Name: Assessing Spatial Learning and Memory in Small Squamate Reptiles (Video) | JoVE Journal
Uploaded: 2017-01-03T14:00:00.000+00:00
Duration: 8 min 44 s
Description: Learn to assess spatial learning and memory in small squamate reptiles using a modified Barnes maze. This method adapts a traditional rodent paradigm for testing non-model species.

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5 minutes

We thank M. Forney, R. Maged, and K. Hellwinkle for data collection and two anonymous reviewers for comments on a previous version of this manuscript. This research was supported by an NSF award to LDL (IOS-0918268).

All procedures were approved by the Penn State University&#160;Institutional Animal Care and Use Committee (IACUC - Protocol ID: 43242) and adhered to all local, state, and federal regulations.
1. Preparation
<ol>
	<li>Purchase or construct the Barnes maze, assuring that goal holes are appropriately sized for the species of interest. For this protocol, use seven adult side-blotched lizards (Uta stansburiana). Determine adequate sample size for studies using other species.
	<ol>
		<li>If constructing the Barnes maze, purchase any standard, nonporous round table (106 cm in diameter), elevated 76.2 cm off the floor.</li>
		<li>Using a tape measure, mark 10 equidistant points spaced 26 cm apart around the perimeter of the table and 6.35 cm from the edge of the table. Use 10 holes whereas rodent mazes typically have more holes. Doing so allows for a reduction in the number of escape options.</li>
		<li>Using a hole saw bit (2.54 cm in diameter), drill out all 10 goal holes and sand any sharp edges.</li>
		<li>Mount four shelf support pegs under each hole with screws, using a home enclosure as a guide for dimensions.</li>
	</ol>
	</li>
	<li>Place the maze in a quiet room with bright overhead lighting and spatial cues available for navigation (doors, cabinets, etc.).</li>
	<li>Mount an auto focus, wide-angle lens webcam (11.3 cm x 3.99 cm) or other suitable camera directly above the maze. Be certain that the field of view from the video feed encompasses the entire maze.</li>
	<li>Place the computer and observer chair at least 1 m from the maze.</li>
	<li>Mark the legs of the maze on the floor with a permanent marker or tape. This allows for correction of any minute movement of the maze over the course of the trials.</li>
	<li>Randomly assign one of the holes as &#34;1&#34; and number the other holes consecutively through &#34;10&#34;. With a permanent marker, write numbers along the side of the outside of the maze for ease of reference. Ensure that the numbers are not visible to an animal within the maze.</li>
	<li>Randomly assign goal holes to the subjects using a random number generator.</li>
</ol>
2. Training Trials
NOTE: Reptiles have a preferred body temperature that can be obtained from the literature. If animals become sluggish over multiple training trials, the maze may be too cool and this may affect behavior. A small space heater or heat tape on the underside of the maze can adequately increase maze surface temperature, the best thermal indication of body temperature, to maintain optimum body temperature and behavioral performance.
<ol>
	<li>If subjects are housed outside of the testing room, bring animals in their home enclosures into the room at least 30 min before testing. Be certain to provide heat in home enclosures (e.g., heat lamps, heat tape) in order to maintain body temperature before testing.
	<ol>
		<li>Open tracking software and webcam application for the video feed. The custom tracking software was written for Matlab and the Image Processing toolbox; other tracking software is commercially available.</li>
		<li>Evaluate the field of view from the video feed. If the maze is not in the field of view, check that the maze is within the marks on the floor.</li>
		<li>Open the worksheet containing information as to the date, start/end time of training trials, subject ID, assigned goal hole, number of training trials run, notes, and observer&#39;s initials.</li>
		<li>Randomize the order in which subjects will be tested. Test subjects in a systematic manner.</li>
	</ol>
	</li>
	<li>Remove the first subject from its home cage and gently place the subject in the middle of the maze. Place a plastic tub over the animal; doing so prevents unconscious orientation bias by the researcher. Allow 30 s to acclimate.</li>
	<li>Mount the animal&#39;s home cage under the maze, directly below the assigned goal hole, by sliding the top of the enclosure into the peg mounts. Be certain that the basking rock in the home cage is directly under the hole, which will serve as a perch when the animal descends into the hole. At this point, the use of thermal or chemical cues of the home enclosure for use in navigation is irrelevant.
