Training level reveals a dynamic dialogue between stress and
memory systems in birds
Flore Lormant1*, Vitor Hugo Bessa Ferreira1,2*, Julie Lemarchand1, Fabien
Cornilleau1, Paul Constantin1, Céline Parias1, Aline Bertin1, Léa Lansade1,
Christine Leterrier1, Frédéric Lévy1, Ludovic Calandreau1
1 INRAE, UMR 85 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly,
France ; CNRS, UMR 7247, F-37380 Nouzilly, France ; Université François Rabelais, F-37041
Tours, France ; IFCE, F-37380 Nouzilly, France.
2 JUNIA ISA, 48 bd Vauban 59046 Lille Cedex, France
* Shared co-first authorship
Correspondence should be addressed to: Ludovic Calandreau, INRAE, UMR 85 Physiologie
de la Reproduction et des Comportements, 37380 Nouzilly, France.
Tel : +33 2 47 42 75 71
Fax : +33 2 47 42 77 43
It is now well-accepted that memory is a dynamic process, and that stress and training level may
influence which memory system an individual engages when solving a task. In this work, we
investigated whether and how chronic stress impacts spatial and cue-based memories according to
training level. To that aim, control and chronically stressed Japanese quail were trained in a task that
could be solved using spatial and cue-based memory and tested for their memory performances after 5
and 15 training days (initial training and overtraining, respectively) and following an emotional
challenge (exposure to an open field). While chronic stress negatively impacted spatial memory in
chronically stressed birds after initial training, this impact was lowered after overtraining compared to
control quail. Interestingly, the emotional challenge reinstated the differences in performance between
the two groups, revealing that chronic stress/overtraining did not eliminate spatial memory. Differences
caused by previous stressors can re-emerge depending on the more immediate psychological state of the
individual. Contrary to spatial memory, cue-based memory was not impaired in any test occasion,
confirming that this form of memory is resistant to chronic stress. Altogether these findings reveal a
dynamic dialogue between stress, training, and memory systems in birds.
Keywords: bird cognition; chronic stress; spatial memory; cue-based memory; overtraining; Japanese
It has long been recognized that chronic stress strongly modulates learning and memory
performances [1–3]. However, the relationship between chronic stress and memory are still
controversial: some studies reported an improving effect of chronic stress on memory performances [4–
6], whereas others reported a negative effect [6–8]. Overall, chronic stress generally provokes memory
impairment for complex tasks and improves simple tasks . These discrepancies support the fact that
memory is not a single unit. Different parts of the brain are responsible for different memory systems
that process different types of information and are differently affected by stress .
Two memory systems have had a considerable amount of scientific interest in the last decades
as they present strong parallels between humans and animals. The first one is spatial memory, a form of
declarative memory based on the hippocampus and used mainly to establish a relationship between
different cues from the environment, which allow the creation of a cognitive mental map [10,11]. As the
cues from the environment are treated in relation to each other, the spatial memory is considered to be
more complex than the cue-based memory, a non-declarative form of memory that results from a simple
association between a salient cue and the target and is based on the striatum [9,12,13]. Studies have
shown that chronic stress has a strong and negative impact on declarative memory, such as spatial
memory [1,3,6,7,14,15], whereas it spares or even improves forms of non-declarative memory
Beyond chronic stress, the training level can also influence the use of multiple memory systems
[9,17–20]. Indeed, rats trained to learn the location of a reward in a maze preferentially use their spatial
memory after a few days of training and shift to a cue-based dominant response with overtraining .
Training and stress levels may also interact: the initial differences in memory performances due to stress
may disappear with overtraining . Other studies suggest training itself may be a source of stress and
reduce hippocampal neurogenesis [22,23], which could explain the shift between memory systems.
While much is known on chronic stress effects on declarative spatial or non-declarative cue-based
memory performances, the influence of the level of training and its interaction with chronic stress
influence remains poorly understood.
