Egocentric Navigation in a Morris Water Maze for Testing Memory Formation in Mice

Mouse models are crucial to understanding not only sensory and cognitive neurological processes, but also diseases, disabilities, and responses to pharmacological or surgical treatments. Behavioral tests are essential assets in the description of these models. The Morris Water Maze is a common behavioral test tool used to measure spatial navigation and memory. We developed our own comparable Morris Water Maze following current standards. Standard tests were replicated including pre-training, spatial acquisition, spatial reference, and spatial reversal in order to determine the validity of our maze. All tests were performed with four C57BL/6 wild type mice. Our newly created maze also included the ability to perform a novel Rotational Cue Test. This test helped determine whether the navigation method used by the mice in the maze was Egocentric or Allocentric. Currently the navigation strategy is debated in the literature and needs to be determined in order to accurately describe behavior of mouse models. The Rotational Cues Test was performed by rotating all visual cues 90 degrees clockwise around the maze to a new quadrant position. Since this alteration did not affect the mice's ability to complete the maze, the mice had no dependence on the cues and the navigational strategy was concluded to be Egocentric. With this information, mouse models tested in the future can have their learning and cognitive patterns identified in accordance to the Egocentric Navigation method they are using to complete the maze.

EGOCENTRIC NAVIGATION IN A MORRIS WATER MAZE FOR TESTING MEMORY FORMATION IN MICE by Andrew Sedley A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Science In Biomedical Engineering Approved: ______________________________ Dr. Dimitri Traenkner Thesis Faculty Supervisor _____________________________ Dr. David Grainger Chair, Department of Bioengineering _______________________________ Dr. Kelly Broadhead Department Honors Advisor _____________________________ Dr. Sylvia D. Torti Dean, Honors College April 2018 Copyright © 2018 All Rights Reserved ABSTRACT Mouse models are crucial to understanding not only sensory and cognitive neurological processes, but also diseases, disabilities, and responses to pharmacological or surgical treatments. Behavioral tests are essential assets in the description of these models. The Morris Water Maze is a common behavioral test tool used to measure spatial navigation and memory. We developed our own comparable Morris Water Maze following current standards. Standard tests were replicated including pre-training, spatial acquisition, spatial reference, and spatial reversal in order to determine the validity of our maze. All tests were performed with four C57BL/6 wild type mice. Our newly created maze also included the ability to perform a novel Rotational Cue Test. This test helped determine whether the navigation method used by the mice in the maze was Egocentric or Allocentric. Currently the navigation strategy is debated in the literature and needs to be determined in order to accurately describe behavior of mouse models. The Rotational Cues Test was performed by rotating all visual cues 90 degrees clockwise around the maze to a new quadrant position. Since this alteration did not affect the mice’s ability to complete the maze, the mice had no dependence on the cues and the navigational strategy was concluded to be Egocentric. With this information, mouse models tested in the future can have their learning and cognitive patterns identified in accordance to the Egocentric Navigation method they are using to complete the maze. ii TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 METHODS 7 RESULTS 13 DISCUSSION 21 REFERENCES 27 iii INTRODUCTION Mouse model behavioral tests are an effective way to study the impact of medicine on humans. These tests may help understand how brain lesions, intellectual diseases, pharmacological injections, and genetic mechanisms may affect a mouse through anxiety, obsessive compulsive disorder (OCD), or memory loss [1]. Though behavior is not easily quantifiable, many tests exist to measure and investigate it in mice. A common experimental apparatus used to quantify spatial learning and memory formation in mice is the Morris Water Maze [2-4]. The Morris Water Maze (MWM) was first created by neurologist Richard G. Morris in 1981, who developed the experiment to test spatial memory formation in mice [2]. A few years later in 1984, he released a set of replicable, standard protocols for other researchers to use for different mouse models [5]. Since then, many alterations have been made to both the maze and the protocol but two main components have remained the same. The maze consists of a large pool filled with opaque water, and a hidden platform submerged beneath the surface for mice to find. Mice are placed at various locations throughout the pool and