Basic behavioral

neuroscience
in rodents
A practical guide

A publication from Noldus Information Technology

 Basic behavioral
neuroscience
in rodents
A practical guide

2 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

 introduction &
content

3 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

content

Housing & husbandry 4

Exploration & locomotion 10

Anxiety & fear 15

Depression 24

Cognition & memory 28

Home cage 57

Concluding remarks & references 64

1 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

introduction
Welcome to the Noldus neuroscience e-book! This Why is (animal)
e-book serves as a stepping stone for your behavioral behavior so essential
research, providing background information and to study?
references per topic. We also included protocol sug- Behaviorally characterizing an ani-
gestions for many behavioral tests, which can help mal model of disease often involves
you in finding the right solution for you. We describe a battery of tests that investigate
the animal’s motivation, locomotor
accompanying arena(s) to the tests and analysis
activity, startle reflex, anxiety, fear
possibilities.
response, social behavior, learning,
Use this e-book to gain information on specific beha- memory, and other emotional and
cognitive traits. Dysfunctions in
vioral research topics, and how these are studied in
these behaviors are used to infer
the current scientific field. Noldus Information Tech-
structural and functional changes
nology strives to provide you with the best solutions in the brain, and the recovery of
and the most accurate information to advance your performance on these tests is used
research. to evaluate the effectiveness of
potential therapeutics.
Enjoy reading, and feel free to contact us if you have
any questions on or suggestions for improving the
contents of this e-book.

animal models
Animal models of human behavior represent a theory
of cognitive or emotional processes, which can be trans-
formed from humans to animals, but is not developed for
specific psychopathology. Models are used to verify these
theories by showing specific symptoms of specific diseases,
which can be achieved through experimental interventions
such as drug administration, genetic manipulation, or
disease modelling. Inducing these symptoms requires that
the underlying physiological and/or neurochemical basis
of these symptoms has been determined.

2 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

advisory note
In this e-book we describe a number of behavioral tests, their scientific background, and present proto-
cols. This information is gathered from years of experience in the field of behavioral neuroscience and
literature research. This information is however not intended as a substitute for academic research. We
advise you to always do your own research. We at Noldus are here to help you in providing solutions for
your behavioral research.

3 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

 husbandry &
housing

4 — So you want to build a core?

Housing & husbandry

housing rodents
Housing is a very common issue when working with rodents. The use of a rodent (rat or mouse) in
a behavioral and/or metabolic study often requires animals to be housed individually. However
by doing this, the animals’ social context is overlooked. As gregarious species, it is widely advised
to avoid housing rats or mice individually. However, housing laboratory mice individually may be
required to collect data at the individual level, such as measurements of food intake and energy
expenditure (often a necessity when performing metabolic studies), or for reasons of social incom-
patibility (including fighting behavior among male mice).

Higher vulnerability to stress is a common finding in individually housed rodents. After an acute
stressor, increased corticosterone levels were found in individually housed mice, while basal corti-
costerone levels remain unchanged [1,2]. In the brain, plastic changes were found after social isolation.
For instance, a decrease in functional BDNF signaling in cortical, thalamic and midbrain areas was
found in chronically individual housed mice compared to socially housed mice [1].

On a behavioral level, increased expression of distress/anxiety-like behaviors has been reported for
individually, compared to socially, housed mice when subjected to standard behavioral tests like
the elevated plus maze (EPM) and open field (OF) test [1,3]. On the other hand, some studies did not

Socially housed BALB/cJ mice in a conventional Makrolon-type cage.

5 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

observe effects on anxiety caused by variations in housing conditions in male mice [4], or even
reported an increase of anxiety levels on an EPM in socially housed mice compared to individually
housed mice [5]. Taken together, these examples indicate that individually and socially housed mice
show different behavior in anxiety paradigms, and perhaps other behavioral tests as well. Such
variation clearly complicates generalization of experimental outcomes between studies using
individually and socially housed mice, and stresses the need for standardization of social housing
conditions within and between experiments.

On the other hand, social housing itself can also cause problems in your behavioral analysis. For
instance, it is generally assumed that rodents housed together within a cage are phenotypically
similar because of their shared cage environment. However, research on social dominance suggests
that groups of rats or mice within cages form social hierarchies, and that their behavior and physio-
logy may differ depending on their dominance-subordinate relationships. Therefore, dominance
relationships may be a confounding factor in animal experiments. For example, it has been found
that subordinate mice show higher levels of anxiety-like behavior compared to dominant mice [6].

Hierarchal structures are more complex in larger groups (e.g. colonies), potentially causing con-
siderable variability in behavioral, metabolic and even physiological outcomes. For example, social
housing with three male mice in the same cage decreases aggressive behavior between mice, where
the subordinate mice have social support from each other, decreasing the distress caused by the
dominant animal [7]. Surprisingly, effects of hierarchy are less often studied in pair-wise housing
conditions, which is becoming standard practice in (metabolic) studies due to increased under-
standing about potential negative effects of social isolation on the wellbeing and performance of
the animals. If we consider experiments in large colonies, many (preclinical) study setups are not
practical/feasible to be performed in such a way. Pair-wise housing would thus be a proper, and
naturally relevant, setup for experiments in rodents compared to individual housing.

which housing method should i Important!

choose? When socially housing
If you summarize all available literature and information, you animals, make sure you
figure out which animals
can find that individual housing basically causes a depressive-
are dominant and sub-
like phenotype leading to body weight/fat increase and beha- ordinate! This is an essen-
vioral changes related to stress, anxiety, and depression. How- tial factor in the analysis
ever although social housing is the more more naturally rele- of your behavioral data.
vant method, it does increase logistical challenges such as This can for example be
done with a tube test. If
identification, hierarchy effects in the cage and even creates
you want to know how to
testing order effects (there will always be an animals that is
perform a tube test, check
tested first, second, third, etc). And if we consider individual out the article by Fan et
housing to be inevitable is some cases, such as with the use of al. that very accurately
cannulas or other implants implants, when animals elicit too describes a protocol in
mice [8].
much fighting behavior, or when there is need for metabolic
cages.

6 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

The key is to be pragmatic in your study design. Your choice of housing should fit wat you actually
need from your data. For example with food intake: do we always need individual data if we are
looking at group effects? Consider measuring food intake of the entire cage. When countering test-
ing order effects, make sure you write out an airtight protocol. Counterbalance groups, time of day,
and especially the order in which you test animals from the same cage. Make sure you habituate
them to a solitary environment before you subject them to a behavioral test.

cage enrichment
Laboratory rodents require proper sensory
and motor stimulation. This is because they
naturally exhibit behaviors such as foraging,
exploring, hiding and building. Environmental
(cage) enrichment can provide this in a laboratory
cage. Earlier, social versus individual housing was
al-ready mentioned, and this indeed is classified
as cage enrichment. Furthermore nest building
materials, knawing wood, hiding places (shelters)
and running wheels can be considered as cage
enrichment. Deprivation of cage enrichment can
lead to serious behavioral abnormalities in your Nest building material such as shredded paper is commonly
provided as cage enrichment.
laboratory animals.

7 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Housing & husbandry

handling
Human contact can be perceived as stressful for a rodent, and even become aversive to the point
that animals can develop anxiety and show exaggerated stress responses when approached. It
is therefore extremely important to appropriately handle your experimental animals. Being calm
and confident is the first major rule. You as a person can show signs of stress, which can be picked
up by the animal. Don’t talk loudly, don’t make sudden movements and in general try to have a
non-threatening approach and an overall caring attitude towards the animals.

how should i handle my animals?

There is more than one way to pick up a rodent. Between rodents there is also a difference, as rats
and mice do not only differ in size, but also in the way they behave towards a handler. As mice are
by far the most commonly used animal in research, most information on common practices are are
available on this species, of which a lot does also apply to other rodents. Mice are small in size, which
makes them vulnerable to predation. For this reason mice often show a strong response towards
capture and/or handling. They are however quick to adapt, and if handled appropriately, might even
voluntarily seek contact.

▪ Picking up by the tail

Picking a mouse up by it’s tail has been a pop-
ular method on handling. It has however been
shown that this method can induce aversion
and anxiety and is advised to be avoided [9,10].
Rats can be picked up more easily by grasping
them around the shoulders.

More non-invasive methods have been developed

such as tunnel handling and cupping.

▪ Tunnel handling
Tunnel handling has become increasingly popu-
lar in the last years. This is basically guiding the
animal into a handling tunnel, and lifting the
tunnel to its desired destination. What makes
this handling technique particularly useful in
Tunnel/tube handling is a way to handle your animals more
the fact that these tunnels can be added to the gently. [9]

8 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

 home cage as cage enrichment. This increases recognition of the tunnel, and also proved to be
particularly useful for more anxious strains.

▪ Cupping
Cupping refers to simply scooping an animal onto the hand. This can be a tricky method, as the
animals (mice) have to habituate being picked up. Naïve and young animals will jump from your
hand, keep this in mind the first few times!

You should also choose your method of handling pragmatically. Use cupping with more docile
strains, and only with animals that are well habituated to handling. Also this is a technique that is
a bit more reserved for experienced animal handlers. Less confidents handlers (and more anxious
mice) should employ the tunnel handling strategy.

Gouveia and Hurst (2013) provide a very informative overview of the benefits of tunnel handling in mice [9].

Videos on how to tunnel handle and cup mice performed can be found here:
https://nc3rs.org.uk/3rs-resources/handling-and-restraint

restraining animals
Restraining animals is a necessary evil in some
cases. Rats are in general easier to handle, but
also mice can accepts physical restraint without
losing tameness towards the handler. Picking
up by the tail should be avoided as stated be-
fore, however with restraining the base of the
tail can be held to properly position the animal
once in/on the hand. To begin restraining the
mouse place it on a surface they can grip, a wire
grid such as the lid of the cage for example,
grab the base of the tail with your dominant
hand. With your non-dominant hand grasp the
loose skin of the mouse at the back of the neck.
Tuck the tail behind your pinkie. Now you have
your dominant hand free to perform any neces-
sary interventions (for example an injection or
An example of a properly restrained mouse.
ear clip). During restraint the animal should be
able to breathe easily.