Using the Japanese quail (Coturnix coturnix) as the animal model, in this work, we investigated
whether and how chronic stress could differentially impact spatial and cue-based memories according
to the level of training. Control and chronically stressed Japanese quail were trained in a dual
spatial/cued task, which could be solved either using the spatial or cue-based memories and tested for
their performances on each type of memory after five and fifteen training days. Additionally, as an
animal's immediate psychological state may interact with chronic stress and impact the relative use of
different memory systems and memory performances [24,25], a series of tests were conducted after an
emotional challenge that consisted of exposing quail to an unfamiliar environment (open field). Finally,
birds were submitted to a test to assess whether they learn to find the target cup by a simple association
between the target cup's colour and the reward or by detecting the difference of colours between the
target cup and the remaining cups. Based on the literature, we expected chronically stressed quail to
show lower performance in the spatial memory test than control ones after initial training (five days of
training). These differences were expected to lower or disappear after overtraining (fifteen days of
training) and reinstated after the emotional challenge. Concerning the cue-based memory, we expected
no differences between chronically stressed and control quail. Since this is a simple form of memory,
we expected the emotional challenge would not disrupt it.
All Japanese quail were bred and maintained at the Pôle d'Expérimentation Avicole de Tours
(UE PEAT, INRAE, 2018. Experimental Poultry Facility, DOI: 10.15454/1.5572326250887292E12)
where the experiment took place. On the day of hatching, chicks were transferred to communal floor
pens. On the 21st day after hatching, chicks were sexed by feather dimorphism, and males were reared
in a single home cage (41×51×25 cm) in a battery under a 12:12h light-dark schedule (light on 08h). A
piece of artificial turf was placed on the cage floor to allow animals to exhibit dust bathing behaviour.
The ambient temperature was maintained at approximately 20±2°C. Unless otherwise specified, food
and water were provided ad libitum.
Animal care and experimental treatments complied with the French Ministry of Agriculture
guidelines for animal experimentation and European regulations on animal experimentation
(86/609/EEC). They were performed following the local animal regulation (authorized C37-175-1) of
the French Ministry of Agriculture under the EEC directive and under ethics committee approval (Val
de Loire, agreement N° 1789 and 1848).
2.2. Chronic stress procedure
At 21th day of age, when quail were transferred in individual cages, they were divided into two
groups, a control and a chronic stress group. Quail from the chronic stress group were submitted to
chronic unpredictable stress (CUS) for 21 days consisting of five to six negative stimulations or stressors
per day. To improve the unpredictability of the stressor delivery and decrease habituation to the stress
procedure, each stressor occurred at unpredictable times each day during both night and day. The CUS
procedure has been fully described and was shown to induce a state of chronic stress in quail [15,26–
30]. Briefly, some stressors were delivered directly in the home cage of the quail: confinement of the
bird in the corner of its home cage for 30min, food frustration by placing transparent devices on the
feeder just before light on for 30min, soft home cage shaking (2×15 min interspaced by 15 min),
unexpected sounds (100 dB) composed of different sounds having or not biological signification for
quail. Disturbances from outside the home cage were also delivered, such as a rapid passage of a plastic
stick on the rods of the home cage twice a day for 2 min, fast sprayings of water or air on feathers (2
sprays interspaced by 2 min), waving of a plastic flag in front of the home cage for 2 min. Other stressors
were also delivered out of the home cage when birds were transferred individually into a new
environment for 30 min. Birds from the same line were also placed together in a transport cage for 30
min or placed on a cart and rolled about in the facility.
The control group was left undisturbed except for routine husbandry procedures similar to those
provided to birds exposed to CUS. The experimenter regularly visited the control quail room to spend
the same amount of time with control and chronically stressed birds. Systematically, when stressors
prevented animals from having access to food, an opaque device was placed on the feeder of control
birds to prevent them from eating food for an equivalent time.
