Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

Summary

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

Abstract

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

Introduction

The purpose of this protocol is to measure spatial learning and memory in mice using the Barnes maze as well as social responsiveness and locomotion using the Damsel-in-Distress paradigm. Commonly used spatial learning and memory tests for rodents include the radial arm maze, which measures a mouse's ability to find hidden food in one arm of a multi-spoked apparatus, and the Morris water maze, which places a mouse in a large tub or pool of water and assesses how long it takes to find a hidden underwater platform. Typically, training for these paradigms spans multiple trials and allows the measuring of both learning acquisition rates and retention through short term and long-term memory trials.

Although the radial-arm maze and Morris water maze are reliable ways to test memory in rodents, they present complications for some researchers. Both mazes use deprivation or strong aversive stimuli as reinforcement¹. The radial arm maze uses food deprivation to ensure that rodents are properly motivated to find a food reward. In the Morris water maze, the effects of stress induced by a forced swim may alter results in the mice².

The Barnes maze is an alternative spatial awareness task that requires rodents to learn the position of a hole in order to escape the bright, open maze surface³. Weak aversive stimulation (light or sound) is then applied to increase the likelihood that mice will escape from the platform. The Barnes maze does not require food deprivation, so the amount of animal preparation is less than in the radial arm maze. It can be used without conflict by researchers who are investigating behaviors or molecules associated with eating, hormonal regulation, or hypothalamic pathways.

The Barnes maze also has advantages over the Morris water maze. It is less stressful than the Morris water maze, causing less elevated levels of corticosterone in mice⁴. In addition, it is much simpler to assemble and requires less dedicated space during the testing procedure and storage.

The Damsel-in-Distress assay is a simple two-part experiment that can assess locomotor activity followed by prosocial behavior. The Damsel-in-Distress assay is designed to assess exploratory behaviors and social responsiveness of a male rodent in response to an entrapped female rodent. A commonly used method to assess sociability (as well as preference for social novelty) is the use of Crawley's three-chamber sociability test, which assesses the free choice of a mouse to spend time near or away from other mice⁵.

Similar to Crawley's three-chamber sociability test, the Damsel-in-Distress assay also measures free choice regarding how to spend time in the presence of another mouse, but it also provides measurements for deeper aspects of social functioning: 1) In the Damsel-in-Distress assay, the trapped female mouse is held in the center of an open field, so the male's potential aversion to an open field is pitted against his drive to socially explore or investigate a distressed female conspecific. 2) The Damsel-in-Distress model also provides a way to assess prosocial and empathetic behavior in response to the trapped mouse, which has not been frequently explored in mice.

Animal empathy is definitely observable and measurable, though not many paradigms exist for this purpose. For example, in rats, a trapped cage mate can induce a state of prosocial motivation, where rats will work to free the trapped animal. In fact, rats will choose to help the trapped animal even before opening a similar container containing a chocolate snack, then access the chocolate and share it with the newly freed rat⁶.

Measures of empathy in mice typically involve the infliction of pain; indeed, mice who watch other mice in pain are more sensitive to pain themselves^7,8. Restraint stress is a characterized phenomenon in rodents and has been coupled with shock to examine stress responses, as one measure of empathy in mice^9,10.

There are ethical considerations associated with induced pain or shock, so stress-inducing alternatives are needed. We developed the Damsel-in-Distress paradigm as a measure of empathetic behavior in the absence of either pain or shock treatment. The trapped mice in our Damsel-in-Distress protocol show overt signs of distress after only several moments as they are unable to turn around within the small container, yet they are unharmed while providing an opportunity for other mice to respond to their distress.

To begin the Damsel-in-Distress assessment, a male mouse is placed in a large novel arena containing a small empty central cylinder. Exploratory behaviors are recorded for several minutes, including how many sections of the arena are crossed and how much time is spent in the central open area. This method provides a quick and easy way to rule out locomotor deficits as a potential confounder in a learning situation that requires coordinated movement for successful completion. It also provides a basic measure of how much aversion to the open center field is present. Both measures could influence Barnes maze performance.

Following the initial exploration, the male is removed from the arena and a female mouse is placed in an enclosed, small clear central cylinder (similar to that used to collect blood from mice). Then, the original male mouse is reintroduced into the arena, and exploratory behavior is again scored. The Damsel-in-Distress paradigm assesses whether a mouse is interested in a social interaction based on changes in behavioral patterns when a trapped female is present (scored by time spent in the center square and the number of digging episodes), and whether the mouse exhibits prosocial behavior towards the trapped female (scored by the amount of cylinder investigations and contact events with the trapped female). The Damsel-in-Distress assay can be used to measure a propensity for social novelty (similar to Crawley's three-chamber sociability test) depending on whether researchers trap a familiar or novel mouse.