9 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

 exploration
locomotion &
10 — So you want to build a core?
Exploration & locomotion

introduction
The behavior of mice and rats is often highly incentivized by their natural curiosity. Curiosity is part
of an essential survival mechanism, which is driven by novelty-seeking and investigative behavior of
the surrounding environments. This so-called motivational state initiates behavior and helps achieve
desired goals. Hunger is another example of a motivational state that initiates (exploratory) beha-
vior, as food seeking and eating behavior are essential for survival.

why do we measure exploratory and locomotor behavior?

In order understand the (behavioral) phenotype of a rodent model, exploratory and locomotor
behavior are commonly studied in behavioral batteries. For example, when comparing different
strains of mice or different effects of drug treatments. If locomotor activity or ability is affected
due to a treatment, then measuring behavior that relies on the ability of the animals to move is
automatically confounded.

how do we measure exploratory behavior?

The open field test is one of the most commonly used platforms to measure
exploratory and locomotor behavior in animals and was introduced by Hall
and Ballachey in 1932 [11]. As a fast and relatively easy test, it provides a variety
of behavioral information ranging from general ambulatory ability to infor-
mation about the emotional state of the animal.

An open field is, simply put, a box with an open area, which makes exploratory
behavior easy to analyze in other spaces as well. Home cage monitoring is a
great example of this. This, however, does differ in interpretation of the be-
havior, as the home cage is a familiar and safe area, whereas a standard open
field is a novel arena.

In other tests (such as the elevated plus maze, three chamber test and Barnes
maze) exploratory behavior can be measured as well. However, interference in
the behavioral readouts may occur due to the enriched environment of these
arenas, whereas a standard open field is as it was stated: an un-enriched open
area solely meant for the exploration by the animal.

11 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Exploration & locomotion

open field

A common first question that comes up when planning an open mouse open field
field is if the arena should be round or square. Ranges from 25 x 25 cm to
80 x 80 cm
Intuitively a round arena seems more appropriate, since the lack 30 - 40 cm raised walls
of corners creates an equal and unenriched space to explore.
Corners can be perceived as safe and sheltered, thus might be rat open field
more attractive to visit for the animals, which could skew results. Ranges from 60 x 60 cm to
120 x 120 cm
In practice this is hardly the case as showed by Grabovskaya and
40 - 50 cm raised walls
Salyha [12], showing virtually no differences in many of the main
readout parameters of the open field, which commonly are: Generally the dimensions
of the open field (smaller or
▪ Total distance moved larger) do not affect locomotor
▪ Time in zone (outer versus inner) behavior, however rats should
be tested in a larger arena
▪ Zone crossings
than mice.
▪ Defecations and urinations
▪ Stretch attend postures
▪ Rearing

An important consideration to make when choosing for a round or square arena, is any further
testing within the same arena. Particularly involving the exploration of objects, such as in the novel
object or novel place recognition test. Specific corner placements of these objects in a square arena
makes its location distinctly recognizable for the animal. A round arena can be used in this context,
but can become tricky when objects have to be placed in a specific place in the arena relating to a

A standard square open field. Credits: He, S. and Corscadden, L. A partitioned open field can also be used, which is used to con-
(2022). Maze Engineers. duct multiple tests simultaneously, increasing the throughput
of your testing. Credits: He, S. and Corscadden, L. (2022). Maze
Engineers.

12 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

specific position in the surrounding area (testing room), since the animals need a point of reference
in order to successfully recognize an object. A square arena could thus be better suited for this pur-
pose.

protocol suggestion
▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

▪ Remove a single animal from the home cage with your preferred handling technique: tail
handling, full hand handling, tube handling. Place the animal in the middle of the open field
arena . Recording/tracking automatically starts in EthoVision XT if this option has been
selected. Otherwise, do not forget to concurrently activate your video recording. It is normal
for the animals to immediately move to the periphery walls of the maze.

▪ Preferably leave the testing room to allow free and uninterrupted movement of the subject
animal. Record/track the animals for 5 or 10 minutes, depending on your preference, previous
experiments, or examples from literature that you wish to replicate.

▪ After the testing time is finished, gently pick up the animal, again using your preferred handling
technique, and return it to its home cage.

▪ Before cleaning the arena, visually count the faecal pellets present and manually record the
numbers for further analysis.

▪ Remove all fecal pellets and wipe up all spots of urination. Spray the floor and walls of the maze
with 30-70% ethanol and wipe down with a clean paper towel. Allow the ethanol solution to
completely dry prior to testing other animals.

setup in ethovision xt
EthoVision XT makes it possible to automatically track all movements and behaviors in the open
field. Drawing zones, and subsequently dividing them accordingly: center, border, etc. This makes
extracting the data from the open field test as simple as a few clicks. In EthoVision XT, two main
zones are of importance: the center of the arena and the borders. With a square shaped arena is
can be advised to also include the corners as zones.

With multiple body point detection in EthoVision XT, additional behavior such as rearing and
grooming can be detected. This gives additional information on the behavioral phenotype in
the open field, and adds resolution to the exploratory and/or locomotor behavioral profile.

13 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

interpretation of the results
▪ Total distance moved - In the open field Total distance moved gives an indication on locomotor
behavior. This is usually presented as an absolute value (in cm). Another indicator that can be
used for this type of behavior is time moving versus time not moving, which can be presented
as a percentage or ratio. For example:

Time moving
(x 100)
Total time

▪ Zone crossings - These crossings also give an indication on locomotor behavior, however this adds
information based on if the animals spends a lot of time exploring one particular zone, or that
this is more distributed over the entire arena. The latter can be visualized in EthoVision XT with
either a heat map or with track visualization.

▪ Time in zone (outer vs inner) - This is a parameter that gives an indication about the willingness
of the animal to spend time in an open area versus an area closer to the walls (giving an indica-
tion on thigmotaxis). Thus depending on your preferred readout, you can present this data either
as time in inner (or center) zone divided by the total time, or the time in the outer zone (close to
the walls) divided by the total time. For example:

Time in center Time in outer zone

(x 100) or (x 100)
Total time Total time

▪ Defecations - Defications are related to emotionality. In general, an increased amount of defe-

cations can be used to indicate increased levels of anxiety in the subject, and are thus presented
as a cumulative value. This can be manually scored within EthoVision XT.

▪ Rearing behavior - Rearing can automatically be detected and scored by EthoVision XT and is
displayed as a cumulative value. This type of behavior consists of subject animals standing on
both hind paws in a vertical upright position. It is considered an exploratory behavior, but has
also been used as a measure of anxiety. Depending on the test situation, rearing can be con-
sidered to be anxiolytic or anxiogenic. It is often used to discriminate anxiety-linked behavior
from simple ambulatory behavior.

14 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

 & fear
anxiety

15 — So you want to build a core?

Anxiety & fear

introduction

Anxiety is a behavioral component that plays a role in any behavioral test, since animals constantly
take decisions while interacting with their environment. Without a clear motivational state, animals
resort to approaching a pleasant situation, and explicitly avoiding unpleasant situations. In some
situations, opposing motivations conflict with each other and have to be resolved by the animal:
approaching a mating partner in a potentially unsecure environment, or staying in a known safe and
sheltered environment. Resolving this conflict between unpleasant versus pleasant is a key compo-
nent of anxiety tests. And although some form of anxiety-like behavior can be measured in almost
any behavioral paradigm, it is important to take validated and robust tests as your main readout
parameter.

Fear is defined as a negative emotional state associated with the perception of imminent or pre-
sent threat to wellbeing or survival. It is a defensive reaction, in order to escape and avoidance
impending identifiable danger. An important difference between fear and anxiety is that anxiety is
associated with the perception of potential or ambiguous threat. Like fear, it is a defensive reaction,
but characterized by a feeling of apprehension, uncertainty, worry, uneasiness, or tension stemming
from the anticipation of potential threat or negative outcomes. When animals (and humans) face
an unambiguous situation; they can avoid the threatening stimulus or escape to safety.

why do we test anxiety and fear?

Anxiety serves as an important characteristic for the general phenotype of the animal. Highly
anxious animals perform any behavioral test much differently compared to animals that display
less anxiety-like behavior. Anxiety tests also often have a distinct learning curve, while fear is highly
preserved in all species of animals (since it plays an essential role in survival). Anxiety is thus an
important characteristic to phenotype in experimental animals, however is does require proper
justification since anxiety (and fear) tests are generally not very pleasant for the experimental
animals. Pharmacological interventions are often aimed at anxiolytic or anxiogenic effects, for
example with a novel drug that is aimed at decreasing anxiety, which has to be validated in a
robust anxiety test.

Like anxiety, fear has a primal role in ensuring survival. However, as these behavioral subsets turn
into a chronic condition, this can interfere with daily functioning and manifest into a disorder.
This poses a large threat to mental health. Treatments for these conditions are primarily tested in
animals (rodent) models. Due to a large degree of cross-species preservation in the neural circuitry
underlying these behaviors, rodent models of fear (and anxiety) provide great translational value for
the human condition.

16 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

what tests can we use to What is
measure anxiety and fear? Thigmotaxis?
Rodents tend to move in contact with
There are several well documented, robust, beha-
vertical surfaces, and prefer to eat in a
vioral tests for anxiety-like behavior which make
corner rather than in an open space. The
use of the choice between an unprotected versus motivational state for these types of
a protected area. behavior is presumably an aspect of taking
cover from predators. The tendency to
▪ Center versus border exploration of the open
remain to vertical surfaces, or “Wall hug-
field test, as described in the previous section, ging”, is referred to as ‘Thigmotaxis’. This
can also be used to measure anxiety-like beha- type of behavior is commonly associated
vior apart from locomotor and/or exploratory with anxiety and serves as a very impor-
tant motivator and interpretation of the
behavior.
results in an open field. This thigmotactic
▪ Aversion towards open spaces and thigmotaxis response makes it so that the animals
(wall hugging) serve as the contrasts in spend more time in the corners and/or
at the border of the open field, making
unpleasant versus pleasant.
this readout an important factor in the
▪ Freezing behavior in the open field is a good analysis of the open field test.
indication of fearful behavior in the open field.

