The authors would like to thank Christopher Newport University for funding the preparation of this manuscript and The University of Vermont for funding the experiments described in the representative results section.

This protocol is approved by the Institutional Animal Care and Use Committee of Christopher Newport University.
1. Construction of the OFTM Apparatus
<ol>
	<li>Construct the boundary wall (40 cm high and 183 cm diameter) from three pieces of white laminate. Construct the circular maze floor (183 cm diameter) from black laminate and glue it to a plastic table top. Secure the boundary wall to the edge of the table with screws and glue.</li>
	<li>Decorate the interior surface of the boundary wall: Leave one quarter solid white; Paint one quarter solid black; Paint one quarter with black stripes; Paint one quarter with black circles.</li>
	<li>Build four &#8220;food towers&#8221; (8 cm x 8 cm x 20 cm; Figure 1A) cut from a square-shaped white PVC post. Glue a piece of black laminate (8 cm x 8 cm) to the top of each tower. Secure them to the maze floor with crazy glue 54 cm apart from each other.</li>
	<li>Build three &#8220;pre-training towers&#8221; (5 cm, 10 cm &#38; 15 cm high respectively; Figure 1B) from a square-shaped white PVC post. Glue a piece of black laminate (8 cm x 8 cm) to the top of each tower. These towers are taped to the maze floor only during the pre-training phase (described below).</li>
	<li>Glue one black opaque plastic food cup with a screw-top lid (= a clear plastic bead container painted black; 1.75 cm high and 3.75 cm diameter) to the top of each food tower. Drill three small venting holes (3 mm diameter) in each lid. The holes ensure that the rat cannot locate the &#8220;correct&#8221; food tower using olfactory cues. The height of the training towers ensures that the rat cannot locate the &#8220;correct&#8221; tower using the uncapped food cup as a visual cue.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53227/53227fig1.jpg" /> 
Figure 1. Photographs of the Open-field Tower Maze (OFTM). (A) The rat is able to find and retrieve a food reward from only one reward-baited &#8220;training tower&#8221; during acquisition, as well as all subsequent training and testing. (B) Three &#8220;pre-training towers&#8221; are placed in the center of the maze arena during pre-training. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53227/53227fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. The OFTM Maze Room
<ol>
	<li>Use a lamp with a 60 W light bulb (Lumens: 800) to dimly illuminate the maze room. The lamp is located 3 ft above the floor and 2 ft to the side of the maze, facing toward the ceiling. Play white noise in the background (~70 dB) using a sound machine.</li>
	<li>Mount a digital video camera to the ceiling above the maze to transmit video to a computer. Hang an opaque curtain to separate the maze from the computer monitoring area.</li>
</ol>
3. Rat Handling, Food Restriction &#38; Housing
<ol>
	<li>Have each experimenter handle all rats for seven days (4 min per day) prior to pre-training.</li>
	<li>Place rats on a food restricted diet (1 hr&#160;of free-feeding per day). This diet ensures that rats will be motivated to retrieve rewards during training, while maintaining a healthy weight (~90% of free-feeding weight).</li>
	<li>Double-house rats (if using females) throughout the duration of the experiment. Pair-housed female rats are less anxious than single-housed female rats12.</li>
</ol>
4. Pre-training Procedures
<ol>
	<li>Bait each uncapped food cup on the seven towers with a food reward (e.g., one piece of fruit cereal) inside.</li>
	<li>Place a rat into the maze in a random location until it finds and consumes all seven food rewards before it is removed.
	<ol>
		<li>If the rat does not retrieve all of the food rewards within 10 min, then remove it from the maze and give pre-training procedures again the following day. Note: some rats will take longer than others to initially learn to retrieve the food rewards from the top of the towers.</li>
		<li>If a rat does not retrieve and consume all seven food rewards from the towers within the first two days of pre-training, give it additional 30 min sessions in the OFTM until the food rewards are retrieved. Typically, the rat will find and consume all the food rewards after one or two of these 30 min sessions.</li>
	</ol>
	</li>
</ol>
5. Place-training &#38; Response-training Procedures
<ol>
	<li>Assign each rat two of four start locations. Counterbalance the following: the start location release point within and between each training day; the type of learning (place vs response); the &#8220;correct tower&#8221; for all the place learners; and the turn each response-learner is trained to make (right vs. left).</li>
	<li>At the start of each training day turn on the noise machine and open the video recording program. Bait all four towers and cover three out of the four food cups, leaving the food reward on the &#8220;correct&#8221; tower exposed.</li>
	<li>On each given trial, start video recording (refer to manufacturer&#8217;s user manual for instructions), then place the rat in the maze at its assigned start location. Then, go behind the curtain to observe the rat&#8217;s behavior on a computer monitor. Remove the rat from the maze immediately after it retrieves the reward or after 3 min&#160;have passed and stop video recording.
	<ol>
		<li>For place-learning rats, always use the same &#8220;correct&#8221; food tower, and place the rat in one of two start locations. A place-learner solves the OFTM by finding a specific location using the spatial cues provided in the maze. For example, bait tower D (= the &#8220;correct&#8221; tower) and release the rat from start location &#8216;2&#8217; or &#8216;3&#8217; (solid arrows in Figure 2A, left), or bait tower A and release a rat from start location &#39;1&#39; or &#39;4&#39; (dashed arrows in Figure 2A, left).</li>
		<li>For response-learning rats, switch the &#8220;correct&#8221; food tower depending on the start location. Response-learners solve the OFTM by learning to make a specific turn (e.g., always turn right), regardless of its starting location in the maze. For example, bait tower D and release the rat from start location &#8216;3&#8217;, or bait tower B and release that same rat from a start location &#8216;2&#8217; (solid arrows in Figure 2B, left). For a different rat, bait tower B and release the rat from location &#8216;1&#8217;, or bait tower A and release that rat from location &#8216;4&#8217; (dashed arrows in Figure 2B, left).</li>
	</ol>
	</li>
	<li>Give all rats a total of 48 training trials across 12 days (four trials per day).</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53227/53227fig2.jpg" /> 
Figure 2. Overhead Schematic Showing Place- and Response-Training &#38; Testing Procedures. This figure shows the start and food reward locations for training place-learners (A, Left) and response learners (B, Left), and also the start and reward locations used during the start location switch test for place-learners (A, Right) and response learners (B, Right). Numbers indicate a rat&#8217;s starting location; gray squares represent food towers; solid and dashed lines point to the baited food tower. Start locations are counterbalanced within each training condition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53227/53227fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Start Location Switch Testing Procedures
<ol>
	<li>After all the rats perform at their asymptotic level (~80% of correct first choice responding) carry out a &#8220;Start Location Switch Test&#8221; to test the persistence of the learned behavior.</li>
	<li>Counterbalance the novel start locations (i.e., the start locations that have not been previously used for the rat).</li>
	<li>Follow the procedures for each training trial described in protocol section 5 with the new start locations.
	<ol>
		<li>For a place-learning rat that originally was released from location &#8216;2&#8217; or &#8216;3&#8217; and had tower D as its &#8220;correct&#8221; tower, place the rat in either of the new start location &#39;1&#39; or &#39;4&#39; , while still baiting tower D (Figure 2A, right).</li>
		<li>For a response-learning rat that was originally released from location &#8216;2&#8217; or &#8216;3&#8217; and learned to turn right to receive the reward (on towers B and D, respectively), place the rat in either of the new start location &#8216;1&#8217; or &#8216;4&#8217; and bait either tower A or C, respectively (which would still require the rat to turn right in order to find the food reward) Figure 2B, right.</li>
		<li>Administer all rats eight start location switch test trials across two days (four trials per day).</li>
	</ol>
	</li>
</ol>
7. Data Analysis
<ol>
	<li>Have an experimenter who has not trained rats and does not know the treatment of each rat score video-taped trials by counting the number of responses a rat makes to find the &#8220;correct&#8221; tower on each trial (indicated by the rat rearing up over the food cup).</li>
	<li>Calculate the percent of times that a rat finds the reward on its first choice (% of first choice correct responses) within each training day. Construct learning curves using the percent of first choice correct responses across training days. Use the percent of first choice correct response for the statistical analysis.</li>
	<li>Calculate the number of trials it took the rat to reach the a priori set criterion to acquire the task (i.e., first choice correct responding on 8 out of 10 consecutive trials). Exclude rats from analysis that do not reach the a priori criterion.</li>
</ol>

