Name: analyzing spatial learning and prosocial behavior in mice using the barnes maze and damsel-in-distress paradigms
Uploaded: 2018-11-17T12:30:00
Description: Watch this Scientific Journal Video about Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms at JoVE.com

We at Hampden-Sydney College give acknowledgement and appreciation to Sean Walden, Zach Leitner, Hunter Lee, and Anton Kheirani for their involvement in testing the protocols for the Barnes maze and Damsel-in-Distress experiments. We would also like to thank James Foster at Randolph-Macon College for the construction of the Barnes maze and the Randolph-Macon College Department of Biology for providing testing space.

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
<ol>
	<li>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 sanitat...

<ol>
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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:
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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&#39;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&#39;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&#39;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&#39;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 themselves7,8. Restraint stress is a characterized phenomenon in rodents and has been coupled with shock to examine stress responses, as one measure of empathy in mice9,10.
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&#39;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&#39;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&#160;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.

<table border="0" cellpadding="0" cellspacing="0" style="width:1391px;" width="1389">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Damsel-in-Distress</td>
			<td style="width:127px;"></td>
			<td style="width:317px;"></td>
			<td style="width:731px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL conical tube</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:317px;">14-432-22</td>
			<td>Any brand of 50 mL conical tube will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rubber bands</td>
			<td>Sprano Brand</td>
			<td>n/a</td>
			<td>Size 62, used to keep caps held to plywood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">hammer</td>
			<td style="width:127px;">Grainger</td>
			<td style="width:317px;">6R252</td>
			<td>Any standard hammer will work</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">nail (size 8D)</td>
			<td style="width:127px;">Grainger</td>
			<td style="width:317px;">4NFE3</td>
			<td>Similarly-sized nails should work just as well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">opaque, topless plastic box</td>
			<td style="width:127px;">AcmePlastics</td>
			<td style="width:317px;">CUT-TO-SIZE-ACRYLIC-CAST-BLACK-SHEET-2025</td>
			<td>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.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">video camera (smartphone)</td>
			<td style="width:127px;">N/A</td>
			<td style="width:317px;">N/A</td>
			<td>Any camera-equipped smartphone will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bite-resistant gloves</td>
			<td style="width:127px;">Kent Scientific</td>
			<td style="width:317px;">GLVDYN02</td>
			<td>Any brand offering appropriate protection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">transparency sheet</td>
			<td style="width:127px;">Staples</td>
			<td style="width:317px;">954145</td>
			<td>Any brand of clear plastic sheet will work, used for scoring&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Barnes Maze</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes</td>
			<td>Corning</td>
			<td>353025</td>
			<td>Spray painted and used as covers for Barnes maze holes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plywood (3/4 in.)</td>
			<td>LP Building Products</td>
			<td>22487</td>
			<td>To contruct Barnes maze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spray Paint</td>
			<td>Krylon</td>
			<td>1274937</td>
			<td>Used to paint petri dish caps black, white paint used to paint plywood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cup Hooks (5/8 in.)</td>
			<td>Ace Hardware</td>
			<td>5360615</td>
			<td>2 used on either side of ventral hole surfaces; rubberband wraps around hooks to hold cap flat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poster Board</td>
			<td>Creatology</td>
			<td>n/a</td>
			<td>Used at edges of maze as extra cues</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light Bulbs</td>
			<td>Phillips</td>
			<td>n/a</td>
			<td>100W light bulb, used to during the trials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rubber bands</td>
			<td>Sprano Brand</td>
			<td>n/a</td>
			<td>Size 62, used to keep caps held to plywood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrasonic noisemaker</td>
			<td style="width:127px;">Victor mini PestChaser</td>
			<td>M753SN</td>
			<td>Used as aversive stimuli</td>
		</tr>
	</tbody>
</table>

analyzing spatial learning and prosocial behavior in mice using the barnes maze and damsel-in-distress paradigms

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.

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...

Watch this Scientific Journal Video about Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms at JoVE.com

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

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.

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...