undergo several trials to find the platform as quickly as possible. Often, distinct shapes on the walls of the tank are provided to serve as distal cues for the mice as they search for the hidden platform. The popularity of the MWM is due to its high reliability across a range of tank configurations, testing procedures, lack of required pre-training, mice and rat compatibility, and its relative immunity to motivational differences across a range of experimental treatment conditions. These treatments include genetic, pharmacological, nutritional, toxicological, or lesional changes to the mice [6]. For these reasons, the MWM is a flexible and useful behavioral tool for various experiments. The MWM has commonly been used to assess learning and memory in mouse models with drug adjusted neurochemical systems [7-11]. The performance of these models in the MWM has been linked to long-term potentiation (LTP) and N-methyl-Daspartate (NMDA) receptor function, both of which are important in hippocampal dependent learning [12,13]. This has made the MWM into a great investigational tool for hippocampal neural circuitry. The MWM has also been used to test mutant mice (i.e. genetically altered) [14-19]. These models have been useful in identifying specific genotypic and phenotypic expressions responsible for observed behavioral traits. For example, mice with a mutation in the alpha-calcium-calmodulin-dependent kinase II (alpha-CaMKII), a synaptic protein enriched in the hippocampus, produced poor MWM performance [20]. This demonstrated that alpha-CaMKII was highly important for spatial navigation processing in the hippocampus, similar to LTP and NMDA receptor function altered pharmacologically. The drug and genetic alterations to mouse models may also be causing memory loss, OCD, or anxiety. These can be measured with the poor performance results in the MWM in which the mice forget the platform location or become overly stressed [8]. All of these examples further demonstrate why the MWM is the premier behavioral test tool used for measuring spatial navigation and reference memory in mouse models [21]. In addition to MWM performance results (as in the previous examples), the navigation strategy used by the mice is also significant. In the study by Gehring et. al., all swimming paths taken in the MWM were classified into one of six categories: 2 Thigmotaxis, Incursion, Scanning, Focused Search, Chaining Response, Self-orienting, Scanning Surroundings, and Target Scanning [22]. Each described a unique swimming path taken by the mice at any given time during the trial. A combination of these can describe the navigation strategy used by the mouse to find the platform [22].When the six possible paths are given numerical values relative to the wall and platform over time, a result can be quantified [22]. While this method is useful to quantify the navigation, the overall navigational strategy can be described in simpler terms. In particular, spatial navigation can be broken down into two possible strategies. All spatial navigation can either be described as Allocentric or Egocentric. While some strategies may use a mix of both, every individual navigational decision can be classified as one or the other [23]. Egocentric is defined as using proximal or internal cues to determine one’s location and direction. This can be thought of as following directions around a neighborhood. To reach your destination, go four blocks, turn left, and continue till you reach the terminus. Allocentric is defined as using distal cues or external cues to compare and triangulate ones position or the targets position [24,25]. This can be visualized as using a mental map of a familiar neighborhood to determine where the destination is. Because I can see the pink house across from the blue house and the church on the corner, I know my destination is in-between the blue house and the church. This depends greatly on familiarity with the environment and targets location in order to triangulate based upon distal location identifiers. A graphic demonstrating the difference between these two navigational strategies can be seen in Figure 1, below [26]. 3 Figure (1) – On the left, an allocentric reference frame represents objects in relation to one another. On the right, an egocentric reference frame represents objects in relation to the location of the self (the observer) [26]. Navigational strategies within the context of the MWM can be isolated by using a specific experimental setup. An allocentric navigation maze must be designed to have ample distal cues and be free of proximal cues [27]. It is often difficult to completely erase proximal cues, but water can often successfully blend together the proximal environment. This is why the MWM is a great tool for testing allocentric navigation because the water and tank have few proximal cues leaving only distal cues placed by the experimenter. Another way to create an allocentric maze is to target the dorsal