2.3. Learning and memory task
At the end of the CUS procedure, 17 control and 20 chronically stressed birds were submitted
to a training procedure followed by a series of memory tests (Figure 1). Briefly, after preliminary phases
dedicated to familiarized birds to the food reward (mealworms), the cups and the arena, control and
chronically stressed birds were submitted to a dual spatial/cued task during five days that consisted of
learning to find food (mealworm) hidden in a single cup (target cup) in an arena that contained eight
opaque cups. On each trial, birds were introduced in the arena by a different starting point among three
possibilities. The location of the target cup was constant among trials and days of training, allowing
animals to solve the task using spatial memory. Moreover, the target cup was black, whereas other non-
target cups were white. Thus, birds could also solve this task using their cue-based memory, based on
the colour of the cup.
The day after the fifth day of training, birds were submitted to a spatial test followed by a
displacement test. During the spatial test, all cups were white, and the task could only be solved by
remembering the spatial location of the target cup during the training phase. During the displacement
test, performed 30 min later, the black target cup was displaced to a new position (different from the
position used during training). Thus, both spatial and cue-based memories could provide a solution to
the test. Quail could go back either to the spatial location of the target cup during the training phase
(spatial memory) or go directly to the displaced black cup (cue-based memory) [15,31,32].
After this first test occasion, birds were trained for ten additional days in the dual spatial/cued
task before being submitted to the second test occasion of spatial and displacement tests. Following the
second test occasion, birds were trained for an additional day (recall training) in the dual spatial/cued
task to avoid that lack of food during the second test occasion reduced bird motivation in subsequent
tests. After this first recall training day, all birds were challenged by exposure to an open field test and
were submitted to the third occasion of spatial and displacement tests.
On the following day, quail were submitted to a second additional training day (recall training)
to keep birds motivated. The day after, birds were submitted to a new test, called "colour test", to assess
whether birds found the target cup by a simple association between its colour (black) and the reward or
by learning a more general rule that the target cup was of a different colour compared to the remaining
During all the memory tests, behaviours of quail were recorded by a camera placed above the
apparatus and computerized by a tracking video system (Ethovision XT; Noldus IT, The Netherlands).
Figure 1: Schedule of the experiment and schematic representation of the apparatus used
for training and testing. At three-weeks-old, quail were submitted to chronic unpredictable stress
(CUS) for three weeks. At the end of the CUS procedure, stressed and control birds were trained in a
dual spatial/cued learning task and submitted to spatial and displacement memory tests after 5 and 15
days of training. After an additional training day, quail were submitted to an emotional challenge
(exposure to an open field) followed by a new series of tests. After the last training day, birds were
submitted to the colour test. Quail could enter the arena from three different starting points (dark arrows),
three starting points during training, and one during testing. The dotted lines represent the division of
apparatus into four different quadrants.
2.4. The dual spatial/cued task
The following procedures and the arena used for the task were previously used and validated by
our group in chickens and quails [13,15,32,33].
2.4.1. Mealworms familiarization
One week before the beginning of training, a brown ceramic cup (6x7cm) was placed in each
quail's home cage. For seven days, three times per day (spaced about 3h), 3 or 4 mealworms were
deposited in the cup to familiarize birds with the cups and mealworms.
The day following the end of the familiarization phase, birds were submitted to the habituation
phase. One hour before each habituation session, food was removed from the home cage of the animals.
During five days, once a day, each quail was introduced into the centre of a beige octagonal arena
(120cm long; 50cm high) surrounded by a blue curtain (1.90m high) and lighted by a bulb at the ceiling
(18 Lux). Four black spatial cues were placed on the walls of the arena and four others on the curtain.
Eight ceramic cups similar to those used for the familiarization were placed in the arena and contained
mealworms. Four cups were covered with white paper, whereas black paper covered another four cups.
Each bird was allowed to explore the arena and the cups in each habituation session until it found and
ate all mealworms or after a maximum time of 600 s. Between each quail and day of habituation, the
position of black and white cups was randomly moved. On each day, the number of cups visited was
recorded to estimate the level of exploration and habituation of the birds (Figure 1).
