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                    The Affective Bias Test and Reward
Learning Assay: Neuropsychological
Models for Depression Research and
Investigating Antidepressant Treatments
in Rodents
Justyna K. Hinchcliffe1 and Emma S. J. Robinson1,2
1

School of Physiology, Pharmacology and Neuroscience, Faculty of Life Sciences,
University of Bristol, Bristol, United Kingdom
2
Corresponding author: emma.s.j.robinson@bristol.ac.uk
Published in the Neuroscience section

The Affective Bias Test (ABT) quantifies acute changes in affective state based
on the affective biases they generate in an associative reward learning task.
The Reward Learning Assay (RLA) provides a control assay for the ABT and
reward-induced biases generated in this model are sensitive to changes in core
affective state. Both tasks involve training animals to associate a specific digging substrate with a food reward. Animals learn to discriminate between two
digging substrates placed in ceramic bowls, one rewarded and one unrewarded.
In the ABT, the animal learns two independent substrate-reward associations
with a fixed reward value following either an affective state or drug manipulation, or under control conditions. Affective biases generated are quantified in
a choice test where the animals exhibit a bias (make more choices) for one of
the substrates which is specifically related to affective state at the time of learning. The ABT is used to investigate biases generated during learning as well
as modulation of biases associated with past experiences. The RLA follows a
similar protocol, but the animal remains in the same affective state throughout
and a reward-induced bias is generated by pairing one substrate with a higher
value reward. The RLA provides a control to determine if drug treatments affect
memory retrieval more generally. Studies in depression models and following
environmental enrichment suggest that reward-induced biases are sensitive to
core changes in affective state. Each task offers different insights into affective
processing mechanisms and may help improve the translational validity of animal studies and benefit pre-clinical drug development. © 2024 The Authors.
Current Protocols published by Wiley Periodicals LLC.
Basic Protocol 1: Bowl digging and discrimination training
Basic Protocol 2: The reward learning assay
Basic Protocol 3: The affective bias test - new learning
Basic Protocol 4: The affective bias test - modulation of affective biases associated with past experiences
Keywords: affective bias r affective bias test r cognitive bias r rat r reward
learning assay

Hinchcliffe and
Robinson
Current Protocols e1057, Volume 4
Published in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cpz1.1057
© 2024 The Authors. Current Protocols published by Wiley Periodicals
LLC. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited.

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INTRODUCTION
Modeling clinically relevant symptoms of major depression disorder (MDD) in animals is
key to understanding the relationships between the biological and experience-dependent
factors that drive the behavioral symptoms of MDD and its treatment. A core feature of
MDD is the prevalence of cognitive processing biases associated with negative affective
states, termed negative affective biases, which may be a key factor underpinning low
mood and negative rumination. In this neuropsychological model of depression, negative affective biases play a causal role in vulnerability, precipitation, and maintenance
of MDD (Godlewska & Harmer, 2021). Human studies suggest healthy controls have
tendencies to overestimate the likelihood of future positive events and underestimate the
negative ones (Bower & Mann, 1992; Korn et al., 2014; Nygren et al., 1996), whereas
people in low, negative mood manifest the opposite (Rude et al., 2002; Sharot, 2011;
Sharot et al., 2011). Moreover, depressed patients exhibit reward-related learning impairments where they attribute less value to rewarding experiences compared to healthy
people (Geugies et al., 2019; Gonda et al., 2015; Lawson et al., 2017).
Conventional animal models and behavioral tests for MDD research have largely focused
on chronic stress manipulations associated with readouts of behavioral despair, such as
immobility time in the forced swim test (FST) and reward sensitivity changes in the sucrose preference test (SPT). While these methods have established some validity in terms
of stress-related behaviors, it is not clear how well they translate to human mood disorders. The FST was also originally developed as a pharmacological screening tool rather
than a test sensitive to changes in phenotypic models, despite it now being quite widely
used for this objective. Although stress is a known risk for MDD, models associated with
other risk factors, such as early life adversity and chronic inflammation, do not consistently result in impairments in these readouts. Furthermore, significant sex differences
have been observed in most of the stress models of depression: learned helplessness,
chronic mild stress, and chronic psychosocial stress, or lipopolysaccharide-induced depression model and in behavioral tests that quantify depressive-like behaviors, such as the
FST or SPT (Dalla et al., 2005, 2010; Kokras & Dalla, 2014; Kokras et al., 2015; Palanza,
2001). Where sex differences have been presented, the manifestation of symptoms or
depressive-like behaviors in females is in the opposite direction to what is observed in
human clinical diagnoses (Lopez & Bagot, 2021; Ma et al., 2019).

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Work over the last 20 years has sought to develop more translational rodent models designed to recapitulate aspects of human neuropsychological deficits that can be objectively measured in different species (Mendl et al., 2009). The first study using the ABT
was published in 2013 (Stuart et al., 2013). The underlying concept of ABT was translated
from clinical observations in depressed patients who exhibit negative affective biases in
relation to reward-related learning and memory. These observations suggest that the affective state during a rewarding experience may bias how the memory of the experience
is encoded and subsequently retrieved, which the ABT aims to quantify in non-human
animals (Robinson, 2018, Fig. 1). The ABT recapitulates in animals a clinically relevant
symptom of MDD and has been shown to achieve constructive, predictive, mechanistic, homological, and translational validity (Hinchcliffe et al., 2017, 2020; Hinchcliffe,

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How to cite this article:
Hinchcliffe, J. K., & Robinson, E. S. J. (2024). The affective bias
test and reward learning Assay: Neuropsychological models for
depression research and investigating antidepressant treatments in
rodents. Current Protocols, 4, e1057. doi: 10.1002/cpz1.1057

Jackson et al., 2022; Stuart et al., 2013, 2015, 2017; Fig. 2). The bowl-digging task is
based on associative learning and memory between a specific digging substrate and a
food reward. During the learning phase, animals undergo two substrate-reward pairing
sessions for each condition: one memory is experienced during an affective state manipulation or test treatment, and the other memory is experienced under control conditions.
The value of each experience is kept equal, and the only factor that drives the bias is the
affective state change induced at the time of learning. The affective biases generated are

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Robinson

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Figure 1 The conceptual framework and hypothesis for the affective bias test are outlined in (A).
This method is derived from the observations of human patients with major depressive disorder
where positive and negative biases in learning and memory have been observed. Depressed patients attribute less value to the memory of positive experiences. In the reward learning assay (B),
a similar protocol to the ABT is used with 4 pairing sessions and a choice test, but animals remain
in the same affective state throughout the 1-week protocol. Animals learn to associate the two
reward-paired digging substrates with either a high (2 pellet) or low (1 pellet) reward to develop
a reward-induced positive bias during the choice test. The affective bias test was developed to
quantify affective biases in rodents by generating two independent memories learned under either
an affective state manipulation or a neutral state (C). This task involves training rats to associate
finding a food reward in a specific digging substrate. The value of each independent learned experience is kept the same, with each animal undergoing four pairing sessions over 4 days followed
by a choice test. During the choice test, the two previously rewarded substrates are presented together for the first time, and the animal’s preferences are quantified over 30 randomly reinforced
trials. Our extensive work with pharmacological and ethological manipulation demonstrated full
validation of the task (Stuart et al., 2013, 2015, 2017, 2019; Hinchcliffe et al., 2017, 2020, 2024;
Hinchcliffe, Jackson et al. 2022). Figure taken and adapted from Hinchcliffe et al. (2024).

quantified using a choice test which takes place at least 24 hr after the last pairing session. During the choice test, the animal is presented with the two previously reinforced
digging substrates at the same time, and biases are observed as an increase or decrease
in the relative value of the treatment-paired experience (Fig. 1).
As part of the validation of the original ABT, we developed a modified version of the
task in which animals remain in the same affective state throughout the protocol, and a
reward-induced bias is generated by pairing one experience with a higher value reward.
The RLA is used as part of the training for all cohorts to ensure they have successfully
learned the rules of the task before proceeding to the test. The method is also useful as a
control for studies that are testing the acute effects of treatment of the retrieval of biased
memories in the ABT. The RLA can be used as a control measure to establish the specificity of treatments to affective state-induced biases or if the treatment causes more general effects on memory. Observations during preparation for ABT studies in a depression
model suggested RLA was sensitive to the core affective state resulting in a reward learning deficit. Further exploration in different rodent depression models, including early life
adversity, chronic corticosterone, chronic interferon-alpha, chronic pro-depressant drug,
and chronic neuropathic pain, have found similar reward learning impairments (Phelps
et al., 2021; Robinson, 2018; Stuart et al., 2019). Alongside our MDD-related research,
we have also used the ABT and RLA to explore animal welfare-related questions using
these readouts as an objective measure of affective state to assess refined housing and
handling methods (Hinchcliffe et al., 2020; Hinchcliffe, Jackson et al., 2022).
In this article, we describe the methods used to train animals in preparation for the ABT
and RLA. Basic Protocol 1 describes the training and basic discrimination learning protocol. Basic Protocol 2 describes the RLA protocol. Basic Protocol 3 describes the ABT
protocol and Basic Protocol 4 describes variations that can be used to investigate modulation of biased memories.

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Robinson

STRATEGIC PLANNING
Animals and equipment require preparation before a study can begin.

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Figure 2 Example validation data of the affective bias test in male Lister Hooded and male and
female Sprague Dawley rats (n = 11-12/group). The graph illustrates a negative affective bias
induced following treatment with the stress hormone corticosterone at a dose of 10 mg/kg and a
positive affective bias following treatment with the serotonin and noradrenaline reuptake inhibitor
venlafaxine at a dose of 3 mg/kg. Data taken and re-drawn from Hinchcliffe et al. (2017) and
Hinchcliffe et al. (2024). Data are shown as mean % choice bias ± SEM (bars) and individual
data points (symbols), one sample t-test against a null hypothesized mean of 0% choice bias, *p
< .05, **p < .01, ***p < .001.

Daily Preparation of Experimental Set Up
10. Perform behavioral procedures and testing during the animals’ active phase between
08:00 hr and 20:00 hr.
11. Print your pairing sheets and have them ready to record each animal’s choices and
latencies to dig (Tables S1 and S2).
12. Use an arena with opaque sides to reduce external distraction (or cover a transparent arena with an external liner). Do not use an arena with high sides that cause
an approaching handler to appear to the rat from above, as this can induce a stress
response.