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The authors have nothing to disclose.

Numerous paradigms can be used to investigate learning and memory in rodents, and each has important steps that experimenters should follow. In the OFTM, it is important that maze conditions (e.g., position of maze, intra- and extra-maze cues, dim lighting and background noise level, daily time of training) are kept constant throughout the duration of the experiment. It is also necessary to minimize stress in rat subjects to ensure continuous performance in the OFTM. Stress can be kept to a minimum in female rats by pair-housing them and also through routine handling by experimenters prior to training. In order to accurately score a rat&#8217;s performance during training and testing (i.e., when a rat makes a correct or incorrect tower choice), it is important to use food towers that are tall enough so the rat must &#8220;climb up&#8221; the tower and rear over the food cup to retrieve a reward. The lid of the food cup is not visible to the rat until it &#8220;climbs&#8221; the tower. The food tower that the rat chooses, therefore, is unambiguous and can be reliably scored by the experimenter. The maze floor must be constructed from material that has high contrast relative to the rat&#8217;s color if the experimenter wants to analyze the rat&#8217;s velocity, distance traveled and/or time spent in a specific portion of the maze using video tracking software.
Long-term memory retention of place and response learning could be examined using the OFTM task. Following the completion of 48 training trials, all rats may spend 21 days in their home cages without any handling. This 21-day interval is commonly used to test for retention of a long-term memory15-17. The rats could be taken off food restriction at the beginning of this memory retention period and subsequently returned to food restriction a week prior to testing. All rats then could receive 12 reacquisition trials across three days (four trials per day) of training (during the reacquisition trials, follow the protocol described in this manuscript). If an experimenter needs to assess long-term memory retention, then rats should not be handled during the retention interval because retrieval cues, such as handling, tend to block long-term forgetting18, 19. Experiments that require handling during the retention interval should be designed to include a retention interval longer than 21 days to avoid this confound. In addition to testing for long-term memory retention, the OFTM can also be used to examine rat&#8217;s ability to learn a new maze rule (e.g., reversal learning).
The OFTM was designed to specifically assess spatial learning (e.g., place- and response-learning in rodents, but only adult Sprague-Dawley rats have been used in actual experiments with this maze. However, it is certainly possible to use other types of rodents in the OFTM, such as other rat strains and mice. It is also possible to test rodents of different ages in the OFTM, although it may be necessary to scale the maze dimensions up or down depending on the size of the subject tested. The OFTM is not currently available for purchase on the open market, so experimenters will need to construct their own maze. It is unlikely that minor differences in construction materials (e.g., texture and/or color) or maze size deviating from the OFTM described in the present protocol will significantly influence how a rat performs in the maze; however, this hypothesis has not been tested. It is also recommended that experimenters conduct pilot work before they begin an experiment.
There are a few notable reasons why the OFTM is a useful paradigm for studying the behavioral and neurobiological mechanisms of spatial learning. The OFTM is similar in complexity to the Morris Water Maze; however, the rat is not forced to swim in this task. Thus, the OFTM can be used to assess learning in a non-stressful situation, unlike the Morris Water Maze which inherently evokes a stress response in rodents10. Additionally, the probability for a rat to make a correct response by chance is 25% lower in the OFTM than in a T-maze. Also, rather than being forced to make one choice from a limited number of predetermined paths (as is required in some spatial paradigms), the rat&#8217;s navigation path is not restricted in the OFTM.
Prior studies have shown that place-learning depends on the hippocampus and response-learning depends on the striatum (reviewed in 5). This brain-region distinction between place- and response-learning has been found using the T-maze apparatus4, a radial-arm task20, as well as the Morris Water Maze3, 21. Future experiments will be conducted to test whether the hippocampus and striatum are necessary for place- and response-learning in the OFTM. If this is the case, additional experiments will be able to confidently test the effects that hormones, drugs, diet, etc. have on hippocampal- vs. striatal-dependent learning pathways in a non-stressful environment.

