1. Set up the fear conditioning chambers to provide a novel environment (Context B): 
   • House light off
   • Switch white light below house light to a red light and turn it on
   • Infrared light on
   • Add white acrylic floor over shock grid
   • Add plastic inserts with different display patterns or textures over test cage walls
   • Clean with 70% isopropanol
   • Add vanilla odor (spread a thin layer of vanilla extract on the drop pan)
2. Adjust camera settings for appropriate video brightness and resolution.
3. Place mice in the chambers and record their behavior using the cued testing program:
   • 0-120 s, baseline period with no tone (pre-CS)
   • 120-150 s, tone on (CS1)
   • 150-210 s, no tone
   • 210-240 s, tone on (CS2) 
   • 240-300 s, no tone
4. Retrieve mice when the program finishes and return them to their holding cages.
5. Clean and prepare chambers for contextual or cued LTM testing of the next set of mice.
6. Analyze percent time spent freezing during the baseline period (pre-CS) and during the two CS presentations to assess cued memory.
    

Fear Extinction

Continuing from the fear conditioning protocol described above, mice are trained to extinguish the cued fear starting on day 7 for three consecutive days with a 35-min session each day. Each training session consists of a 120-s baseline period, followed by twenty 30-s CS exposures at varying intervals. On day 10, extinction LTM was assessed like cued LTM by re-exposing mice to the CS in a novel context using two 30-s CS presentations given 1 min apart in a 5-min session. On day 20, renewal of conditioned fear was performed by re-exposing mice to the CS in the original training context using the same protocol as for training except no US was delivered.
 

Extinction training: 

1. Continue the procedure for Everyday (described above in Fear Conditioning).
2. Set up the fear conditioning chambers with Context B.
3. Adjust camera settings for appropriate video brightness and resolution.
4. Set stereo volume to 60 (Sony Amplifier); use Sd/cb (85-dB white noise).
5. Place mice in the chambers and record their behavior using the extinction training program:
   • 0-120 s, baseline period with no tone (pre-CS)
   • CS1-CS20, tone on for 30 s each with randomized intertrial intervals of 20-120 s in duration
6. Retrieve mice when the program finishes and return them to their holding cages.
7. Prepare chambers for the next set of mice. 
8. Analyze percent time spent freezing during the baseline period (pre-CS) and during each CS presentation.
    

Extinction testing: 

1. Follow Everyday procedure (under Fear Conditioning).
2. Set up the fear conditioning chambers to provide a novel environment (Context C): 
   • House light on
   • Switch to white light below house light and turn it on
   • Use a wire mesh non-shock floor
   • Add white plastic inserts over test cage walls
   • Clean with 70% ethanol
   • Add lavender odor (Dr. Bronner’s soap) to each drop pan (enough to coat the surface in a thin layer) and place beneath wire mesh floor
3. Adjust camera settings for appropriate video brightness and resolution.
4. Set stereo volume to 60 (Sony Amplifier); use Sd/cb (85-dB white noise).
5. Place mice in the chambers and record their behavior using the extinction testing program:
   • 0-120 s, baseline period with no tone (pre-CS)
   • 120-150 s, tone on (CS1)
   • 150-210 s, no tone
   • 210-240 s, tone on (CS2) 
   • 240-300 s, no tone
6. Retrieve mice when the program finishes and return them to their holding cages.
7. Prepare chambers for the next set of mice. 
8. Analyze percent time spent freezing during the baseline period (pre-CS) and during the CS presentations.
    

Renewal testing: 

1. Follow Everyday procedure (under Fear Conditioning).
2. Set up the fear conditioning chambers with Context A.
3. Adjust camera settings for appropriate video brightness and resolution.
4. Set stereo volume to 60 (Sony Amplifier); use Sd/cb (85-dB white noise).
5. Place mice in the chambers and record their behavior using the renewal program:
   • 0-120 s, baseline period with no tone (pre-CS)
   • 120-150 s, tone on (CS1)
   • 150-210 s, no tone
   • 210-240 s, tone on (CS2) 
   • 240-300 s, no tone
6. Retrieve mice when the program finishes and return them to their holding cages.
7. Prepare chambers for the next set of mice. 
8. Analyze percent time spent freezing during the baseline period (pre-CS) and during the CS presentations.

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