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Animals
1. Studies involving living animals require ethical approval from the relevant National/Institutional Animal Ethics Committee and must follow local regulations for
the care and use of laboratory animals.
2. After animals have been delivered to the facility, provide 1 week for acclimatization
to the new environment and recovery from transportation before starting the behavioral experiments. Transportation stress can lead to significant alterations in the immune, endocrine, cardiovascular, nervous, and reproductive systems, as well as behavior (Obernier & Baldwin, 2006; Tuli et al., 1995). During this acclimatization period,
the animals can begin habituation to handling and rewards. If the study plan involves
changing the light:dark cycle, time is needed for the animals’ circadian rhythms to
adapt. The adaptation period should be no less than 7 days for a 12 hr shift in cycle.
3. House rats in suitable cages with adequate ventilation, e.g., metal mesh lid and at least
18 cm height to allow rearing and at least the minimum floor space area for age and
weight (<600 g = 800 cm2 and ∼1500 cm2 for animals over 600 g). Rats also benefit
from access to playpens, which can help maintain a more positive affective state and
provide more normal control subjects (Hinchcliffe, Jackson et al., 2022).
4. The number of rats per cage should avoid overcrowding stress (no more than 4 rats per
cage), which can lead to an increase of corticosteroids and anxiety-related behaviors
(Gavrilov et al., 2022), but avoid individual housing whenever possible, as rats benefit
from social contact (Harper et al., 2002).
5. Animal holding rooms should be maintained under controlled temperature (21.5 ±
1°C) and humidity (55 ± 10%) conditions. The light:dark cycle should be programmed for 12 hr:12 hr with lights off in the morning (8:00 am), to enable testing of
the animals during their active phase.
6. Clean the home cages once a week to ensure a clean environment, but transfer enrichment to maintain the olfactory cues within the cage. Cage changing should be
conducted after the animals have finished behavioral procedures for the day.
7. Provide food and water ad libitum (unless the specific experimental design requires
animals to be kept on a food-restricted regimen).
8. Reserve an appropriate amount of time (at least 1 week) to handle the animals prior to
training and testing to reduce their handling stress and aversion to the experimenter.
Animals should be calm and easy to handle, without overt signs of distress (e.g.,
audible vocalizations, signs of struggle when picked up, fecal pellets). For our refined handling protocols, see the 3Hs Initiative: Housing, Handling, and Habituation
(www.3hs-initiative.co.uk). Rats are unlikely to perform the ABT correctly if they remain stressed by human contact even if food restriction motivates them to forage for
rewards.
9. Plan your study design carefully in advance, including counterbalancing of all the factors (e.g., substrate, bowl position, treatments). Tables 2, 3, and 4, presented in Basic
Protocols 2, 3 and 4, detail a counterbalanced design for the ABT and RLA. Whenever
possible, the experimenter should be blind to treatment to avoid unintentional biases
influencing the animal’s behavior.

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13. Put a clean paper liner inside the arena and keep it for 5 days of training or testing,
only removing fecal pellets. Remove the liner only if it gets very dirty. It is best to
avoid sawdust or other substrates that can distract the animal from the bowls.
14. Use two identical ceramic bowls. Place both bowls filled with appropriate substrates
inside the arena and against the back wall, about 5 cm (1 rat width) apart (Fig. 3A).
15. Always choose a trio of digging substrates (reward-paired substrates - ‘A’ or ‘B’
versus unrewarded substrate - ‘C’) matched for digging effort and with rewarded
substrates counterbalanced across experimental subjects (see Table 1).

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Figure 3 Photographs illustrate in panel (A) the ABT arena with bowls and rat position; (B) a
gentle rat hold during the task; (C) rat transport box; (D) bowls with sawdust used during digging
training; (E) bowls with paper bedding and shredded dishcloth used during discrimination training;
(F) bowls with example three substrates: sparkly pompons, raffia ribbon, balloons; (G) bowls with
example three substrates: hessian sack, moss, matchsticks; (H) bowls with example three substrates: fur, polyester, bath sponge; and (I) bowls with example three substrates: absorbent fiber,
string, foam shapes.

Substrate ‘A’

Substrate ‘B’

Substrate ‘C’

Test 1

Felt

Shredded dishcloth blue

Exfoliating gloves

Test 2

Absorbent fiber

String

Foam shapes

Test 3

Dusters

Tissue paper balls

Yellow bath sponge

Test 4

Black satin

Cardboard

Rope

Test 5

Fur

Polyester

Pompoms

Test 6

Cellulose sponge

Corrugated paper

Perlite

Test 7

Purple ribbon

Green raffia ribbon

Sparkly pompoms

Test 8

Brown pet bedding

Cork

Hessian sack

Test 9

Cotton wool balls

Stringy cloth

Hairbands

Test 10

Organza

Silk

Shredded paper

Test 11

Bin liner

Plastic scourer

Straws

Test 12

Cotton mix

Leather

Balloons

Test 13

Chubby wool

Shoelaces

Velcro

Test 14

Brown partition paper

Dishcloth squares

Polyester lining

Test 15

Aspen

Cypress

Colored matchsticks

Test 16

Christmas ribbon

Umbrella

Tights

Test 17

Towel

Canvas

Pipe cleaners

Test 18

Newspaper

Paper pet bedding

Confetti

Test 19

Suede

Chenille strands

Yellow fleece

Test 20

Poster squares

Polystyrene

Sequins

Test 21

Crepe paper squares

Scarf yarn

Sparkling fiber

Test 22

Denim

Rucksack strap

Foam pad

Test 23

Maple tree leaves

Moss

Black lining

Test 24

Silver birch leaves

Silver birch bark

Hemp bedding

Test 25

Swimming suit

Straw hat

Suede fabric

The digging substrate provides a multidimensional cue, i.e., texture and olfactory cues,
which the animals learn to associate with a food reward. Before starting an experiment,
prepare the digging substrates (see Table 1), such as by cutting them to a size small
enough to provide good coverage of the food reward when placed in the ceramic bowls.
16. Use a lid to cover at least half of the arena (whole arena for 1st habituation) to prevent
the animals from jumping onto the rim.
17. For each trial, place the rat near the bowls in the middle of the arena to help reduce
any side bias (Fig. 3A).
18. Do not remove the non-selected bowl until the animal has shown a committed choice
(when the animal has obviously and actively started to dig or search within one of
the substrates), then remove the bowl in a single action. It is better to be too slow
than too fast.
19. Randomly place pellets in locations equivalent to clock positions 10, 12, and 2. Remember to bait the bowl, position it in the arena, and then press down the substrates
in both bowls from left to right to ensure no differences in handler-related olfactory
cues.
20. Between trials, either place the rat in a holding box (Fig. 3C) while you reset the
bowls or gently hold the animal on your chest (Fig. 3B).

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Table 1 Example of the Substrate Trios Routinely Used in Our Group

To induce a negative affective bias in Basic Protocol 4, the most frequently used manipulations are corticosterone (stress hormone, 10 mg/kg subcutaneous) or FG7142 (anxiogenic benzodiazepine partial inverse agonist, 3 mg/kg subcutaneous). For both treatments, a pre-treatment time of 30 min before the pairing session induces a reliable negative affective bias in the choice test (Hinchcliffe et al., 2024). Both compounds can be
purchased from commercial suppliers (we purchase them from Merck, previously SigmaAldrich, UK). We dissolve corticosterone first in 5% DMSO (VWR Chemicals, UK) and
then add 95% sesame oil (Merck, UK). FG7142 is suspended in sterile saline with one
drop per 1 ml of Tween 80 (Merck, UK). For both drugs, use a 1 ml/kg dose volume.
Make sure the drugs are well mixed and vortex before dosing, especially the FG7142
suspension. Always prepare the drugs fresh on the day of use and, if required, store them
at 4°C.
NOTE: All protocols involving animals must be reviewed and approved by the appropriate Animal Care and Use Committee and must follow regulations for the care and use of
laboratory animals.
BASIC
PROTOCOL 1

BOWL DIGGING AND DISCRIMINATION TRAINING
This protocol describes how to first train rats to retrieve rewards contained in ceramic
bowls and presented in a testing arena. Once animals have learned to reliably retrieve
buried rewards from a bowl containing substrate versus an empty bowl, a single discrimination learning session is used to train the animals to associate one digging substrate
with a reward and the other with no reward, and to select the correct substrate to the
required learning criteria. A final training stage for all cohorts involves the RLA protocol, which is used to check the cohort is performing the assay correctly and develops a
reward-induced bias (see Basic Protocol 2).

Materials
Rats (In our laboratory, we routinely use male Lister Hooded rats; RR
ID:RGD_2312466, Envigo, UK) that weigh approximately 275-300 g at the
beginning of the study; however, albino strains Sprague Dawley (RR
ID:RGD_734476, Charles River, UK) (Hinchcliffe et al., 2017, 2020) and
Wistar (RR ID:RGD_2312504, Charles River, UK) (Phelps, unpublished) and
females have also been used (Hinchcliffe et al., 2017, 2020)
Standard laboratory rat chow (e.g., TestDiet, UK, or equivalent)
Rodent reward pellets (e.g., 45 mg purified rodent tablets, containing sucrose,
casein, maltodextrin, corn starch, corn oil, minerals, silicon dioxide, vitamins,
magnesium stearate, DL-methionine, Test Diet, Sandown Scientific, UK, or
equivalent)
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Nontoxic detergent for cleaning (e.g., non-fragranced Anistel, UK, or equivalent)
Water bottles for drinking water (500 ml, from e.g., Techniplast, UK, or equivalent)

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Pharmacological Studies
Carefully consider the dose range when using pharmacological treatments in the ABT
or RLA, as higher doses can lead to non-specific effects. Ideally, pharmacokinetic data
should be used to inform dose selection based on predicted receptor occupancy, but where
this is not available, estimations based on clinical data and scaling for animal weight can
be useful (Nair & Jacob, 2016). It should be noted that previous studies with antidepressant drugs suggest effective doses are lower than those used in conventional models of
depression and align more closely with clinical doses (Stuart et al., 2013). Higher doses
of antidepressants tend to induce non-specific effects in the task (Hinchcliffe et al., 2024).
Compounds that bind irreversibly with the target receptor are not suitable for the induction of affective biases in the ABT due to the within-subject design, although they can
be tested in the retrieval protocol of the ABT or RLA using a between-subject design.

Housing
1. After the animals arrive at the animal facility, allow them to habituate to their new
environment, diet, and light-dark cycle for at least a week before beginning any
procedures or experimental manipulations.
2. During the acclimatization period, provide water and standard laboratory chow ad
libitum for all animals.
3. House animals in pairs in standard enriched laboratory cages under a 12:12 hr reverse
light-dark cycle (e.g., lights off at 08:00 hr).