The open-field tower maze (OFTM) can be used to examine the behavioral and neurobiological mechanisms of spatial learning in rodents. Importantly, the OFTM allows researchers to study both &#8220;place-based&#8221; and &#8220;response-based&#8221; learning types. During place-learning an animal navigates through a maze using the spatial cues in the surrounding environment (e.g., a food reward is located next to a tall beacon). During response-learning the animal makes a specific turn response regardless of its position relative to spatial cues (e.g., make a left or right turn to find a food reward). It has been previously reported that response-learning and place-learning rely on two different memory systems3, 4 and mapping their neural mechanisms has been an ongoing scientific pursuit2, 5-7. The design of the OFTM was adapted from the one previously described8.
Open field paradigms (a component of the OFTM) are commonly used to investigate anxiety in rodents9. During the typical open field anxiety test rats that spend more time in the center of an open field have lower stress and anxiety levels than rats that spend more time near the perimeter. In the OFTM, a rat must enter the center of the maze to find the food reward. Therefore, correct navigation in the OFTM indicates that these rats are non-stressed. One reason why the rats are non-stressed during the training is because unlike standard open-field tests of anxiety training the OFTM requires pre-training phase during which the rat is pre-exposed to the maze over a long period of time. In addition, the training occurs in a dimly lit environment further reducing the anxiety-inducing aspect of an open field which is typically brightly lit.
Another unique feature of the OFTM, compared to some other mazes, is how learning is measured. Experimenters using the Morris Water Maze or Barnes Maze typically measure learning by analyzing the speed and/or path length a rat travels to reach its target location. These dependent measures can be affected by non-learning factors, such as changes in a rat&#8217;s general motor activity. However, the OFTM measures learning using the percent of first-choice correct response a rat makes to find the food reward. This dependent measure is not vulnerable to changes in motor performance and therefore, more accurately assesses learning than measurements of speed and distance. Nevertheless, the experimenter can also measure a rat&#8217;s speed and distance traveled in the OFTM using video tracking software.
Other maze-types, such as the T-maze, Morris Water Maze and Barnes Maze can be used to study place- and response-learning. However, the OFTM is unique in a few notable ways. First, the OFTM is a more sensitive behavioral assay than the T-maze because the OFTM has a chance performance of 25%, whereas the T-maze has a chance performance of 50%. Second, a rat can only turn in one of two ways in the T-maze, but has unrestricted route possibilities when navigating in the OFTM. Third, unlike the Morris Water Maze and Barnes Maze tasks (which have unrestricted route possibilities like the OFTM), experiments using the OFTM do not require rats to navigate through an aversive situation. Rats are motived to navigate in the Morris Water Maze to escape swimming in water and they are motivated to navigate in the Barnes Maze to avoid being in an open, brightly-lit environment. It has been demonstrated that exposure to the Morris Water Maze or Barnes Maze induces the release of stress hormone (i.e., corticosterone) in rodents10. However, rats navigate in the OFTM because they are motivated to find a food reward and training is conducted with dim ambient lighting. Thus, the OFTM is a non-aversive appetitive task. Results from experiments that use a stressful maze paradigm to examine the effects of manipulations such as drug, hormone, exercise, or diet on learning and memory mechanisms can be difficult to interpret because &#8220;stress&#8221;, by itself, may influence cognitive function11.

