Handling Techniques to Reduce Stress in Mice

Podsumowanie

This paper describes a handling technique in mice, the 3D-handling technique, which facilitates routine handling by reducing anxiety-like behaviors and presents details on two existing related techniques (tunnel and tail handling).

Streszczenie

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects on animal well-being and can also be a confounding factor for experimental variables such as anxiety measures. Over the years, handling techniques that minimize handling-related stress have been developed with a particular focus on rats, and little attention to mice. However, it has been shown that mice can be habituated to manipulations using handling techniques. Habituating mice to handling reduces stress, facilitates routine handling, improves animal wellbeing, decreases data variability, and improves experimental reliability. Despite beneficial effects of handling, the tail-pick up approach, which is particularly stressful, is still widely used. This paper provides a detailed description and demonstration of a newly developed mouse-handling technique intended to minimize the stress experienced by the animal during human interaction. This manual technique is performed over 3 days (3D-handling technique) and focuses on the animal's capacity to habituate to the experimenter. This study also shows the effect of previously established tunnel handling techniques (using a polycarbonate tunnel) and the tail-pick up technique. Specifically studied are their effects on anxiety-like behaviors, using behavioral tests (Elevated-Plus Maze and Novelty Suppressed Feeding), voluntary interaction with experimenters and physiological measurement (corticosterone levels). The 3D-handling technique and the tunnel handling technique reduced anxiety-like phenotypes. In the first experiment, using 6-month-old male mice, the 3D-handling technique significantly improved experimenter interaction. In the second experiment, using 2.5-month-old female, it reduced corticosterone levels. As such, the 3D-handling is a useful approach in scenarios where interaction with the experimenter is required or preferred, or where tunnel handling may not be possible during the experiment.

Wprowadzenie

Mice and rats are essential assets to preclinical studies^1,2 for multiple purposes, including endocrinal, physiological, pharmacological or behavioral studies². From the increasing number of studies involving animals, it arose that uncontrolled environmental variables including human interaction influence various outcomes in biomedical research^3,4,5. This is responsible for significant variability observed across experiments and research laboratories^4,5, posing a major caveat in animal research.

Various approaches have been implemented with the goal of limiting the impact of environmental stressors and reducing reactivity to human interaction. For example, to limit the impact of environmental stressors, standardization of housing conditions and automated housing systems^6,7 have been implemented across laboratories. Regarding interaction with human beings, commonly used approaches for handling and transporting animals had little regard for animal discomfort and stress. For instance, picking up animals by their tail or using forceps⁸ increases baseline anxiety^9,10,11, reduces exploration^9,12 and contributes greatly to inter-individual variability within and across studies^13,14. As a result, other approaches were developed, such as the cup handling technique, which is applicable to mice and rats. In this approach, the animals are "cupped" out of their cage, and held by the experimenters with their hands forming a cup^9,10,11. Another useful alternative to tail handling involves the use of a polycarbonate tunnel to transfer mice^9,10,15. This approach eliminates direct interaction between the mouse and the experimenter. Both the cup and tunnel approaches showed efficacy in reducing anxiety-like behaviors and fear of the experimenter that can be exaggerated by aversive handling techniques, such as tail pick up/tail handling^9,10.

Therefore, increasing evidence demonstrates the usefulness of proper mouse handling for reducing variability between individuals^9,11, and improving animal welfare¹⁰. However, the techniques mentioned above are still faced with limitations. The cup handling technique has been implemented with schedules ranging from 10 days (10 sessions over 2 weeks¹⁶) up to 15 weeks¹⁷, which is a considerable amount of time for facility staff and experimenters. Additionally, the effectiveness of cup handling varies by strain⁹ and conventional cup handling in open hands may lead to naïve mice or particularly jumpy strains to jump from the hand^9,18. Tunnel handling results in more consistent and generally quicker results in gentling¹⁹. Tunnels are also used as home cage enrichment. They help animals habituate to handling quickly and provide the added benefits of enrichment. Tunnel handling, however, has limitations when transferring animals between apparatuses. Interestingly, Hurst and West⁹, and Henderson et al.²⁰ demonstrated that using gentle and brief manual handling to transfer animals from the tunnel to the apparatus does not affect their phenotype.

