1. Spatial Learning by mice on a Three-dimensional radial maze
Reference Memory task
Benjamin James
ABSTRACT
Navigating 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.

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Introduction
Navigating through the world is vital to the survivalof animals. Neural
encoding of the world has survivaland 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 abouttheir currentposition and the relative
position of important objects and food sources. Currentwork supports the
theory that animals hold an internal mechanismand 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 horizontaland vertical plane.
Moving through three-dimensional space has additional computational
requirements, which requires complex perception and encoding of spatial
cues. Animals have to move againstgravity, which increases energy
demands in three-dimensional movements compared to two-dimensional
movements. The additional requirement for cognitively mapping height and
horizontaldistance in three-dimensional environments, as opposed to
planar environments, is much greater.
The ability to synthesiseknowledgeof spatial cues and locations in three-
dimensional space, rather than two-dimensionalspacewould provide more
in depth information to the animal. This would aid in the precision of
navigating around local environments and finding food sources.
Ithas been suggested fromcurrent neural studies in rodents that vertical
and horizontalspaces are encoded differently (Jeffery et al. (2011). The
separation of vertical and horizontalinformation has also been discovered
in fish (Holbrook & Burt de Perera, 2009).

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This would suggestthat vertebrate use a dual coded cognitive map for
encoding vertical and horizontalplanes and that representation of space is
flat in the plane of locomotion (Jeffery et al. 2012).
Itis thought that the hippocampus in humans has a major role in spatial
navigation and memory for life events. It is hoped fromstudies 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 thatwill 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 haveto 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-termmemory), to remember
wherethey havebeen during the task and which arms the food reward is
on and not on.
We expect to see over the courseof five weeks mice making fewer
mistakes, learning wherethe food rewards arelocated 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 courseof the experiment.
Prior to starting the research placement I had to read about the subject.
This was so I understood whatwe would be trying to understand during the
experiment and the currentknowledge surrounding spatialencoding. 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 currentresearch into spatial navigation in a 3D world
that gave me an overview of research surrounding this area.
My supervisor gavemesome usefularticles that gaveme a basic
understanding aboutthe subject. Oneof the papers that I read was about
Anisotropic encoding of three-dimensionalspace by place cells and grid cells
(Jeffery et al. (2011)). This articlegave 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 whatwe were hoping to achieve. I also looked into some of
the meanings of the scientific languageused in the literature and asked my
supervisor aboutany words thatI was unsureabout.
Methodology
We would be using a 14 arm three-dimensional Radiolarian mazeto 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
ensuremice would want to eat the reward.
8 male mice would be used on the maze. We used 8 mice so we could
compareperformances and ensure wehad enough mice to collect enough
data to work outaverages and draw conclusions.
We trained the mice during habituation for 5 days beforewe started the
real experiment. This was to ensurethe mice would be comfortableto 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 phaseof 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 freefeeding body weight.

5. 5
We then handled mice in the roomwe would be carrying outthe
experiment in as partof 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 cyclewith stimulated dawn at 23:30
and simulated dusk at 11:30. Allmice 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 beforethe second trial started (2:30-
3:30). Between trials raw data was input into an Excel spreadsheet, to work
out averages and analyseresults for each trial and day. This was important
to do to ensureall 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 frompotential diseases and preventthe
development of an allergy. We woregloves, 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 werebaited 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 fromthe upper halve, three arms fromthe
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. Onetrial started at 12:30 (during dark cycle)
and once we had completed the firsttrial, mice weregiven a break and we
then started the second trial at 2:30. Two trials were carried out instead of
one so wecould have as many repeats as possibleand to compare
performances between the firstand 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 werescored 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
constantto improvereliability. A video camera was also used that took a
clip of each training session in case any visits were missed.
Once wehad collected our data we placed our scores into an excel
spreadsheetfor raw reference memory scores and day trials.
The scores we collected enabled us to work outthe averages for each trial
for total visits, omission (the number of baited arms missed out),
commission (total visits - (6 - Omission), referencememory 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 werevisited. From this we created line
graphs to presentresults.
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-armthree-dimensionalradial armmaze (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) wereexcluded fromthe reference memory task. The
spherewas 30cmin diameter with 14 evenly spaced cylindrical arms
protruding fromthe sphere. The arms were3.5cmin diameter. A three-
dimensional maze was used instead of a two-dimensionalmaze. 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 theweight 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.
Figure 1-Radiolarian maze
Figure 2-Radiolarian maze
with labels 1-12
0
3
2
5
4
61
7 11
12
8 109
-1