striatum and connected structures responsible for egocentric processing in the brain [28]. If these are impaired, the only form of navigation remaining is allocentric. By contrast, an egocentric navigation maze needs to be designed to have ample proximal cues and minimal distal cues [27]. The easiest way to achieve this is to limit the subject’s vision and force the use of only proximal/internal cues. Since the MWM is usually much larger compared to the subject, 4 the distal cues can be reduced by lowering the light in the experimental set up. Another way to create an egocentric maze is to target the hippocampus, entorhinal cortex, and surrounding structures in the brain responsible for allocentric processing [28]. If these are impaired, only egocentric navigation is left [28]. While experimental set up can be useful in making only one navigational strategy possible, sometimes a preferred MWM configuration needs to have its navigation strategy investigated. Currently, there is disagreement between what navigation strategy is being used in similar MWM configurations. In the study of Rodgers et. al., Allocentric navigation was determined to be the primary method of navigation [29]. Specific experiments highlighting certain cues during different stages of the experiment showed that individual visual cues played a significant role in the navigation of the mice through the maze. Dependency on these distal cues confers evidence that the navigation method is allocentric. While the methods of this study were not clearly stated, their MWM setup and protocol contained similar elements to that of a different study. In the study of Vorhees and Williams, their own protocol determined the spatial navigation strategy as Egocentric [30]. Experiments had been performed with distal cues and trends of learning were observed. However, when the distal cues were removed, the mouse’s ability to complete the maze did not change at all. This concluded that either the mice were using both allocentric and egocentric navigation initially and simply reverted to egocentric when the distal cues were removed, or they were only using egocentric from the start. Either way, the mice were not using distal cues to navigate so the method at some point must have been egocentric as there were no other distal cues provided in the maze. Both of these studies used similar MWM configurations and protocols but 5 yielded different conclusions about the navigation strategy being used. For this reason, it is important that we test our own developed MWM and mouse strain for what navigation strategy is being used. We developed a comparable Morris Water Maze that includes the ability to perform a Rotational Cue Test to determine whether the navigation method is Egocentric or Allocentric. To demonstrate the compatibility of the constructed MWM, a standard experimental protocol was followed as established in the study by Vorhees and Williams [30]. The following tests were performed from the protocol: Pre-Training, Submerged Platform Training, Spatial Acquisition, Spatial Reference, and Spatial Reversal. By performing a similar analysis of the results, the mazes were comparable. To determine whether the mice are using the distal cues or internal cues, a new protocol will be introduced in which the cues are rotated for a trial after the normal training. Comparing this Rotational Cues Test and the normal training will determine whether distal cues are used for navigation. This novel experiment will identify the navigation methodology used to complete the maze. Correspondingly, the behavioral response of the model to an alteration in the navigation tool can be quantified and used to describe the memory loss, OCD, or anxiety that may be occurring in the mouse model. 6 METHODS Setup of the Maze A 1.3-meter (m) diameter aluminum tub with a depth of 0.61 m was positioned on a short platform to serve as the arena for the Morris Water Maze. A framework of adjustable lighting equipment was set above the tub and consisted of five adjustable LED light fixtures and a camera. Light and camera adjustments could be made for various experiments. The entire setup was enclosed by black curtains on rods that could be slid back and forth to reveal or hide the maze from the experimenter. This curtain ensures no external objects can be seen by the mice if all the curtains are closed. Figure 2 below shows the experimental setup. Figure (2) – Overview of the Morris Water Maze setup. 7 A square white platform with top dimensions of 108 millimeter (mm) by 108 mm was secured on top of a stand to give the overall apparatus a height of 76.2 mm. This platform served as the destination for the mice while swimming in the maze. An additional 19.05 mm tall white training platform with the same dimensions as the previous square platform was placed on top of the apparatus. The tub was filled with water up to the surface of the training platform. For submerged tests, the training platform was removed, leaving the white platform and stand of the apparatus, 19.05 mm underwater. For surface level experiments, the training platform was kept in place. 