Please visit the previous chapter for the testing protocol of the open field test.

17 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Anxiety & fear

elevated
plus maze

The elevated plus maze arena is entirely raised from mouse elevated plus maze
the floor (hence the elevated component) and shaped Ranges from 25 x 25 cm to
as a plus-sign. This creates four arms, of which two 50 x 50 cm
15 - 30 cm raised walls
opposing arms are enclosed with raised walls, and the
Raised ±50 cm from the floor
other two arms are completely open. The elevated plus
maze is thought of as one of the most robust and reliable rat elevated plus maze
anxiety tests. Although it makes use of the same beha- Ranges from 25 x 25 cm to
vioral contrasts as the open field (aversion of open spaces/ 50 x 50 cm
30 - 40 cm raised walls
thigmotaxis), the spaces are much more defined.
Raised ±65 cm from the floor

protocol suggestion
▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

▪ Remove a single animal from the home cage with your preferred handling technique: tail
handling, full hand handling, tube handling. Place the animal in the center square of the
elevated plus maze. Recording/tracking automatically starts in EthoVision XT if this option
has been selected. Otherwise, do not forget to concurrently activate your video recording.
It is normal for the animals to immediately move into an enclosed arm of the maze.

A standard elevated plus maze.

Credits: He, S. and Corscadden, L. (2022). Maze Engineers.

18 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

▪ Preferably leave the testing room to allow free and uninterrupted movement of the subject
animal. Record/track the animals for 5 minutes.

▪ After the testing time is finished, gently pick up the animal, again using your preferred handling
technique, and return it to its home cage.

▪ Before cleaning the arena, visually count the faecal pellets present and manually record the
numbers for further analysis.

▪ Remove all faecal pellets and wipe up all spots of urination. Spray the entire maze with 30-70%
ethanol and wipe down with a clean paper towel. Allow the ethanol solution to completely dry
prior to testing other animals.

setup in ethovision xt
In arena settings, 5 zones are required:
central square, open arm 1, open arm 2,
closed arm 1 and closed arm 2.

Group the open and closed arms together

with a zone label to automatically extract
a total time on open versus closed arms.

A simple EthoVision arena setup in an elevated plus maze with zones

for the open arms, closed arms and a central square.

interpretation of the results

Time spent on the open arms of the maze is related to lower anxiety-like behavior. Time spent in the
closed arms is related to an increase in anxiety-like behavior, since this part of the maze is enclosed
and sheltered with raised walls. However, this test should preferably only be performed once, since
rats and mice show less exploration of the open arms upon repeated testing. This avoidance can
also not be rescued by anxiolytics compounds and thus reflects a lack of motivation as opposed to
increased anxiety. Visualizing time spent in open versus closed arms is generally done with an open
arm ratio:

Time spent in open arms

(x 100)
Total time in all arms

Total arm transitions says something about general activity on the maze, while additional behaviors
such as rearing and freezing can also be automatically detected and scored in EthoVision XT. These
behaviors give additional information on the anxiety-like and fearful phenotype.

19 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Anxiety & fear

elevated
zero maze

The elevated zero maze is a variation on the elevated plus mouse elevated zero maze
maze, and makes use of the same behavioral contrast. This 50 cm diameter
shape was created to eliminate the center region of the plus 15 cm raised walls
Raised 60 cm from the floor
maze. It is an elevated ring-shaped arena with two open and
two closed quadrants (with elevated walls).
rat elevated zero maze
Tucker and McGabe describe an experiment in which they 100 cm diameter

compare the elevated plus maze and elevated zero maze 30 cm raised walls
Raised 60 cm from the floor
in male and female C57BL/6J mice [14]. They found that the
elevated zero maze encourages greater exploration of the
anxiogenic regions (open quadrants), while also finding consistent outcomes over multiple sessions,
while the elevated plus maze is advised to be only performed once per mouse to avoid learning and
habituation to the maze, potentially masking anxiety-like behavior. One obvious difference between
the elevated zero maze and elevated plus maze is the starting point. The central square is removed
in the elevated zero maze taking away the availability of a logical starting point while also removing
any ambiguity in the interpretation of the time spent in the maze.

protocol suggestion
▪ Transport the animals, preferably in their home
cages, into the testing room and allow the
animals to acclimate to this room for a mini-
mum of 30 minutes prior to starting the test.

▪ Remove a single animal from the home cage

with your preferred handling technique: tail
handling, full hand handling, tube handling.
Place the animal in the center square of the
elevated zero maze. Recording/tracking auto-
matically starts in EthoVision XT if this option
has been selected. Otherwise, do not forget to
concurrently activate your video recording. It is A standard elevated zero maze.
Credits: He, S. and Corscadden, L. (2022). Maze Engineers.
normal for the animals to immediately move
into an enclosed arm of the maze.

20 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

▪ Preferably leave the testing room to allow free and uninterrupted movement of the subject
animal. Record/track the animals for 5 minutes.

▪ After the testing time is finished, gently pick up the animal, again using your preferred handling
technique, and return it to its home cage.

▪ Before cleaning the arena, visually count the faecal pellets present and manually record the
numbers for further analysis.

▪ Remove all faecal pellets and wipe up all spots of urination. Spray the entire maze with 30-70%
ethanol and wipe down with a clean paper towel. Allow the ethanol solution to completely dry
prior to testing other animals.

setup in ethovision xt
In arena settings, 4 zones are required: open quadrant 1, open quadrant 2, closed quadrant 1 and
closed quadrant 2. Group the open and closed quadrants together with a zone label to automatically
extract a total time on open versus closed quadrants.

interpretation of the results

Like the elevated plus maze, the total time spent on the open quadrants of the maze is related
to a lower anxiety-like behavior. Additionally, total distance moved is also a common parameter
measured in the zero maze, especially since this test can be performed multiple times within the
same mouse.

Total quadrant transitions says something about general activity on the maze, while additional be-
haviors such as rearing and freezing can also be automatically detected and scored in EthoVision XT.
These behaviors give additional information on the anxiety-like and fearful phenotype.

21 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Anxiety & fear

light-dark
box

The light dark box uses the aversion of rodents towards mouse light-dark box
brightly illuminated areas as a behavioral contrast to test 60 cm long x 20 cm wide
unconditioned anxiety. Originally described in 1980 by 20 - 26 cm high

Crawley and Goodwin [4], and since then adopted as one of

rat light-dark box
the most used tests to measure anxiety-like behavior. The
105 cm long x 35 cm wide
box consists of a small (one third of the box) dark safe com-
35 - 46 cm high
partment and a larger (two thirds of the box) illuminated
compartment, a sliding door separates the two compart- Removable roof & sliding door
Black IR material
ments.

protocol suggestion
▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

▪ Remove a single animal from the home cage with your preferred handling technique: tail
handling, full hand handling, tube handling.

▪ Animals can be placed either in the middle of the lit chamber [5,6], or of the dark chamber [7,8].
However, since latency to enter the light chamber can be used as an anxiety-index, the door
between the chambers can be used to prevent the animal immediately moving to the other
chamber. In this case, place the animal in the dark chamber with the door closed.

▪ Recording/tracking automatically starts in EthoVision XT if this option has been selected.

Otherwise, do not forget to concurrently activate your video recording.

▪ After placement in the box, or after the door opens, allow the animal to move freely between
the two chambers for 10 minutes. Preferably leave the testing room during this time.

▪ After the testing time is finished, gently pick up the animal, again using your preferred handling
technique, and return it to its home cage.

▪ Before cleaning the arena, visually count the faecal pellets present and manually record the
numbers for further analysis.

▪ Remove all faecal pellets and wipe up all spots of urination. Spray the entire maze with 30-70%
ethanol and wipe down with a clean paper towel. Allow the ethanol solution to completely dry
prior to testing other animals.

22 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

setup in ethovision xt
In EthoVision XT, in the arena settings tab, draw two zones: light compartment and dark compart-
ment.

interpretation of the
results
▪ Latency to enter the dark or the light com-
partment for the first time and the total
time spent in the lit versus dark compart-
ment are used as readouts for bright-space
anxiety.

▪ Total transitions (from light to dark com-

partment and vice versa) gives an indi-
cation of activity/exploration because of
habituation over time.

In general, control mice spend more time in

An example of a light-dark box for mice.
the bright chamber compared to anxious mice. Credits: He, S. and Corscadden, L. (2022). Maze Engineers.
Similarly control mice generally show
a higher number of transitions between the
chambers, while the latency to enter the
bright chamber is lowest in this group.

23 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

 depression

24 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Depression

introduction

Depression is the world’s most predominant mental health problem, affecting approximately over
300 million people worldwide [13]. It is of upmost relevance to be able to investigate this in a preclini-
cal model. Depression in rodents is modeled through a number of ways, but mainly characterized by
its core symptoms: episodes of depressed mood, decreased drive and loss of interest and pleasure,
with various accessory symptoms. Evidently modeling such a heterogenous phenotype is very
difficult, if not impossible.

why do we test depression?