<table><tbody><tr><td>OFTM construction supplies</td><td></td><td></td><td></td></tr><tr><td>Safety Glasses, Clear, Uncoated</td><td>Grainger</td><td>4EY97</td><td>Used for general eye protection during maze construction</td></tr><tr><td>Masking Tape, Beige, 18&amp;#160;mm x 55&amp;#160;m</td><td>Grainger</td><td>15F739</td><td>General supply</td></tr><tr><td>Wiss All-purpose Scissor</td><td>Home Depot&amp;nbsp;</td><td>37103278647</td><td>General supply</td></tr><tr><td>Lancaster 6 foot Round Folding table - Heavy Duty white granite plastic - 29&quot;&amp;nbsp;</td><td>www.webstaurantstore.com</td><td>384YCZ72RND</td><td>Used as base of the maze</td></tr><tr><td>Laminate Countertop sheet in White Matte Finish (48 x 96 x 0.039)</td><td>Home Depot&amp;nbsp;</td><td>1573603504896</td><td>Used as walls for the maze</td></tr><tr><td>Laminate Countertop sheet in Black Matte Finish (48 x 96 x 0.039)</td><td>Home Depot&amp;nbsp;</td><td>1595603504896</td><td>Used as floor of the maze</td></tr><tr><td>Adhesive, Spray, 24&amp;#160;oz, 17.6&amp;#160;oz Net. 3M</td><td>Grainger</td><td>3MA16</td><td>Used to adhere the laminate floor to the table top and pieces of laminate to each other</td></tr><tr><td>16.6 oz #90 hi-strength S</td><td>Lowe&#039;s</td><td>193252</td><td>Used to adhere the laminate floor to the table top</td></tr><tr><td>Superflex RTV, Silicone Adhesive Sealant, Loctile</td><td>VWR</td><td>300040-068</td><td>Used to seal around the baze (between the floor and walls of the maze)</td></tr><tr><td>Industrial Sealant; 10.1&amp;#160;oz</td><td>Grainger</td><td>4UH06</td><td>Used to seal around the baze (between the floor and walls of the maze)</td></tr><tr><td>Painter&#039;s Masking Tape; Shurtape; 2&amp;#160;in x 60&amp;#160;yd</td><td>Grainger</td><td>6FET7</td><td>Used to mark out visual patterns on maze walls</td></tr><tr><td>Mini/Trim paint tray</td><td>ACE Hardware</td><td>1365105</td><td>Used to hold paint&amp;nbsp;</td></tr><tr><td>Foam mini roller 4&quot; 6&amp;#160;pk</td><td>ACE Hardware</td><td>1494871</td><td>Used for painting patterns&amp;nbsp;</td></tr><tr><td>Val Sample Set Nbase paint</td><td>ACE Hardware</td><td>1505031</td><td>Used as paint for pattern on the walls of the maze</td></tr><tr><td>HP Paint Black</td><td>Walmart</td><td>72450405005</td><td>Used to paint pattern on the walls of the maze</td></tr><tr><td>Brush</td><td>Walmart</td><td>81773801110</td><td>Used to paint pattern on the walls of the maze</td></tr><tr><td>Duct Tape, 2&amp;#160;in x 60&amp;#160;yd, 10.5&amp;#160;mil. Black</td><td>Grainger</td><td>15F767</td><td>Used to wrap the exterior of the maze</td></tr><tr><td>4x4 8&amp;#160;ft PT pine Vinyl-CTD Post W/Cap</td><td>Home Depot&amp;nbsp;</td><td>40933070541</td><td>Used to build towers for the maze</td></tr><tr><td>20 gram Pro Super Glue</td><td>Lowe&#039;s</td><td>125870</td><td>Used to glue tower tops, towers to the floor of the maze and food cups to the tower tops</td></tr><tr><td>Bead Containers 9 - 1/2</td><td>Michaels</td><td>652695766978</td><td>Used as food cups on top of towers</td></tr><tr><td>Spray Paint, Black, 12&amp;#160;oz</td><td>Grainger</td><td>6KP22</td><td>Used to paint food cups &amp;amp; covers</td></tr><tr><td>Room prepration</td><td></td><td></td><td></td></tr><tr><td>Conair SU1W Sound Machine; 10 Sounds</td><td>Home Depot&amp;nbsp;</td><td>204493600</td><td>Used for background noise</td></tr><tr><td>Lamp</td><td>Walmart</td><td>5027698392</td><td>Used for dim lighting of maze room</td></tr><tr><td>ES 14&amp;#160;W DayLight Spiral Bulb 4&amp;#160;pk</td><td>Home Depot&amp;nbsp;</td><td>762148208433</td><td>Used for dim lighting of maze room</td></tr><tr><td>Shower Curtain</td><td>Walmart</td><td>3429910901</td><td>Used for separation between the maze and the experimenter</td></tr><tr><td>Curtain Rod</td><td>Bed Bath &amp;amp; Beyond</td><td>76598645104</td><td>Used for separation between the maze and the experimenter</td></tr><tr><td>Froot Loops Cereal</td><td>Walmart</td><td>3800039122</td><td>Used as reward</td></tr><tr><td>Recording equipment</td><td></td><td></td><td></td></tr><tr><td>Media Recorder 3:4 video</td><td>Noldus</td><td>NMR4-0200</td><td>Used to record training trials</td></tr><tr><td>Monochrome GigE Camera</td><td>Noldus</td><td>XCFW-BG62</td><td>Used to record training trials</td></tr><tr><td>Precision M4800 Laptop</td><td>Dell</td><td>N/A</td><td>Used to run video software</td></tr></tbody></table>

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 examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how &#8220;stress&#8221; might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function1. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats2. Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