Together, the Barnes maze and Damsel-in-Distress experiments allow the accurate evaluation of mouse learning capabilities and social responsiveness in the absence of extremely stressful stimuli. As with all behavioral assays, these experiments should be performed with great sensitivity towards the animal experience, minimizing the discomfort experienced by the animal.

Like most mazes, differences in locomotor activity may influence performance on the Barnes maze, so researchers should also assess locomotor activity, especially when using the Barnes maze to assess learning in mice with mutations that may impair movement (such as those found in Huntington's disease mouse models, or those exposed to toxins that may induce hyperactivity or retard movement, such as ethanol). In addition, maze and chamber surfaces should be thoroughly cleaned and bedding changed between each animal to avoid scent cue confounds.

Importantly, all materials can be fabricated on site with minimal financial investment and the small physical footprint of these assays means that these experiments can be replicated in almost any setting, allowing for great flexibility. This type of accessibility allows good science to be performed at smaller institutions with limited resources or in situations where pilot data needs to be collected quickly in the absence of substantial support.

Protocol

All methods described here were approved by the Institutional Animal Care and Use Committee (IACUC) of Hampden-Sydney College or Randolph-Macon College (where some work was previously performed).

1. Basic Housing of Mice

1. House the mice in plastic cages with solid bottoms and sides, and a layer of soft bedding and nesting material such as paper shredding. Use bedding that is composed of shredded corn cobs or wood shavings, and ensure that the bedding is changed regularly for sanitation.
2. Provide access to food and water ad libitum. The top of the plastic cage houses a food hopper. Dispense properly formulated mouse food pellets from the food hopper and provide a water dispenser. Ensure that the cage top features an air filter to protect mice from outside contaminants.
3. Maintain the natural circadian rhythms of the mice by following a 12-hour light-dark cycle in the housing facility. Conduct behavioral testing at the same time of day, ideally during the animal's dark cycle, such as during the evening hours.
   NOTE: Care should be taken to distinguish mice in the housing. Various methods for distinguishing mice are available, such as ear punches, ear tags, and tail markings.

2. Barnes Maze Testing: Construction

1. Obtain a circular wooden board that is 120 cm in diameter.
2. Cut 20 circular holes (4.5 cm in diameter) around the perimeter of the circle. Position each hole to be 2.5 cm away from the maze edge and 13 cm apart from neighboring holes.
3. Smooth the surfaces and paint them glossy white (a bright color is recommended).
4. Designate one side of the maze board for insertion of small cup hooks roughly 2-3 cm away from each of the 20 holes and place two hooks for each hole (one on either side of each hole).
5. Use shallow black (plastic) disks for covering holes on side of maze with hooks. Use hooks to secure thick rubber bands in order to hold the disks onto the bottom of the maze.
6. Securely mount the Barnes maze 120 cm above the ground and away from other similarly tall objects like tables or chairs. A box or stool can be used to support the middle of the maze.
7. Place large white posters with one shape on each wall (triangle, circle, and cross) as extra-maze cues on 3 sides of the maze. Maintain the posters on the walls around the maze for each trial.
8. On last side of maze, set up a solid black curtain to hide observers so that data can be recorded accurately without the researchers being visible to mice on the maze.
9. Suspend a video camera over the arena with a bird's-eye view of the entire maze surface.
10. Clean every surface of the maze (black disks and target box included) with water then a 70% ethanol solution before and after each mouse trial.
11. Place a 100 W light source 25 cm over center of maze (turn on/off at the start/finish of trial) and be sure to have the disks and hooks facing the floor.
    NOTE: All other overhead lights in the room should be turned off during testing. The addition of an ultrasonic noise maker hung next to 100 W light is recommended. Turn it on/off at the start/finish of each trial.

3. Barnes Maze Testing: Procedure

NOTE: Ensure that all the maze components are cleaned with water and 70% ethanol solution before and after each trial, allowing for time to dry completely before testing resumes. Be sure to have cleaning supplies ready as well as timers for the trials.