400 mL of Non-Toxic Crayola Washable White Paint was added to make the water opaque and hide the white platforms. Figure 3 below shows the platform apparatus without the training platform. Figure (3) – Platform apparatus without the training extension for submerged tests. 8 Spatial cues were created using white and black construction paper. The black paper was cut into four distinct shapes and glued to white paper for contrast. These shapes were a triangle, ring, x and a set of three parallel horizontal bars. The sheets were laminated to be waterproof. Magnetic directions North, South, East, and West were labeled on the top edges of the tub and divided the tank into four magnetic referenced quadrants. The laminated spatial cues were randomly taped to the walls of the tank relative to the quadrant for all experiments. A top view schematic that shows the location of the cues can be seen in Figure 4(a). A top down picture of the experimental set up can be seen in Figure 4(b). (a) (b) Figure (4) – (a): Schematic of the MWM with distal cue locations. (b): Top view of experiment apparatus with the training platform visible. 9 Recording System An IMAGINGSOURCE™ DMK 22AUCO3 camera was positioned 4 feet above the maze using a series of lever arms. The light fixtures above the maze outputted a total luminance of 100 lux evenly throughout the maze. ANY-maze™ software was used to record the video feed and save important parameters such as the duration, total distance traveled, path efficiency, and time spent in each quadrant from each trial. Protocol Protocols from previous MWM studies, specifically the study by Vorhees and Williams, were used as templates in the applied experiments [30]. Four C57BL/6 wild type mice (n=4) from the Mario Capecchi Lab were used with IACUC approval for the duration of all experiments. The first step was to create a training regimen for the mice to acclimatize to the experimental conditions, setup, equipment etc. During this phase, the distal cues had been removed. Training consisted of placing the mice in a random quadrant, facing the wall of the tank. The training platform was placed 15 centimeters from the wall, in the middle of the East quadrant. The mice were permitted to search for and sit on the platform for the 3 minutes (mins) of the trial. If the mice did not find the platform within the allotted time, they were placed directly on the platform, and allowed to sit for 15 seconds to be shown that the platform exists. This was repeated for 5 days with each mouse (n=4) performing 3 trials per day (m=3). 10 While the mice may have been comfortable sitting on the pre-training platform, removal of the extension for the submerged tests may mean the mice might swim over it. It is vital that the mice be comfortable sitting on the submerged platform in the water, so, an additional training day was added in which each mouse was placed directly on the submerged platform and allowed to sit for 2 mins. This was performed for 3 trials (m=3) for each mouse (n=4) with the training platform removed but still located in the East quadrant. After all the training, a day of rest was provided before moving on to the next test. Prior to the Spatial Acquisition experiment, the distal cues were inserted and the platform was moved to the South quadrant; the training platform was still absent. The mice were once again placed in a random quadrant facing the wall of the tank and were permitted to search for and sit on the submerged platform for 3 mins of the trial. Much like in the training phase, if the mice did not find the platform within the allotted time of 3 mins, they were placed directly on the platform and allowed to sit for 15 secs to show that the platform existed. This was repeated for 5 days, 3 trials (m=3) for each mouse (n=3) per day. Spatial Reference is often referred to as the probe trial. After Spatial Acquisition had occurred, a 2-day break was taken to prepare for Spatial Reference since it requires the mice to swim the whole time while also recalling the platform location from days prior. The platform was removed from the maze and the mice were tested for 1 trial in 1 day (m=1). The start was still in random quadrants and the mice searched for the platform for 3 mins. 11 For Spatial Reversal, everything was kept the same as Spatial Acquisition except the platform was moved to the North quadrant. This was done in order to look at the effects of overwriting the memory of Spatial Acquisition. This test was once again performed for 5 days with 3 trials (m=3) for each mouse (n=4) per day. For the Rotational Cues Test, everything was kept the same as the Spatial Reversal, except for 1 alteration. All visual cues were rotated 90 degrees clockwise around the maze to a new quadrant position. This test was run for 1 day, 3 trails (m=3) per mouse (n=4) and concluded the experiments. Data Analysis All data was exported in comma separated text files (CSV) and then imported into Microsoft Excel. MATLAB™ was used to create plots and perform a Standard Error of the Mean calculation, a breakdown of percent time spent in a variety of locations in the maze, time to platform (delay), and a t-test resulting in a p-value. All behavioral observations were made by reviewing the recorded video feed in the ANYMAZE™ program. Tracking plots were directly exported from ANYMAZE™ for individual trials of interest for further analysis. 