The representative results described here have been previously published in the Neurobiology of Learning &#38; Memory, Elsevier2. In this experiment the OFTM was used to test the effects of chronic and cycling estradiol treatments on place- and response-learning in female Sprague-Dawley rats (250 &#8211; 300 g; N = 48). There were two important reasons why the OFTM was used to conduct this experiment. First, estradiol has been shown to induce differential effects on place- and response-learning7. Second, it was necessary to test the effects of estradiol on learning in a non-stressful environment, because gonadal hormones have differential effects in humans exposed to stressful situations13. In the Lipatova et al. (2014) experiment ovariectomized female rats were administered daily subcutaneous injections of estradiol (10 &#181;g/kg) or vehicle solution (peanut oil). Rats in the &#8220;Continuous&#8221; condition received daily injections of estradiol and rats in the &#8220;Vehicle&#8221; condition received daily injections of the vehicle solution. Rats in the &#8220;Cycling&#8221; condition were given injections of estradiol every 4th day and vehicle solution on the other days. Half the rats in each condition were trained with place-training procedures and half were trained with response-training procedures. Six rats did not reach the a priori acquisition to criterion and thus were not included in the analysis. The final groups were: Vehicle-Place (Vehicle-P; n = 9), Vehicle-Response (Vehicle-R; n = 6), Continuous-P (n = 7), Continuous-R (n = 7), Cycling-P (n = 7) and Cycling-R (n = 6).
Acquisition to Criterion: Place-Training vs. Response-Training
After pre-training, each rat was given 48 acquisition trials on the OFTM. A 2 x 3 (training type x treatment) ANOVA was performed using the number of trials the rats took to reach the a priori acquisition criterion, which revealed a significant main effect of treatment [F(2, 36) = 5.6, p &#60; 0.01] and training type [F(1, 36) = 5.09, p &#60; 0.04]. A follow up test (HSD) showed that rats given cycling estradiol-replacement took fewer trials to reach the acquisition criterion for place-learning than rats given cycling injections or vehicle injections, p &#60; 0.01. Estradiol treatment had no effect on the number of trials it took the rats to reach the acquisition criterion for response learning (Figure 3).
<img alt="Figure 3" src="/files/ftp_upload/53227/53227fig3.jpg" /> 
Figure 3. Trials to Criterion for Place- and Response-training. This graph shows the average number of trials for rats given place (black bars) and response (white bars) training needed to reach 8 out of 10 consecutive first choice correct responses. Female rats given cycling estradiol replacement required fewer trials to reach criterion for place-learning than rats given continuous estradiol replacement and vehicle-treated rats. There was no significant difference in acquisition of response learning across the different treatment conditions. [Modified from Lipatova et al. 2014 and reprinted with permission from Elsevier] <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53227/53227fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Acquisition in Place-Learners
Figure 4 shows acquisition curves for the vehicle and estradiol-treated rats that were given place-training. A 3 x 12 (treatment x trial block) repeated-measures ANOVA revealed a significant within-subject effect of trial block [F(11, 220) = 22.47, p &#60; 0.01], indicating that performance improved during training. Additionally, the analyses revealed a significant between-subject effect of treatment [F(1,20) = 3.76, p &#60; 0.05] (Figure 4). A follow up test (HSD) showed that rats given cycling estradiol treatment (Cycling-P) had higher percentages of 1st choice correct trials than vehicle (Vehicle-P) and continuously (Continuous-P) estradiol treated rats, p &#60; 0.02. There was no difference in the percent of 1st choice correct responses between the Vehicle-P and Continuous-P group. These results showed that cycling estradiol treatment enhanced acquisition of place-learning.
<img alt="Figure 4" src="/files/ftp_upload/53227/53227fig4.jpg" /> 
Figure 4. Acquisition Rate for Place-Learning. This graph shows the rate of acquisition for rats given place-training based on the percentage of first choice correct responses made within blocks of four trials. After 48 trials all rats reached ~80% first choice correct responding. Cycling estradiol replacement rats (Cycling-P; white triangles) acquired place-learning significantly faster than continuous estradiol replacement rats (Continuous-P; white squares) and vehicle treated rats (Vehicle-P; white circles). [Modified from Lipatova et al. 2014 and reprinted with permission from Elsevier] <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53227/53227fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Acquisition in Response Learners
Figure 5 shows acquisition curves for the vehicle and estradiol-treated rats given response-training. A 3 x 12 (treatment x trial block) repeated-measures ANOVA revealed a within-subject effect of trial block [F(11, 176) = 12.59, p &#60; 0.01], showing that performance improved throughout training. However, treatment type had no significant effect on the rate of response learning.