Husbandry, handling, and feeding
4. Prior to any behavioral experiments with laboratory animals, experimenters need
to undertake the appropriate training, including theoretical background, handling,
daily care, and obtain any necessary approvals to work with animals for scientific
purposes.
5. Following a week of acclimatization, weigh and tail mark each animal. Gently transfer each rat from its home cage to a scale, weigh it individually, and then record its
body weight in a weight record book. Before returning it to the home cage, mark
the rat’s tail with a unique and easy-to-identify number, code, or letter using a nontoxic, permanent marker. Continue to weigh the rats at least weekly to monitor their
weight gain and mark their tails on the same day. As animals reach maturity, it can
be useful to use body condition scores to help maintain a healthy weight.
6. Three days before the start of training, restrict food for all rats to initiate a regime to
maintain body weight at approximately 90% of their free-feeding weights matched
to the normal growth curve (∼18 g food per rat/day laboratory chow) and provide
water ad libitum. Once they reach maturity, use body condition scores and maintain
the animals in good condition (4/5) but not obese. Greater levels of food restriction
can overly motivate the animals for food and impact the reliability of the behavior.
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Type opaque NK cages (55 × 35 × 21 cm, from e.g., Techniplast, UK, or
equivalent), with metal mesh lids and food hoppers, lined with bedding (such as
aspen woodchip) and a handful of nesting material such as paper wool
Environmental enrichment cages (In our laboratory, we house rats with paper
bedding, cotton rope, wood chew, aspen balls, cardboard tube, and a red
Perspex® house (30 × 17 × 10 cm))
Scale for weighing the rats (e.g., Weighwell, UK, or equivalent)
Nontoxic, permanent marker pen for tail marking (e.g., Pentel N850)
Perspex® testing arena with removable lid (40 × 40 × 25 cm)
Three ceramic identical bowls (10 cm, e.g., pet drinking bowls)
Absorbent paper-based liner cut to the size of the arena (40 × 40 cm)
Small plastic container for reward pellets
Appropriate personal protective equipment (face mask, hair net, gloves,
scrubs/animal facility gown, clogs/overshoes, etc.)
Laboratory notebook (for manual recording of the experimental details) and folder
for storage of printed pairing sessions and choice test sheets
Personal computer to use Microsoft Excel package (to prepare experimental design
plans, dosing sheets, sheets for data acquisition, and processing) and GraphPad
Prism (data processing, visualization).
Data handling software (e.g., Microsoft Excel, RR ID:SCR_016137, GraphPad
Prism; RR ID:SCR_002798)
Statistical analysis software (e.g., GraphPad Prism; RR ID:SCR_002798 or SPSS;
RR ID:SCR_002865)

7. At the end of the acclimatization period, habituate rats to reward pellets in a ceramic bowl. For 3 days, place a small number of pellets and one bowl in their home
cage and observe to make sure all rats have eaten the pellets. Ensure all rats are
habituated to handling with positive reinforcement and familiarized with the travel
box/holding cage (Fig. 3C) before they start training. Handling-induced stress will
confound results and limit the reliability of both the ABT and RLA.

Training
The first 2 days comprise habituation to the testing arena. The animals are then introduced to digging training for at least the next 5 days with increasing levels of difficulty.
On each day, bring rats (with cage mate(s)) into in the behavioral room for 5-10 min,
either in a transport box or cage or in their home cage. Return animals to the holding
room once all the subjects for that cage have completed the session for that day.
8. Habituation 1 (usually on Thursday of week 1)
a. Place two rats from the same cage in the arena together with both bowls (no
substrate) containing pellets and allow them to explore.
b. Observe animals and record if they both eat pellets and score the number of fecal
pellets.
c. Leave both animals in the arena for 10 min.
9. Habituation 2 (usually on Friday of week 1)
a. Place one rat in the arena with both bowls (no substrate) containing a few pellets
and allow to explore.
b. Observe the animal: record if it eats pellets and score the number of fecal pellets.
c. Rebait bowls each time pellets are eaten for up to 10 min.
10. Shuttle training (usually on Monday of week 2)
a. Use two bowls with no digging substrate.
b. Place one pellet in one of the three reward locations in one bowl (10, 12, and 2
on a clock).
c. Place one rat in the arena with both bowls (no substrate) and allow to explore.
d. Once the rat has consumed the pellet in the bowl, place a pellet into the left-hand
corner of the arena and wait for the animal to find it, then pick up the animal and
remove it from the arena to a holding box (or gently hold) between trials.
e. Re-bait the bowl with one pellet in one of the three locations and repeat the trial.
Bait either the left or the right bowl in a pseudo-random spatial order to avoid
animals developing a side bias and to train animals that either bowl could contain
a reward.
f. Repeat for 12 trials. Omissions do not count toward the total number of trials.
g. If animals fail to collect pellets (explore bowls) after 30 s, remove them from the
arena and place them in a holding box for 5 s before restarting.
h. Record trial outcomes (‘correct’ or ‘omission’) and latencies.
i. At the end of the day, feed ∼2/3 of the normal food ration.
j. Replace tray liner and wash bowls with water. Let it dry to be ready for the next
day.

Hinchcliffe and
Robinson

Criteria for progression: The rat performs at least 10 trials consuming the pellets from
the bowl and the left corner of the arena. Any animal that fails to achieve the criteria
should be re-run later in the day or the next day. Do not progress animals to the next
stage until criteria have been met.

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Healthy animal growth curves can be found in the laboratory rodents’ supplier materials
(e.g., Envigo Research Models and Services Catalogue).

Trial 1
d. Use two bowls with no digging substrate.
e. Place one pellet in one of the three locations in one bowl.
f. Place an individual rat in the arena and wait until the pellet is found and consumed.
Do not remove the other bowl from the arena.
g. Once the rat has consumed the pellet in the bowl, place a pellet in the left-hand
corner of the arena and wait for the animal to find it, then pick up the animal and
remove it from the arena to a holding box (or gently hold) between trials.
Trial 2-3
h. Place the pellet in the same location as trial 1, but cover it with 1 cm sawdust (Fig.
3D).
i. Leave the other bowl empty with no sawdust.
j. Place an individual rat in the arena and wait until the pellet is found and consumed.
k. If the rat does not start digging in the bowl after ∼30 s, remove the animal and place
it in the holding box (or gently hold it) for 5 s and mark the trial as an ‘omission’.
The cut-off time may need to be increased if rats are still exhibiting any fear-related
behaviors. For animals that fail to dig, repeat trials 2-3 until they dig within 30 s.
Placing the pellet on top of the sawdust for one trial before burying may re-engage
animals not performing the task.
l. Once the rat has consumed the pellet in the bowl, place a pellet into the left-hand
corner of the arena and wait for the animal to find it, then pick up the animal and
remove it from the arena to a holding box (or gently hold) between trials.
Trial 4-xx
m. Continue with 1 cm sawdust in one bowl and place one pellet in one of the three
locations. Randomize the position of the baited bowl with sawdust between the left
and right positions.
n. Continue to leave the other bowl empty.
o. Place an individual rat in the arena until the pellet is found and consumed.
p. Once the rat has consumed the pellet in the bowl, place a pellet in the left-hand
corner of the arena and wait for the animal to find it, then pick up the animal and
remove it from the arena to a holding box (or gently hold) between trials.
q. If the rat does not start digging in the bowl for 15 s, remove the animal to a holding
box (or gently hold) for 5 s and mark trial as an ‘omission’.
r. If you get two consecutive omissions, consider increasing the latency cut-off for a
couple of trials e.g., 30 s.
s. Repeat until 12 trials are completed (‘omissions’ do not count as completed trials).
t. Record trial outcomes (‘correct’, ‘incorrect’, or ‘omission’) and latency to dig (time
from placing in the arena to time to dig).
If the rat has 5 consecutive ‘omissions’, repeat trial 1 and trial 2.
Criteria for progression: The rat performs at least 12 trials. Any animal that fails to achieve
the criteria should be re-run later in the day or the next day. Do not progress animals to
the next stage until criteria have been met.

12. Digging training 2 (usually on Wednesday of week 2)

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11. Digging training 1 (usually on Tuesday of week 2)
a. Bring the assigned rats (with cage mates) from their holding room. While you
are setting up your bowls with substrates and the pairing paper sheets, the rats
can habituate to the testing room.
b. Continue to use the pellet in the left-hand corner to train recall (animal return)
and remove the animal from the arena between trials.
c. Record trial outcomes (‘correct’, ‘incorrect’, or ‘omission’) and latencies.

Trial 1
d. Bait one bowl with one pellet and no sawdust. Leave the other bowl empty.
e. Let the rat explore both bowls until the pellet is collected.
f. Once the rat has consumed the pellet in the bowl, place a pellet in the left-hand
corner of the arena and wait for the animal to find it, then pick up the animal and
remove it from the arena to a holding box (or gently hold) between trials.
Trial 2
g. Place the pellet in the same location and place 1 cm sawdust in that bowl. Leave the
other bowl empty.
h. Remove the empty bowl once the rat has committed to digging in the sawdust bowl.
It is very important that bowl removal occurs after the animal has started digging.
For the first couple of trials, bowl removal may distract the rat, so leave them for
long enough to return to the baited bowl and find the reward.
i. Once the rat has consumed the pellet in the bowl, place a pellet in the left-hand
corner of the arena and wait for the animal to find it, then pick up the animal and
remove it from the arena to a holding box (or gently hold) between trials.
j. If the rat does not start digging within ∼30 s, remove the animal to a holding box
(or gently hold) for 5 s and mark trial as an ‘omission’.
Trial 3
k. Place pellet in the same location and place 2 cm sawdust in that bowl. Leave the
other bowl empty.
l. Remove the empty bowl once the rat has committed to digging in the sawdust bowl
and eaten the pellet.
m. Once the rat has consumed the pellet in the bowl, place a pellet in the left-hand
corner of the arena and wait for the animal to find it, then pick up the animal and
remove it from the arena to a holding box (or gently hold) between trials.
n. If the rat does not start digging within ∼10 s, remove the animal to a holding box
(or gently hold) for 5 s and mark trial as an ‘omission’.
Trial 4-xx
o. Continue with 2 cm sawdust in one bowl and place one pellet in one of the three
locations.
p. Randomize the position of the baited bowl with sawdust between the left and right
positions.
q. Continue to leave the other bowl empty.
r. Wait for the rat to find the reward. If the rat does not start digging ∼10 s, remove
the animal to the holding box (or gently hold) for 5 s, then repeat the trial and mark
the first attempt as an ‘omission’.
s. Once the rat has consumed pellet in the bowl, place a pellet in the left-hand corner
of the arena and wait for the animal to find it, then pick up the animal and remove
it from the arena to a holding box (or gently hold) between trials.
t. Repeat until 12 trials are completed; ‘omissions’ do not count as completed trials.
If the rat has 3 consecutive omissions, the cut-off time may need to be adjusted but should
be adapted for each rat.
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a. Bring the assigned rats (with cage mates) from their holding room. While you are
setting up your bowls with substrates, the rats can habituate to the testing room.
b. Continue to use the pellet in the left-hand corner to train recall and remove the
animals from the arena between trials.
c. Record trial outcomes (‘correct’, ‘incorrect’, or ‘omission’) and latencies to dig.

13. Digging training 3 (usually on Thursday of week 2)
a. Repeat Digging training 2 , but stop using the pellet in the left-hand corner (rats
should be trained to return to hand by now).
b. Continue to remove the animal from the arena between trials.
c. If the rat does not start digging within ∼5 s, remove the animal to the holding box
or gently hold for 5 s, then replace it in the arena (mark trial as an ‘omission’).
d. If the rat has 3 consecutive omissions, the cut-off time may need to be adjusted
but should be adapted for each rat.
e. Record trial outcomes (‘correct’, ‘incorrect’, or ‘omission’) and latencies.
The cut-off time is used to encourage the animals to approach the bowls quickly and dig. It
is gradually reduced through the training sessions but may need to be adjusted for each
animal based on their confidence in the task and performance. If animals have a slow
average latency (on the border of the cut-off time) from the previous session, it may be
necessary to use a longer cut-off time than described in this protocol. If >5 consecutive
omissions occur, the animal can lose interest and stop performing the trials.
Before progressing further with training, it is important that the rat has learned the trial
sequence (approach bowl – choose substrate – dig for reward – return to the handler) and
is reliably approaching the bowls, digging for reward, eating the reward, and returning
to the handler. They may have the odd trial when they are distracted, but any animal that
is not reliably performing the trial sequence will need further digging training before
they progress to the discrimination stage. If animals are not returning to the handler,
use additional training sessions with a reward placed in the left corner after the bowl is
removed. Although it can be frustrating waiting for animals to leave the bowls and return
to the handler, patience at this stage will benefit running the task in the longer term.
In our lab, more than 95% of rats will learn the task with one day of training at each stage
and meet criteria after a single session. If rats are not meeting criteria the most likely
cause is the handler-animal interaction and relates more to the pre-training habituation
than training.