	<ol>
		<li>Rodent mazes&#160;use a clean enclosure as an escape for the subject. However, encourage entry into the hole during training trials by mounting the animal&#39;s home enclosure under the maze.</li>
	</ol>
	</li>
	<li>Gently remove the plastic tub from the top of the maze.</li>
	<li>Start recording of the training trial with the webcam application.
	<ol>
		<li>Allow the subject to explore the maze for 10 min. If anything atypical occurs, note this in the worksheet.</li>
		<li>If the subject falls or runs off the table, gently return the subject to the middle of the maze. Continue timing and recording if the animal is out of the maze for 30 s or less. If the subject is out of the maze for more than 30 s, abort the trial. Record this information in the notes section of the workbook.</li>
		<li>If the subject descends into the goal hole unassisted, consider the trial as over at that time.</li>
		<li>If the subject does not descend into the goal hole, consider the trial as over after 10 min have elapsed.</li>
	</ol>
	</li>
	<li>If the subject does not descend into the goal hole after 10 min, by hand gently guide the animal, head first, into the appropriate goal hole.</li>
	<li>Stop video and tracking software. Save video footage.</li>
	<li>Allow the subject to rest for at least 2 min in its home enclosure while the enclosure is still mounted under the maze.</li>
	<li>Using a spray bottle and paper towels, clean the top of the maze with a diluted soap mixture of 1:10 soap to water. Doing so precludes the use of chemical cues when navigating the maze. Some species, particularly those that make heavy use of chemosensory information, necessitate additional rinsing of the maze with hot water to eliminate all scent cues.</li>
	<li>Remove the enclosure from under the maze and either repeat the training trial with the same individual or a new individual. Each subject can be run in up to 5 training trials per day, with 30 min&#160;interval between an individual&#39;s trials.</li>
	<li>Repeat this procedure until a subject descends into its goal hole unassisted, in 3 different training trials, indicating that learning of the goal location has occurred. The three training trials do not need to be consecutive. If a subject reaches this criterion, the subject moves to the probe trial in order to assess spatial memory.</li>
</ol>
3. Probe Trials
NOTE: Once a subject reaches criterion, the subject has learned how to navigate to the goal. However, at this point, it is still unclear if the subject is navigating using a spatial strategy or some other navigational strategy. Probe trials test for this and should be performed the day after the subject reaches criterion in the training trials.
<ol>
	<li>Rotate the maze 180&#176;, assuring that the legs of the maze are within the marks on the floor. Rotation of the maze allows for local cues that directly identify the goal to be in direct conflict with the more stable spatial cues.</li>
	<li>Repeat steps 2.1 - 2.5.2, omitting step 2.3 of mounting the subject&#39;s home enclosure under the maze. Doing so precludes the use of olfactory or other cues emanating from the home enclosure during maze exploration.</li>
	<li>During probe trials, subjects are free to explore the maze for the full 10 min to assess exploratory behavior. After 10 min, stop video and tracking software.</li>
	<li>Remove the subject from the maze and return it to its home cage.</li>
	<li>Clean the top of the maze with the diluted soap mixture.</li>
</ol>
4. Behavioral Measures
NOTE: For training trials, include behavioral measures such as latency to arrive at the goal hole, number of errors made (investigation of a nongoal hole; the animal&#39;s snout must be within 1 cm of the hole), and proportion of time spent in the correct quadrant of the maze.
<ol>
	<li>For training trials, determine a search strategy. A direct strategy is defined as descending into the goal hole with fewer than 3 errors. A serial strategy is examination of 3 or more consecutive maze holes along the perimeter of the maze. A random strategy is non-serial examination of maze holes with 3 or more errors. 
	NOTE: For probe trials, behavioral measures include latency to arrive at the spatially correct hole, number of errors made before investigating the spatially correct hole, and the proportion of time spent in the spatially correct quadrant of the maze.</li>
	<li>For a more stringent assessment of non-random choice of the spatially correct goal hole, employ sampling without replacement when calculating chance rate.</li>
</ol>

