Spatial Learning by mice on a Three-dimensional radial maze Reference Memory task

Benjamin James

ABSTRACTNavigating strategies and the processes of exploration of rodents through two-dimensional space has been widely studied. However the real world is three-dimensional and little is known about how three-dimensional spaces are encoded and navigated in animals and humans. Using a Three-dimensional radiolarian maze task, which consists of a central spherical core from which 14 arms are projected in all directions, we hope to test Reference memory (Long term memory) in Three-dimensional space. Mice are required to explore the maze and retrieve food from the ends of the baited arms without missing out or revisiting baited arms they have already been too. During the reference memory task only a few of the arms will be baited. From previous findings we hope to show that during a three-dimensional task mice do not confuse the arms in the spatial learning task. This will give us an insight into the encoding of three-dimensional space and whether mice can navigate the vertical and horizontal parts of the task in their representation of space.

Introduction

Navigating through the world is vital to the survival of animals. Neural encoding of the world has survival and evolutionary advantages. This ability requires the perception and encoding of spatial cues associated with important locations within the environment, cues such as food locations. It is thought that the hippocampus is central to the cognitive map and spatial mapping of complex space.

Research has shown that animals, especially rodents, are well adapted to processing information about their current position and the relative position of important objects and food sources. Current work supports the theory that animals hold an internal mechanism and representation of space. This is often referred to as the cognitive map (Jeffery et al. 2012). Previous Studies have focused on spatial navigation in two-dimensional environments. However the real world is Three-dimensional, with animals moving in a horizontal and vertical plane.

Moving through three-dimensional space has additional computational requirements, which requires complex perception and encoding of spatial cues. Animals have to move against gravity, which increases energy demands in three-dimensional movements compared to two-dimensional movements. The additional requirement for cognitively mapping height and horizontal distance in three-dimensional environments, as opposed to planar environments, is much greater.

The ability to synthesise knowledge of spatial cues and locations in three-dimensional space, rather than two-dimensional space would provide more in depth information to the animal. This would aid in the precision of navigating around local environments and finding food sources.

It has been suggested from current neural studies in rodents that vertical and horizontal spaces are encoded differently (Jeffery et al. (2011). The separation of vertical and horizontal information has also been discovered in fish (Holbrook & Burt de Perera, 2009).

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This would suggest that vertebrate use a dual coded cognitive map for encoding vertical and horizontal planes and that representation of space is flat in the plane of locomotion (Jeffery et al. 2012).

It is thought that the hippocampus in humans has a major role in spatial navigation and memory for life events. It is hoped from studies into the spatial navigational systems and mapping of three-dimensional spaces by rodents, we will be able to understand episodic memory (The memory for life events) in humans, in which it is thought a spatial framework system is used. Understanding episodic memory will help us understand the things that go wrong with it in many neurodegenerative conditions such as Alzheimer’s disease (Jeffery et al. 2014).

Our study aims to see how complex Three-dimensional spaces are represented and used in navigation by mice. In our experiment we will be conducting a simple 3D navigation task, in which mice will explore and receive food rewards that will be placed at vertical and horizontal coordinates. We will be using a radiolarian maze to test the mice with. This is an unfamiliar environment, which the mice will have to navigate and cognitively map to find food rewards.

We will be focusing on reference memory (Long term memory) in mice. Although our study will primarily be focusing on reference memory, mice will have to use working memory (short-term memory), to remember where they have been during the task and which arms the food reward is on and not on.

We expect to see over the course of five weeks mice making fewer mistakes, learning where the food rewards are located and only visiting these arms, with the number of errors decreasing and the mice eventually making no errors, or the number of errors plateauing for a number of days during the course of the experiment.

Prior to starting the research placement I had to read about the subject. This was so I understood what we would be trying to understand during the experiment and the current knowledge surrounding spatial encoding. From Dr. Kate Jeffery’s lab page (http://www.ucl.ac.uk/jefferylab/research) I read

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about the research carried out at the lab and the aims of this research. I also read a review of current research into spatial navigation in a 3D world that gave me an overview of research surrounding this area.