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Results
Results wereentered into an Excel spreadsheet as below. We then worked
out the averages for each Trial and the standard error, so wecould create
line graphs to presentour results that included error bars. Averages and
standard errors werealso worked out for each day. This was also done for
the probetrials.
Using IBMSPSS Statistics 21 we conducted repeated measures ANOVA
Statistical analysis for both trial, day and probetrial. 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), referencememory errors, revisits, timeand 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 courseof the 50 trials (25 days). Any resultthat was below p=≤
0.05 was seen as statistically significant.
For the reference memory task we weremostly 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 consecutivedays,
showing no improvement. Over the courseof the 25 days of trials the
reference memory errors as a percentage of the total number of visits

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decreased from 54.46% to 30.00%(F (24,168) =5.82, p<. 001). Reference
memory errors decreased from5.75 to 3.38 over the 25 days of trials (See
Fig. 12).
The time taken for the mice to complete the task decreased from296.94
seconds to 148.25 seconds (F (24,168) =10.51, p=<.001) over thecourseof
the 25 days of trials (See Fig. 14). Working memory errors as a percentage
of total visits decreased from19.43% to 6.18% (F (24,168)= 2.78, p<.001)
over the courseof the 25 days of trials (seeFig. 16).
Omission decreased from2.19 to 0.00 (F(24,168) =11.07, p<.001) over the
25 days of trials (See Fig.17). Totalvisits remained around 9.88 ±4.00
(F(24,168) =2.38, p=.001)over the25 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 from6.06 to 4.06 over the 25 days of trials (See Fig.
18).
Two probetrials wereconducted where no arms werebaited 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 firstday of trials, the last day of trials and the two probe trials to
confirmif there had been significant referencememory learning. Fromthe
probetrials 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 probeday there was a decrease of
27.67% (seeFig. 19). This was significantaccording 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 supportthe idea that mice can hold and store a visual
representation of food sources in complex three-dimensionalspace over a
long period of time.

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Figure 3. Average
number of total
visits (Trial).
Figure 4. Average
working memory
errors as a
percentage of the
total number of
visits (Trial)
Fig 5. Average of
Reference memory
errors as a
percentage of the
total visits (Trial).
Fig 6. Average For
the time taken to
complete the task
(Trial).
Trial Graphs

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Fig.7 The average
number of revisits
(Trial).
Fig 8. Average
Omission (Trial).
Fig 9. Average
Commission
(Trial).
Fig 10. Average of
Reference memory
Errors (Trial).

12. 12
Day Graphs
Fig. 11.
Average Total
Visits (Day).
Fig 12. Average
for Reference
memeory
errors as a % of
total visits.
(Day)
Fig 13. Average
for Reference
memeory
errors (Day).
Fig 14. Average
for Time (Day).

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Fig 15. Average
Revisits (Day).
Fig 16. Average
% Working
Memory Errors
(Day).
Fig 17. Average
Omission
(Day).
Fig 18. Average
Commission
(Day).

14. 14
ProbeTrial Graph
Evaluation
The findings of this study highlight the ability of mice to locate and
representpositions of food rewards within a three-dimensionalspace.
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 measurethe 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 wecould have used a radiolarian maze of a different size, or a mazewith
a different three-dimensional shape, such as a cube (seeFig. 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 wecould have placed food rewards onto lollipop
sticks or used a food reward that is less likely to drip off the end of the pins.
Fig 19. Average
Reference
Memory Errors
(Probe day).