Rodent models of depression have been focused on a Early life stress
specific aspect of its pathophysiology, accompanied Early life stress is a way to model
by a specific behavioral test. Given the multifactorial adult depression in rodents. Gene-
nature and heterogeneity of depressive-like symp- rally, adverse early life experiences
are an important risk factor for the
toms, it can be debated that this is not the most
development of depression (and
clinically-relevant approach. However rodents were
other mental disorders). This can be
never meant to be a one-on-one comparison to achieved by maternal separation;
humans, but rather used as a tool to model specific prolonged separation of pups from
disease-related traits that can be tested and treated the mother, which causes significant
in a pre-clinical setting. This principle highly applies amounts of stress [20]. The conse-
quences of this early life stress are
to the case of modeling depression: By representing a
expressed in adulthood and persist
specific feature of depression in a model, and testing for life particularly through the HPA
this in a behavioral setting, it enables us to achieve (hypothalamic-pituitary-adrenal)
a better understanding of an essential biological axis.
mechanism, basically serving as a piece of the puzzle.

what tests can we do to measure depression?

The sucrose preference test is a test that measures anhedonia in rodents [21], which is the inability
to experience pleasure from rewarding or enjoyable activities, a core depressive symptom. This
test is basically a two-bottle choice test that measures the intake ratio of a sucrose (sugar) solution
to water, relying on rodents’ natural tendency to prefer sweet food. Depressed rodents show a
decreased preference towards the sucrose solution [22].

25 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Depression

the forced
swim test

A behavioral paradigm to test depression is the forced swim mouse test cylinder
test, also known as the behavioral despair test, which was 25 cm high
introduced by Porsolt et al. in the 1970s . This test involves
[23,24] 13 cm diameter

placing a rat or mouse inside a cylinder filled with (lukewarm)

rat test cylinder
water, which the rodent naturally tries to escape.
50 cm high
The test is designed to obviously let the animal fail to escape, 30 cm diameter
and as animals give up after a certain period of time, and
become immobile, they are removed from the container. Depressed animals give up earlier than
non-depressed animals, while it has also been found that swimming behavior is generally increased
after administration of (clinically used) antidepressants [25,26], which indicates a positive result in this
test.

protocol suggestion
Preparation
▪ Fill the swim cylinder about 2/3rd with water, and make sure the water is around 23-25°C
(check with a thermometer!)

▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

Pretest
▪ Place the animals individually into the swim cylinders (up to four cylinders can be utilized for
appropriate throughput) and start the timer (video recording is optional in this phase). After 15
minutes of swimming, remove the animals from the water, dry them with a towel and return
them to their home cages. If the animals are group housed, ensure that no post-swim animal
is placed back into a home cage with animals that still have to undergo the preswim. In such
instances, a temporary dry cage with fresh bedding is recommended until the animal can be
returned to its home cage.

Swim test
▪ 24 hours after the pretest, the swim test can be performed by again placing the animals in the
swim cylinder (which is 2/3rd filled with fresh lukewarm water). It is important to record this
session! This session takes 5 minutes, remove the animals from the cylinders after this time
and dry them before returning to their home cage or a temporary dry cage.

26 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

setup in ethovision xt Notice!
Due to the severity of the forced
Video tracking with EthoVision XT allows you to
swim test and its impact on animal
detect some behaviors automatically, such as
welfare, this test has come under
immobility, which is indicative of freezing. The three considerable scrutiny in recent years.
variations of mobility – immobile, mobile, and highly It’s scientific utility as a depression
mobile – indicate the three most important beha- model is debated, as there is a
lack of neurobiological correlation
viors in the forced swim test:
between the behaviors measured in
▪ Floating (immobility this test, and the human situation.
▪ Swimming It is however important to compart-
mentalize the discussion surround-
▪ Climbing/escaping-
ing the forced swim test, as it still
There are two ways to detect this with EthoVision remains an important scientific tool
XT, and they can be used alongside each other. First in academia, drug discovery and
the research industry where high
is by tracking the centre point of the animal and
throughput screening of novel com-
determining if it is immobile or not. Thresholds can
pounds is essential. The forced swim
be defined by the user. Second, you can use activity test is still a test with a considerable
detection. This method might even give you more level of predictive validity, however
robust data as it focuses on changes in the arena should definitely not be considered
a full spectrum analog of human
from one video frame to the next.
depression, as discussed before. The
outcomes of the forced swim test
are one-dimensional, however the
interpretation of the results implementation is also relatively
simple and inexpensive. This is an
In general, a higher immobility score represents a extremely important trade-off to
more depressive-like phenotype. This can be treated consider, but should not devalue the
with an antidepressant, like Imipramine, as seen usefulness of the forced swim test as
a drug discovery and validation tool.
in an example given by Slattery and Cryan (2012),
decreasing immobility and increasing climbing
(escape behavior) in Sprague Dawley rats [27].

27 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

 cognition

&
memory

28 — So you want to build a core?

Cognition & memory

introduction

Cognition is defined as ‘the mental action or Why only talk about

process of acquiring knowledge and under- mouse transgenic
standing through thought, experience, and the models?
senses. In essence, it is the ability to perceive and Although the rat has been the animal
react, process and understand, store and retrieve of choice for drug development and
information, make decisions and produce appro- fundamental research for decades, it
progressively faded away in favor of
priate responses. Memory refers to the storage
mice, a species in which genetic mani-
and subsequently retrieval of this encoded data/
pulation is much easier and for which
information. there is a greater variety of research
reagents available.
There are multiple forms of cognition and
Non-transgenic rat models are also
memory, each relating to, and/or responsible for
studied, but rely (for example) on
their specific behaviors or actions. These forms
pharmacological induction of the
are however highly intertwined, making them disease, relating more the sporadic
challenging to distinctly measure. By investigating form of Alzheimer’s disease. Check
separate cognitive domains through behavioral out the study from Lecanu and
Papadopoulos on that topic [29].
batteries scientists are able to tear apart distinct
cognitive functions.

In animal research, cognition and memory are a popular, though challenging, subject to study.
Understanding the development and/or progression of neurodegenerative diseases, like Alzheimer’s
disease, requires studies in these neurocognitive domains. Pre-clinical studies thus serve as an
essential step to better understand the underlying (neural) mechanisms of such diseases. Trans-
genic mouse models that mimic a specific subset of pathophysiology of Alzheimer’s disease for
example are amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2). These are
Alzheimer’s disease-linked mutations discovered in humans, which function in the mouse much as
they do in humans. Obviously, these mutations on itself do not fully phenocopy the full spectrum
of the human disease, but like before (in other models) provide great insight in specific molecular
mechanisms which are responsible for specific pathophysiology.

A great overview of animal models of neurodegenerative disease is described by Dawson et al. (2018) in
Nature Neuroscience [28].

29 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Cognition & memory

forms of cogni-
tion & memory

allocentric and egocentric spatial memory

Spatial learning refers to the association or representation of an organism in a three-dimensional
environment. In animal research terms: An animal learning its position in a given space. This task
highly relies on visual cues and/or landmarks. Allocentric refers to encoding information about the
location of one object relative to the location of other objects. While egocentric represents the
location of objects in space relative to your own body [30].

Want to read more about the importance of distinguishing allocentric and egocentric search strategies in
rodents? This review article by Grech et al. (2018) provides relevant information and additional literature [30].

working memory
Working memory is generally defined as short term memory for an object, stimulus, or location that
is used withing a testing session, but not typically between sessions. In simple terms: keeping in
mind everything that is required while performing a task.

short term memory

Short term memory is crucial for many basic tasks in life. In animals, short-term memory plays a
critical role in understanding new environments, allowing goal-directed behavior, and generally
speaking, providing a substantial survival advantage. Without it, animals couldn’t successfully
avoid predators and search for prey, build nests, and assimilate and understand novel environ-
ments within their existing models of the world.

long term memory

Long term memory is basically information that is retained for a longer period of time, being
classified as relevant to store (sometimes indefinitely). Where you live, your name and of your loved
ones, where you went to high school, your first kiss etc. Episodic memory is a category of long-term
memory, and refers to an episode of your past experiences. This usually involves a when and a where.
The latter is quantifiable in rodents, usually involving training to a specific location linked to a
reward or aversive event.

30 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

associative learning
Associative learning is a process in which a new response becomes associated with a particular
stimulus. Thus, Pavlovian learning is a form of associative learning, since this is typically induced in
subjects that associate stimuli with a negative (aversive) stimulus or situation: such as a light cue
followed by a foot shock. Basically, associative learning is the ability of an animal to connect a
previously irrelevant stimulus with a particular response which mainly occurs through the process
of conditioning. Reinforcement of this response strengthens this behavioral pattern even further.

cognitive flexibility / reversal learning

Cognitive flexibility is the ability to rapidly change, or adapt, behavior while facing specific (changed)
circumstances. Successful discrimination learning is necessary, since a reversal learning task relies
on reversing the two stimuli and assessing whether (or how fast) the subjects learn the new posi-
tioning or object or other stimulus. Basically: when a subject is used to a specific situation, how fast
can it adapt when specific environmental cues are changed [31].

Want to read more on the neural basis of reversal learning? Check out this paper by Izquierdo et al. [31]
from 2017.

31 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Cognition & memory

novel object
recognition

The novel object recognition test is designed to study recog- mouse open field
nition memory in rodents using one or more familiar objects Ranges from 25 x 25 cm to
and one novel object, and are typically performed in an open 80 x 80 cm
30 - 40 cm raised walls
field arena. Animals tend to spend more time investigating
the novel object as a result of the natural curiosity of rodents. rat open field
This must be compared to a baseline or training environment, Ranges from 60 x 60 cm to
where animals are habituated to the testing arena and a set 120 x 120 cm
of familiar objects. If during the novel object recognition test, 40 - 50 cm raised walls

exploration of the novel and familiar objects is the same, this

can be interpreted as a memory or cognitive deficit. In addi-
tion to the novel object recognition test, the novel location recognition test can also be performed,
which measures spatial memory. Since in the latter test the object is moved (not replaced by an-
other object), studies suggest that while object recognition is hippocampus-independent, location
recognition is, in fact, dependent on hippocampal functioning. This has long been the general con-
sensus, however, as we gain a larger understanding in the neurobehavioral field due to technological
advances, recently studies have shown, like one study by Cinalli Jr et al., that the mouse hippocampal
CA1 region and Perirhinal cortex play complementary roles in spontaneous object recognition [32].

protocol suggestion - square arena

Phase 1 - Learning objects and locations, same day as the open field test
▪ Transport the animals, preferably in their home cages,
into the testing room and allow the animals to accli- Important!
mate to this room for a minimum of 30 minutes prior This test requires that the
to starting the test. animals are already habituated
to the arena they are tested in.
▪ Place two similar objects in opposing corners of the This is usually done in an open
arena, randomize these corners for all animals tested. field arena, thus this protocol
Objects used can be anything from Lego Duplo blocks follows the protocol of the
and small toys that are easy to clean, to simple glass or open field test in chapter 1.