14. Discrimination (usually on Friday of week 2)
The discrimination session is used to train animals to associate the reward location
with one of two different digging substrates not previously encountered. This stage
also enables the experimenter to establish whether the rats have met the required
learning criteria to progress.
a. Bring the assigned rats (with cage mates) from their holding room. While you are
setting up your bowls with substrates and the pairing paper sheets, the rats can
habituate to the testing room.
b. Fill one bowl with 2 cm of substrate ‘A’, e.g., chopped tissue bedding, and the
other bowl with 2 cm of substrate ‘B’, e.g., shredded dishcloth (Fig. 3E).
c. Mix finely crushed reward pellets into both substrates to reduce animals’ ability
to use olfactory cues to locate the reward.
d. Bait substrate ‘A’ bedding (odd-numbered animals) or substrate B (evennumbered animals) with one pellet as ‘rewarded’ substrate.
e. Use the same bowls and substrates for all rats that day.
f. Trials are run using the same method established during digging training, and
animals should approach and dig in the bowl within ∼ 5 s and return to the handler
after consuming the reward or failing to find a reward. Animals that fail to show
robust trial behavior may still be experiencing a degree of stress and may benefit
from additional handling habituation and digging training. Remove animals from
the arena between trials.

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Criteria for progression: The rat performs at least 12 trials. Any animal that fails to achieve
the criteria should be re-run later in the day or the next day. Do not progress animals to the
next stage until criteria have been met.

g. Record trial outcomes (‘correct’, ‘incorrect’, or ‘omission’) and latencies to dig.
Choice of the reward-paired substrate is marked as a ‘correct’ trial, digging in the
unrewarded substrate is classified as an ‘incorrect’ trial, and if an animal fails to
approach and explore the bowls within 10 s, the trial is recorded as an ‘omission’.

Trial 1
h. Allow the animal to explore both bowls, find the pellet, and leave the bowl. Do not
restrict to 30 s if the animal is still exploring. If the animal is not engaging with the
task for more than 30 s, remove for 5 s, then repeat.
Trial 2-xx
i. Place rat in the arena (Fig. 3A), wait for the animal to ‘choose’ a substrate, then
remove the other bowl (Movie 1).
When has the animal made a choice? When the animal has obviously started to dig or
search within one of the substrates. The animal should be allowed to investigate both bowls
and should not be rushed. If the animal does not make an obvious choice after ∼10 s,
remove the rat from the arena to a holding box or hold for 5 s, then re-run the trial.
When you place a rat to approach a bowl and make a choice, do not give away any cues
(e.g., lifting your arm to remove the other bowl) to the animal until it has made a choice.

j. Use ∼5 s cut-off time for removing the animal and marking it as ‘omission’ but be
prepared to adapt to each subject.
k. If a rat has 3 consecutive omissions, use a longer cut-off for the next trial before
going back to the 5 s cut-off time.
l. Omissions do not end a series of correct trials (if a rat has 4 correct trials followed
by an omission, they still only need 2 more correct trials to meet the criterion)
m. Run until the animal achieves 6 consecutive correct trials (maximum 20 trials).
Criteria for progression: If an animal achieves 6 consecutive correct trials in less than 20
trials in total, it is considered trained and correctly performing the task. The probability of
achieving this criterion by chance is 0.0156. Once animals successfully reach the criteria
in the discrimination session, they are considered trained and can progress to testing in the
RLA.
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Video 1 Example of one trial performed by a rat during pairing sessions. Video taken under white
light for illustrative purposes only.

The RLA protocol is carried out over 5 days (usually week 3 for an experienced experimenter) and is used to check that the cohort is correctly performing the task and, at the
population level, a reward-induced positive bias should be observed before progressing
the cohort to testing. Using a within-subject design, animals learn to associate one of
the two reward-paired digging substrates with either a high (2 pellet) or low (1 pellet)
value reward. Three objectives can be achieved using the RLA: a) to determine whether
the animals correctly perform the task post-discrimination training, b) to investigate the
animals’ core affective state, and c) to test for any acute, non-specific effects on memory
following an acute drug treatment prior to the choice test (Fig. 4). The results for the cohort are analyzed statistically to verify that a significant positive bias is observed. At the
end of this protocol, the animals are trained and ready to undergo testing following acute
pharmacological or non-pharmacological manipulations in the ABT (Basic Protocols 3
and 4).

BASIC
PROTOCOL 2

See Basic Protocol 1 for details on materials, housing, husbandry, handling, feeding, and
training.

Pairing sessions
1. Using Table 3, run substrate-reward pairing sessions using a fully counterbalanced
design that follows that of the basic discrimination training stage but does not include
an exploratory first trial.
2. Fill bowl 1 with substrate ‘A’, bowl 2 with substrate ‘B’, and bowl 3 with substrate
‘C’.
3. Use the same substrates and bowls for the whole week but remove contaminants.
4. Mix finely crushed reward pellet into substrate ‘C’ to prevent animals from sniffing
out the reward pellet.
5. Record trial outcomes (‘correct’, ‘incorrect’, or ‘omission’) and latencies to dig.
Choice of the reward-paired substrate is marked as a ‘correct’ trial, digging in the

Figure 4 Overview of the reward learning assay protocol to test if animals correctly perform the
task post-discrimination training (design A), to investigate the core affective state of animals (design
B), and to test for any acute, non-specific effects on memory following an acute drug treatment prior
to the choice test (design C). In this study design, animals are in the same affective state throughout
pairing sessions but learn to associate one of the substrate-reward cues with a higher-value reward
(2 reward pellets) or a low-value reward (1 reward pellet). The reward-induced bias is quantified
using a choice test (design A and B) or with the drug or vehicle administered 1 hr before testing
(for example) to investigate the acute effects on retrieval (design C). Figure taken and adapted from
Hinchcliffe et al. (2024).

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THE REWARD LEARNING ASSAY

Day 1

Day 2

Day 3

Day 4

Day 5

Pairing 1

Pairing 2

Pairing 3

Pairing 4

Choice test

Group 1

A vs. CDrug

B vs. CVehicle

A vs. CDrug

B vs. CVehicle

A vs. B,30 trials

Group 2

B vs. CDrug

A vs. CVehicle

B vs. CDrug

A vs. CVehicle

A vs. B,30 trials

Group 3

A vs. CVehicle

B vs. CDrug

A vs. CVehicle

B vs. CDrug

A vs. B,30 trials

Group 4

B vs. CVehicle

A vs. CDrug

B vs. CVehicle

A vs. CDrug

A vs. B,30 trials

a

Each animal receives manipulation/drug treatment or control/vehicle counterbalanced over the four substrate-reward pairing sessions. Substrate
(reward-paired substrates—“A” or “B” versus unrewarded substrate—“C”) and day are also counterbalanced, resulting in four different groups.

Table 3 Standard Experimental Design Testing in the Reward Learning Assaya

Day 1

Day 2

Day 3

Day 4

Day 5

Pairing 1

Pairing 2

Pairing 3

Pairing 4

Choice Test

Group 1

A vs. C2 pellets

B vs. C1 pellet

A vs. C2 pellets

B vs. C1 pellet

A vs. B,30 trials

Group 2

B vs. C2 pellets

A vs. C1 pellet

B vs. C2 pellets

A vs. C1 pellet

A vs. B,30 trials

Group 3

A vs. C1 pellet

B vs. C2 pellets

A vs. C1 pellet

B vs. C2 pellets

A vs. B,30 trials

Group 4

B vs. C1 pellet

A vs. C2 pellets

B vs. C1 pellet

A vs. C2 pellets

A vs. B,30 trials

a

Each animal receives 2 pellets or 1 pellet counterbalanced over the four substrate-reward pairing sessions. Substrate (reward-paired substrates—“A”
or “B” versus unrewarded substrate—“C”) and day are also counterbalanced, resulting in four different groups.

unrewarded substrate is classified as an ‘incorrect’ trial, and if an animal fails to
approach and explore the bowls within 5 s, the trial is recorded as an ‘omission’.
6. Bring the assigned rats (with cage mates) from their holding room. While you set up
your bowls with substrates and the pairing session paper sheets, the rats can habituate
to the testing room.
7. Place an individual rat in the arena and run the pairing session.
8. Make sure the two pellets used for the high reward value substrate are placed close
together and check animals are finding and consuming both pellets. Some rats may
leave briefly and then return to find the second pellet before returning to the handler.
9. Remove the animal from the arena between trials.
10. If a rat has 3 consecutive omissions (trial >5 s), use a longer cut-off for the next trial
before going back to the 5 s cut-off time.
11. Omissions do not end a series of correct trials (if a rat has 4 correct trials followed
by an omission, it still only needs 2 more correct trials to meet the criterion).
12. Run until the animal achieves 6 consecutive correct trials (maximum 20 trials).
13. Return all animals to their holding room immediately after the pairing session.
Animals should complete two pairing sessions for each substrate over days 1-4, with one
substrate used on days 1 and 3 and the other on days 2 and 4 counterbalanced across the
cohort. Failing to meet the learning criterion for at least one session for both rewarded
substrates is rare and can be used as an exclusion criterion.
Animals should learn the reward-paired substrate in 10-15 trials, showing improvement
over the 4 days of the experiment and from pairing sessions 1-3 and 2-4. Even if an
individual animal does not meet the learning criterion within 20 trials, continue with the
protocol.
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Table 2 Standard Experimental Design for Testing Manipulation-Induced Affective Bias Versus Control Manipulationa

14. Run individual animals using the same procedure as the pairing sessions but bait
bowls using random reinforcement as per schedule. Template suitable for recording
data
15. Mix finely crushed reward pellets into both substrates ‘A’ and ‘B’ to reduce the
likelihood of the animal using odor to find the reward.
16. Use only 1 pellet on each rewarded trial.
17. Make sure animals are given enough time to make a choice (let them briefly explore
both bowls but avoid letting them sniff out the pellet), but aim to keep to <5 s as
much as possible, taking each rat’s behavior into consideration.
18. Place a rat in the arena and wait for the animal to choose a substrate, then remove
the other bowl.
19. When you place a rat to approach a bowl and make a choice, do not give any cues
(e.g., lifting your arm to remove the other bowl) to the animal until it makes a clear
choice.
20. Record the animals’ choices and latency to dig. There are no ‘correct’ or ‘incorrect’
trials, only choices for either substrate ‘A’ or ‘B’. If an animal fails to make a choice
within 10 s, record trial as an ‘omission’. If ‘omissions’ occur, mark them down, but
do not count into 30 trials that animals need to complete.
21. Return all animals to the holding room.
22. For testing animals’ core affective state, a between-subject study design is used with
animals from each treatment group being trained and tested in the RLA in a randomized study design. For studies involving the induction of a depression-like phenotype
using a chronic manipulation such as repeated stress or a pro-depressant drug, it is
better to pre-train the animals and undertake an RLA before allocating them to treatment groups. At the end of the planned treatment period, a repeat of the RLA can
be run using new substrates. Normally, treatment continues throughout the second
RLA, but this will depend on the specific experimental objectives (Fig. 4B).
23. For testing the acute, non-specific effects of a drug, dose animals with the drug or
vehicle and required pre-treatment time prior to the choice test (Fig. 4C).