<img alt="Figure 5" src="/files/ftp_upload/53227/53227fig5.jpg" /> 
Figure 5. Acquisition Rate for Response-Learning. This graph shows the rate of acquisition for rats given response training based on the percentage of first choice correct responses made within blocks of four trials. After 48 trials of training all rats reached ~80% first choice correct responding. There was no significant difference in acquisition rate across the different treatment conditions (Cycling-P; black triangles vs. Continuous-P; black squares vs. Vehicle-P; black circles). [Modified from Lipatova et al. 2014 and reprinted with permission from Elsevier] <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53227/53227fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Start Location Switch in Place-Learners
Place-learners were initially impaired when the start location was switched, regardless of treatment. A 2 x 3 (last trial block on the original start location vs. the first trial block on the switch start location test x treatment) repeated measures ANOVA revealed a significant effect of start location [F (1,18) = 9.68, p &#60; 0.01] (Figure 6). There was no significant interaction or between-subjects effect. However, previous findings have shown that vehicle-treated place-learners are less impaired than the estradiol-treated rats during a start location switch test14. Therefore, pairwise comparisons were performed to compare percent of 1st choice responses between each group&#8217;s last block on the original start location and the first trial block on the switch start location. Estradiol-treated rats had lower 1st choice response percentages during the switch start test compared to their original start location, p &#60; 0.05. As previously reported, there was no significant decline in performance found for vehicle-treated rats. The difference in performance between vehicle-treated and estradiol-treated rats became larger on the second trial block of the switch start location test (Figure 6). A 2 x 3 (last block on the original start location vs. the second trial block on the switch start location x treatment) repeated measures ANOVA revealed a significant effect of start location [F(1,18) = 20.69, p &#60; 0.01] (Figure 6), showing that performance in vehicle-treated rats on the second block of the switch start location test was the same as their performance on the original start location and estradiol-treated rats displayed a prolonged deficit. Pairwise comparisons showed that Continuous-P and Cycling-P rats had significantly reduced performance on the second day of the new start location compared to their performance during the last day of original start location, p &#60; 0.01. All rats returned to pre-switch test levels of performance when they were subsequently trained on their original start location (Figure 6). This finding was confirmed with a 2 x 3 (original start location before switch vs. original start location after switch x treatment) repeated measures ANOVA that revealed no significant within-subject or between-subjects effect.
<img alt="Figure 6" src="/files/ftp_upload/53227/53227fig6.jpg" /> 
Figure 6. Start Location Switch Test for Place-Learners. This graph shows the percentage of first choice correct responses in four-trial blocks for place-learners on the last day before the start location switch (left panel), during the start location switch test (middle panel) and back with the original start location (right panel). Estradiol-treated rats (white squares &#38; white triangles) had overall lower percentages of 1st choice correct responses than vehicle-treated rats (white circles). [Modified from Lipatova et al. 2014 and reprinted with permission from Elsevier] <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53227/53227fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Start Location Switch in Response Learners
Response-learners were impaired when given the switch start location test. A 2 x 3 (last block of the original start location vs. first trial block of the new start location x treatment) repeated measures ANOVA revealed a significant effect of start location [F(1,16) = 10.28, p &#60; 0.01], but no interaction or main effect of treatment. Therefore, the decline in performance during the first block of the switch start test was equivalent across each treatment condition (Figure 7). However, unlike in the case of place-learners, pairwise comparisons revealed that performance in estradiol-treated response-learners recovered to pre-switch test performance level on the second trial block of the switch start location test, while vehicle-treated rats continued to show impaired performance, p &#60; 0.04. Therefore, vehicle-treated response-learners were more impaired on the switch start location test than estradiol-treated response-learners. All rats returned to pre-switch test levels of performance when they were subsequently tested with their original start location (Figure 7). This finding was confirmed with a 2 x 3 (original start location before the switch test vs. original start location after the switch test x treatment) repeated measures ANOVA did not reveal any significant within-subject or between-subjects effects.