My supervisor gave me some useful articles that gave me a basic understanding about the subject. One of the papers that I read was about Anisotropic encoding of three-dimensional space by place cells and grid cells (Jeffery et al. (2011)). This article gave me a basic understanding of research into spatial encoding in rodents and enabled me to understand the aims of spatial encoding research.

These articles were found online so I could read them. This helped me in writing my report and meant that I could understand the true aims of the experiment and what we were hoping to achieve. I also looked into some of the meanings of the scientific language used in the literature and asked my supervisor about any words that I was unsure about.

Methodology

We would be using a 14 arm three-dimensional Radiolarian maze to test reference memory. In this study only 12 of the arms would be recorded. We would bait 6 arms with condensed milk. Condensed milk was used as the food reward and not food pellets. The food needed to be a treat to ensure mice would want to eat the reward.

8 male mice would be used on the maze. We used 8 mice so we could compare performances and ensure we had enough mice to collect enough data to work out averages and draw conclusions.

We trained the mice during habituation for 5 days before we started the real experiment. This was to ensure the mice would be comfortable to carry out the task and got used to the experimental environment. We had to weigh and handle the mice on the first day of the habituation phase of the experiment. Handling continued on the second day. On the Third day of habituation mice were placed onto a restricted diet to maintain mice within 85-90% of free feeding body weight.

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We then handled mice in the room we would be carrying out the experiment in as part of habituation. We did this on day two of habituation. On the remaining three days we placed the mice on the radiolarian maze we would be using during the experiment. There was no food reward (Condensed milk) on the maze at this point. This was to get the mice used to the environment, so the mice would be happy to explore the radiolarian maze when it came to the real experiment. Mice explored the maze for 10 minutes each. We took records of the mice using a recording sheet. After each trial we input data into an excel spreadsheet.

The mice were on a 12-hour light/dark cycle with stimulated dawn at 23:30 and simulated dusk at 11:30. All mice were trained during the dark cycle between 12:30 and 15:00. Each trial took around an hour (12:30-1:30) with the mice being given an hour break before the second trial started (2:30-3:30). Between trials raw data was input into an Excel spreadsheet, to work out averages and analyse results for each trial and day. This was important to do to ensure all data was input correctly and so we could see the progress of results between trials and days.

As we were handling mice we needed to wear protective clothing to prevent contamination from potential diseases and prevent the development of an allergy. We wore gloves, facemask, overshoes, head cap and gown. We also had to wash are hands after each session as a safety precaution.

During the experiment six of the 12 arms were baited during each trial. The position of the baited arms was randomly assigned to each mouse; this was to ensure that the upper and lower arms on the radiolarian maze were equally baited (Three arms from the upper halve, three arms from the lower halve). Trials would last 5 minutes each or until all the baited arms had been visited and the rewards received.

We conducted 2 trials a day. One trial started at 12:30 (during dark cycle) and once we had completed the first trial, mice were given a break and we then started the second trial at 2:30. Two trials were carried out instead of one so we could have as many repeats as possible and to compare performances between the first and second trial. The maze was rotated by

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180 degrees every trial to control for any cues there may be within the maze.

Mice were scored on working memory errors (revisits to arms), reference memory errors (a visit to a non-baited arm) and visits to a baited arm. The order of arm visits and time taken to complete the maze were also noted. Two experimenters scored the recordings to make sure all recordings were constant to improve reliability. A video camera was also used that took a clip of each training session in case any visits were missed.

Once we had collected our data we placed our scores into an excel spreadsheet for raw reference memory scores and day trials.

The scores we collected enabled us to work out the averages for each trial for total visits, omission (the number of baited arms missed out), commission (total visits - (6 - Omission), reference memory errors, revisits, time and Reference memory errors as a percentage of the total number of visits and working memory errors as a percentage of total visits. We also listed the order in which the arms were visited. From this we created line graphs to present results.

Mice would be trained on this task for five weeks or until the number of reference memory errors as a percentage of total visits remained constant for at least 3 days.