THE AFFECTIVE BIAS TEST - NEW LEARNING
The ABT protocol is used to investigate the neuropsychology of affective state-induced
biases associated with specific cue-reward memories. This protocol can be used in different ways to investigate different aspects of affective bias modification. To investigate
affective biases induced at the time of learning, the affective state manipulation or drug
treatment is combined with the learning experience and the arising choice bias generated
is quantified at least 24 hr after the last treatment.

BASIC
PROTOCOL 3

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Choice test
Reward-induced biases generated in this protocol are quantified during a choice test performed at least 24 hr after the last pairing session. During the choice test, the two previously rewarded substrates (‘A’ and ‘B’) are presented at the same time over 30 trials (Fig.
4). In order to keep rats motivated to dig, but without providing new associative information, a single 45 mg food pellet is placed in either substrate using a random schedule
with a probability of one in three, so that rats randomly receive a reward (i.e., substrate
‘A’ contained a pellet on 10 of the 30 trials, and likewise for substrate ‘B’; and no trials
were both bowls baited).

For details on materials, housing, husbandry, handling, feeding, and training, see Basic
Protocol 1.

Pairing sessions
Each week is comprised of four pairing sessions (one per day, usually Monday to Thursday) to generate two independent cue-specific memories (Fig. 5A) and a choice test performed not less than 24 hr after the last pairing session. Using a within-subject design,
each animal acquires two substrate-reward association memories under different treatment or control conditions. The value of the reward is kept the same for both experiences,
and the experiment design counterbalances all other factors, so any biases observed during the subsequent choice test can be attributed to the treatment at the time of learning.
During the pairing sessions, each trial involves presenting the rat with a choice between
two bowls containing two different digging substrates (make sure they are matched for
a digging effort), one of which is reward-paired (substrate ‘A’ or ‘B’, counterbalanced
across subjects and manipulations) and containing a single 45-mg reward pellet, and the
other of which is unrewarded (substrate ‘C’). Substrate ‘C’ is kept the same for all four
pairing sessions. Either substrate ‘A’ or ‘B’ is presented during pairing sessions on days
1 and 3, and the other is presented on days 2 and 4, with the order counterbalanced across
subjects (see Table 2).

Hinchcliffe and
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New trios of substrates are used for each week of the study, and animals can complete
multiple studies as long as new substrates are used. Where a study involves comparing
multiple treatments or doses of a drug, the same trio of substrates is used for all animals for that week, but the treatments are counterbalanced over the course of the study.
Templates suitable for recording data from pairing sessions are provided in Table S1 and
Table S2.

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Figure 5 Overview of the affective bias test protocol to investigate the effects of positive or negative manipulations on new learning (design A) and acute or sustained effects of treatment on a
negative affective bias (design B). In the first study design, animals undergo four pairing sessions
under two conditions: psychosocial manipulation or pharmacological treatment versus control manipulation or vehicle treatment. On day 5, the affective biases are quantified over 30 trials in the
choice test (design A). In the study design illustrated in design (B) animals are treated with either
FG7142 (FG, 3 mg/kg) or corticosterone (CORT, 10 mg/kg) to induce a negatively biased memory.
Affective biases are quantified using a choice test with the drug or vehicle administered, e.g., 1
or 24 hr before testing, to investigate the acute or sustained effects on retrieval. Figure taken and
adapted from Hinchcliffe et al. (2024).

2. Dose animals with the drug or vehicle at the required pre-treatment time prior to
pairing sessions or subject animals to non-pharmacological manipulation prior to
and/or after pairing sessions.
3. Fill bowl 1 with substrate ‘A’, bowl 2 with substrate ‘B’, and bowl 3 with substrate
‘C’.
4. Use the same substrates and bowls for the whole week but remove contaminants.
5. Mix finely crushed reward pellet into substrate ‘C’ to prevent animals from sniffing
out the reward pellet.
6. Record trial outcomes (‘correct’, ‘incorrect’, or ‘omission’) and latencies to dig.
Choice of the reward-paired substrate is marked as a ‘correct’ trial, digging in the
unrewarded substrate is classified as an ‘incorrect’ trial, and if an animal fails to
approach and explore the bowls within 5 s, the trial is recorded as an ‘omission’.
7. Bring the assigned rats (with cage mates) from their holding room. While you are
setting up your bowls with substrates and the pairing sessions paper sheets, the rats
can habituate to the testing room.
8. Place the rat in the arena (Fig. 3A), wait for the animal to choose a substrate, and
then remove the other bowl.
9. When you place a rat to approach a bowl and make a choice, do not move or give
any cues (e.g., lifting your arm to remove the other bowl) to the animal until it makes
a clear choice.
10. Remove the animal from the arena when it returns to the handler, as in training and
between trials.
11. If a rat has 3 consecutive omissions, increase the cut-off time for the next trial before
returning to the 5 s cut-off time.
12. Omissions do not end a series of correct trials (if a rat has 4 correct trials followed
by an omission, they still only need 2 more correct trials to meet the criterion)
13. Run independent trials with the baited bowl placed in either the left or right location
to maintain a pseudo-random spatial order until the animal achieves 6 consecutive
correct trials (maximum 20 trials if the animal does not achieve the criterion within
a total of 20 trials).
14. Return all animals to their holding room immediately after the pairing session.
15. Between animals, clear the arena of any fecal pellets or bits of remaining substrate.
16. Animals should complete 2 pairing sessions for each substrate over days 1-4 with
one substrate used on days 1 and 3 and the other on days 2 and 4 and counterbalanced
across the cohort. Failing to meet learning criteria for at least one session for both
rewarded substrates is rare and used as an exclusion criterion.
Animals should learn the reward-paired substrate in around 10-15 trials showing improvement over the four days of the experiment and from pairing session 1 to pairing
session 2.

Choice test
Affective biases generated by this protocol are quantified during the choice test on day 5
when the two previously rewarded substrates (‘A’ and ‘B’) are presented at the same time

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1. Following the trial details in Table 2, perform substrate-reward pairing sessions on
days 1-4.

17. Run individual animals using the same procedure as per pairing sessions but bait
bowls using random reinforcement as per schedule.
18. Mix a finely crushed reward pellet into both substrates ‘A’ and ‘B’.
19. Use just 1 pellet on each rewarded trial.
20. Make sure animals are given enough time to make a choice but remain engaged with
the task.
21. Place a rat in the arena and wait for the animal to choose a substrate, then remove
the other bowl.
22. When you place a rat to approach a bowl and make a choice, do not give any cues
(e.g., lifting your arm to remove the other bowl) to the animal until it makes a clear
choice.
23. Record the animals’ choices and latency to dig. There are no ‘correct’ or ‘incorrect’
trials, only choices for either substrate ‘A’ or ‘B’. If an animal fails to make a choice
within 10 s, record trial as an ‘omission’. If ‘omissions’ occur, mark them down but
do not count into 30 trials that animals need to complete.
24. Return all animals to their holding room.
BASIC
PROTOCOL 4

THE AFFECTIVE BIAS TEST - MODULATION OF AFFECTIVE BIASES
ASSOCIATED WITH PAST EXPERIENCES
To investigate modulation of biased memories, an affective-state-induced bias is first
generated and then the treatment is given either shortly before (acute) or 24 hr (sustained)
before the choice test. The same protocol as New Learning (Basic Protocol 3) is used to
generate a biased memory using a treatment known to induce the desired affective bias
e.g., the benzodiazepine inverse agonist FG7142 at 3mg/kg subcutaneously induces a
reliable negative affective bias. The protocol is run over four consecutive pairing sessions
(one per day, usually Monday to Thursday) with a choice test on day 5 (acute modulation)
or day 6 (sustained modulation). Using a within-subject design, each animal is tested with
or without drug pre-treatment counterbalanced over the weeks of the study. For studies
involving the 24 hr (or later) timepoint, the treatment is given in the home cage. In a recent
study, we also combined the drug treatment with a cue reactivation protocol to further
investigate the interactions between the treatment and memory reactivation (Hinchcliffe
et al., 2024).
For details on materials, housing, husbandry, handling and feeding, training, and Reward
Learning Protocol see Basic Protocol 1.

Pairing sessions
1. Pairing sessions are run using the same protocol as in New Learning protocol detailed above (see Basic Protocol 3, section Pairing sessions) but with an additional
intervention step post-learning but pre-choice test (Table 4, Fig. 5B).

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Choice test
2. To test acute modulation, on day 5, animals were dosed with the drug or vehicle, and
pre-treatment time prior to the choice test was also required on day 5 (Fig. 5B).

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for 30 trials. In order to sustain the rat’s engagement during the choice test, place a single
45 mg food pellet is placed in either bowl using a random schedule with a probability of
one in three, so that rats randomly receive a reward (i.e., substrate ‘A‘ contained a pellet
on 10 of the 30 trials, and likewise for substrate ‘B’; and no trials where both bowls are
baited). An example of a choice test data sheet is given in Table S3.

Day 1

Day 2

Day 3

Day 4

Day 5 or 6

Day 5 or 6

Group

Pairing 1

Pairing 2

Pairing 3

Pairing 4

Treatment group

Choice test

1

A vs. CFG7142

B vs. CVehicle

A vs. CFG7142

B vs. CVehicle

Drug

A vs. B,30 trials

2

B vs. CFG7142

A vs. CVehicle

B vs. CFG7142

A vs. CVehicle

Vehicle

A vs. B,30 trials

3

A vs. CVehicle

B vs. CFG7142

A vs. CVehicle

B vs. CFG7142

Drug

A vs. B,30 trials

4

B vs. CVehicle

A vs. CFG7142

B vs. CVehicle

A vs. CFG71422

Vehicle

A vs. B,30 trials

a

Each animal receives drug (FG7142/corticosterone) or vehicle counterbalanced over the four substrate-reward pairing sessions. Substrate (rewardpaired substrates - “A” or “B” versus unrewarded substrate - “C”) and day are also counterbalanced resulting in four different groups. On day 5, each
animal receives treatment prior to the choice test (on day 5) to investigate the acute modulation of the negative biases, while to test sustained modulation,
each animal receives treatment on day 5 and is tested on day 6 in the choice test.