(3D printed) plastic objects.

▪ Remove a single animal from the home cage with your preferred handling technique: tail
handling, full hand handling, tube handling. Place the animal in the middle of the (open field)

32 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

 arena. Recording/tracking automatically starts in EthoVision XT if this option has been selected.
Otherwise, do not forget to concurrently activate your video recording.

▪ Preferably leave the testing room to allow free and uninterrupted movement of the subject
animal. Record/track the animals for 5 or 10 minutes, depending on how long they were tested
in the open field test.

▪ After the testing time is finished, gently pick up the animal, again using your preferred handling
technique, and return it to its home cage.

▪ Before cleaning the arena, visually count the faecal pellets present and manually record the
numbers for further analysis.

▪ Remove all fecal pellets and wipe up all spots of urination. Spray the floor, walls and objects of the
maze with 30-70% ethanol and wipe down with a clean paper towel. Allow the ethanol solution
to completely dry prior to testing other animals.

Phase 2, option 1 - Novel object recognition test, approximately 24 hours after phase 1
▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

▪ Again place two objects in opposing corners of the arena (these must be the same corners as
phase 1!), one object is the same as in phase 1, but the other object must be of a different shape
(similar in size). Randomize which object is replaced for all animals tested.

▪ Remove a single animal from the home cage with your preferred handling technique: tail
hand-ling, full hand handling, tube handling. Place the animal in the middle of the (open field)
arena. Recording/tracking automatically starts in EthoVision XT if this option has been selected.
Otherwise, do not forget to concurrently activate your video recording.

▪ Preferably leave the testing room to allow free and uninterrupted movement of the subject
animal. Record/track the animals for 5 or 10 minutes, depending on how long they were tested
in the open field test.

▪ After the testing time is finished, gently pick up the animal, again using your preferred handling
technique, and return it to its home cage.

▪ Before cleaning the arena, visually count the faecal pellets present and manually record the
numbers for further analysis.

▪ Remove all fecal pellets and wipe up all spots of urination. Spray the floor, walls and objects of the
maze with 30-70% ethanol and wipe down with a clean paper towel. Allow the ethanol solution
to completely dry prior to testing other animals.

Phase 2, option 2 - Novel object recognition test, approximately 24 hours after phase 1
▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

▪ Again place the same objects in the arena (these must be the same objects as phase 1). However,
one object must remain in a similar corner/position of the arena, while the other object must be
moved to a different location. Randomize which object is moved for all animals tested.

33 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

▪ Remove a single animal from the home cage with your preferred handling technique: tail
handling, full hand handling, tube handling. Place the animal in the middle of the (open field)
arena. Recording/tracking automatically starts in EthoVision XT if this option has been selected.
Otherwise, do not forget to concurrently activate your video recording.

▪ Preferably leave the testing room to allow free and uninterrupted movement of the subject
animal. Record/track the animals for 5 or 10 minutes, depending on how long they were tested
in the open field test.

▪ After the testing time is finished, gently pick Important!

up the animal, again using your preferred If you want to test novel object recog-
handling technique, and return it to its home nition and novel location within the
cage. same group of animals: phase 1 must
be repeated before going on to either
▪ Before cleaning the arena, visually count the of the options. Thus if you have done
faecal pellets present and manually record the novel object recognition, you have to
numbers for further analysis. re-train the animals as was done in phase
1 before performing a novel location task
▪ Remove all fecal pellets and wipe up all spots (and vice versa).
of urination. Spray the floor, walls and objects
of the maze with 30-70% ethanol and wipe down with a clean paper towel. Allow the ethanol
solution to completely dry prior to testing subsequent animal.

setup in ethovision xt
General arena zones are similar to the open field. Exploration of the object is usually defined as the
animal’s nose being in the zone of 2-3 cm around the object. Some researchers exclude data points
in which both the nose and the center point were detected in this zone, effectively excluding climb-
ing behavior and exploration around the object instead of the object itself. Other researchers use a

Arena setup in EthoVision XT

for a novel object recognition
test. Image credits: Alicia
Brantley, PhD. Mouse Beha-
vior Core, Scripps Research.

34 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

different approach, describing donut shaped object zone between 0.75 and 1.5 times the radius of
the object defined as the zone of exploration.

EthoVision XT allows you to define areas and objects within your video image by simply drawing
zones around them. If you want object exploration to be defined as being within 2 cm of the object,
for example, you can draw a zone that is the same shape as your object, plus a 2 cm circumference.
Donut shapes are also possible by excluding the center of the circle. When zones are defined,
EthoVision XT automatically registers when the animal enters the zone, and which body points
are in that zone. You can also filter out data points in which the animal has both its nose and body
center in the zone (e.g. indicating the animal is climbing on the object).

interpretation of the results

The most important results from the novel object or novel location test include the exploration
times of the objects and the frequency of each object exploration. These are generally expressed as
the percentage of exploration time spent on novel object:

Time spent exploring novel object

(x 100)
Time spent exploring any object

The preference for novelty is a positive value if there is preference for novelty, and is zero if there is
no preference:

Time novel - Time familiar

(x 100)
Time novel + Time familiar

35 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Cognition & memory

the t/y
maze

The Y-maze and T-maze look and function very similarly to mouse t/y-maze
each other, although some differences in readouts exist. The 3 Equal arms: 35 cm length
T-maze is constructed in the shape of a T and presents the 15 cm raised walls
3 doors between the arms
animals with two choice options: a 90-degree left or right
manual operation
turn. Using different motivational cues, either working
memory or short term spatial memory can be measured. rat t/y-maze
Internal and/or external visual cues can be used to probe 3 Equal arms: 52 cm length
spatial memory, while positive/negative reinforcement in 30 cm raised walls
3 doors between the arms
one of the choice arms can be utilized to test intact working
manual operation
memory.
important
The Y-maze is very similar to the T-maze. The name already
The T-maze has arms set at 90
gives it away considering the construction/layout of this degrees of each other, the Y-maze
maze compared to the T-Maze: the Y-maze is shaped in a Y. at 120 degrees (equal corners)
This minimal distinction does change the arm-choice com-
ponent of the test, since in this setup there are three equal arm choices at 120-degrees. Popular
paradigms in the Y-maze thus do not consist of baiting/motivating the arm choice, but rather rely-
ing on the animal’s innate preference to explore previously unexplored areas. Short-term memory
can be tested by blocking access to one of the arms in the first phase of the test and observing the
time spent in that arm in the second phase where all three arms can be accessed. This novel arm
preference task is a test for allocentric spatial memory as the animals use cues from both inside and
outside of the maze to remember the location of this unexplored arm.

Spontaneous alternation is a unique readout that can be measured with the Y-maze (which can also
in theory be measured with the T-maze, although requiring constant investigator interaction). This
is a paradigm that tests working memory, and involves the animals freely exploring all three arms
and observing if they chose to enter the arm most recently explored or they alternate and enter the
more novel arm.

protocol suggestion
Spontaneous alternation in the Y-maze
▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

36 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

▪ Remove a single animal from the home cage with T-maze or Y-maze?
your preferred handling technique: tail handling,
It depends on what you would
full hand handling, tube handling. Place the animal like to measure. As a test for
in at the same end of one arm and allow the to spatial learning and memory
move freely through the maze during an 8-minute retrieval, the T-maze presents a
clear left-or-right choice, in which
session. Recording/tracking automatically starts in
this choice could be motivated by
EthoVision XT if this option has been selected.
placing a bait.
Otherwise, do not forget to concurrently activate
When opting for spontaneous
your video recording. alternation, the Y-maze is the

▪ Preferably leave the testing room to allow free and more common choice, since all
three arms present an equal
uninterrupted movement of the subject animal.
choice (equal 120 degree corners).
▪ After the testing time is finished, gently pick up
the animal, again using your preferred handling
technique, and return it to its home cage.

▪ Before cleaning the arena, visually count the faecal pellets present and manually record the
numbers for further analysis.

▪ Remove all faecal pellets and wipe up all spots of urination. Spray the entire maze with 30-70%
ethanol and wipe down with a clean paper towel. Allow the ethanol solution to completely dry
prior to testing other animals.

Suggested protocol for the T-maze

A T-maze protocol is very similar to the Y-maze. A crucial difference is the arm choices. With spon-
taneous alternation is the Y-maze, free moving arm choices is of utmost importance. However in
a T-maze often a left-or-right choice is preferred towards a baited arm (or with another form of
reward/learning). Thus the animal is placed in a starting arm, and makes a left or right choice.
This is repeated multiple times to obtain a score.

A standard T-maze with sliding doors. A standard see-through Y-maze.

Credits: He, S. and Corscadden, L. (2022). Maze Engineers. Credits: He, S. and Corscadden, L. (2022). Maze Engineers.

37 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

setup in ethovision xt
In EthoVision XT three main zones should be drawn, representing the three arm choices, let’s call
the arms a, b, and c.

interpretation of the results

In the analysis profile the total number of arm entries must be counted, which is a measurement
of activity and locomotion during the testing session and is also be used to calculate the percen-
tage of alternation. Alternation is defined as successive entries into the three arms, of which the
order does not matter per se, but that an alternation is only achieved in a set of three different
arm entries. For example, successive entries into arms a, c and b is considered an alternation, while
entries into arms a, b, and a again is not considered an alternation. This measure of spontaneous
alternation can be expressed as an absolute number, or as a percentage, and is a measure of short
term spatial memory.