To provide an alternative to existing methods, with achievable habituation in a short period of time, this article describes a novel technique that expands on the cup handling technique, therefore requiring no particular equipment. This approach uses milestones to gauge the level of comfort mice have with the handling process. It shows efficacy at decreasing mouse reactivity and stress (at the behavioral and hormonal levels), facilitates routine handling and contributes to reducing variability between animals. Details of this technique are provided here, and its efficacy at reducing anxiety-like behaviors, improving interaction with experimenters, and limiting peripheral stress-hormone (corticosterone) release are demonstrated in two separate studies (male and female mice), in comparison with tunnel handling (positive control) and tail handling techniques (negative control).

Protokół

Procedures involving animal subjects were approved by the CAMH animal care committee and conducted in compliance with the Canadian Council on Animal Care guidelines.

NOTE: The handling method described herein can be used in various mouse strains, including non-transgenic (C57/BL6, BalbC, CD1, SV129, etc.) and transgenic lines. It can also be used with young or old mice, noting that young adult (4-6 weeks old) mice tend to be slightly more active than adult or old mice, especially on day 1.

1. Experimental preparation

1. Prior to study initiation, as per ARRIVE guidelines²¹, randomly assign mice to each handling group (3D-Handling, Tunnel Handling or Tail Handling).
2. Identify the room to perform the handling. It can be performed in the housing room, or in a separate room. If the handling is performed in a separate room, which requires the animals to be moved on a moving cart, allow the animals to habituate to the new room for 20-30 min prior to initiation of the handling protocol.
3. For group housed-animals, use a temporary cage to house mice after the handling, before regrouping them all in their initial home cage. This reduces potential fights between animals prior to handling (particularly in males).
4. Work on a counter (preferably a cleared countertop) or in a biosafety cabinet, with the housing cage away from the animal being handled. Close proximity to the housing cage increases the risk of jumping. If animals are group-housed, jumping of the mouse being handled into the home cage may cause stress to cage-mates.
   ​NOTE: Working in a biosafety cabinet limits the risk of mice jumping on the floor, and can be required in certain facilities. This technique can be used in a biosafety cabinet, making sure to always perform all steps inside the biosafety cabinet, and avoiding mice walking on handler forearms.

2. DAY 1: 5 min per mouse

1. Gently open the cage and place the lid on the side, remove nesting materials, and other enrichment such as running wheels or shelters.
2. Introduce a gloved open hand to the home cage, slowly placing the hand along one side of the cage wall (the wall closest to the handler, Figure 1A).
   1. Do not immediately try to pick up the mouse.
3. Remain immobile and allow the animal to habituate to the presence of the hand in the cage for about 30 s.
4. Attempt to pick up the mouse in the palm of the hand (i.e., avoid picking up the animal by its tail).
   1. If the mouse is not easily picked up after 3 attempts, guide the mouse to a corner and cup with both hands.
   2. Gently move the cupped hands towards the mouse to try to pick it up.
   3. If unsuccessful after a maximum of 3 attempts with both hands, pick up the mouse gently by the base of its tail, and transfer it to your forearm or flat hand.
5. With the mouse in the hand, keep the hand as flat and open as possible.
   NOTE: This provides a flat platform for the mouse to step onto, and limits the risk of bites.
6. Holding the hand open and flat with palm up, place the other hand adjacent to the hand holding the mouse and allow the mouse to move freely from hand to hand without any restraint (Figure 1B).
7. Let the mouse explore and move between hands for 1 min.
   1. At this point mice may try to jump away. Position the hands such that if the mouse jumps, it will land on a countertop rather than the floor.
   2. If a mouse looks like it is preparing to jump (moving towards the edge of the hand and rearing on hind legs), slowly place the other hand in front of it and try to guide it into walking onto this hand. Avoid sudden movements as it increases their risk of jumping.
   3. If a mouse does jump, attempt to pick it up avoiding tail handling and resume the handling session. If the mouse stays on the floor or out of the hands for more than 10 s, add additional time to the handling session to make up for any time the mouse was out of the hands.
   4. Take notes of the jump. Total number of jumps can be used to assess potential variability between animals.
8. After 1 min of handling with flat hands, relax the palm of the hand, and slightly cup the mouse in the hand, prior to gently rolling the mouse between hands (Figure 1C).
   1. To "roll", position the mouse in the palm of the hand, on a flat hand, perpendicular to fingers.
   2. Slowly close the hand, placing the fingers on the back of the mouse.
   3. Place the free hand directly under the hand holding the mouse.
   4. Slowly turn/rotate the hand with the mouse to gently transfer the mouse to the other hand (180° flip).
   5. Repeat this back and forth between hands.
9. Alternate from gentle rolling between hands and free exploration on open hands for 60 s, alternating between techniques about every 20 s.
10. Perform a "shelter test" (Figure 1D).
    1. Let the mouse move to the edge of the hand then bring the 2 hands together.
    2. Very slowly, cup them so the mouse fits inside a "shelter" formed by the hands. Leave an opening so the mouse can escape if needed.
    3. Aim to keep the mouse in the shelter for 5-10 s, without any restraint.
    4. Alternate between the shelter test, roll between hands and free exploration of open hands for another 60 s, being sure to perform the shelter step 3 or more times.
11. In all procedures described in 2.10, do not rush the process. If the mouse appears stressed (i.e., tentative to escape, jumps from the hands, avoiding contact with hands etc.) by being confined inside the hands, continue with rolling between hands and free exploration for 20 s, and then retry.
12. Milestone: Perform at least 1 successful shelter test of 10 s for completion of Day 1.
    1. Consider a shelter test successful when the mouse stays in the hands. If the mouse pops its head out and returns to the shelter, it is still a successful test. If the animal entirely exits from the shelter, it is a failure.
13. Allow free exploration in hands for 30 s.
14. Gently replace the mouse in its cage. If group housed, place the mouse in the temporary cage until all cage mates are handled. Return the mice to their original cage by picking them up in the palm of the hand. Do not use a tail pick up.
15. Clean the bench top of potential feces and urine with 70% ethanol.
16. Rinse gloves thoroughly with 70% ethanol (or appropriate cleaning solution) or change gloves prior to handling the next mouse (it is possible to keep the same gloves for cage mates).
    ​NOTE: It is recommended to perform the handling with a reasonable number of animals to avoid fatigue from the handler. Handling 24 mice takes around 2 h and it is recommended to not exceed 24 mice per handler. If more animals need to be handled, it is recommended either to have multiple handlers, or to split the handling procedures into subgroups, over multiple days.