1. House all the mice in groups while using a reliable identification method. Keep mice outside of the testing room when they are not actively running on the maze. This is to ensure they are not subjected to the ultrasonic noisemaker prematurely.
2. Handle mice by gently picking up and holding them from the base of the tail, with the paws kept on one's hand.
3. For training, have the mice run on the maze daily once per day for 7 days in a row to assess learning/acquisition.
   NOTE: A single long-term trial can be done on a later date to assess memory/retention. The current protocol trained adolescent mice beginning at postnatal day 32 and a long-term trial was performed in adults at 4 months of age.
4. Randomly assign each mouse to a target hole to use throughout the testing period. The holes can be labeled 1-20 on the bottom of the maze or on the outside of the maze perimeter, where they are not visible to the mouse in the maze.
5. Replace the assigned hole disk with a small black box (23 x 11 cm). Firmly attach it to the Barnes maze using rubber bands connected to the nearest hooks.
   NOTE: The target box is shallow enough so that the mouse can easily step down into it or contains a step to ensure that the mouse does not have to jump down into it.
6. Place the mouse onto the center of the maze beneath a cup to acclimate for 30 seconds until the test begins. Begin video recording.
7. Turn on the light and ultrasonic noisemaker. Lift the cup via a string mechanism to avoid biasing the initial heading of the animal. Start a timer and sit behind the curtain to observe.
8. After the mouse enters the target hole, cover the hole with the same opaque cup used at the start of the trial or a binder and turn off the ultrasonic noisemaker. If the mouse has not entered the target after 5 minutes have elapsed, corral the mouse into the target hole.
   1. Ensure the mouse enters the hole. When mouse is inside the target box, cover the hole and turn off the noisemaker. Allow the mouse to remain in the target box for 1 minute undisturbed.
9. Although the target hole will remain constant for each mouse throughout the training period, run mice in a random order each day to ensure that they are not following any cues/scents left by the previous mouse.

4. Barnes Maze Testing: Data Analysis

1. During the Barnes maze trials, record the following timed data: total time spent on maze, time to find target, and time to enter target hole if the target is entered.
2. Track the overall movement of the mouse to determine the number of errors (number of incorrect holes explored) before and after the target is found. Also record the distance from the target hole to the first hole explored (distance is measured in number of holes away), along with any notable grooming behavior.
3. Track the movements of each animal on a piece of paper with a diagram of the maze during each trial and use it to analyze search strategies, as well as determine the number of holes that were explored in the quadrant opposite the target. Use video analysis to confirm the paths.

5. Damsel-in-Distress Testing: Constructing the Restraint Chamber

1. Cut a clear cylinder to a length equal to ¾ of the length of the female mouse from the base of the tail to tip of the nose. Ensure that the dimensions of the cylinder are such that the mouse placed inside cannot turn around.
   NOTE: We cut the cone end of a 50 mL conical tube down to make a 4 cm long tube for 1-month-old mice and an 8 cm long tube for 4-month-old mice.
2. Cap both ends of the cylinder such that one of the caps can be removed and reattached. Punch 3-4 holes, approximately 0.5 cm each, in each cap. Ensure that the holes are large enough to allow nose-touching between mice and breathing¹².
   NOTE: Alternatively, a device for mouse restraint during blood collection can be used.

6. Damsel-in-Distress Testing: Preparing the Arena

1. Ensure that the arena is an opaque topless plastic square box of 38 x 38 cm with 19 cm walls. Fill it uniformly with corn bedding to a height of approximately 2.5 cm.
   NOTE: The corncob bedding makes digging events easier to detect.
2. Suspend a video camera over the arena so that the entirety of the arena is in view.

7. Damsel-in-Distress Testing: Exploratory Behavior Measure

1. Place the closed and empty stress chamber into the center of the arena.
2. Begin recording with the suspended camera.
3. Select a male mouse. If the mice are marked for identification, note this male's identity; if not, mark this mouse so that it can be distinguished after returning to the cage.
4. Gently place the male mouse beneath a cup at the lower left corner in the arena. After 30 seconds, remove the cup via a string mechanism.
5. Allow the mouse to explore for 10 min, taking care to stay out of its field of view during recording. At the end of the 10 min, remove the mouse from the arena and return it to a holding cage for the next 5 min¹².
6. Stop recording and save the video file with an appropriate identifier.