3. To test sustained modulation, on day 5, dose animals with the drug or vehicle ∼24 hr
prior to the choice test, which should be conducted on day 6 (Fig. 5B).
4. The choice test is performed as described for the new learning protocol (see Basic
Protocol 3, section Choice test).

COMMENTARY
Background Information
It is widely acknowledged that conventional methods for quantifying depressionrelated behavior and predicting antidepressant
efficacy are limited (Gururajan et al., 2019;
Planchez et al., 2019). The approach we and
others have taken involved looking to neuropsychological impairments observed in patients that are quantified using objective measures which could be translated to non-human
species. Development of the rodent tests involves a shift from using emotional stimuli,
such as faces, words, or sentences, to cues that
are suitable for non-human animals. Building from the observation that cognitive processes such as learning and memory, decisionmaking, and attention are negatively biased in
MDD (Mendl et al., 2009; Paul et al., 2005),
two key areas have emerged: decision-making
biases using a judgement bias task (Harding
et al., 2004) and reward learning and memory
biases using an affective bias test (Stuart et al.,
2013).
The first cognitive bias task for rodents was
reported by Mendl’s group in 2004 when they
developed an operant version of a go/no-go
judgment bias task for rats (Harding et al.,
2004). Rats were trained to respond to a specific auditory cue to obtain a reward and refrain from responding to avoid punishment
(white noise). Rats subjected to a chronic mild
stress regime were less likely to anticipate a
positive/rewarding outcome in response to intermediate ambiguous cues suggesting a negative bias (Harding et al., 2004). Studies in

phenotypic models suggest a good correlation between predicted affective state changes
and optimistic or pessimistic choices in the
judgment bias task (Enkel et al., 2010; Harding et al., 2004; Papciak et al., 2013). However, the pharmacological findings published
so far are less consistent with the human emotional interpretation task, particularly in terms
of the time course of antidepressant effects
(Joormann & Gotlib, 2006; Harmer, Goodwin et al., 2009; Harmer, O’Sullivan et al.,
2009; Lagisz et al., 2020; Neville et al., 2020).
The disparity might be linked to the difference in the nature of the tasks; human studies rely on emotional stimuli that provoke innate thoughts and responses, while animal task
uses long training procedures to teach animals
the reference cue-affective associations. Although there are inconsistent pharmacological
findings from judgement bias tasks, they have
shown some promise as a tool to investigate
the decision-making process under ambiguity
associated with changes in core affective state
(Lagisz et al., 2020), particularly for animal
welfare research.
The ABT and RLA were developed to
quantify affective-state or reward-induced
biases associated with learning and memory.
The ABT is suitable for quantifying shortterm changes in affective state associated
with a specific reward memory (substrate cuereward association), while the RLA has been
shown to be sensitive to changes in the core
affective state. The ABT is normally run in
healthy subjects but has been combined with a

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Table 4 Standard Experimental Design for Testing Acute (5-Day Protocol) and Sustained Manipulation (6-Day Protocol)
of Drug-Induced Negative Affective Bias Versus Vehiclea

ticosterone treatment also affects performance
in the SPT, suggesting the reward learning impairments in this assay may be a more reliable
translation method to study reward-related impairments in MDD. Negative affective states
are associated with other psychiatric disorders and a study using sub-chronic phencyclidine (PCP) treatment to model Schizophrenia
found similar reward learning impairments in
the RLA (Sahin et al., 2016). In our most recent work investigating RAADs, we have focused on the retrieval of biased memories and
used the RLA as a control assay to establish
if the observed effects are specific to an affective state-induced bias or involve more general
disruptions in memory processes (Hinchcliffe
et al., 2024). Studies utilizing female rats and
different strains revealed a consistent picture
in terms of both positive and negative affective bias modification across both pharmacological and psychosocial manipulations of the
affective state in both ABT and RLA (Hinchcliffe et al., 2017).
Having established the translational validity of both the ABT and RLA, we have been
able to use these methods to explore novel
hypotheses relating to antidepressant efficacy
as well as the fundamental neurobiology of
affective bias modification. These studies
have revealed important differences between
conventional delayed-onset antidepressants
and RAADs in terms of the way they interact
with affective biases. Conventional delayed
onset antidepressants positively bias new
learning consistent with the human neuropsychological model of antidepressant efficacy
(Godlewska & Harmer, 2021; Harmer, Goodwin et al., 2009; Harmer, O’Sullivan et al.,
2009). RAADs such as ketamine and psilocybin attenuated negative biases associated with
past experiences (Hinchcliffe et al., 2024; Stuart et al., 2015). When the choice test is carried
out 24 hr after treatment with ketamine and
psilocybin, the negative bias becomes positive
consistent with a re-learning effect (Hinchcliffe et al., 2024). The conventional antidepressant venlafaxine has no effect on biases associated with past experiences, and ketamine
lacks the ability to positively bias new experiences (Stuart et al., 2015). The psychedelic
psilocybin positively biased new experiences,
suggesting its neuropsychological effects
combined features of both conventional and
RAADs (Hinchcliffe et al., 2024). These
differences may be important factors contributing to the time course of their effects on
mood. This contrasts with the FST, where both

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phenotypic model to investigate vulnerability
to the acute effects of short-term affective state
manipulations. This type of study involves
a more complex design with phenotype as
a between-subject factor and acute affective
state manipulation as a within-subject factor.
Pharmacological and psychosocial manipulations of affective states have been used to
demonstrate the validity of the new learning
protocol for the ABT. These included testing
acute treatment with a variety of conventional
delayed onset antidepressants, social and environmental enrichment, psychosocial stress,
pro-depressant drugs, and immune challenges, with the direction of the affective bias
included consistent with the predicted effects
on animals’ affective state (Hinchcliffe et al.,
2017, 2020, 2024; Hinchcliffe, Jackson et al.,
2022; Stuart et al., 2013, 2015, 2017, 2019).
Drugs of abuse (cocaine, amphetamine, nicotine), an antidepressant that failed in clinical
trials (aprepitant, a neurokinin 1 antagonist),
and diazepam (benzodiazepine anxiolytic) did
not produce any effects in the ABT (Stuart
et al., 2013). Whether the treatment was given
prior to or immediately after substrate-reward
learning did not change the observed bias for
the antidepressant venlafaxine or psychosocial stress (Stuart et al., 2013) suggesting a
complex integration of experiences and affective state. The magnitude of an affective bias
amplifies with each successive experience
(Stuart et al., 2013), and negative affective biases induced by FG7142 can be attenuated by
post-treatment induction of a positive affective
state (Hinchcliffe, Jackson et al., 2022).
The reward learning assay (RLA), described in Basic Protocol 2 is similar to the
ABT and was used in the initial validation
experiments conducted while developing the
ABT protocol. Animals remain in the same
affective state throughout the pairing sessions
and choice test, but one experience is paired
with a higher value reward, and in normal animals, this results in more choices for high
reward-paired substrate and a reward-induced
positive bias (Hinchcliffe et al., 2017, 2024;
Stuart et al., 2013, 2015). Running the RLA
is helpful during training to check animals are
correctly performing the task, but the rewardinduced positive bias has also been found to be
impaired in animals in a depression-like state
associated with risk factors including stress,
pro-depressant drug treatment, and early life
adversity (Stuart et al., 2017, 2019). In these
studies, we also quantified reward sensitivity
using the SPT and found that only chronic cor-

Critical Parameters
The paramount factors to achieve reliable
data from the ABT are refined handling techniques, habituation facilitated with positive
reinforcement, and providing standardized
enriched housing. Include enough time in
the study design to handle animals prior to

training and testing to minimize distress and
aversion to the experimenter and minimize the
use of physical restraint. Animals should be
calm and easy to handle, without overt signs
of distress e.g., audible vocalizations, signs of
struggling when picked up, fecal pellets. The
handler should be the experimenter and should
be the same person for the cohort throughout
the experiment. Changing handlers within a
study is not recommended, and if a change is
needed, animals should be habituated to a new
handler before starting further experiments.
For more details on our refined handling,
habituation, and housing protocols, see our
website 3Hs Initiative: Housing, Handling,
and Habituation (www.3hs-initiative.co.uk).
Acute stress is a potential confound in both
the ABT and RLA as the experience of stress
can generate a negative affective state and
negative affective bias. This is particularly
an issue with substance administration procedures. Oral dosing in palatable solutions
is the route of administration least likely to
generate a stress response but is not suitable
for all compounds, and where intraperitoneal
and/or subcutaneous injection procedures are
required, these should be performed using a
low-restraint method. In our group, injections
are performed using a low-stress, minimal
restraint method such as the one developed
in our research group (Stuart & Robinson,
2015, 3Hs website protocols). All animals are
habituated to the holding position required
for injection for five days prior to the experiments. Subcutaneous injections are performed
with minimal physical restraint and applied
to the left or right flank (changing daily) (3Hs
website protocols). In all experiments, the
experimenter should be blind to treatment
with treatments administered for withinsubject studies using a fully counterbalanced
experimental design, where all animals receive a vehicle (0 mg/kg), and all drug
doses.
The handler, i.e., the human factor, can
have a very large effect on the animal’s behavior and requires experience with animal behavior to be able to successfully train animals and
achieve reliable performance. It is important
that the handler is calm and patient and does
not rush the animals during training and testing. It is crucial to assign enough time to run
the pairing sessions and choice testing and adjust the size of the cohort to achieve this. Care
should be taken not to provide inadvertent cues
to the animal about the location of the reward
or try to rush the animal. By rushing to run rats

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conventional and RAADs similarly reduce
immobility time. Further exploring the relearning effects at 24 hr, we found that the
effects of ketamine were dependent on protein synthesis localized to the rat medial
prefrontal cortex and could be modulated by
cue-reactivation, consistent with experiencedependent neural plasticity (Hinchcliffe et al.,
2024). These findings potentially link their
effects on neural plasticity with affective
bias modification and experience-dependent
learning and memory.
We have also begun to investigate the underlying neural mechanisms that modulate affective biases and how these are affected by
different classes of antidepressants. Studies
using targeted brain lesions or temporary pharmacological lesions suggest the involvement
of both the amygdala and medial prefrontal
cortex in the modulation of affective biases
(Stuart et al., 2015). Amygdala lesions inhibited the formation of a venlafaxine-induced
positive bias, and FG7142- or stress-induced
negative bias was also reduced. Temporary inactivation of the mPFC with muscimol microinfusion, ketamine, or the 5-HT2A agonist DOI
resulted in attenuation of FG7142-induced
negative bias (Hinchcliffe et al., 2024; Stuart
et al., 2015). These findings align favorably
with human imaging studies, which suggest
both prefrontal and amygdala regions are involved in affective bias modification and exhibit changes in activity in patients with MDD
(Mayberg, 2003; Ressler & Mayberg, 2007).
The ABT method does not require expensive equipment and the training period is
much shorter than in tasks based on traditional
instrumental-based learning protocols. The
ABT and RLA are more refined methods than
the FST/TST, particularly when combined
with refined methods for substance administration. The ABT is useful for differentiating
the effects of delayed versus rapid-acting
antidepressants, including novel compounds.
The RLA has shown construct validity as a
phenotypic model and to study changes in the
core affective state, including for assessing the
animal welfare of laboratory rodents (Hinchcliffe et al., 2020; Hinchcliffe, Jackson, 2022).