# spontaneous alternations
Spontaneous alternation % = x 100
total number of arm entries - 2

38 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Cognition & memory

the radial
maze

In 1976, Olton and Samuelson [33] devised a classic task for mouse radial arm maze
assessing working memory and spatial memory in rodents: 8 Equal arms: 35 cm length
the radial arm maze. This maze has eights arms, which radiate 15 cm raised walls

from a central platform. To test working memory, all arms of

rat radial arm maze
the maze are baited (food reward), the animal is placed on the
8 Equal arms: 52 cm length
center platform and is allowed to explore freely. Retrieving the 30 cm raised walls
food reward from each arm, and not re-entering a previously
visited arm is a key readout for this. Re-entering a previously
visited arm is considered a working memory error. To test spatial memory, one arm (or multiple) can
be baited, and the animal is trained in multiple sessions to this location. In each following session
the speed to reach the arms should increase, indicating healthy spatial memory. Removing the food
reward (or placing it in another arm) can subsequently be used to also test spatial memory and
cognitive flexibility (how fast it will test new locations, or adapt, to find the reward).

protocol suggestion
Pre-training (can be done across several days prior to the experiment)
▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

▪ Allow animals to acclimatize to the maze and encourage exploration: place multiple animals
(preferably cage mates) on the maze and let explore freely for 20 minutes with food rewards
scattered throughout the maze.

▪ On the subsequent days only place food rewards at the ends of the arms.

A standard radial arm maze. An elevated radial arm maze with sliding doors to seal off
Credits: He, S. and Corscadden, L. (2022). Maze Engineers. specific locations can also be used. Credits: He, S. and
Corscadden, L. (2022). Maze Engineers.

39 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Training phase (this can be repeated over the course of 10 to 20 days)
▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

▪ Bait all arms of the maze (in the end of the arms).

▪ Remove a single animal from the home cage with your preferred handling technique: tail
handling, full hand handling, tube handling. Place the animal in in the center of the maze
and allow the animal to move freely through the maze. The session is terminated when the
subject has visited all 8 arms and has eaten the reward after 16 arm visits are made (regardless
of which arms) or after a maximum of 15 minutes. Recording/tracking automatically starts in
EthoVision XT if this option has been selected. Otherwise, do not forget to concurrently activate
your video recording.

Testing phase
▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

▪ Bait some of the arms of the maze, while the remaining arms remain un-baited.

▪ Remove a single animal from the home cage with your preferred handling technique: tail
handling, full hand handling, tube handling. Place the animal in in the center of the maze and
allow the animal to move freely through the maze. The session is terminated when 8 minutes
have passed or until all baited arms are entered. Recording/tracking automatically starts in
EthoVision XT if this option has been selected. Otherwise, do not forget to concurrently activate
your video recording.

▪ After the testing time is finished, gently pick up the animal, again using your preferred handling
technique, and return it to its home cage.

▪ Before cleaning the arena, visually count the faecal pellets present and manually record the
numbers for further analysis.

This protocol is adapted from Maze Engineers:

https://conductscience.com/maze/portfolio/radial-arm-maze/

40 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

setup in ethovision xt
In EthoVision XT, an arena with 8 arms (or more/less depending on the size of the maze) should be
drawn, and a center zone.

A simple EthoVision arena setup in the radial arm maze: all

arms are defined, as well as the central area of the arena.

interpretation of the results

The following parameters are essential to measure from the radial arm maze:

▪ Total numer of arm entries (all four paws in an arms)

▪ Total correct arm entries (a novel arm that has not previously been entered)
▪ Total wrong arm entries/errors (a previously visited or un-baited arm)

With this you can create a so-called memory score:

(correct arm entries) - (incorrect arm entries)

Memory score =
(correct arm entries) + (incorrect arm entries)

A memory score of 1 reflects a perfect memory score of entering only novel arms. This score is
likely to improve over several tests.

Also the time between retrieving food rewards can be scored as a measure of activity and
willingness to explore.

41 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Cognition & memory

the morris
water maze

Developed by Richard G. Morris in 1984, the Morris water maze mouse water maze
is a test of spatial learning, and relies on swimming (navigating) Diameter is ±120 cm
from a start location, and locating a submerged escape platform. ± 55 cm raised walls

Usually this is assessed over multiple trials, and spatial learning

rat water maze
is ultimately assessed in a trial with the platform removed, which
Diameter is ±180 cm
will trigger a preference for the previous location of this platform. ± 95 cm raised walls
In a similar fashion, this can also be used to measure cognitive
flexibility (as explained before). In this case the original location escape platform
of the platform (which the animal has learned) is moved to a ± 8 cm in diameter

different location in the maze. The Moris water maze has proven
to be a robust and reliable test that is strongly correlated with
hippocampal synaptic plasticity and NMDA receptor function.

protocol suggestion
Preparation
▪ Fill up the maze with regular tap water, the temperature should be brought up to approximately
26°C (this can take a while!). Use non-fat dry milk, or non-toxic white paint to make the water
opaque. This will make the platform invisible to the animals in the testing phase, but also
improve contrast of animal to background for video recording and analysis in EthoVision XT.

▪ Place the escape platform in the

center of the pool. During the
training phase, it must be ex-
posed, one inch above the water.
This teaches the animal that
there is a platform, and that it is
the way to get out of the water.

▪ Place three or four distal visual

cues (length/width ~30 cm)
surrounding the arena, visible to
the animals while subjected to the
A Morris water maze is basically a small pool which is then filled with water.
test. Make sure you can properly drain it after use! Credits: He, S. and Corscadden, L.
(2022). Maze Engineers.

42 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Training phase - Each animal will undergo three consecutive trials
▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

▪ Remove a single animal from the home cage with your preferred handling technique: tail
handling, full hand handling, tube handling and place the animal on the platform in the
center for twenty seconds.

▪ After this period take the animals to one of the 4 quadrants of the maze: north, south, east or
west: gently lower the animal into the water, and let the animal search for the platform for a
maximum of 60 seconds. If the animal doesn’t find the platform withing this timeframe, record
the maximum amount of time for the trial (60 seconds), and gently guide the animals to the
platform with your hand, and let it sit there for 15 seconds. The animals needs to be taught to
swim to the platform.

▪ Repeat the same procedure for two more trials, starting at a different quadrant for each trial.
Dry the animals afterwards before returning them to their home cage.

Testing phase - Each animal will undergo up to 12 trials.

▪ Fill the water maze with a bit more water, to submerge the platform about an inch below the
surface. Make sure the lighting and water temperature are the same as in the training phase.

▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

▪ Remove a single animal from the home cage with your preferred handling technique: tail
handling, full hand handling, tube handling and place the animal in a quadrant, facing the wall
of the maze. Each consecutive trial should begin in a different quadrant (starting direction), but
randomize the order! Recording/tracking automatically starts in EthoVision XT if this option has
been selected. Otherwise, do not forget to concurrently activate your video recording.

▪ Observe the animal until it reaches the platform,

and record the time it took. If the animal doesn’t Optional: reversal
reach the platform in 60 seconds, guide it to the learning or probe trial
platform, as in the training phase. Either way, let After these 12 trials you have two
the animal sit on the platform for 10 seconds, and options: either do a probe trial with
then dry it off and return the animal to a holding the platform removed entirely, and
observing/verifying that the animals
cage and continue with the next animal.
understands/remembers the location
▪ Repeat this until all 12 trials are performed this of the platform. Or move the plat-
wat, while periodically checking the platform, form to a different location, and do
a reversal learning task. Also observe
temperature and cleaning the maze.
if the animal indeed remember the
▪ When all trials are complete, dry off the animals, previous location, but now also score
return them to their home cage and housing whether the animal can find the new
location of the platform.
room and drain the pool.

43 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

setup in ethovision xt
In EthoVision XT draw 4 main zones, which are the quadrants of the arena. Also draw a zone around/
on the location of the platform. In analysis profiles we want to acquire distance moved, time spent
in each quadrant and latency to find platform.

A standard arena setup for the morris water maze in

EthoVision XT consists of drawing 4 quadrants. Don’t
forget to draw the area of the escape platform!

interpretation of the results

The primary and most used outcome of the Morris water maze is the escape latency. This is the
time it take for the animal find the platform. This can be influences by swimming speed however,
which some researchers overcome by (instead of escape latency), take the path length between the
starting point and escape platform. With the probe/reversal trial, the time spent around the old
platform location is measured within a cut-off timeframe (e.g. 60 seconds), or the number of
platform (location) crossings.

44 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Cognition & memory

cincinnati
water maze

The Cincinnati maze is a type of water maze that is specifically cincinnati water maze
aimed at testing egocentric spatial memory. This maze consists Length is 120 cm
of interconnected T-intersections (basically multiple T-mazes) in Width is 120 cm
Corridor width is ± 14 cm
which the animals have to navigate from start to finish [22]. This
Wall height is 37 cm
test requires egocentric navigational ability because of the lack
of visual cues: the animal has to rely on it’s own memory of the
maze to make the correct choices (which is a left-or-right choice). The Cincinnati water maze is often
filled with water as a motivation to escape. Motivation by food reward can be problematic if a treat-
ment for example causes differences in body weight, appetite or reward salience.

protocol suggestion
Preparation
▪ Partially fill up the maze with
regular tap water, the tempe-
rature should be brought up
to approximately 26°C (this
can take a while!). You can use
non-fat dry milk, or non-toxic
white paint to make the water
opaque. This will improve con-
trast of animal to background
for video recording and analysis
in EthoVision XT. A Cincinnati water maze.
Credits: He, S. and Corscadden, L. (2022). Maze Engineers.