3. DAY 2: 3 to 5 min per mouse

1. Attempt to pick up the mouse in the palm of the hand. At this stage, it should be already feasible and mice should not jump out of the hand.
2. Start with palm open as on Day 1, allowing the mouse to explore freely for 20 s.
3. Then, roll the mouse between hands a few times (4-5 times).
4. Perform the "shelter test" for 5 s.
5. Repeat the shelter test several times (~5-6) over a 2 to 3 min period.
6. During the same 2 to 3 min period, alternate with the roll between hands and free exploration of open hands step from day 1 to improve habituation.
   1. Touch the mouse on its head and back (Figure 1E), 5-6 times. A sign of habituation is when the mouse lets you touch it without attempting to escape.
   2. Perform a "Nose poke": Try to touch the snout of the mouse, 2 to 3 times (Figure 1F).
      1. If the mouse attempts to bite or shows obvious signs of stress at being touched, do not immediately attempt the nose poke again. Instead, alternate with flat hand exploration and roll. "Habituation" is reflected by the animal not running away or turning its head in cases of human contact.
7. In all procedures described in 3.4-3.6, do not rush the process. If the mouse appears stressed by being confined inside the hands or does not want to be touched, continue with rolling between hands for 20-30 s and then retry.
8. Milestones: Perform at least 1 successful nose poke for 2-3 s for completion of Day 2.
9. Stop this session after about 3 min of handling if the animal reacts well to the "shelter", "head petting", "nose poke", and if the mouse appears to be willing to explore the hands without signs of stress.
10. If the mouse continues to exhibit signs of stress or is not reacting well to the "shelter test" or "nose poke" test, continue the session until reaching 5 min as in Day 1.
11. Replace the mouse in its cage, clean the bench top and gloves as in Day 1.

4. DAY 3: Around 3 min per mouse

1. On the third day, proceed through the same steps as in Day 2, for 2 to 3 min.
   1. Pick up the mouse in the palm of the hand.
   2. Transfer and roll the mouse between hands
   3. Perform a shelter test.
   4. Try to pet the mouse on the back and head.
2. Alternate between these steps over approximately 1 to 2 min.
3. Continue the procedure until the mouse is relaxed enough to sit in the palm of the hand without attempting to escape.
4. Before the end of Day 3, repeat the shelter test and nose poke test as a test of habituation.
   1. If both tests can be completed on their first attempt, the habituation process is complete. Continue gently handling the mouse for 30 s to a minute.
   2. If the mouse is initially resistant to either test, repeat steps 4.1-4.3 for 20-30 s before reattempting the nose poke and shelter test.
   3. If the mouse remains resistant to these tests after 3 min, the third day may be repeated.
5. Milestones: Perform at least 2 successful shelter tests of 10 s each, and 2 successful nose poke test for completion of Day 3, and completion of the entire 3D-handling procedure.
6. Return the mouse to its cage, clean the bench top and gloves.