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The authors declare that they have no competing financial interests.

When experimentally testing for spatial learning and memory, there are several important conceptual issues that are addressed in some of the major steps in the protocol. First, subjects must demonstrate that they are learning the location of the goal hole over the course of the training trials. Attaining the preset criterion demonstrates that learning of the goal hole location has occurred. If subjects do not learn the location of the goal hole, there is no feasible way to then determine a navigational strategy. If anima...

Many neurodegenerative diseases such as Alzheimer&#39;s present with a progressive decline in cognitive ability, typically concurrent with degradation of the brain1-4. To test for the influence of brain injury and degradation on cognitive processes, clinical research has leveraged the advantages of model rodent species and standardization of testing apparatus and protocol. In particular, spatial learning and memory processes have been assessed via several standard paradigms such as the Morris water maze, Barnes maze, and radial arm maze (for a comprehensive review of these and other paradigms, see 5,6). The rich history of these spatial learning and memory paradigms has proven quite successful, allowing researchers to understand many of the facets and nuances of the relationship among human memory, brain function, and disease.
While assessment of cognitive processes has been examined in clinical research for quite some time, research directed towards the cognitive abilities of nonmodel species is relatively new. Researchers studying cognition in nonmodel species are typically interested in the ecological and evolutionary relevance of cognitive processes, particularly in the context of survival and reproduction. Some studies in reptiles have suggested that advanced cognitive abilities, in particular spatial memory, may underlie some behaviors, particularly those concerning navigation and orientation. However, while many studies have demonstrated that reptiles can reorient after displacement7,8, the cognitive mechanisms underlying reorientation behavior have not yet been teased apart. Because of this, some studies have attempted to experimentally assess the importance of spatial learning and memory during navigation9-17. The methodology in these studies are predominantly modeled after rodent paradigms and protocols, sometimes modified for use in reptiles, but these studies have had variable success in assessing spatial memory. Some studies have demonstrated spatial learning and memory in some species11-17 while other studies found no evidence of such9,10. Thus, the role or existence of spatial learning and memory during navigation in reptiles is still unclear.
One issue that may be problematic when experimentally assessing spatial learning and memory in reptiles is the ecological relevance of the task. Reptiles are a special taxonomic group quite distinct from rodents, demonstrating large variation in ecology, behavior, and physiology. Differences in behavior across reptilian species could possibly impact assessment of spatial cognitive abilities, particularly if the paradigm used does not tap into a natural behavior. For instance, in a species that typically seeks refuge in small crevices, spatial abilities may be easily assessed using a Barnes maze whereas this maze may not be the ideal paradigm choice in a species that typically remains motionless. Similarly, most squamate reptiles are not aquatic and thus the Morris water maze may not be a suitable choice for testing spatial learning and memory (but see15); however, this maze may be an ideal choice for testing spatial abilities in turtles16. Finally, the physiology of this group must be accounted for, as reptiles are ectothermic and proper temperature maintenance, particularly of the substrate, must be considered during the testing procedure.
The protocol and paradigm presented here were used to probe for spatial learning and memory in adult side-blotched lizards (Uta stansburiana)13, a small lizard that&#160;typically flees from predators into small crevices in rocks18. Knowing this aspect of the natural history and behavior of the species, we used a modification of the traditional Barnes maze to test for spatial learning and memory. The Barnes maze is a dry-land maze and typically used for testing spatial cognition in rodent models. We modified our maze in several ways from the rodent maze, in both design and protocol (described below). Our maze consisted of a circular platform with 10 holes equidistant from each other along the perimeter of the platform (Figure 1). The protocol described here involves a subject participating in training trials to learn the location of a goal hole, then, once the subject learns the location of the goal hole, a probe trial is used to ascertain spatial memory use during navigation to the goal.

<table><tbody><tr><td>Barnes maze</td><td>TSE Systems</td><td>302050-BM/M</td><td>Available from other vendors. Alternatively, a Barnes maze can be constructed from a standard, non-porous round table.</td></tr><tr><td>Heat tape</td><td>Big Apple Pet Supply</td><td></td><td>May also use a small space heater situated on the floor under the maze.</td></tr><tr><td>Pet keeper for small animals</td><td>Petco</td><td>1230204</td><td>Housing enclosure that can be mounted under the maze.</td></tr><tr><td>Nickel plated shelf support pegs</td><td>Newegg</td><td>241941</td><td>Pegs attached to underside of maze. Secures enclosure to maze during trials.</td></tr><tr><td>LifeCam Studio webcam</td><td>Microsoft</td><td>Q2F-00013</td><td>Available from other vendors. Other brands of webcams may also be used.</td></tr><tr><td>Tracking software</td><td>Code custom written for Matlab&lt;br/&gt; and the Image Toolbox</td><td></td><td>Video tracking software. Other tracking software such as VideoMot 2 from TSE Systems can be used.</td></tr></tbody></table>

assessing spatial learning and memory in small squamate reptiles

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This protocol allows for the experimental assessment of spatially based navigation in small lizards. A previous study successfully used this protocol to probe for spatial navigation in male side-blotched lizards13. In that particular study, males were trained to navigate to a goal hole and, once criterion was reached, progressed into a probe trial to assess the cues prioritized when navigating to a goal hole.
The repr...