Trial phase - 15 days, 2 trials per day

▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

▪ Remove a single animal from the home cage with your preferred handling technique: tail
handling, full hand handling, tube handling and place the animal at the start of the maze.

▪ Allow the animals to explore the maze for 5 minutes. If the exit was not found within this
time remove the animal without showing the exit, record the maximum time.

45 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

▪ Dry off the animal and return the animal to a holding cage and continue with the next animal.

▪ Perform two trials per day, leaving a minimum of 15 minutes between trials.

setup in ethovision xt
In EthoVision XT the correct path should be drawn throughout the arena. Incorrect arm choices
should be drawn as separated zones. This process takes a little more time compared to other mazes.
But once drawn, analysis can go very quick. The parameter for an arm entry must be set (in order to
properly count errors). Generally this is set at head and shoulder entry into a zone, but can also be
more strict such as all 4 paws (front and hind legs) enter a zone. Total errors and latency to escape
are ultimately the readouts.

interpretation of the results

Schaefer at al. presents a nice overview of their outcomes with the Cincinnati maze [23]. Over time a
clear decrease in escape latency and errors can be seen, indicating a learning process. The outcomes
of the two trials per day are averaged per day. A repeated measures ANOVA (with post hoc testing)
can be used to determine the differences between groups per timepoint.

46 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Cognition & memory

the barnes
maze

The Barnes maze is a dry-land environment for testing learning, mouse barnes maze
memory and cognitive flexibility. Carol Barnes designed this beha- Diameter is 100 cm
vioral paradigm in 1979 to test memory deficits in aging rats [35]
, 20 holes, 5 cm diameter

while only being adapted to mice as late as 1995 . This test is

[36]
rat barnes maze
regarded as an alternative to the Morris water maze, which relies on
Diameter is 130 cm
swimming behavior potentially confounding learning and memory 20 holes, 10 cm diameter
readouts. Although being coined as an alternative, there are some
The performance in the Barnes
clear advantages and disadvantages towards using either a Barnes
maze, and the Morris water
maze or Morris water maze to test learning and memory which will maze, are also highly sensitive
be listed here. to anxiety in rodents, which can
be induced (or of greater levels)
Generally the Barnes maze is a well-established behavioral para- in animals subjected to for
digm, and is used, as stated, to test learning and memory in rodent example pharmacological and/
models for, for example, Autism Spectrum Disorder [37], Alzheimer’s or genetic manipulation. This
can be mitigated for example
disease [38] and ageing [39], to name a few. Performance in this test
by decreased light intensity.
is primarily linked to allocentric spatial learning, since the animals
learn the position of an ‘escape box’ relative to other positions on
the maze. This is made clear by the construction of the Barnes maze, which is a circular table with
circular holes around the circumference. As mentioned, the goal of this test is that the subject (rat
or mouse) reaches an escape box that is positioned beneath one of these holes. This task relies on
visual cues, which thus prompts spatial learning and memory. Learning the position of this box
beforehand is essential to this test, which we thus call the acquisition phase. This can be done in a
number of ways, food reward is a popular choice. However it has been seen that motivating animals
to indeed look for the escape box can be challenging. Non-responders are not uncommon in this
test. These are animals that simply ignore the tendency to reach the escape box. Oversaturation
can also take place when there are a large number of acquisition trials.

A C57BL/6J mouse looking down into the escape hole of a Barnes maze.

47 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

 Barnes maze or Morris water maze, which one should I choose?
Advantages of the Barnes maze Disadvantages of the Barnes maze
1. The Barnes maze does not involve swimming, 1. A lack of stressful stimuli in the Barnes maze
which can be perceived as stressful and can result in slow learning. To increase the
increases corticosterone levels . [53]
motivation to escape a mild stressor such as
2. Swimming in the Morris water maze also white noise or a buzzer sound can be played [36].
causes reductions in core body temperature 2. The Barnes maze requires a bit more training/
which can affect performance [54], this can be trials than the Morris water maze. Where a
circumvented somewhat by regulating the Barnes maze generally takes 15-20 acquisition
water temperature properly. trials, even having protocols of up to 40 trials.
3. While in a swimming task, rodents can take The Morris water maze is generally performed
to floating, which is thought to represent a over ± 12 trials.
state of behavioral despair, a common readout 3. It is said that the Barnes maze is somewhat
of depressive-like behavior in the forced swim limited to the use of young (3-5 months of
test. age) adult C57BL/6J mice [40,41].

Concluding
Both the Barnes maze and the Morris water maze are well validated behavioral paradigms for studying
learning and memory. Both have clear advantages and disadvantages. It should thus be pragmatically
considered which one is the best for you. Do you have a validated setup of one or the other in your lab?
Have you previously published with one of these tests? Or do you simply want to avoid swimming, for
example in mice which are less-than-natural swimmers compared to rats? Think practically about these
things when writing/planning your study design.

protocol suggestion (multiple days)

Preparation
▪ Place the maze in your testing room at the appropriate lighting intensity for your tests. You might
have to decrease this intensity a bit, since this will improve performance on this test in animals
with higher levels of anxiety.

▪ Place three or four distal visual cues (length/width ~30 cm) surrounding the arena, visible to the
animals while subjected to the test

Habituation (day 1)
▪ Transport the animals, preferably in their home cages, into the testing room and allow the
animals to acclimate to this room for a minimum of 30 minutes prior to starting the test.

▪ Attach the escape box to the platform.

▪ Remove a single animal from the home cage with your preferred handling technique: tail
handling, full hand handling, tube handling and place the animal in the escape box for 1 minute.

▪ After this, place the animal in the center of the maze, allow it to explore for 5 minutes or until it
reaches the escape box.

48 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Training/acquisition phase (days 1 - 10)
▪ Allow an interval of at least 1 hour between the habituation trial and the first training session.

▪ Attach the escape box to the maze, at a different location than the habituation trial! This position
will remain the same for all training/acquisition trials.

▪ Daily two training session can be performed with an average length of 3-5 minutes per session
(your preferred choice).

▪ Remove a single animal from the home cage with your preferred handling technique: tail
handling, full hand handling, tube handling and place the animal in the center of the maze

▪ The trial is over when the maximum

time for the test has elapsed, or if the
animals has found the escape box. If
the animal does not find the escape
box within the given timeframe, place
the animal in the escape box for 15
seconds after the trial and note the
maximum time.

▪ After each trial remove all fecal pellets

and wipe up all spots of urination.
Spray the maze with 30-70% ethanol
and wipe down with a clean paper
towel. A standard Barnes maze is always elevated from the floor.
Credits: He, S. and Corscadden, L. (2022). Maze Engineers.

Probe trial
▪ Three days after the final session of acquisition training, perform a probe trial for 1 minute. In this
trial the escape box should be removed from the apparatus.

setup in ethovision xt
In EthoVision XT there is a predefined template for the Barnes maze, making tracking and analyzing
as easy as dragging and dropping the circles (for the escape holes) to the correct place in the video
image. Specify which hole has the goal box underneath, and you are ready to acquire your data.
Record the following parameters: latency to locate the escape box, latency to enter the escape box,
number of (incorrect) holes checked before locating and escaping to the box and distance traveled
prior to locating the escape box (and total distance traveled).

interpretation of the results

Investigating the animal’s searching strategy over consecutive trials is an interesting readout of the
Barnes maze. For example, some animals randomly search for the correct hole while in the acquisi-
tion phase, while other animals stick to a certain pattern when systematically checking each hole.
These search strategies are classified as random, serial, or direct (or spatial).

49 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

In general, with repeated testing, rodents typically progress through the search strategies in the
order listed (random, serial, and direct). This progression over time can be used to visualize the
increase in searching performance during the acquisition phase of this test. However some animals,
even with direct visual markers at the escape box, keep preferring the serial search strategy over
the direct (spatial strategy) [42]. However these strategies aren’t necessarily slower and/or faster
compared to each other. Rodents, particularly mice, can walk rapidly over the edge of the maze,
employing the serial search strategy, and showing no particular difference in time to reach the
escape box, and keep using this method in all trials. This does however result in significantly more
errors, since they generally check more incorrect holes. Visualizing and classifying this strategy is
thus of upmost importance for the quantification of this test, since total number of errors will give
a skewed representation of the animals’ performance.

random serial spatial

Visualizing searching strategies on the Barnes Maze with EthoVision XT is critical for the interpretation of this test.

Want to read more about search strategies in the Barnes maze? Check out this interesting publication by
Harrison et al. [42].

50 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

 home cage
behavior

51 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Home cage behavior

introduction

Standard tests, such as the open field, elevated plus maze (and others named in this e-book) only
provide brief snapshots of animal behavior at pre-determined time points. This way you can speci-
fically evoke a type of behavior that is of interest. For example when you specifically want to look
at acute anxiety. However a more ethologically relevant approach is to look at behavior in the home
cage.

why do we test in a home cage?

Monitoring behavior in the home cage eliminates stress in the animal that can be induced by for
example handling and exposure to a new environment. Home cage monitoring also adds the option
of monitoring behavior over time, be it food/water intake or behavioral changes over time after a
(novel) manipulation (e.g. pharmacological or genetic). This can also be utilized to directly increase
resolution in behavioral models that tend to show subtle abnormalities.

Also reproducibility is essential, though often still an overlooked aspect. All the behavioral tests
described in this e-book rely on standardizing testing and assay protocols for reproducible data.
A lot of variation still exists between labs, but also within labs which can be attributed to many
factors (even the people that execute the test). The more variables that can be controlled, and
standardized within the scientific community, the more reproducible the data becomes. This
reproducibility can be increased in standardized home cage setup

how do we test in a home cage

Home cage monitoring can be done with a self-constructed setup, like mounting cameras above
each cage. Also systems with IR sensors are implemented, or beam breaks to determine whether the
animals has crossed a specific zone and even under-cage capacitive plates. The difficulty in these
setups arises when the data, specifically the timing, needs to be integrated. This is where home
cage monitoring systems come in. These systems, like Noldus’ PhenoTyper, have specifically been
developed for this purpose and contain a number of hardware modules that can be controlled and
integrated within EthoVision XT.