<img alt="Figure 7" src="/files/ftp_upload/53227/53227fig7.jpg" /> 
Figure 7. Start Location Switch Test for Response-Learners. This graph show the percentage of first choice correct responses in four-trial blocks for response learners on the last day before the start location switch (left panel), during the start location switch test (middle panel) and back with the original start location (right panel). Estradiol-treated rats (black squares &#38; black triangles) overall had higher percentages of 1st choice correct responses than vehicle-treated rats (black circles). [Modified from Lipatova et al. 2014 and reprinted with permission from Elsevier] <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53227/53227fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Place and Response Learning in the Open-field Tower Maze at JoVE.com

Place and Response Learning in the Open-field Tower Maze

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

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 ...

place-and-response-learning-in-the-open-field-tower-maze

Nanyang Technological University

Research

JoVE Journal

Behavior

10.3K Views.  Christopher Newport University. The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

Video: Place and Response Learning in the Open-field Tower Maze

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 (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.&#160;bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.&#160;distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.&#160;random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.&#160;shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g.&#160;latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

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 ...

Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have developed visual cognitive tasks for rats using the Floor Projection Maze1,2&#160;that are optimized for visual abilities of rats permitting stronger comparisons of experimental findings with other species.
In order to investigate neural correlates of learning and memory, we have integrated electrophysiological recordings into fully automated cognitive tasks on the Floor Projection Maze1,2. Behavioral software interfaced with an animal tracking system allows monitoring of the animal's behavior with precise control of image presentation and reward contingencies for better trained animals. Integration with an in vivo electrophysiological recording system enables examination of behavioral correlates of neural activity at selected epochs of a given cognitive task.
We describe protocols for a model system that combines automated visual presentation of information to rodents and intracranial reward with electrophysiological approaches. Our model system offers a sophisticated set of tools as a framework for other cognitive tasks to better isolate and identify specific mechanisms contributing to particular cognitive processes.