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

The overall goal of this procedure is to assess spatial memory and learning in small squamate reptiles using a modified Barnes maze. This method can help answer key questions concerning the spatial cognitive processes of non-model species, particularly in small squamate reptiles. The main advantage of this technique is that we have modified a traditional rodent paradigm, the Barnes maze, to allow for testing of spatial learning and memory in small lizards.It's possible that the maze could be similarly altered to be applicable to other species as well. For this protocol, it is necessary to purchase or construct the Barnes maze. Make certain that the goal holes are appropriately sized for the species of interest.For this demonstration, fence lizards are in use, so the holes are sized to match. Position the maze in a quiet room with bright overhead lighting and spatial cues around the maze, such as doors and cabinets, to help the animals navigate. Above the maze, mount an auto-focus wide-angle lens webcam or other suitable camera.The key is for the field of view to contain the entire maze. At least one meter to the side of the maze, position the computer used for animal tracking. Once positioned, mark the legs of the maze on the floor with a permanent marker or tape, so it can be adjusted back into its original position with ease.Regarding the maze, randomly assign one of the escape holes as number one, and number the other holes consecutively up to number 10. Mark the numbers along the outer wall of the maze, so they do not serve as visible cues for the animals. Under the maze, if needed, use a small space heater or attach heat tape to increase the maze surface temperature so that the animals will be at their optimum body temperature for metabolic activity.To begin, randomly assign goal holes to each animal. If the animals are housed outside of the testing room, bring their home cages into the room at least 30 minutes before testing. Be certain to provide them heat, if needed, so that they are at their ideal body temperature when tested.On the computer, open the custom MATLAB tracking software and webcam application to get the video feed. Then, evaluate the field of view again and adjust the maze's position as needed. Next, open the worksheet to record the basic experimental parameters, which should include the date, start and end times, animal IDs, assigned goal holes, number of training trial runs, notes, and observer's initials.Then, randomize the animals'testing order and start testing them in a systematic fashion. First, remove an animal from its home cage and gently place it in the middle of the maze. Then, cover it with an opaque plastic tub and allow the animal to acclimate for 30 seconds to the tub.Meanwhile, mount the animal's home cage under the maze directly below the assigned escape hole. Slide the top of the enclosure into the peg mounts, and put the basking rock in the cage, directly under the hole. Now, gently remove the plastic tub from the top of the maze and start recording the trial on the computer.Allow the animal to explore the maze for ten minutes, and if anything atypical occurs, note it in the worksheet. If the animal falls or runs off the table, gently return it to the middle of the maze, provided it has not been out of the maze for more than 30 seconds. Otherwise, abort the trial and record this result in the notes section.If the subject descends into the escape hole unassisted, the trial ends at that time. If the subject does not escape into the hole within 10 minutes, end the trial and gently guide the animal to its escape hole by hand, moving it head first. Once in its home cage under the maze, allow the animal to rest for at least two minutes.Meanwhile, clean the top of the maze with a diluted soap mixture and paper towels. Some species, particularly those who make heavy use of chemosensory information, necessitate additional rinsing of the maze with hot water to eliminate all scent cues. Once the maze is cleaned, remove the enclosure from under the maze and continue testing the animals.Each can run the maze up to five times per day, with a half-hour interval between trials. Repeat this training procedure until the subject has descended into its goal hole unassisted on three different trials. These success do not need to occur consecutively, and there are no limits on the number of trials needed.The number of training trials to reach criterion can be burdensome. Alteration of the maze in an ecologically relevant manner, taking into consideration a species'natural history and behavior may help to alleviate this. Then, proceed with the probe trial on the following day to further test that animal.Once the animals have learned their escape hole, use the probe trial to determine if it is navigating using a spatial strategy or some other navigational strategy. For this trial, rotate the maze 180 degrees, so the local cues within the maze will be in direct conflict with the spatial cues outside the maze. Then, test the animal using the same procedure as a training trial.However, no cage is mounted under the escape hole. Thus, no olfactory cue from the cage can help guide the animal to its escape. If the animal fails to find the escape hole, just remove it from the maze and return it to its home cage.Do not teach it where the hole is located. As before, always clean the maze before testing the next animal. Seven adult side-blotched lizards were tested as described.Individuals took between 17 and 81 trials to advance to the probe trial, with the average near 50. It did take significantly less time for the group to find the escape hole in the fourth quartile of their trials than the first, which suggests learning. During the probe trials, individuals went to the spatially correct goal hole at a rate well better than chance and spent a majority of their time in this quadrant of the maze.Thus, spatial cues in the room were being prioritize over local cues in the maze. After watching this video, you should have a good understanding of how to modify a Barnes maze to accommodate testing of a small squamate reptile, as well as how to run animals in training and probe trials. While attempting this procedure, it's important to remember to take into account the natural history and behavior of the species of interest when designing a task to probe for spatial memory and learning.Following this procedure, more detailed applications would be appropriate, such as decreasing reliability of spatial cues or making local cues more obvious. Doing so may reveal how the stability of different cues can modulate cue prioritization and memory.

assessing-spatial-learning-and-memory-in-small-squamate-reptiles

Research

JoVE Journal

Neuroscience

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Learn to assess spatial learning and memory in small squamate reptiles using a modified Barnes maze. This method adapts a traditional rodent paradigm for testing non-model species.

Assessing Spatial Learning and Memory in Small Squamate Reptiles

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