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>

<ol>
	<li>Adriano, F., Caltagirone, C., Spalletta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+volume+reduction+in+first-episode+and+chronic+schizophrenia:+A+review+and+meta-analysis.">Hippocampal volume reduction in first-episode and chronic schizophrenia: A review and meta-analysis.</a> Neuroscientist. 18, 180-200 (2012).</li><li>Karl, A., Schaefer, M., Malta, L. S., Dorfel, D., Rohleder, N., Werner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+meta-analysis+of+structural+brain+abnormalities+in+PTSD.">A meta-analysis of structural brain abnormalities in PTSD.</a> Neurosci. Biobehav. Rev. 30, 1004-1031 (2006).</li><li>Shi, F., Liu, B., Zhou, Y., Yu, C., Jiang, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+volume+and+asymmetry+in+mild+cognitive+impairment+and+Alzheimer's+disease:+Meta-analyses+of+MRI+studies.">Hippocampal volume and asymmetry in mild cognitive impairment and Alzheimer's disease: Meta-analyses of MRI studies.</a> Hippocampus. 19, 1055-1064 (2009).</li><li>Videbech, P., Ravnkilde, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+volume+and+depression:+A+meta-analysis+of+MRI+studies.">Hippocampal volume and depression: A meta-analysis of MRI studies.</a> Am. J. Psychiatry. 161, 1957-1966 (2004).</li><li>Sharma, S., Rakoczy, S., Brown-Borg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+spatial+memory+in+mice.">Assessment of spatial memory in mice.</a> Life Sci. 87, 521-536 (2010).</li><li>Vorhees, C. V., Williams, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+spatial+learning+and+memory+in+rodents.">Assessing spatial learning and memory in rodents.</a> ILAR J. 55, 310-332 (2014).</li><li>Jenssen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+awareness+by+the+lizard+Anolis+Cristatellus:+Why+should+a+non-ranging+species+demonstrate+homing+behavior.">Spatial awareness by the lizard Anolis Cristatellus: Why should a non-ranging species demonstrate homing behavior.</a> Herpetologica. 58, 364-371 (2002).</li><li>Pittman, S. E., Hart, K. M., Cherkiss, M. S., Snow, R. W., Fujisaki, I., Smith, B. J., Mazzotti, F. J., Dorcas, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homing+of+invasive+Burmese+pythons+in+South+Florida:+evidence+for+map+and+compass+senses+in+snakes.">Homing of invasive Burmese pythons in South Florida: evidence for map and compass senses in snakes.</a> Biol. Lett. 10, (2014).</li><li>Day, L. B., Crews, C., Wilczynski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+reversal+learning+in+congeneric+lizards+with+different+foraging+strategies.">Spatial and reversal learning in congeneric lizards with different foraging strategies.</a> Anim. Behav. 57, 393-407 (1999).</li><li>Day, L. B., Crews, C., Wilczynski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+medial+and+dorsal+cortex+lesions+on+spatial+memory+in+lizards.">Effects of medial and dorsal cortex lesions on spatial memory in lizards.</a> Behav. Brain Res. 118, 27-42 (2001).</li><li>Holtzman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Slither+to+Hither:+Orientation+and+Spatial+Learning+in+Snakes.">From Slither to Hither: Orientation and Spatial Learning in Snakes.</a> Integr. Biol. 1, 81-89 (1998).</li><li>Holtzman, D. A., Harris, T. W., Aranguren, G., Bostock, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+learning+of+an+escape+task+by+young+corn+snakes,+Elaphe+guttata+guttata.">Spatial learning of an escape task by young corn snakes, Elaphe guttata guttata.</a> Anim. Behav. 57, 51-60 (1999).</li><li>LaDage, L. D., Roth, T. C., Cerjanic, A. M., Sinervo, B., Pravosudov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+memory:+are+lizards+really+deficient.">Spatial memory: are lizards really deficient.</a> Biol. Lett. 8, 939-941 (2012).</li><li>Nobel, D. W. A., Carazo, P., Whiting, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+outdoors:+male+lizards+show+flexible+spatial+learning+under+semi-natural+conditions.">Learning outdoors: male lizards show flexible spatial learning under semi-natural conditions.</a> Biol. Lett. 8, 946-948 (2012).</li><li>Fo&#224;, A., Basaglia, F., Beltrami, G., Carnacina, M., Moretto, E., Bertolucci, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orientation+of+lizards+in+a+Morris+water-maze:+roles+of+the+sun+compass+and+the+parietal+eye.">Orientation of lizards in a Morris water-maze: roles of the sun compass and the parietal eye.