5. Optional approach for animals to be subjected to restraint for injection or gavage

NOTE: On Day 3, if the animal will be restrained for experimental purposes (oral gavage, intra-peritoneal injection, etc.), the mice can be subjected to the neck pinch test.

1. Grasp the nape of the neck between the thumb and forefinger (Figure 1G).
2. Lift the mouse 3-5 cm above the hand for 2-3 s.
   NOTE: This is normally a non-natural position for adult mice, and if the mice remain near immobile, they are well habituated to handling and will be easy to restrain for experimental purposes.
3. Place the mouse back on in the flat hand, or if the mouse is reactive to the neck pinch, consider placing it on the experimenter's sleeve, cage lid or countertop
   NOTE: If working in a biosafety cabinet, do not place the mouse on the sleeve or it could walk up and exit the biosafety cabinet. Prefer placing the mouse on the countertop inside the biosafety cabinet.
4. Leave the mouse to freely explore the experimenter's hand for 1 min.

6. Optional approach for additional days of handling

1. In the eventuality of a highly stressed mouse line, add additional days to decrease the reactivity and stress level of the animals, using the methods described in Day 2/3.
   ​NOTE: Many factors can affect baseline stress of the animals including strain, presence of transgenic modification, age, sex and housing conditions. If these factors are not consistent between groups such as aged animals being tested against young controls or transgenic animals being tested against wild type controls, it is recommended that the same number of days of habituation are used for each group.

7. Tunnel handling

NOTE: This technique is applicable only to the Tunnel-handled mice. Tunnels are polycarbonate tubes approximately 13 cm in length and 5 cm in diameter.

1. Place the tunnel in the cage of the mouse.
2. Leave the tunnel in the cage for 7 days prior to handling.
3. Open the cage and place the lid on the side.
4. Gently guide the mouse into the polycarbonate tunnel (already in the cage).
5. Lift the tunnel from the cage, horizontally. If necessary loosely cover the ends of the tunnel to prevent the animal from jumping/falling out of the tunnel, potentially falling back in its cage or on the floor.
6. Move the animal in the tunnel away from the home cage and hold it away from any surfaces for 30 s.
7. Place the tunnel back in the home cage, allowing the mouse to exit the tube.
8. Wait for 60 s and then repeat steps 7.4-7.7 once.
9. Rinse gloves thoroughly with 70% ethanol or change gloves prior to habituating the next mouse.
10. Repeat this procedure for 10 consecutive days.

8. Tail handling

NOTE: This technique is applicable only to the Tail-handled mice. It is used to transfer mice from their cage to an apparatus, and vice-versa.

1. Open the cage and place the lid on the side.
2. Grasp the mice by the base of the tail between thumb and forefinger.
3. Lift the mouse from the cage.
4. In 2-3 s, transfer mouse to the experimenter's opposite forearm while maintaining a grip on the tail to avoid the mouse dangling.
5. When tail handling is required in the implementation of this experiment (e.g., before blood draws for cortisol testing) animals are transferred to the experimenter's forearm by tail handling and held for 15 s before being returned to their cage.

9. Elevated Plus Maze

1. Room setup
   1. Place the maze in the middle of the room, under a digital camera equipped with a memory card.
   2. Set up the light of the room at ~60 Lux using 2 standing lamps placed behind the maze.
   3. Turn off any overhead lighting to avoid direct light on the maze that creates reflection and disrupts the detection of the animals in the maze.
   4. Once all the equipment is set up, transfer the animals to the room and let them acclimatize to the light settings and the new environment for 30 minutes.
2. Testing
   1. Clean the maze with 70% ethanol to prevent smells from dust or from the animal that was tested previously.
   2. Start the camera.
   3. Use a piece of paper with the animal ID to record the ID on the video, prior to placing the animal in the maze (this will facilitate the proper identification of which mouse is being filmed on each video).
   4. Use the appropriate handling technique to each animal to transfer it to the maze.
   5. Place the mouse on the central platform, facing an open arm.
   6. Allow the mouse to explore the apparatus for 10 min, undisturbed.
   7. After 10 minutes, stop the camera.
   8. Retrieve the mouse from the maze and put it back in its cage.
   9. Clean feces and urine from the maze with 70% ethanol.
   10. Once testing is complete with all mice, transfer videos from the memory card to a computer for video tracking.
   11. Using automated animal tracking software, track the number of entries to the open and closed arms, and the time spent in open or closed arms (here Ethovision XT 14).