This method can help answer key questions in the behavioral neuroscience field about whether a genetic mutation or environmental variable can impact social behaviors or the acquisition and retention of spacial memories. The main advantages of these techniques are that both the studies can be simply constructed with minimal financial investment and that neither protocol requires the animals to experience extreme or stressful stimuli. The Damsel in Distress method can provide insight into mouse pro-social behaviors, but it can also be used as an empathy measure in other mammalian model organisms.Before beginning the Barnes Maze Testing Procedure, clean all of the maze components with water and 70%ethanol. When the components are dry, assemble the maze and randomly assign each mouse to a target hole to use throughout the testing period. The holes can be labeled one to 20 on the bottom of the maze or on the outside of the maze perimeter where they are not visible from within the maze.To start a training test, replace the assigned hole disc with a 23 by 11 centimeter black box and use rubber bands and hooks to firmly attach the box to the maze. Then, place a mouse onto the center of the Barnes Maze beneath a cup and allow the mouse to acclimate for 30 seconds. At the end of the acclimation period, begin the video recording and turn on the light and ultrasonic noise maker.Lift the cup via a string mechanism to avoid biasing the initial heading of the animal and start a timer behind an observation curtain. Track the animal's movements on a piece of paper with a diagram of the maze and use the diagram to analyze the mouse's search strategies as well as to determine the number of holes that were explored in the quadrant opposite the target. When the mouse enters the target hole, cover the hole with a heavy opaque binder and turn off the ultrasonic noise maker.If the mouse has not entered the target after five minutes, corral the animal into the target hole. Then, allow the mouse to remain in the target box for one minute undisturbed, making sure to turn off the ultrasonic noise. Then, return the animal to its cage.When all of the mice have been tested, manually review their recording to track the overall movement of the mouse to determine the number of incorrect holes explored before and after the target is found, as well as the number of holes from the target hole to the first hole explored and any notable grooming behavior. For exploratory behavior Damsel in Distress Testing, place a closed and empty stress chamber into the center of an empathy arena and begin recording with the suspended camera. Gently place a male mouse beneath a cup at the lower left corner in the arena for 30 seconds.Then, remove the cup via a string mechanism and allow the mouse to explore for 10 minutes taking care to stay out of the field of view during the recording. At the end of the exploration period, move the mouse from the arena into a holding cage for five minutes and stop and save the recording. For social responsiveness Damsel in Distress Testing, gently manually restrain a female mouse and lower the mouse into a restraint chamber.Close the open end of the chamber behind the mouse and confirm that the mouse is unable to turn around. Begin recording with the suspended camera and place the restraint chamber inside in the center of the arena. Allow the animal to acclimate for 10 minutes taking care to remain out of the field of view.Then, transfer the male mouse from the holding area back into the empathy arena for 10 seconds and allow the male mouse an additional five minutes to explore the arena taking care to remain out of the field of view. At the end of the exploration period, stop and save the video file and return both mice to their cages. Then, replace the corn bedding and sanitize both the arena and the stress chamber with 70%ethanol.To analyze the data, open the video file in the appropriate tracking software and place a transparent sheet over the screen. Outline the square of the arena with a marker and divide the arena square into nine equal compartments. Review the video data for the first five minutes of the initial exploratory measure recording the number of compartments crossed, the time spent in the center square of the arena, the number of digging and grooming episodes, and the number of times the mouse touched the empty center restraint chamber.Then, review the video data for the five minutes of the social responsiveness measure immediately after the male is reintroduced into the arena with the trapped female recording the same data as for the initial exploratory measure, but adding tracking of the number of times the male mouse touched noses with the trapped female mouse. When Barnes Maze trained neonates are run as adults in a single long term trial, the ethanol injected mice have a harder time remembering where the target hole was located than saline injected control animals. Ethanol treated mice also explore significantly more holes in the opposite quadrant from the target, indicating a failure to remember the correct general spacial location and use a sequential hole to hole search in a ring shaped fashion until the hole is found.In the Damsel in Distress model, older mice cross more squares than younger mice. In the presence of a trapped female however, male mice of both ages spend less time exploring the arena. Juvenile males spend more time digging during their time in the arena in the presence of a trapped female mouse than when alone suggesting anxiety or pro-social behavior toward the trapped female.By contrast, male mice touch the tube containing the trapped female and initiate trapped female contact significantly more frequently as adults than as juveniles. While attempting this procedure, it's important to remember to keep the conditions constant between and within the trials to ensure that the environmental variables do not confound the results of the study. Following the initial use of these behavioral assays, the same animals can be retested at various time points during development to answer questions about long term memory and possible changes in social behaviors.

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JoVE Journal

Behavior

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land ...

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for ...

Novel Object Recognition Test for the Investigation of Learning and Memory in Mice

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is ...

Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and ...

The (Spatial) Memory Game: Testing the Relationship Between Spatial Language, Object Knowledge, and Spatial Cognition

The Spatial Memory Game: Testing the Relationship Between Spatial Language, Object Knowledge, and Spatial Cognition

The memory game paradigm is a behavioral procedure to explore the relationship between language, spatial memory, and object knowledge. Using two different ...

An Automated T-maze Based Apparatus and Protocol for Analyzing Delay- and Effort-based Decision Making in  Free Moving Rodents

Many neurological and psychiatric patients demonstrate difficulties and/or deficits in decision making. Rodent models are helpful to produce a deeper ...

Analysis of Learning and Memory Ability in an Alzheimer's Disease Mouse Model using the Morris Water Maze

A Morris water maze (MWM) experiment forces experimental animals to swim and learn to find a platform hidden in the water. It is widely used in scientific ...