12 RESULTS All of the results presented in this section were acquired during a 2-week period using the same four mice for each experiment. All of the experiments were performed inside the Capecchi/Traenkner Lab behavioral room located at the University of Utah. The results are presented in the same chronological order as the experiments. No breaks were taken between tests unless specified in the provided protocol. Pre-Training Pre-Training familiarized the mice with the Morris Water Maze along with the objective of finding a platform. During this phase, the parameter of interest was the percent time spent by the mice on the platform. It was expected to be high since the platform was visible above the surface. Figure 5, below, shows that the mice became more comfortable with the maze and finding the platform over the 5-day Pre-Training period. All the mice spent more than 80% of their time on the platform so we concluded that they understood that the objective of the maze was to find the platform. In fact, most mice had already met this criterion after day 2, and were ready for more complex experiments, without needing the additional 3 days of Pre-Training. Mice that did not achieve at least 80% affinity over the entire Pre-Training period would have been removed from the experiment. 13 Percent Time Spent on Platform (%) 100 75 50 25 0 1 2 3 4 5 Days Figure (5) – Percent Time spent on the platform during each trial was calculated and averaged together for all trials each day. This result is graphed over the 5 days of PreTraining (n=4; m=3; mean ± SEM). Over the 5 days of Pre-Training, the percentage of time spent on the platform increases above an 80% affinity. Submerged Platform Training was performed to ensure that the mice would be comfortable standing on the platform submerged underwater. If they were not, the mice may swim over the top of the platform, and the results would no longer accurately display their ability to find the platform. The important parameter for this test was how many times the mice jumped off the submerged platform after being directly placed on it, during the trial. For all 12 trials performed by the 4 mice, only in one instance did a mouse jump off the platform. This result was considered an outlier, and we proceeded with experiments, confident that mice were comfortable standing on the submerged platform after they found it. 14 Spatial Acquisition tested the mices’ abilities to find a hidden platform within the Morris Water Maze. Visual Cues were added to the maze and the submerged platform was moved to the South quadrant. The parameter of interest used to gauge how quickly the mice completed the maze was time taken to reach the platform, or delay. This was used because once the mice found the platform, they rarely left. Trials of all mice in a day were averaged together and the standard error of the mean was calculated to measure the variance in the results. Results for all 5 days of Spatial Acquisition can be seen in Figure 6, below. A t-test between mean times of day 1 (155 secs) vs. day 5 (29 secs) gave a pvalue of 0.00098. Since the p-value is significantly smaller than 0.05 (95% confidence interval), the results were found to be statistically significant. In other words, the mice demonstrated learning over the 5-day period as evidenced by the decrease in maze completion times from day 1 to day 5. 15 Time to Platform - Delay (sec) 200 150 100 50 0 1 2 3 4 5 Days Figure (6) – Time to the platform (delay) was calculated for each trial, then averaged together for all trials each day. This result is graphed over the 5 days of Spatial Acquisition (n=4; m=3; mean ± SEM). Over the 5 days of Spatial Acquisition, the time to platform drops significantly. Spatial Reference is often referenced as the probe trial because it examines any memories or tasks learned in previous tests. In this case, the platform was removed from the maze to test the mice’s ability to “find” the missing platform. For this experiment, the percentage of time spent in each quadrant of the maze was calculated for each minute of the 3-minute trial. Trials of all the mice were averaged and the results are shown in Figure 7. We expected the mice to spend the majority of their time searching in the South quadrant where the platform should be, but instead, we observed no bias towards this quadrant at any time point during the trial. Consequently, the spatial reference test was inconclusive. 