10. Experimenter Interaction (derived from Hurst and West⁹)

1. Room setup
   1. Place a table in the middle of the testing room under a digital camera equipped with a memory card.
   2. Set up the light at 50-70 Lux with 4 light bulbs placed in the corner of the room facing up to the ceiling. Turn off overhead lighting to avoid direct light on the maze that creates reflection and disrupt the detection of the animals in the arena.
   3. Bring the animals to the room.
   4. Let them acclimatize to the room for 30 minutes.
2. Experiment
   1. Place the home cage under the digital camera.
   2. Remove the lid.
   3. Remove nesting material and other enrichment that might interfere with tracking of animals.
   4. Start the camera.
   5. Use the cage card with the animal ID to identify the animal on the video.
   6. Place a hand in the home cage along the wall of the cage in the front right side.
      1. Ensure that the handler's head is not blocking the camera to film the mouse.
   7. Start a timer.
   8. Keep the hand immobile for 2 minutes, and let the mouse explore the hand.
   9. Remove the hand from the cage for 15 s.
   10. Attempt to pick up the mouse using cupped hands and record whether the mouse flees.
   11. Repeat the last step up to five times, every 5 seconds, or until the mouse allows itself to be picked up.
   12. Record the number of attempts required to pick up the mouse.
   13. Return nesting material and enrichment to the cage.
   14. Clean gloves with 70% ethanol or change gloves before proceeding to the next animal.
   15. After testing, transfer videos from the memory card to a computer.
   16. Using automated video tracking software, divide the cage into four equal quadrants and record the time spent by the mouse in each quadrant (here, Ethovision XT 14).

11. Novelty Suppressed Feeding

1. Food deprivation
   1. 3 days prior to the test, perform a full cage change, and single house the animals (single housing is preferable to perform the home cage testing).
      NOTE: Providing fresh bedding removes potential dust or little pieces of food accumulating in the bedding since last cage change.
   2. The day before testing, weigh all animals around 6 pm.
   3. Remove all food from the food hopper, and ensure that there are no pieces of food in the cage or in the bedding.
2. Room setup
   1. Place the NSF chamber on a table.
   2. Fill the chamber with a thin layer of corn bedding (or other bedding that is different from bedding used in animals home cage).
   3. Set up the light at 70 Lux with 4 light bulbs placed in the corner of the table where the chamber stands, facing up to the ceiling. Turn off overhead lights to maintain low room lighting.
   4. Place one pellet of standard chow used in the facility, on the side of the chamber facing the experimenter (≈10 cm from the wall).
3. Testing
   1. In the morning after food deprivation, bring the animals to the room 30 minutes prior to testing to let them acclimatize to the light settings and the new environment.
   2. Weigh all animals in order to measure their weight loss based on the weight measured the previous day. Animals should lose 8-12% overnight to be able to perform the task properly.
   3. Sort the animals per weight loss, and screen them starting from the mouse that lost the most to the mouse that lost the least weight.
   4. Ensure that the chamber is filled with bedding and with a single pellet.
   5. Place the animal on the opposite side of the chamber, away from the food pellet.
   6. Start the timer immediately.
   7. Let the mouse explore the chamber for up to 12 minutes.
   8. Measure the latency to approach and feed (animal must bite and eat) on the food pellet.
      1. Consider it to be an approach when the animal comes close to the pellet, smells it and does not bite.
      2. Define a bite as when the animal starts consuming the pellet.
   9. Record the latency to approach and feed on the pellet in seconds.
   10. Once the mouse has fed on the food pellet, remove the mouse from the chamber.
   11. Discard the bedding but save the pellet that will be used for testing appetite drive in the mouse home cage.
   12. Reset the chamber for the next animal and proceed with the next animal.
   13. 15 min after completion of the test in the chamber, drop the pellet used during the test, inside the home cage of the mouse, against the wall at the front of the cage.
   14. Measure the latency to feed on the pellet when the pellet is in the home cage. This is a measure for appetite drive.
       1. It is preferable to remove the nesting material to ensure that the mouse sees the pellet being dropped in its cage.