Apparatus

We used a 14-arm three-dimensional radial arm maze (See figure 1 and 2). Only 12 of the arms would be used in the study. The top arm (0) and the bottom arm (-1) were excluded from the reference memory task. The sphere was 30cm in diameter with 14 evenly spaced cylindrical arms protruding from the sphere. The arms were 3.5cm in diameter. A three-dimensional maze was used instead of a two-dimensional maze. A three-dimensional maze would be a more realistic representation of the real world (the real world is three-dimensional).

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Bandages were wrapped around the body and arms of the maze; this would enable the mice to be able to climb and the maze with ease and not fall off. The maze was placed into an empty rack and suspended with nylon wire. Arms would be baited with condensed milk. Condensed milk would act as the food reward. This was placed onto the head of pins (Only six pins were baited at any one time).

We would be using 8 male mice in our study that would be placed onto a restricted diet two days into the habituation phase of five days. This is to maintain the mice within 85-90% of the weight the mice were on when on unrestricted diet.

We used Mice because they are lightweight natural climbers that would easily manage to explore the maze. Other rodents such as rats would be too large to be placed onto the maze.

Results

Results were entered into an Excel spreadsheet as below. We then worked out the averages for each Trial and the standard error, so we could create line graphs to present our results that included error bars. Averages and

Figure 1-Radiolarian maze

Figure 2-Radiolarian maze with labels 1-12

-1

9 108

12

117

1 6

4

5

2

3

0

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standard errors were also worked out for each day. This was also done for the probe trials.

Using IBM SPSS Statistics 21 we conducted repeated measures ANOVA Statistical analysis for both trial, day and probe trial. This showed that there had been statistically significant learning across total visits, omission (the number of baited arms missed out), commission (total visits - (6 - Omission), reference memory errors, revisits, time and Reference memory errors as a percentage of the total number of visits and working memory errors as a percentage of total visits, by all mice on the radiolarian maze over the course of the 50 trials (25 days). Any result that was below p=≤ 0.05 was seen as statistically significant.

For the reference memory task we were mostly interested in the reference memory errors as a percentage of the total number of visits. We conducted 50 trials before the percentage of reference memory errors remained around 31-±3% (Below chance levels of 56%) for 3 consecutive days, showing no improvement. Over the course of the 25 days of trials the reference memory errors as a percentage of the total number of visits decreased from 54.46% to 30.00% (F (24,168) = 5.82, p<. 001). Reference memory errors decreased from 5.75 to 3.38 over the 25 days of trials (See Fig. 12).

The time taken for the mice to complete the task decreased from 296.94 seconds to 148.25 seconds (F (24,168) = 10.51, p=<.001) over the course of

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the 25 days of trials (See Fig. 14). Working memory errors as a percentage of total visits decreased from 19.43% to 6.18% (F (24,168) = 2.78, p<.001) over the course of the 25 days of trials (see Fig. 16).

Omission decreased from 2.19 to 0.00 (F(24,168) = 11.07, p<.001) over the 25 days of trials (See Fig.17). Total visits remained around 9.88 ±4.00 (F(24,168) = 2.38, p=.001) over the 25 days of trials (See Fig 11). Revisits decreased from 2.13 to 0.81 over the 25 days of trials (See Fig. 15). Commission decreased from 6.06 to 4.06 over the 25 days of trials (See Fig. 18).

Two probe trials were conducted where no arms were baited to see if the mice would visit the previously baited arms to prove the mice had learnt over the 25 days of trials. Repeated measures ANOVA was used to analyse the first day of trials, the last day of trials and the two probe trials to confirm if there had been significant reference memory learning. From the probe trials the analysis confirmed that over the 25 days there had been significant learning for the reference memory errors as a percentage of total visits. Between the first day and last day there was a significant decrease in reference memory errors as a percentage of total visits of 23.69%. Between the first day and probe day there was a decrease of 27.67% (see Fig. 19). This was significant according to repeated measures ANOVA, F(2,14) = 45.27, p<.001. The results from repeated measures ANOVA would indicate that there had been significant learning by all the mice over 25 days of trials (Two trials a day). This would support the idea that mice can hold and store a visual representation of food sources in complex three-dimensional space over a long period of time.