16 Percent Time Spent on Platform (%) 60 40 West South 20 North East 0 1 2 Minute of Trial 3 Figure (7) – Percentage of time spent in each quadrant during each minute of the trial was calculated, then averaged together for all trials for the day (n=4; m=1; mean ± SEM). No significant amount of time was spent in the south quadrant so the results are inconclusive. Spatial Reversal tested the ability of the mice to reverse their memory of finding the hidden platform from the Spatial Acquisition phase of the experiment. The platform was moved from the South Quadrant to the North Quadrant. The parameter of interest was how quickly the mice completed the maze by reaching the platform, or delay. Trials of all mice in a day were averaged together and the standard error of the mean was calculated to measure the variance in the results. Results for all 5 days of Spatial Reversal can be seen in Figure 8, below. A t-test between mean times of day 1 (70 secs) vs. day 5 (38 secs) gave a p-value of 0.168018. Since the p-value is larger than 0.05 (95% confidence interval), the results were found to not be statistically significant. However, the mice still demonstrated a strong correlation of learning over the 5-day period as evidenced by the decrease in maze completion times from day 1 to day 5. 17 Time to Platform - Delay (sec) 90 60 30 0 1 2 3 Days 4 5 Figure (8) – Time to the platform (delay) was calculated for each trial, then averaged together for all trials each day. This result is graphed over the 5 days of Spatial Reversal (n=4; mean ± SEM). Over the 5 days of Spatial Reversal, the mice learn to overwrite their memory and find the new platform location faster over time. The Rotational Cues test was a novel experiment that was created to determine the navigation strategy used by the mice in the Morris Water Maze. Everything was kept the same as Spatial Reversal except the visual cues were rotated 90o clockwise around the maze to a new quadrant position. If the mice were dependent on these distal cues to navigate, they were expected to think that the platform was now in the East quadrant, thereby increasing the time taken to complete the maze. If the mice were not dependent on the distal cues for navigation, they would be unaffected by the change and would complete the maze in the same or less time as the previous Spatial Reversal test. Dependence on the visual cues suggests an Allocentric navigation method while no dependence on the visual cues indicates an Egocentric navigation method. Results for this test can be seen in Figure 18 9. Comparing the mean time of 38 secs to find the platform on the last day of the Spatial Reversal test, and the mean time of 20 secs in the Rotational Cues Test, the decrease in time indicates that the navigation method is Egocentric. No statistical calculations were performed since the Rotational Cues test was completed faster than the Spatial Reversal test and showed no dependence on the distal cues for navigation. Time to Platform - Delay (sec) 90 60 30 0 1 2 3 4 5 6 Days Figure (9) – The time to platform for the 5 days of Spatial Reversal are shown again in blue. The 6th day in red shows the time to platform during the rotational cues test (n=4; m=3; mean ± SEM). The mice found the platform faster during the Rotational Cues Test compared to day 5 of Spatial Reversal, concluding their navigation method is Egocentric because they are not dependent on the distal cues for navigation. 19 Tracking plots are an effective way to look at the navigational path the mouse took during their trial. In Figure 10, below, the tracking plot from Mouse 2 is shown for both the last day of Spatial Reversal and The Rotational Cues Test. The tracking plots for all other mice and trials follow the same trend. This plot helps confirm that the mouse did reach the platform just as quickly after the cues had been rotated and that the paths taken followed similar trends. The behavior of this path is classified as random since there is no apparent clear searching strategy such as circling. (a) (b) Figure (10) – (a): Tracking plot from Mouse 2 during Spatial Reversal. (b): Tracking plot from Mouse 2 during The Rotational Cues Test. 20 DISCUSSION We have developed a comparable Morris Water Maze (MWM) with the ability to perform a Rotational Cue Test to determine whether the navigation method is Egocentric or Allocentric. As seen in the results section, rotation of the visual cues had no effect on the ability of the mice to navigate the maze indicating that either the mice are indifferent towards the shapes of the cues, or they do not use the cues to navigate the maze. Regardless, the navigation method was Egocentric since the mice did not use the individual separate cues to navigate the maze. The validity of our findings, however, rely greatly on the comparability of our Morris Water Maze to those used currently. Each of the performed protocols taken from the