Troubleshooting
See Table 5 for common problems and troubleshooting solutions.

Statistical Analysis
Analyze the data using appropriate statistical software such as SPSS 28 or GraphPad Prism 10.0 (GraphPad Software, USA).
Figures should summarize mean and variance
and illustrate individual data points.
To calculate a choice bias for each animal,
sum the number of choices made for the drugpaired (affective bias test) or 2 pellets-paired
(reward learning assay) substrate, divide by
the total number of trials, and multiply by 100
to give a percentage value. Then subtract 50
to give a score where a choice bias toward
the drug-paired substrate gives a positive value
and a bias toward the control-paired substrate
gives a negative value.

Choice bias =

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To calculate the % side bias, manually
score the number of times the animal chose the
bowl located on the left (or right, reciprocally)
side and use the equation:
Side bias
=

number of choices made for the left bowl
total number of trials

×100
To calculate the % substrate bias, manually
score the number of times the animal chose the
bowl with substrate A (or B reciprocally) and
use the equation:
Substrate bias
=

number of choices made for substrate A
total number of trials

×100
To calculate the number of pellets collected during the choice test, manually score
the number of times the animal chose the rewarded substrate.
Depending on the number of groups and
factors included in the study design, use a suitable statistical test such as a two-tailed t-test
(for two treatment groups) or Repeated Measures ANOVA (for three or more treatment
groups) with treatment as the within-subject
factor. A 2-factor ANOVA is used for studies
involving a between-subject and withinsubject factor e.g., phenotype and drug treatment. As a post-hoc analysis, perform pairwise comparisons by using two-tailed paired
t-tests (for two treatment groups) or Dunnett’s
test (for three or more treatment groups). To
analyze choice bias data against a theoretical
mean of 0% choice bias for each manipulation, use a one-sample t-test against a null
hypothesized mean of 0% choice bias for each
manipulation.

Understanding Results
The example data in Figure 6 and Basic
Protocol 3 illustrate a significant preference
for the control-paired substrate representing
a negative affective bias in rats following
psychosocial stress, restraint stress, and social


number of choices made for the manipulation - paired subtrate
× 100 − 50
total number of trials

Calculate the side bias, substrate bias, and
the number of pellets collected during the
choice test to control for non-specific effects
and test whether animals are performing the
task correctly.

isolation. If animals prefer the manipulationpaired substrate, this is a positive affective
bias, e.g., following an antidepressant or social play (Hinchcliffe et al., 2017; Stuart et al.,
2013). Response latencies for well-trained

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in the pairing sessions, there is a higher likelihood of making an error or causing a stress
response in the animal.
Following the principles of the 3Rs, calculate your sample size with sufficient statistical power for the predicted effect size. The
sample size (N = 12-16) for our experiments
is based on our previous affective bias test
studies and power calculation with α = 0.05
and β = 0.8 (Stuart et al., 2013, 2015). A
meta-analysis suggested a medium to large effect size for the drug-induced negative bias
and reward-induced bias in Lister Hooded and
Sprague Dawley rats (Hinchcliffe et al., 2017).
Another factor to consider is the likelihood of
exclusion from the study. This is low for experienced handlers but may be higher for inexperienced handlers. Exclusion criteria used
for training and testing are: when an animal
fails to meet the training criterion, fails to learn
the substrate-reward association during at least
one pairing session, fails to complete 30 trials
in the choice test (usually only seen with acute
manipulations before the choice test), has a %
choice bias more than 2 SDs from the mean,
or the positive control did not generate the predicted affective bias suggesting a cohort level
issue.

Problem

Possible cause

Solution

Animals do not dig for
rewards

Animals not habituated enough and are
showing a stress response.

Do additional handling sessions and increase
food restriction for the next couple of
training sessions.

Animals not returning
to the handler

Animals not habituated enough and are
showing a stress response.

Do additional handling sessions.

Animals are rushed and not given time to
learn the trial sequence.

Give animals time to find the reward pellet in
the left corner and then pick up. Continue to
reward the left corner until the trial sequence
is well established.
You can try to “wiggle” your fingers at the
closer edge of the arena to encourage
returning to the starting point and/or picking
up the animal after the return cut-off time is
∼1 min. For trained animals, run reminder
discrimination session with new pair of
substrates and reward with pellet in the
corner to encourage returning to the starting
point.

Animals developing
substrate bias or side
bias in the choice test

This can confound the result and studies with
a substrate or side bias should be excluded.
These biases are usually seen when animals
are trying to solve the reward location rule
within the choice test rather than making
choices based on their memories.

Re-run with new substrates and preferably
ones that have worked for previous studies
(see Table 1 for examples). If a cohort has a
side or substrate bias in the first RLA at the
end of training, it suggests they have not
learned the task correctly.

Animals do not
complete choice test

Either they are not motivated enough to dig
and forage for pellets, or they experience
sedative/non-specific effects of the drug
manipulations.

Lower the dose of the drug used. Make sure
animals are mildly food-restricted to
maintain their motivation in the task.

Problems with animals’
low performance
during training sessions
or pairing sessions

Animals are not motivated enough to dig or
too anxious and not habituated to the
experimenter or ABT arena. In the acute and
sustained modulation studies make sure you
use the doses previously validated and
reliable to induce the negative bias i.e.,
corticosterone 10 mg/kg or FG7142 3
mg/kg. Increasing the dose will induce
non-specific effects in animals and e.g.,
FG7142 at higher doses 5-6 mg/kg carrying
risk of triggering seizures in rats.

Mildly increase food restriction and/or spend
more time to handle and habituate rats.

An animal consistently
achieves more than 10
rewards during a choice
test or cohort mean
shows progressive
increase above chance.

Animals may be using olfactory cues to
locate rewards.

Monitor performance of individual animals
and check choice latencies. If animal has a
mean latency higher than the population
mean, use a training session with reduced
cut-off time to speed up decision-making
and encourage the animal to use the
substrate cue. This may need to be repeated
over several sessions.

rats should be in the range of 1-5 seconds
(Fig. 6B). Differences in response latencies
during pairing sessions and choice tests, as
well as increased omissions and number of
trials to criteria during pairing sessions, can

be observed in drug studies where pharmacological manipulations can cause non-specific
effects, like sedation, changes in motivation or
disrupted learning rate (Fig. 6B and Tables 6
and 7). Figure 6C shows the number of pellets

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Table 5 Troubleshooting Guide

Table 6 Example Data for Basic Protocol 3, the Effects of Restraint Stress and Psychosocial Isolation (RSSI) on New
Learning In The Affective Bias Testa

ID rat

Substrate
A

Substrate
B

Substrate
paired with
RSSI

Pellets
earned

Mean
latency
[s]

% Substrate
bias (A)

% Side bias
(Left)

% Choice bias

1

13

17

A

11

1.86

43.33333

43.33333

−6.67

2

21

9

B

10

1.71

70

56.66667

−20.00

3

11

19

A

13

1.27

36.66667

50

−13.33

4

19

11

B

14

1.56

63.33333

46.66667

−13.33

5

12

18

A

10

1.45

40

43.33333333

−10.00

6

20

10

B

10

2.19

66.66667

63.33333333

−16.67

7

21

9

B

8

2.90

70

56.66667

−20.00

8

12

18

A

10

1.66

40

60

−0.00

9

17

13

B

11

1.74

56.66667

40

−6.67

10

14

16

A

8

1.31

46.66667

46.66667

−3.33

11

19

11

B

9

1.56

63.33333

70

−13.33

12

15

15

A

15

1.57

50

53.33333

0.00

a

Choice bias data: number of substrate A choices, number of substrate B choices, substrate paired with manipulation, i.e., RSSI, number of pellets
earned, mean latency to make a choice (s), % substrate bias, % side bias, and % choice bias. Data are shown as individual animals.

Hinchcliffe and
Robinson

consumed during the 30 trials of the ABT
choice test, used to verify that the performance of the cohort remains at chance (10/30
trials). Cohorts earning, on average, more
than 14 pellets per session may be using other
cues, such as olfaction, to find reward. No
significant substrate and side bias (Figure
6D and E) indicate rats performing the task
correctly and the affective bias arises only
from changes in their emotional experience.

In case of animals demonstrating the substrate
or side bias, see Troubleshooting.
Healthy animals develop a significant
reward-induced positive bias at the population level in the RLA (Fig. 7A). If rats fail to
develop a reward-induced positive bias, they
are not performing tasks correctly and require
further digging training. When the RLA is
used in an animal model of depression, two
weeks of chronic treatment with interferon

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Figure 6 Sample raw data of the effects of restraint stress and social isolation on new learning in
the affective bias test in male Sprague Dawley rats (n = 12/group). (A) % choice bias, (B) latency
to make a choice, (C) number of pellets earned, (D) % substrate bias, and (E) % side bias. Data
taken and re-drawn from Hinchcliffe et al. (2017). Data are shown as mean % choice bias ± SEM
(bars) and individual data points (symbols), one sample t-test against a null hypothesized mean of
0% choice bias, ***p < .001.

Restraint stress and
psychosocial isolation

Response latency (s)

Number of trials to criterion

Rat ID

Vehicle

Manipulation

Vehicle

Manipulation

1

2.0289

1.4431

8

7

2

2.5300

2.2453

8

7.5

3

1.7020

2.2560

9

10

4

1.6367

1.5424

6

11

5

1.8615

1.9123

6.5

6.5

6

2.9129

3.4896

9

12.5

7

2.0125

4.1850

12.5

10

8

1.9710

1.4920

8.5

8.5

9

1.8876

1.8233

7

7.5

10

2.3454

1.3640

9.5

6.5

11

2.9663

2.6226

6.5

10.5

12

3.1778

1.6625

8

7

a

Pairing session data: response latency to make a choice (s) and number of trials to criterion. Data is shown as mean from
the two pairing sessions for each substrate-reward association, vehicle, and manipulation for individual animals.