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Fig 6. Average For the time taken to complete the task (Trial).

Fig 5. Average of Reference memory errors as a percentage of the total visits (Trial).

Figure 4. Average working memory errors as a percentage of the total number of visits (Trial)

Figure 3. Average number of total visits (Trial).

Trial Graphs 10

Fig 10. Average of Reference memory Errors (Trial).

Fig 9. Average Commission (Trial).

Fig 8. Average Omission (Trial).

Fig.7 The average number of revisits (Trial).

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Day Graphs

Fig 14. Average for Time (Day).

Fig 13. Average for Reference memeory errors (Day).

Fig 12. Average for Reference memeory errors as a % of total visits. (Day)

Fig. 11. Average Total Visits (Day).

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Fig 18. Average Commission (Day).

Fig 17. Average Omission (Day).

Fig 16. Average % Working Memory Errors (Day).

Fig 15. Average Revisits (Day).

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Probe Trial Graph

Evaluation

The findings of this study highlight the ability of mice to locate and represent positions of food rewards within a three-dimensional space. However, further research would need to be conducted into the mechanisms of three-dimensional representations to further understand the cognitive map within the brain. This could be found out by electrophysiology studies of rodents to measure the electrical properties of cells and tissues thought to be involved in spatial navigation such as place or grid cells.

Variations of the radiolarian maze could be used to further investigate this. The radiolarian maze could have been adapted to include more or less arms or we could have used a radiolarian maze of a different size, or a maze with a different three-dimensional shape, such as a cube (see Fig. 20).

When placing the food reward (Condensed milk) on the arms, the condensed milk would often drop off the pinheads. To improve the design of the radiolarian maze we could have placed food rewards onto lollipop sticks or used a food reward that is less likely to drip off the end of the pins. Other adaptations of the maze could be to design a small platform onto

Fig 19. Average Reference Memory Errors (Probe day).

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which food could be placed, thus preventing food rewards from failing off. Ear buds were used to apply the condensed milk to the pinheads, as the condensed milk easily stayed on the ear buds and could easily be applied.

We could have conducted our experiment differently by comparing Two-dimensional and three-dimensional mazes to see differences in the way mice learn spatial tasks and the speed and ease at which they learn them.

The study could have been conducted with other Rodents such as rats or we could have used other mammals such as cats or dogs to make comparisons about spatial navigation. Further studies would have to be conducted using a range of vertebrate, to confirm the findings about spatial navigation in rodents and the cells that are involved.

A larger radiolarian maze could have been used with more obstacles as this would be a more realistic representation of complex space and give a greater insight into spatial navigation in three-dimensional space.

Overall the experiment was conducted well. Over the course of the 5 weeks of training, the mice did learn the location of all the baited arms and all successfully completed the radiolarian maze. This gave us an insight into the ability of mice to navigate the position of objects in three-dimensional space. Variations of the radiolarian maze might give us greater insight into the ability of mice to navigate three-dimensional environments. The use of electrophysiology experiments would have given us an understanding of the cells that are being used in navigational memory and cognitive mapping.

I have learnt about the cells that are believed to be responsible for navigational memory such as grid cells and place cells. I have also been given insight in the ability of mice to be able to learn the locations of food rewards.

Fig. 20 Variations of the Radiolarian arm maze.

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Conclusions

Using the Three-dimensional radiolarian arm maze, we could see whether mice could represent locations in complex three-dimensional spaces, locating food rewards and holding this spatial memory over time.

The mice learnt the reference memory task, learning food reward locations and holding these representations over long periods of time. The mechanisms behind this ability are still unclear.

It is hoped from research into spatial navigation and its mechanisms that we can understand what happens when this goes wrong in patients suffering from neurodegenerative diseases such as Alzheimer’s. Three-dimensional spatial navigational studies have shed light on areas of the brain that are affected by neurodegenerative decline and the ways in which spatial navigation and memory are affected.

There are declines in navigational skills in normal ageing with patients with dementia. This decline is a result of structural and functional alterations in the neural network (Lithfous et al. 2013).