literature will now be discussed in how they were useful in showing our maze was equivalent. The Pre-Training exercise was necessary to ensure that the mice were competent for the experiment. While 5 days are recommended, it was found that by day 2, all the mice had already met the criterion of spending more than 80% of the trial time on the platform. The number of Pre-Training days could be reduced in future experiments, without affecting the results of the study. This experiment was also a successful first step in demonstrating the capabilities of our Morris Water Maze; the basic principle mechanics of our maze (i.e. camera tracking software, platform, lighting, and aluminum tub) performed as expected. The Submerged Platform Training was an extra regimen added to ensure the mice would be comfortable in experiments with a submerged platform. This was important as mice in previous studies without this training would sometimes swim right over the platform, unaware of its presence. [31]. Only 1 mouse in 12 trials jumped off after being 21 placed on the submerged platform. Though this may indicate that training might not have been required, it is safer to spend one day training in order to improve subsequent results. Spatial Acquisition was the most important experiment for demonstrating the comparability of our MWM. By producing meaningful learning trends over multiple test days, our MWM was shown to be a viable spatial navigation test tool. The average time to find the platform, across all mice for the first day, was 154 secs, and by the last day this number had dropped to 28 secs. Statistical analysis showed that these values were significantly different (up to a 95% confidence interval). In a study by Brandeis et. al., this level of learning was achieved in 4 days with 8 wild-type mice doing 3 experiments per day [3]. This study is good to compare to since they used the same type of mice and had a similar protocol. Since our study achieved the trend of learning with half the sample number with only one more day of testing, it is concluded that our maze is equivalent. This demonstrates that our MWM is a competitive behavioral tool to measure spatial memory in mice. Spatial Reference served as the probe trial to ascertain what was learned in Spatial Acquisition. Removal of the platform from the maze was expected to prompt the mice to continue searching for the platform at its previous location. Consequently, we expected the percentage of time spent by the mice in the south quadrant to be significantly higher than others. Our results showed no bias towards this quadrant at any point during the trial. Instead, the mice seemed to spend roughly equal amounts of time in all quadrants, making the results insignificant towards determining any characteristics about the spatial navigation of the mice. Future experimental designs in this MWM should be wary of performing Spatial Reference tests and may benefit from altogether skipping this test. 22 Spatial Reversal was a useful test to examine over-writing of memory. For this test the platform has been moved to the North quadrant and the average time to find the platform (all mice) on the first day of Spatial Reversal was 70 secs. This was significantly longer compared to the last day of spatial acquisition that only took an average of 28 secs, indicating that the mice are adjusting to a new platform location. By the last day of Spatial Reversal, the average time to find the platform reduced to 38 seconds, and statistical analysis between the first and last days of Spatial Reversal showed the two groups of data were significantly different up to an 80% confidence interval. In the study by Brandeis et. al., a 95% confidence learning trend for a Spatial Reversal test was achieved in 4 days with 8 wild-type mice doing 3 experiments per day [3]. Though our Spatial Reversal result was not as significant as in our Spatial Acquisition or in the Brandeis study, it nevertheless shows an improvement over the 5 days of Spatial Reversal. This was expected as the mice had already become quite proficient at finding the platform; all they had to do was overwrite their old memory of the platform location and learn a new one. Our result may have been more significant if more mice were used like in the Brandeis study. This test not only re-emphasizes that our MWM can be used to test spatial navigation, but that it can also be used to test the over-writing of memory for a new spatial navigation task. The Rotational Cues test was the novel part of our testing paradigm. It was designed in order to determine whether the navigational strategy used in our MWM was Allocentric or Egocentric. The mean time to find the platform on day 5 of Spatial Reversal was 38 secs, but in the Rotational Cues Test, it was 20 secs. Since the mice reached the platform faster after the distal cues had been rotated, it was concluded that the mice were not dependent on the cues for navigation. Since these rotated