On each day of training, birds were submitted to 2 training trials per day with an inter-trial
interval of 1 hour. On each trial, birds were introduced in the arena by a different starting point among
three possibilities (Figure 1). Only one cup was rewarded and contained three mealworms (target cup).
The target cup was always black and at a fixed position on each trial, whereas the seven other cups were
non-rewarded and white. The trial ended when the bird reached the target cup and ate the mealworms,
or after a maximum time of 300 s. If the bird did not find the target cup, it was gently guided to it and
allowed to eat the mealworms before being removed from the arena. Between each trial, the bird returned
to its home cage. The latency to find and eat the food in the rewarded black cup was recorded on each
2.4.4. The spatial test
This test was used to assess the impact of chronic stress on spatial memory. Each bird was
allowed to freely explore the arena and the 8 cups for 1 minute. All cups were empty and, more
importantly, of similar colour (white). The number of cups visited (number of errors) before reaching
the target cup was scored. Moreover, the arena was virtually divided into four equal quadrants, and the
time spent in each quadrant (spatial quadrant and three other quadrants) during the test was measured
using the tracking video system (Figure 1). The time spent on the three other quadrants was averaged.
The spatial test was conducted after 5 and 15 days of training (initial training and overtraining,
henceforth) and after an emotional challenge by exposing birds to an open field (emotional challenge,
2.4.5. The displacement test
The displacement test was used to assess the impact of chronic stress on the use of spatial and
cue-based memory systems. It was systematically conducted 30 min after the spatial test. During this
test, similarly to the training phase, the arena was equipped with one black cup and seven white cups,
but all cups were empty of food. The black cup was displaced to a new position (Figure 1). The new
position was changed for each test conducted after initial training, overtraining, and emotional challenge.
All birds were allowed to explore the arena and the cups for 1 min freely. The number of errors before
reaching the spatial and displaced cup was scored. Similar to the spatial test, the arena was virtually
divided into four equal quadrants (spatial quadrant, displaced cup quadrant, and two other quadrants),
and the time spent in each quadrant during the test was measured using the tracking video system. The
time spent on the two other quadrants was averaged.
2.4.6. Tests after an emotional challenge
The third occasion of both tests (spatial and displacement tests) was conducted after an
emotional challenge. This emotional challenge consisted of exposing the birds to an open field test,
which is known to provoke anxiety-like quail behaviors [15,34,35]. Each quail was individually placed
in the centre of a square arena (80 × 80 × 80 cm) of white wood with a beige linoleum floor. The arena
was placed into a new experimental room, surrounded by a green curtain and strongly lighted (50 lux).
Each quail was allowed to explore the arena for 5 min before being withdrawn from the arena. Thirty
minutes after the open field test, quail were submitted to a spatial test followed by a displacement test.
2.4.7. The colour test
This test aimed to assess whether birds learn to find the target cup by a simple association
between its colour (black) and the reward or by detecting the difference of colours between the target
cup and the remaining cups (Figure 1). Each bird was allowed to freely explore the arena and the eight
cups for 1 minute. All cups were empty and black, except for one white cup and not located in the spatial
position used for training. The number of visits before reaching the white cup was scored. Moreover,
the number of visits before reaching the black cup located in the spatial location used during training
was scored and compared to those to reach the white cup and other black cups. As for previous tests, the
time spent in the four equal quadrants (white cup quadrant, spatial black cup quadrant, and two other
black cup quadrants) of the arena was measured. This test was performed after a second recall training
day conducted the day after the third test occasion.
2.5. Statistical analysis
Data from habituation (number of cups visited) and from training (latency to reach the target
cup) were analysed by parametric analyses of variance (ANOVA repeated measures) with stress group
(control and chronically stressed quail) as between-subject factors and days (mean values for both
training trials within days) as within-subject factors.