Spatial navigational studies have given insights into the way the brain maps its surroundings and have shown that navigational training programs can improve spatial performances in navigational tasks with patients suffering from dementia.

Further animal studies need to be conducted before we can fully understand the physiological mechanisms that are responsible for spatial navigation and mapping in the brain. Three-dimensional and two-dimensional studies in rodents as well as electrophysiology experiments have helped us understand more about spatial mapping and navigation.

Fig. 20 Variations of the Radiolarian arm maze.

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Appendix

Buzsaki, G., Moser, E.I (2013) 'Memory, navigation and theta rhythm in the hippocampal-enthorhinal system', nature neuroscience, Vol 16, no.2, pp. 130-138.

References

Hayman, R., Verriotis, M.A., Jovalekic, A., Fenton, A.A, Jeffery, K.J (2011) ‘Anisotropic encoding of three-dimensional space by place cells and grid cells’, nature neuroscience, Vol. 14, no.9, pp.1182-1188.

Holbrook, R.I. and Burt de Perera, T. (2009) Separate encoding of vertical and horizontal components of space during orientation in fish. Animal Behaviour, 78(2), 241-245.

Lithfous et al. (2013) 'spatial navigation in normal aging and the prodromal stage of Alzheimer's disease: Insights from imaging and behavioral studies', Ageing research reviews, Vol. 12,Issue 1, pp. 201-213.

Jovalekic A, Hayman R, Becares N, Reid H, Thomas G, Wilson J, Jeffery KJ (2011) Horizontal biases in rats’ use of three-dimensional space. Behavioral Brain Research, 222: 279-288.

Jeffery et al. (2012) Navigating in a 3D world (online). Available at: http://www.ucl.ac.uk/jefferylab/publications/2013_Jeffery_BBS_preprint.pdf (Accessed 4th August 2014).

Jeffery et al. (2014) Research (Online). Available at: http://www.ucl.ac.uk/jefferylab/research (Accessed 4th August 2014)

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Bibliography

Grobéty, MC. & Schenk, F. (1992) ‘Spatial learning in three-dimensional maze’, Animal behavior, Vol. 43, no.6, pp.1011-1020.

Hayman, R., Verriotis, M.A., Jovalekic, A., Fenton, A.A, Jeffery, K.J (2011) ‘Anisotropic encoding of three-dimensional space by place cells and grid cells’, nature neuroscience, Vol. 14, no.9, pp.1182-1188.

Holbrook, R.I. and Burt de Perera, T. (2009) Separate encoding of vertical and horizontal components of space during orientation in fish. Animal Behaviour, 78(2), 241-245.

Lithfous et al. (2013) 'spatial navigation in normal aging and the prodromal stage of Alzheimer's disease: Insights from imaging and behavioral studies', Ageing research reviews, Vol. 12,Issue 1, pp. 201-213.

Jovalekic A, Hayman R, Becares N, Reid H, Thomas G, Wilson J, Jeffery KJ (2011) Horizontal biases in rats’ use of three-dimensional space. Behavioral Brain Research, 222: 279-288.

Muller, R. (1996) ‘A Quarter of a Century of Place Cells’. Neuron, Vol.17, 979-990.

Jeffery et al. (2012) navigating in a 3D world (online). Available at: http://www.ucl.ac.uk/jefferylab/publications/2013_Jeffery_BBS_preprint.pdf (Accessed 4th August 2014).

Jeffery et al. (2014) Research (Online). Available at: http://www.ucl.ac.uk/jefferylab/research (Accessed 4th August 2014)

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Acknowledgements

With thanks to the Nuffield foundation and Emma Newall for providing the placement. Many thanks to Kate Jeffery Lab UCL and Jonathan Wilson for agreeing to allow me to work with them in their lab for my placement and giving me an insight into behavioral neuroscience.

The project has confirmed to me that I want to pursue a career in neuroscience and has shown me how a real lab operates and the exciting research that goes on at the UCL institute of Behavioural neuroscience. This has opened my mind to considering a career in neuroscience research.

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