8. Damsel-in-Distress Testing: Social Responsiveness Measure

1. Use the same arena and recording setup as in the exploratory behavior measure.
2. Select a female mouse. If the mice are marked for identification, note this female's identity; if not, mark this mouse so that it can be distinguished after it is returned to the cage.
   NOTE: A littermate, cage mate, or novel mouse can be used, depending on the research question. If multiple trials are being performed, maintain the type of relationship across all trials.
3. Hold the female mouse gently by the base of the tail, or by neck restraint, if necessary, and lower it into the restraint chamber. Close the open end behind it and ensure that it is unable to turn around.
   NOTE: Consider wearing bite-resistant gloves, as the female mouse will be resistant to entering the restraint chamber.
4. Begin recording with the suspended camera.
5. Place the restraint chamber with the trapped female mouse inside in the center of the empathy arena and allow the female to acclimate for 10 min. Take care to remain out of the female's field of view.
6. After the female mouse has been in the restraint chamber for 5 min, place the marked male mouse back into the empathy arena using the same process as before. Allow the male mouse an additional 5 min to explore the arena, again taking care to remain out of the field of view of the mice.
7. Stop recording and save the video file with an appropriate identifier.
8. At the end of the 5 min, remove both mice from the arena and place them into their cages. Replace the corn bedding and sanitize both the arena and the stress chamber with 70% ethanol¹².

9. Damsel-in-Distress Testing: Data Analysis

1. Tracking software can be used, but the video can also be analyzed manually. Once the video file is visible on the computer, overlay a transparent sheet over the screen and outline the square of the arena using a marker. Divide the arena square into nine equal compartments.
2. Review the video data for the first 5 min of the initial exploratory measure. Record the number of compartments crossed (locomotor activity/exploratory behavior), time spent in the center square of the arena (open field aversion), number of digging and grooming episodes, and number of times the mouse touched the empty center restraint chamber.
   NOTE: Scoring is not done for the second 5 min of the male's 10-minute initial exploration, nor for the 10 min the female is trapped in the central chamber in the absence of the male.
3. Review the video data for the 5 min of the social responsiveness measure immediately after the male is reintroduced into the arena with the trapped female. Record the same data as for the initial exploratory measure, but additionally record the number of times the male mouse touched noses with the trapped female mouse.

Results

The Barnes Maze

To illustrate how the Barnes maze can be used, we investigated whether a single early ethanol exposure caused a difference in learning over the course of mouse development. C57Bl6/J mice were either injected with a 2.5 g/kg ethanol solution (n = 8) or with saline (n = 6) twice, two hours apart, at postnatal day 6. We trained the animals on the Barnes maze during adolescence (P30), then p...

Discussion

The Barnes maze and the Damsel-in-distress experiments are inexpensive, quick, and relatively easy ways to evaluate spatial learning, locomotor activity, and prosocial behavior in mice. Other advantages include the absence of overt stressors, pain, or food restriction for the animal. Like most learning/memory paradigms, a disadvantage of the Barnes maze is the number of trials required for the animals to learn where the target hole is located and enter.

Data collection:

Materials

Name	Company	Catalog Number	Comments
Damsel-in-Distress
50 mL conical tube	Fisher Scientific	14-432-22	Any brand of 50 mL conical tube will work
Rubber bands	Sprano Brand	n/a	Size 62, used to keep caps held to plywood
hammer	Grainger	6R252	Any standard hammer will work
nail (size 8D)	Grainger	4NFE3	Similarly-sized nails should work just as well
opaque, topless plastic box	AcmePlastics	CUT-TO-SIZE-ACRYLIC-CAST-BLACK-SHEET-2025	Opaque plastic, cut to size (30 cm L x 19 cm W x 3-6 cm H). Step may be added to ensure no more than a 3.5 cm entrance depth for the mouse.
video camera (smartphone)	N/A	N/A	Any camera-equipped smartphone will work
bite-resistant gloves	Kent Scientific	GLVDYN02	Any brand offering appropriate protection
transparency sheet	Staples	954145	Any brand of clear plastic sheet will work, used for scoring
Barnes Maze
Petri Dishes	Corning	353025	Spray painted and used as covers for Barnes maze holes
Plywood (3/4 in.)	LP Building Products	22487	To construct Barnes maze
Spray Paint	Krylon	1274937	Used to paint petri dish caps black, white paint used to paint plywood
Cup Hooks (5/8 in.)	Ace Hardware	5360615	2 used on either side of ventral hole surfaces; Rubber band wraps around hooks to hold cap flat
Poster Board	Creatology	n/a	Used at edges of maze as extra cues
Light Bulbs	Phillips	n/a	100W light bulb, used to during the trials
Rubber bands	Sprano Brand	n/a	Size 62, used to keep caps held to plywood
Ultrasonic noisemaker	Victor mini PestChaser	M753SN	Used as aversive stimuli