We describe protocols for training rats for chronic electrophysiological recordings in fully automated cognitive tasks on a Floor Projection Maze.

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have ...

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 most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

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 behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

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 ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

Utilizing a Reconfigurable Maze System to Enhance the Reproducibility of Spatial Navigation Tests in Rodents

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze shape, thus requiring several separate mazes in different configurations. The maze geometry cannot be reconfigured in a single environment to accommodate scalability and reproducibility. The reconfigurable maze is a unique approach to address the limitations, allowing quick and flexible configurations of maze pathways in a repeatable manner. It consists of interlocking pathways and includes feeders, treadmills, movable walls, and shut-off sensors. The current protocol describes how the reconfigurable maze can replicate existing mazes, including the T-shaped, plus-shaped, W-shaped, and figure-eight mazes. Initially, the T-shaped maze was constructed inside a single experimental room, followed by modifications. The rapid and scalable protocol outlined herein demonstrates the flexibility of the reconfigurable maze, achieved through the addition of components and behavioral training phases in a stepwise manner. The reconfigurable maze systematically and precisely assesses the performance of multiple aspects of spatial navigation behavior.

The present protocol describes a reconfigurable maze, a unique system for testing spatial navigation and behavioral phenotypes in rodents. The adaptability of this maze system enables the execution of various experiments in a single physical environment. The ease of structural rearrangement generates reliable and reproducible experimental results.

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze ...

Understanding Spatial Learning in Mice Using the Active Place Avoidance Test

The Active Place Avoidance (APA) Test, an Effective, Versatile and Repeatable Spatial Learning Task for Mice

Hippocampus-dependent spatial learning in rodents has been tested using a variety of methods. These include the Morris water maze (MWM), Y-maze, and novel object location (NOL) tasks. More recently, the active place avoidance (APA) task has been developed as an alternative to these more traditional approaches. In the APA task, mice must use spatial cues placed around a rotating arena to avoid a stationary shock zone. Due to the multiple parameters that can be adjusted, the APA task has been demonstrated to be a very versatile approach. It lends itself to being used longitudinally and repeatedly for the same cohort of mice. Here, we provide a detailed protocol to successfully conduct the APA task. We also highlight alternative APA approaches that can be used to examine different components of spatial learning. We describe the data collection and analysis processes. Critical steps during the APA task are discussed to increase the likelihood of successfully conducting the test. The APA task has several advantages over more traditional spatial navigation tests. It is appropriate to use with aged mice or those with disease phenotypes such as Alzheimer's disease. The complexity of the task can be easily altered, allowing a wide range of mouse strains to be tested. Further, the APA task is suitable for testing animals that have undergone surgery or experimental interventions that may have affected motor or neural function, such as stroke or traumatic brain injury.

Author Spotlight: Investigating the Impact of Aging on Hippocampal-Dependent Spatial Learning

Here, we present a protocol for the active place avoidance test, a hippocampus-dependent spatial learning paradigm designed for rodents. Altering key parameters allows for re-testing of animals before and after treatments or over time.

Hippocampus-dependent spatial learning in rodents has been tested using a variety of methods. These include the Morris water maze (MWM), Y-maze, and novel ...

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 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, ...

Neuroscience

Spatial Memory Testing Using Mazes

Spatial learning and memory are neurological functions that allow us to remember important details associated with our environment. Scientists test this phenomenon in rodents using different types of mazes like Morris water maze, Radial water maze, and Barnes maze. By investigating spatial memory in rodents, neurobehavioral scientists can gain valuable understanding of how these processes are altered following brain damage in humans.
Here, the neurobiology behind spatial memory is briefly reviewed and some general principles behind maze tests are discussed. Then, the video explains generalized protocols on how to conduct trials with specific types of mazes. Lastly, it will explain how behavioral researchers are using these tools to conduct some specific experiments.

Spatial Memory Tests Using Morris Water, Radial Water &amp; Barnes Mazes

Spatial learning and memory are neurological functions that allow us to remember important details associated with our environment. Scientists test this ...

Education

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Psychology

Behavioral Science

Barnes Maze: A Method For Assessing Spacial Learning and Memory in Lab Animals

Source: Ingersoll, J. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/58008/analyzing-spatial-learning-prosocial-behavior-mice-using-barnes-maze">Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms</a>. J. Vis. Exp. (2018).
This video shows how to use the Barnes Maze with a mouse. The Barnes Maze testing procedure is used by researchers to assess spatial learning and memory in laboratory animals following repeated trials.

Source: Ingersoll, J. et al. Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms. J. Vis. ...