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Other adaptations of the maze could be to design a small platform onto
which food could be placed, thus preventing food rewards fromfailing off.
Ear buds wereused 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-dimensionalmazes 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 haveused other mammals such as cats or dogs to make
comparisons aboutspatial navigation. Further studies would haveto be
conducted using a range of vertebrate, to confirmthe findings about spatial
navigation in rodents and the cells that are involved.
A larger radiolarian maze could havebeen 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.
Overallthe experiment was conducted well. Over the courseof 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. Theuse 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 responsiblefor
navigational memory such as grid cells and place cells. I havealso been
given insight in the ability of mice to be able to learn the locations of food
rewards.

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Fig. 20
Variations of
the
Radiolarian
arm maze.

17. 17
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.
Itis hoped fromresearch into spatial navigation and its mechanisms that
we can understand whathappens when this goes wrong in patients
suffering from neurodegenerativediseases such as Alzheimer’s.
Three-dimensional spatial navigationalstudies haveshed 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 structuraland functional alterations in
the neuralnetwork (Lithfous et al. 2013).
Spatial navigational studies have given insights into the way the brain maps
its surroundings and haveshown thatnavigational training programs can
improvespatial performances in navigational tasks with patients suffering
fromdementia.
Further animal studies need to be conducted before we can fully
understand the physiologicalmechanisms that are responsiblefor spatial
navigation and mapping in the brain. Three-dimensional and two-
dimensional studies in rodents as well as electrophysiology experiments
have helped us understand moreabout spatial mapping and navigation.

18. 18
Appendix
Buzsaki, G., Moser, E.I (2013) 'Memory, navigation and theta rhythmin the
hippocampal-enthorhinalsystem', nature neuroscience, Vol16, 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 spaceby place cells and grid
cells’, nature neuroscience, Vol. 14, no.9, pp.1182-1188.
Holbrook, R.I. and Burtde Perera, T. (2009) Separateencoding of vertical
and horizontalcomponents of spaceduring orientation in fish. Animal
Behaviour, 78(2), 241-245.
Lithfous et al. (2013) 'spatialnavigation in normalaging and the prodromal
stage of Alzheimer's disease: Insights fromimaging and behavioralstudies',
Ageing research reviews, Vol. 12,Issue1, pp. 201-213.
Jovalekic A, Hayman R, Becares N, Reid H, Thomas G, Wilson J, Jeffery KJ
(2011) Horizontalbiases in rats’ useof 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.p
df (Accessed 4th August2014).
Jeffery et al. (2014) Research (Online). Availableat:
http://www.ucl.ac.uk/jefferylab/research (Accessed 4th August2014)

19. 19
Bibliography
Grobéty, MC. & Schenk, F. (1992) ‘Spatiallearning 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 spaceby place cells and grid
cells’, nature neuroscience, Vol. 14, no.9, pp.1182-1188.
Holbrook, R.I. and Burtde Perera, T. (2009) Separateencoding of vertical
and horizontalcomponents of spaceduring orientation in fish. Animal
Behaviour, 78(2), 241-245.
Lithfous et al. (2013) 'spatialnavigation in normalaging and the prodromal
stage of Alzheimer's disease: Insights fromimaging and behavioralstudies',
Ageing research reviews, Vol. 12,Issue1, pp. 201-213.
Jovalekic A, Hayman R, Becares N, Reid H, Thomas G, Wilson J, Jeffery KJ
(2011) Horizontalbiases in rats’ useof 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.p
df (Accessed 4th August2014).
Jeffery et al. (2014) Research (Online). Availableat:
http://www.ucl.ac.uk/jefferylab/research (Accessed 4th August2014)

20. 20
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 wantto pursuea career in
neuroscienceand 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 neuroscienceresearch.