52 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

what makes a cage a home cage
The definition, and ultimately design, of a home cage monitoring system is key to approaching a
more natural relevant environment to test in. Key features include:

▪ Bedding material
▪ Chow and drinking water identical to vivarium conditions
▪ Environmental enrichment
▪ Adequate cage ventilation
▪ Cage size

Additionally the home cage monitoring system can be expanded with things like running wheels
to measure activity, continuous food and water intake measurements, recording ultrasonic vocali-
zations and a cognition wall to measure learning and memory in a home cage environment.

how long can we test in a home cage

Continuous monitoring of behavior in a home cage monitoring system can range anywhere from
3 days to 4 weeks. When opting for longer periods of observation, it can be beneficial to use larger
cages as they permit less frequent cage cleaning. Prolonged observation of behavior, for example
continuous monitoring and/or longitudinal studies has proven to be indispensable to gain insights
in the interplay of genetic factors and time. For example, studies of locomotor activity in genetic
mouse models for autism uncovered consistent hypoactivity in the dark phase (the perceived active
phase of rodents) with multiple-week home cage monitoring [43]. This is in line with abnormalities
in rest and activity rhythms in Autism Spectrum Disorder patients [44,45]. Previous studies in acute
testing situations like the open field for example however reported either hyperactivity [46,47] or
hypoactivity [48,49]. This shows that home cage monitoring proves to be of great value in such
delicate behavioral models.

Home cage monitoring systems, and the benefits they bring to reproducibility, is extensively reviewed by
Grieco et al. in a large collaborative study.

53 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

Home cage behavior

what to measure?

activity, mobility, circadian rhythmicity

Locomotor activity is a key component in many behavioral tests, suggesting that genetic diffe-
rences in activity levels may be a critical consideration when comparing mouse strains.

The first chapter of this e-book described exploratory and locomotor behavior as a tool to gain
an understanding of the (behavioral) phenotype of a rodent model. A test like the open field
approaches this in an acute situation for a short period of time, which has benefits such as to
measure the acute response to a novel environment. However to add translational value long
term measurements prove to be an ideal tool. The example of Autism Spectrum Disorder, as
studied in a home cage by Angelakos et al. [33], shows this.

Long term measurements of activity, mobility and the circadian rhythmicity simply give an in-
sight in the daily routine of the animal. In a standard test we look at the spontaneous reaction to
a specific situation. You can imagine this also translates to different situations considering humans.

Wheel running in a home cage has been the original measure for home cage activity as wheel run-
ning counters were easy to implement, and provided data files that were not very large in size, while
being able to accumulate data over the period of weeks or even months. Wheel running however
presents a problem when stating the want or need to measure normal home cage activity, since this
tends to increase activity and food intake, while also being able to modulate the circadian regulation
of metabolism. Bains et al. present the differences between wheel running and home cage activity
in a very insightful review [50].

wheel running

home cage

Wheel running behavior versus standard home cage behavior do not show the same activity patterns.
This is why wheel running behavior cannot be considered a proxy of standard home cage behavior.

54 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

anxiety
Anxiety is an extremely important parameter to assess, as also explained in the second chapter
of this e-book. It is however a very sensitive readout, since a rodent can easily be stressed. Often
a rodents’ response to a specific situation can be hard to interpret and/or classify as anxiety (or
another behavioral response). This is the reason why, with anxiety, we tend to want to stick to
validated test setups and protocols. The elevated plus maze is the prime example of this.

Anxiety in the home cage is however also measurable. The most popular approach to this is the
light spot test[51]. During this test a bright light is programmed to be targeted at the drinking and
feeding zone during the dark phase. Rodents are nocturnal animals, they want to avoid lit environ-
ments. Control animals versus animals that have been treated with Diazepam, an acute anxiolytic
drug, show decreased exploration outside of the shelter (present in the home cage).

food and water consumption

Food and water consumption are important biomarkers of general wellbeing, but can also be linked
to spontaneous behavior that may vary with genetic and pharmacological interventions. Traditio-
nally food and water has been assessed either for a short period of time, or by fasting animals and
assessing intake shortly after this period. Home cage observation offers the advantage of continu-
ous monitoring of this intake, without the need of handling the animals or having to move them
to a novel cage.

Food intake assessment is also important in metabolic and/or reward studies. Does a specific inter-
vention prompt animals to eat more/less? Also the response to a novel (high fat) diet can be closely
monitored, or even things such as stress eating. Water consumption can be used to, for example,
perform a sucrose preference test. Here two water sources are presented, one plain water, the other
sucrose water, and hedonic-like behavior can be assessed. The integration with lick-o-meters makes
data integration extremely robust.

social behavior
Classically social behavior is tested in a three chamber arena. More recent studies have performed
prolonged observation in social groups of rodents using a home cage recording. Discriminating
between animals can be realized with RFID chips combined with video tracking. Peleh et al. found
that, with this method, BTBR mice (a mouse model for Autism Spectrum Disorder) engaged in fewer
social interactions compared with C57BL/6J mice [52].

cognition, learning and memory

Although cognition, learning and memory are classically measured in standard, short term, test set-
ups, recent advancements has also seen the use of cognition walls in home cage setups. Previously
learning and memory testing was quite restricted to operant condition tasks, such as the pressing of
a lever for a food reward, this however often requires either labor-intensive animal handling, or food

55 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

restriction in order to motivate the animals to press such a lever. The Sylics CognitionWall can be
placed in front of a traditional reward dispenser, after initial assessment of basal behavior. Animals
then need to learn to earn a reward by passing through one of the holes, while entering through the
other two holes does not result in a reward. The difficulty of this task can be varied by adjusting the
number of entries required to receive the reward.

PhenoTyper home cage monitoring system can come equipped with a number of hardware solutions. Including a Cognition Wall
(by Sylics) and a food pellet dispenser. Photo credits: Sylics.

56 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

 conclusions
references &
57 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology
conclusions
This e-book presented a basic overview of behavioral neuroscience practices and protocols. The
world of behavioral neuroscience is much bigger than presented here, however this e-book does
hopefully serve as a low threshold stepping stone into further research.

At Noldus we aim at providing the best tools and information to advance your research. One of our
most powerful tools, as mentioned on multiple occasions, is our EthoVision XT tracking software.
This software allows you to automate testing. EthoVision XT utilizes a special algorithm that detects
the nose point, body center, and tail base of rats and mice. Additionally, deep learning neural net-
works have also been incorporated, in order to increase nose-point detection of rodents even further.
This does not only provide you with accurate detection of exploration and other behaviors, it also
provides information about the animal’s relative position, heading, and rotational movement.

EthoVision XT is embedded in the scientific community of behavioral neuroscience, and included in

methodology in over 18.000 publication, of which many in high impact journals. It has truly contri-
buted to the advance in behavioral tracking over the last few decades.

We hope you enjoyed reading, and if there are any question or concerns regarding the contents of
this e-book, feel free to contact us!

For more information, visit www.noldus.com

58 — Basic behavioral neuroscience in rodents A publication by Noldus Information Technology

a list of
references
1. Berry, A.; Bellisario, V.; Capoccia, S.; Tiras- 6. Horii, Y.; Nagasawa, T.; Sakakibara, H.;
sa, P.; Calza, A.; Alleva, E.; Cirulli, F. (2012). Takahashi, A.; Tanave, A.; Matsumoto, Y.;
Social deprivation stress is a triggering Nagayama, H.; Yoshimi, K.; Yasuda, M.T.;
factor for the emergence of anxiety- and Shimoi, K.; Koide, T. (2017). Hierarchy in the
depression-like behaviours and leads to home cage affects behaviour and gene
reduced brain BDNF levels in C57BL/6J expression in group-housed C57BL/6
mice. Psychoneuroendocrinology, 37 (6), male mice. Sci. Rep., 7 (1)
762–772
7. Van Loo, P.L.P.; Van Zutphen, L. F. M.;
2. Bartolomucci, A.; Palanza, P.; Sacerdote, P.; Baumans, V. (2003) Male management:
Ceresini, G.; Chirieleison, A.; Panerai, A. E.; Coping with aggression problems in male
Parmigiani, S. (2003). Individual housing laboratory mice. Laboratory Animals, 37
induces altered immuno-endocrine (4), 300–31
responses to psychological stress in male
8. Fan, Z.; Zhu, H.; Zhou, T.; Wang, S.; Wu, Y.;
mice. Psychoneuroendocrinology, 28 (4),
Hu, H. (2019). Using the tube test to
540–558
measure social hierarchy in mice. Nat.
3. Lander, S. S.; Linder-Shacham, D.; Gaisler- Protoc., 14 (3), 819–831
Salomon, I. (2017). Differential effects of
9. Gouveia, K.; Hurst, J. L. (2013). Reducing
social isolation in adolescent and adult
Mouse Anxiety during Handling: Effect of
mice on behavior and cortical gene
Experience with Handling Tunnels. PLoS
expression. Behav. Brain Res., 316, 245–254
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4. Arndt, S. S.; Laarakker, M. C.; van Lith, H. A.;
10. Hurst, J. L.; West, R. S. (2010). Taming
van der Staay, F. J.; Gieling, E.; Salomons,
anxiety in laboratory mice. Nat. Methods
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photo credits
▪ He, S. and Corscadden, L. (2022). Maze Engineers.
Retrieved from https://conductscience.com/maze/

▪ Sylics, Research Solutions in Neuroscience (www.sylics.com)

▪ Alicia Brantley, PhD. Mouse Behavior Core, Scripps Research,

Florida Campus, U.S.A. (www.scripps.edu)

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