For spatial, displacement, and colour tests, the number of errors (or visits) before reaching the
target cup(s) and the time spent in the different quadrant of the arena were analysed by ANOVA with
groups (control and stressed) as between-subject factors and test occasions (test after initial training, test
after overtraining and test after the emotional challenge) as within-subject factors. Additional within-
subject factors (e.g., quadrants or cups considered) were also included, dependent on the variables under
consideration. When main effects or interactions were significant, analyses were followed by multiple
comparisons corrected by Tukey HSD.
When quail did not find a target cup and visited less than 4 cups, they were considered non-
explorer. For these birds, a number of 8 errors and an equivalent time of 15 sec in each quadrant were
attributed. All statistical analyses were performed using IBM SPSS 21 and R version 3.6.1. Statistical
significance was set at p < 0.05.
3.1. Effect of chronic stress on habituation and training in the dual spatial/cued task
During habituation, both groups visited more cups and ate significantly more mealworms over
days without any differences between control and chronically stress quail (effect of days: F4,140=18.15,
p<0.001; effect of stress: F1,35=0.13, p=0.72; interaction days x stress: F4,140=1.11, p=0.35; Figure 2a).
During the initial training period, both groups similarly learned the location of the target cup
since the latency to find the target cup significantly decreased over days (effect of days: F4,140=19.60,
p<0.001; effect of stress: F1,35=0.28, p=0.60; interaction days x stress: F4,140=0.93, p=0.45; Figure 2).
Similarly, during the overtraining period, the latency to reach the target cup significantly decreased
without any effect of chronic stress (effect of days: F9,315=5.89, p<0.001; stress effect: F1,35=0.13,
p=0.73; interaction days x stress: F9,315=0.58, p=0.81; Figure 2b).
The latency between the last day of overtraining (the fifteenth day of training) and the recall
period between tests (last two additional days) was not different between days, nor between control and
chronically stressed quail (effect of days: F2,70=1.12, p=0.33; effect of stress: F1,35=0.65, p=0.42;
interaction days x stress: F2,70=1.10, p=0.33), indicating that animals reached a learning plateau.
Figure 2: Effects of chronic stress on habituation and training performances in the dual
spatial/cued task. a) Number of visited cups over days of habituation in control and chronically stressed
quail. During habituation, all eight cups were rewarded with mealworms. b) Latency to reach the
location of the target cup over days of training in control and chronically stressed quail. Mean ± SEM
3.2. Effect of chronic stress during the spatial test
The number of errors made before reaching the spatial cup was significantly increased in
chronically stressed quail (effect of stress: F1,35=5.62, p=0.023, Figure 3a), independently of the test
occasion and the interaction with stress (effect of test occasion: F2,70=1.45, p=0.24; interaction test
occasion x stress: F2,70=2.72, p=0.072).
The time spent in the spatial quadrant compared to other quadrants was affected by the quadrant
considered, stress, and the interaction quadrant, test occasion and stress (effect of quadrant: F1,35=7.58,
p=0.009; effect of stress: F1,35=6.13, p=0.018; interaction quadrant x test occasion x stress: F2,70=3.36,
p=0.040). Post-hoc analyses revealed that, during the tests after the initial training and the emotional
challenge, control birds spent significantly more time in the spatial quadrant compared to other
quadrants (p<0.001 and p=0.001, for the tests after the initial training and the emotional challenge,
respectively). In contrast, chronically stressed birds spent an equivalent time in the spatial quadrant
compared to other quadrants (p=1 and p=1 for the tests after the initial training and the emotional
challenge, respectively, Figure 3b). No differences were found for the test after overtraining: both
control and chronically stressed quail spent an equivalent time in the spatial quadrant compared with
other quadrants (p=1 and p=1, for control and chronically stressed quail, respectively, Figure 3b).
Figure 3. Effects of chronic stress in spatial memory tests performed after initial training (5 days),
overtraining (15 days), and after an emotional challenge (EC, exposure to an open field). a)
Number of errors before reaching the spatial location of the target cup, and b) Time (in seconds) spent
in different quadrants of the arena (spatial and other quadrants). † p≤0.05, significant difference between
control and chronically stressed quail. ** p≤0.01; *** p≤0.001, significant difference between the spatial
quadrant and other quadrants. Mean ± SEM are given.