12. Serum Collection and Corticosterone Measurement

1. Handle animals for 1 min using the assigned technique, 15 min prior to blood collection (this can be done with group housed or single housed animals, keeping in mind the risk for fights when regrouping mice).
   1. For the tunnel handled mice, guide them to the tunnel, lift the tunnel from the cage for 1 min, and replace the mouse in its cage.
   2. For tail handled mice, grab the tail base of the mouse and remove the mouse from its cage. Transfer the mouse to the experimenters sleeve for 1 min, and return the mouse to its cage by tail handling.
   3. For 3D-handled mice, use cupped hands to remove the mouse from its cage. Hold the mouse in cupped hands for 1 min, and return it to its cage.
2. 15 min after handling, proceed with blood collection from the submandibular vein²².
3. Firmly scruff the mouse such that the head of the mouse is securely immobilized.
4. Locate the site of puncture.
   1. There is a small hairless dimple along the mandible of the face that can be used as a landmark to locate the puncture site. Drawing a line between the base of the jaw and this dimple the puncture site lies behind this dimple towards the ear by roughly 5 mm, just behind the hinge of the jaw.
5. Hold a clean 23 G needle perpendicular to the puncture site and use a quick firm lancing motion. The tip of the needle should penetrate to a depth between 1-2 mm, blood will flow immediately as soon as the vein is punctured.
6. Collect ~150 µL of blood in EDTA coated collection tubes and store on ice.
7. Apply slight pressure with a sterile gauze pad to the puncture site for 5 s or more to allow the blood to clot.
8. Once blood has clotted, return the mouse to its home cage.
9. Centrifuge blood at 4 °C 3,500 x g for 10 min.
10. Decant the supernatant.
11. Store the supernatant at -20 °C for downstream analyses.
12. Measure corticosterone levels using a corticosterone ELISA kit following manufacturer's protocol.
13. Use a spectrophotometer to read the ELISA outcomes.

Wyniki

Two separate studies were performed with C57BL/6 mice. Study #1 included 6-month-old males and Study #2 included 2.5-month-old females (N=36/study) from Jackson Laboratories (Cat #000664). Mice arrived in the facility at the age of 2 months. While Study #2 females were handled and tested two weeks after arrival, Study #1 males were only handled and tested at the age of 6 months (delay due to global pandemic shutdown). During this time, one mouse from Study #2 died, prior to starting handling experiments. The Study #1 mal...

Dyskusje

This study and method development are based on the observation that handling techniques in mice are still overlooked by the scientific community, and that some labs are still reluctant to implement habituation or handling techniques to reduce stress and reactivity of their animals prior to experiments. While representing a time commitment, animal handling provides beneficial effects to the animals that may contribute to the success of the experiments to be performed and prevents experiments from having to be performed mu...

Materiały

Name	Company	Catalog Number	Comments
23 G x 1 in. BD PrecisionGlide general use sterile hypodermic needle. Regular wall type and regular bevel.	BD	2546-CABD305145	Needles for Blood collection
BD Vacutainer® Venous Blood Collection EDTA Tubes with Lavender BD Hemogard™ closure, 2.0ml (13x75mm), 100/pk	BD	367841	EDTA Coated tubes for blood collection
Bed’o cobs ¼” Corn cob laboratory animal bedding	Bed-O-Cobs	BEDO1/4	Novel bedding for novelty suppressed feeding
Centrifuge	Eppendorf	Centrifuge 5424 R	For centrifugation of blood.
Corticosterone ELISA Kit	Arbor Assays	K003-H1W
Digital Camera	Panasonic	HC-V770	Camera to record EPM/Experimenter interactions
Elevated Plus Maze	Home Made	n/a	Custom Maze made of four black Plexiglas arms (two open arms (29cm long by 7 cm wide) and two enclosed arms (29 cm long x7 cm wide with 16 cm tall walls)) that form a cross shape with the two open arms opposite to each other held 55 cm above the floor
Ethanol	Medstore House Brand	39753-P016-EA95	Dilute to 70% with Distilled water, for cleaning
Ethovision XT 15	Noldus	n/a	Automated animal tracking software
Laboratory Rodent Diet	LabDiet	Rodent Diet 5001	Standard Rodent diet
Memory Card	Kingstone Technology	SDA3/64GB	For video recording and file transfer
Novelty Suppressed Feeding Chamber	Home Made	n/a	Custom test plexiglass test chamber with clear floors and walls 62cm long, by 31cm wide by 40cm tall .
Parlycarbonate tubes	Home Made	n/a	13 cm in length and 5cm in diameter
Purina Yesterday’s news recycled newspaper bedding	Purina	n/a	Standard Bedding
Spectrophotometer	Biotek	Epoch Microplate Reader