cues were the only distal cues in the MWM 23 setup, the only navigational method possible was Egocentric. The ways in which this navigation method may have worked include the use of common distance to the wall and feeling for the platform underneath the feet. The proximal cue of distance between the platform and the wall may have promoted the mice to swim in a circle around the maze at approximately that distance until the platform was found. The other method may have been the internal cue of feeling for the platform with the feet. This feeling was directed towards finding the platform and could easily have been used to complete the maze. The latter strategy was more likely to have been used by the mice since the pathing of the mice was determined to be random instead of circular as seen in Figure 10. This is an important finding as it re-emphasizes the idea that the mice are not using the cues to triangulate a position and head directly there. This random search pattern is more of what would be expected from an Egocentric navigation method [24]. While several methods of Egocentric navigation exist, the importance of this experiment was to identify the overall strategy. Future experiments can investigate the specificity of the navigation method employed. The above results must be contextualized within the limits of this study. The first and primary limitation was the small sample number of animals used. Though all four animals in our study seemed to demonstrate significant learning trends through Spatial Acquisition and Spatial Reversal, compared to other studies that had a minimum of six animals, this sample size was insufficient [7-19]. It should be noted that six animals is considered the minimum required according to the study by Vorhees and Williams [30]. Having a higher sample number of mice in our study would have increased the accuracy of our results as was seen in the study by Brandeis [3]. 24 Another limiting factor of this study was that it only tested C57BL/6 wild type mice. While many other studies have used a similar mouse strain [29,30], there are so many other mouse strains that may behave differently in the MWM and perhaps use a different navigational strategy [7-19]. The MWM was also originally designed for rats, as seen in the original Morris experiments [2,5]. In these original studies, the pathing to the platform became nearly a direct line by the end of the learning period of 5 days. From this it was conferred the rats were using some type of Allocentric Navigation to triangulate the platform location. The rat species may have been able to use the distal cues given in the maze as part of their navigational strategy to accomplish this because of their improved eyesight over mice [30]. The navigational strategy should be determined using similar testing methods as presented in this paper for each individual mouse/rat model because as shown here, one set of results may not be relevant across all species or mouse strains. Despite the limitations of this study, the results collected demonstrate that an Egocentric navigation strategy is being used by our mouse model in our Morris Water Maze. Specific experiments can now be designed to alter this navigation method. For instance, the dorsal striatum and connected structures can be targeted to study their function and responsibility in egocentric processing in the brain [28]. When tested in our MWM, the performance of reaching the platform will either increase or decrease depending on how the Egocentric navigation is affected in these models. In a study by Serino et. al., it was found that egocentric impairments are one of the earliest manifestations of Alzheimer’s Disease [32]. This could be studied more extensively in our MWM using the same methodology as the previous example. This could help link certain pharmacological or genetic alterations that are made in mouse models to the early onset of Alzheimers 25 Disease. Insufficient information about the navigation strategy may only allow for conjectures about the methods being used by the mice to navigate the maze. For example, changing a sign to a different shape would not affect Egocentric navigation but would alter Allocentric navigation of mice. An example future experiment to examine the Egocentric navigation strategies in the MWM more closely would be to remove the distal cues and place physical cues within the maze. Physical cues would serve as reference points for the mice to physically relate their position in the maze. Experiments in which these proximal cues are altered, rotated, or removed could lead to further understandings of how egocentric navigation works. Our developed MWM has rigorously been tested as a behavioral tool and is now usable by other researchers in Capecchi/Traenkner Laboratory, at the University of Utah. This study examined a simplistic configuration of the MWM and the corresponding navigation strategy. 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