Figure 7 The reward learning assay in male Lister Hooded rats (n = 12-16/group). (A) % choice
bias from post-discrimination training, (B) % choice bias following chronic treatment with interferon
alpha (IFNA), (C) % choice bias from RLA assessing the specificity of effects of acute treatment on
memory retrieval. Data taken and re-drawn from Hinchcliffe et al. (2017) (A), Hinchcliffe, Thomas
et al. (2022) (B), and Hinchcliffe et al. (2024) (C). Data are shown as mean % choice bias ± SEM
(bars) and individual data points (symbols), one sample t-test against a null hypothesized mean of
0% choice bias, ***p < .001 and paired t-test, ### p < .001.

alpha-induced reward learning impairments
and animals failed to develop a positive
bias toward the high reward cue during the
choice test (Fig. 7B). The results for substrate bias, side bias, pellets consumed, response latencies, number of pairing sessions to criteria during the RLA choice test

are analyzed the same way and with the
same interpretation as the ABT choice test
(Table 8).
The results in Figure 8 illustrate the effects of the RAAD ketamine administered at 1
mg/kg, 1 hr (panel A) or 24 hr (panel B) before
the choice test. All animals receive FG7142

Hinchcliffe and
Robinson

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Table 7 Example Data for Basic Protocol 3, the Effects of Restraint Stress and Psychosocial
Isolation on New Learning in the Affective Bias Testa

ID rat

Substrate
A

Substrate
B

Substrate
paired with Pellets
2 pellets
earned

Mean
latency [s]

% Substrate
bias (A)

% Side bias
(left)

% Choice bias

1

12

18

B

11

2.32

40

40

10

2

19

11

A

13

1.88

63.3333

50

13.3333

3

17

13

A

11

2.43

53.3333

50

6.66667

4

13

17

B

11

1.91

46.6667

46.6667

6.66667

5

14

16

B

12

2.17

46.6667

53.3333

3.33333

6

18

12

A

12

2.00

53.3333

46.6667

10

7

17

13

A

8

2.36

53.3333

60

6.66667

8

13

17

B

14

1.94

50

40

6.66667

9

14

16

B

9

1.78

40

43.3333

3.33333

10

18

12

A

7

1.68

66.6667

50

10

11

18

12

A

12

1.67

63.3333

50

10

12

11

19

B

12

1.63

50

53.3333

13.3333

a

Choice bias data: number of substrate A choices, number of substrate B choices, substrate paired with manipulation, i.e., 2 pellets, number of pellets
earned, mean latency to make a choice (s), % substrate bias, % side bias, and % choice bias. Data are shown as individual animals.

Figure 8 The effects of 1 mg/kg ketamine on acute and sustained modulation in the affective bias
test in male Lister Hooded rats (n = 12-15/group). (A) % choice bias of acute modulation following
ketamine treatment and (B) % choice bias of sustained modulation following ketamine treatment.
Data taken and re-drawn from Hinchcliffe et al. (2024). Data are shown as mean % choice bias
± SEM (bars) and individual data points (symbols), one sample t-test against a null hypothesized
mean of 0% choice bias, ***p < .001 and paired t-test, ### p < .001.

Hinchcliffe and
Robinson

during the pairing sessions and would be
expected to exhibit a negative affective bias
during the choice test (Fig. 8A and B). This is
what is observed in the vehicle-treated groups
at both time points, with a significant negative
affective bias observed with a one-sample
t-test. Ketamine significantly attenuates this
negative bias at 1 hr with no bias observed in
the one-sample t-test and pairwise comparisons revealing a significant attenuation (Fig.
8A, Table 9). At 24 hr, a positive bias is observed with the one sample t-test and pairwise
comparisons (or ANOVA if more than one test

group) show a significant difference from the
vehicle control (Fig. 8B). Treatments that fail
to modulate biased memories exhibit similar
negative biases to the vehicle group and no
difference from vehicle treatment is observed.
To investigate the specificity of any affective
bias modulation observed similar analyses
are used for the same treatments in animals
performing the RLA. Where treatments have
no effects in the RLA they are considered to
selectively modulate affective state-induced
biases (Fig. 7C). If treatments impair performance in the RLA, they are considered to

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Table 8 Example Data for Basic Protocol 2, the Reward Learning Assaya

Week 1

ID
rat

Substrate
A

Substrate
B

Substrate
paired
with
CORT

1

15

15

A

13

2.29

50

56.66667

Ket

0

2

14

16

B

14

2.06

46.66667

43.33333

Ket

3.3333

3

14

16

B

12

2.60

46.66667

36.66667

Ket

3.3333

4

15

15

A

11

2.76

50

60

Ket

0

5

14

16

A

13

2.81

46.66667

63.33333

Veh

−3.3333

6

17

13

B

11

2.58

56.66667

46.66667

Veh

−6.6667

7

18

12

B

11

2.77

60

56.66667

Veh

−10

8

14

16

A

12

2.27

46.66667

43.33333

Veh

−3.3333

9

17

13

A

12

2.31

56.66667

40

Ket

6.6667

10

14

16

B

8

2.32

46.66667

50

Ket

3.3333

11

14

16

B

14

2.05

46.66667

53.33333

Ket

3.3333

12

18

12

A

9

2.50

60

46.66667

Ket

10

13

12

18

A

7

2.34

40

56.66667

Veh

−10

14

16

14

B

12

2.63

53.33333

43.33333

Veh

−3.3333

15

14

16

A

12

2.46

46.66667

50

Veh

−3.3333

ID
rat

Substrate
A

Substrate
B

Substrate
paired
with
CORT

Pellets
earned

Mean
latency
[s]

% Substrate
bias (A)

% Side bias
(left)

Drug
treatment
prior to
choice test

%
Choice
bias

1

13

17

A

12

2.06

43.333333

40

Veh

−6.6667

2

19

11

B

12

2.31

63.333333

56.66667

Veh

Pellets
earned

Mean
latency
[s]

% Substrate
bias (A)

% Side bias
(left)

Drug
treatment
prior to
choice test

%
Choice
bias

Week 2

−13.3333
3

18

12

B

7

2.47

60

53.33333

Veh

−10

4

14

16

A

8

2.15

46.666667

50

Veh

−3.3333

5

18

12

A

11

2.42

60

53.33333

Ket

10

6

12

18

B

15

2.29

40

50

Ket

10

7

13

17

B

10

2.65

43.333333

53.33333

Ket

6.6667

8

17

13

A

10

2.65

56.666667

63.33333

Ket

6.6667

9

15

15

A

12

2.47

50

53.33333

Veh

0

10

18

12

B

12

2.52

60

43.33333

Veh

−10

11

16

14

B

11

2.45

53.333333

43.33333

Veh

−3.3333

12

14

16

A

11

2.61

46.666667

56.66667

Veh

−3.3333

13

16

14

A

10

2.08

53.333333

50

Ket

3.3333

14

14

16

B

13

1.96

46.666667

53.33333

Ket

3.3333

15

15

15

A

5

2.44

50

63.33333

Ket

0

a

Choice bias data: number of substrate A choices, number of substrate B choices, substrate paired with manipulation, i.e., corticosterone (CORT)
treatment, number of pellets earned, mean latency to make a choice (s), % substrate bias, % side bias, drug treatment prior to choice test, and % choice
bias. Data are shown as individual animals.

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Table 9 Example Data for Basic Protocol 4, the Acute Modulation of the Drug-Induced Negative Affective Biases by
Ketamine (Ket) 1 mg/kga

Time Considerations
The training of animals in preparation
for the ABT or for studies involving manipulations in the RLA takes 3 weeks for an
experienced handler and habituated animals.
Because animals must reach the criterion before progressing through the different stages
of training, the time required for each animal
and handler can vary. The maximum duration
for a session should be 30 min, and animals
that are not performing the task can be terminated earlier than this, particularly if they are
showing signs of stress. The session is then
repeated later that day. The animals should
only be trained and tested during their active
phase.
The size of the cohort is key to how long
a training or testing session will last each day.
In our laboratory, we typically run 12-16 animals in one cohort with one experimenter.
The initial training sessions can take ∼6 hr
for a cohort of 12 animals but get quicker
each session. By the end of training an animal should complete a pairing session in ∼510 min and a choice test in 20-30 min. The
time it takes for the animal to complete a session will vary between handlers and is usually
much quicker for those experienced in running
the task. Learning to set up the bowls and substrates, the order of presentation, and recording the data during the session all take time to
learn and get quicker with experience.
Intervention studies require 1 week per manipulation (treatment versus control would require 2 weeks, and 2 doses of treatment vs
control would require 3 weeks). For the ABT,
the treatments are counterbalanced across the
weeks of the study. For the RLA, separate
groups are required for phenotypic studies. For
ABT involving phenotypic models or chronic
treatments, separate treatment groups are required for the between-subject factor, and each
group experiences the acute manipulations in
a counterbalanced design and one manipulation per week. For both the RLA and ABT
studies involving a between-subject factor, it
is usually necessary to run balanced replicates,
which are then pooled at the end of the study.

Acknowledgments

Hinchcliffe and
Robinson

This work was supported by the BBSRC
grants (BB/V015028/1, BB/N015762/1) and
an MRC grant (MR/L011212/1) awarded to
ESJR. The authors would like to thank all staff
members and students involved over the years

in the development and optimization of the affective bias test and reward learning assay: Julia Bartlett, Abigail Benn, Ben Clarke, Charlie
Clarke-Williams, Nadia Crellin, Chloe Hardcastle, Claire Hales, Megan Jackson, Katie
Kamenish, Caroline Phelps, Fruzsina Rabi,
Oda Moe Sorensen, Sarah Stuart, Matthew
Wilkinson, Christian Wood.

Author Contributions
Justyna Hinchcliffe: Conceptualization;
data curation; formal analysis; investigation; methodology; validation; visualization;
writing—original draft; writing—review and
editing. Emma Robinson: Conceptualization;
data curation; formal analysis; funding acquisition; investigation; methodology; project
administration; resources; software; supervision; validation; visualization; writing—
original draft; writing—review and editing.

Conflict of Interest
ESJR has obtained research grant funding through PhD studentships, collaborative
grants, and contract research from Boehringer
Ingelheim, Compass Pathways, Eli Lilly, IRLab Therapeutics, MSD, Pfizer, and Small
Pharma, and acting as a paid consultant for
Compass Pathways and Pangea Botanica.

Data Availability Statement
The data that support the protocol
are openly available in Open Science
Framework at https:// osf.io/ w5ufe/ , DOI
10.17605/OSF.IO/W5UFE.

Supporting Information
cpz11057-sup-0001-SuppMat.xlsx
Table S1. Pairing sheet for recording data for
ABT protocol pairing sessions (Left).
cpz11057-sup-0002-SuppMat.xlsx
Table S2. Pairing sheet for recording data for
ABT protocol pairing sessions (Right).
cpz11057-sup-0003-SuppMat.xlsx
Table S3. Data entry template for ABT choice
test sessions.

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Key References
Stuart et al. (2013). See above.
Validation of the affective bias test and reward
learning assay, new learning studies
Stuart et al. (2015). See above.
Validation of the affective bias test and reward
learning assay, new learning and acute modulation of negative affective biases studies
Hinchcliffe et al. (2017). See above.

Validation of the affective bias test and reward
learning assay
Stuart et al. (2017). See above.
Validation of the affective bias test and reward
learning assay, new learning studies
Robinson (2018). See above.
Validation of the affective bias test and reward
learning assay
Stuart et al. (2019). See above.
Validation of the affective bias test and reward
learning assay, new learning studies
Hinchcliffe et al. (2020). See above.
Affective biases in animal welfare studies
Hinchcliffe, J. et al. (2022). See above.
Affective biases in animal welfare studies
Hinchcliffe (2024). Rapid-acting antidepressant
drugs modulate affective bias in rats. Science
Translational Medicine, 16(729), eadi2403.
https://doi.org/10.1126/scitranslmed.adi2403
Acute and sustained modulation of the negative affective biases, new learning studies and reward
learning assay

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Robinson

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Stuart, S. A., & Robinson, E. S. (2015). Reducing
the stress of drug administration: Implications
for the 3Rs. Scientific Reports, 5, 14288. https:
//doi.org/10.1038/srep14288