</a> J. Exp. Biol. 212, 2918-2924 (2009).</li><li>L&#243;pez, J. C., Vargas, J. P., G&#243;mez, Y., Salas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+non-spatial+learning+in+turtles:+the+role+of+medial+cortex.">Spatial and non-spatial learning in turtles: the role of medial cortex.</a> Behav. Brain Res. 143, 109-120 (2003).</li><li>Petrillo, M., Ritter, C. A., Powers, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+acetylcholine+in+spatial+memory+in+turtles.">A role for acetylcholine in spatial memory in turtles.</a> Physiol. Behav. 56, 135-141 (1994).</li><li>Zani, P. A., Jones, T. D., Neuhaus, R. A., Milgrom, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+refuge+distance+on+escape+behavior+of+side-blotched+lizards+(Uta+stansburiana).">Effect of refuge distance on escape behavior of side-blotched lizards (Uta stansburiana).</a> Can. J. Zool. 87, 407-414 (2009).</li><li>Mason, R. T., Gans, C., Crews, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reptilian+Pheromone.">Reptilian Pheromone.</a> Hormones, Brain, and Behavior: Biology of the Reptilia. , 114-228 (1992).</li><li>Crawley, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotypes+of+inbred+mouse+strains:+implications+and+recommendations+for+molecular+studies.">Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies.</a> Psychopharmacology. 132, 107-124 (1997).</li><li>Schellinck, H. M., Cyr, D. P., Brown, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Many+Ways+Can+Mouse+Behavioral+Experiments+Go+Wrong?+Confounding+Variables+in+Mouse+Models+of+Neurodegenerative+Diseases+and+How+to+Control+Them.">How Many Ways Can Mouse Behavioral Experiments Go Wrong? Confounding Variables in Mouse Models of Neurodegenerative Diseases and How to Control Them.</a> Adv. Stud. Behav. 41, 255-366 (2010).</li><li>O'Leary, T. P., Brown, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+apparatus+design+and+test+procedure+on+learning+and+memory+performance+of+C57BL/6J+mice+on+the+Barnes+maze.">The effects of apparatus design and test procedure on learning and memory performance of C57BL/6J mice on the Barnes maze.</a> J. Neurosci. Methods. 203, 315-324 (2012).</li><li>O'Leary, T. P., Brown, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+apparatus+design+and+behavioral+measures+for+the+assessment+of+visuo-spatial+learning+and+memory+of+mice+on+the+Barnes+maze.">Optimization of apparatus design and behavioral measures for the assessment of visuo-spatial learning and memory of mice on the Barnes maze.</a> Learn. Mem. 20, 85-96 (2013).</li><li>Patil, S. S., Sunyer, B., H&#246;ger, H., Lubec, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+spatial+memory+of+C57BL/6J+and+CD1+mice+in+the+Barnes+maze,+the+Multiple+T-maze+and+in+the+Morris+water+maze.">Evaluation of spatial memory of C57BL/6J and CD1 mice in the Barnes maze, the Multiple T-maze and in the Morris water maze.</a> Behav. Brain. Res. 198, 58-68 (2009).</li><li>Roth, T. C., Krochmal, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+age-specific+learning+and+experience+for+turtles+navigating+a+changing+landscape.">The role of age-specific learning and experience for turtles navigating a changing landscape.</a> Curr. Biol. 25, 333-337 (2015).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="851">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Barnes maze</td>
			<td style="width:239px;">TSE Systems</td>
			<td style="width:176px;">302050-BM/M</td>
			<td style="width:183px;">Available from other vendors. Alternatively, a Barnes maze can be constructed from a standard, non-porous round table.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="42">
			<td height="42" style="height:42px;">Tracking software</td>
			<td style="width:239px;">Code custom written for Matlab 
			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

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...

Watch this Scientific Journal Video about Assessing Spatial Learning and Memory in Small Squamate Reptiles at JoVE.com

Assessing Spatial Learning and Memory in Small Squamate Reptiles

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, ...

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

Research

JoVE Journal

Neuroscience

7.1K Views.  Penn State Altoona. 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.

Video: Assessing Spatial Learning and Memory in Small Squamate Reptiles

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...