3.3. Effect of chronic stress during the displacement test
Analyses on the number of errors made before reaching the target cups (either the spatial cup or
the displaced cup) showed that animals were more performant to reach the displaced cup, independent
of their treatment group. There was also a significant interaction between the test occasion and the target
cups (effect of target cup: F1,35=100.71, p<0.001; effect of test occasion: F2,70=3.22, p=0.046; effect of
stress: F1,35=0.010, p=0.92; interaction target cup x stress: F1,35=0.27, p=0.6; interaction target cup x test
occasion: F2,70=8.26, p=0.001; interaction target cup x test occasion x stress: F2,70=0.87, p=0.42). Post-
hoc analyses revealed animals did significantly fewer errors before reaching the displaced cup in all test
occasions, compared to the errors before reaching the spatial cup (p=0.001, p<0.001 and p<0.001, for
the tests performed after initial training, overtraining, and just after the emotional challenge,
respectively, Figure 4a). Between test occasions and within-target cup, post-hoc comparisons revealed
that animals made significantly fewer errors before reaching the displaced cup during the tests after
overtraining and after the emotional challenge, compared to the test after initial training (p=0.001 and
p=0.005, respectively, Figure 4a).
Analyses on time spent in different quadrants showed that animals did not spend their time
evenly between quadrants. They spent significantly more time than the chance level in the displaced cup
quadrant independent of their treatment group. There was also significant a significant interaction
between the test occasion and the time spent in different quadrants (effect of quadrants: F2,70=67.19,
p<0.001; effect of test occasion: F2,70=0.82, p=0.44; effect of stress: F1,35=3.81, p=0.059; interaction
quadrants x stress: F2,70=1.09, p=0.34; interaction quadrants x test occasion: F4,140=3.25, p=0.014.
interaction quadrants x test occasion x stress: F4,140=0.42, p=0.78). Post-hoc analyses revealed that the
time spent in the displaced cup quadrant was higher for all test occasions compared to those spent on
the spatial quadrant and other quadrants (p<0.05, for the tests performed after initial training,
overtraining, and just after the emotional challenge, respectively, Figure 4b).
Figure 4. Effects of chronic stress in the displacement tests performed after initial training (5
days), overtraining (15 days), and after an emotional challenge (EC, exposure to an open field). a)
Number of errors before reaching the target cups (spatial cup or displaced cup), and b) Time (in seconds)
spent in different quadrants of the arena (either the spatial quadrant, SQ, the displaced cup quadrant,
DQ, or the other quadrants, OQ). * p≤0.05; ** p≤0.01; *** p≤0.001, significant difference between the
spatial cup and displaced cup or between spatial quadrant, displaced cup quadrant, and other quadrants.
Mean ± SEM are given.
3.4. Effect of chronic stress during the colour test
During the colour test, independently of chronic stress, the number of visits made before
reaching a cup was different depending on the cup considered (effect of cup: F2,70=18.60, p<0.0001;
effect of stress: F1,35=0.03, p=0.87; interaction cup x stress: F2,70=0.06, p=0.95; Figure 5). Post-hoc
analyses showed that the number of visits before reaching the spatial black cup was significantly lower
than those performed before reaching other black cups or the white cup (p=0.02 and p<0.001,
respectively, Figure 5a).
Finally, the time spent in the different quadrants was also significantly dependent on the
quadrant considered (effect of quadrants: F2,70=3.62, p=0.032). Post-hoc analyses revealed a
significantly larger amount of time spent in the black spatial cup quadrant compared to other black cups
and white cup quadrants (p=0.01). No differences were found between the treatment groups, nor for the
interaction quadrants and treatment groups (effect of stress: F1,35=1.02, p=0.31; interaction quadrants x
stress: F2,70=0.59, p=0.55, Figure 5b).
Figure 5. Effects of chronic stress on the colour test. a) Number of visits before reaching the white
cup, the black spatial cup, or other black cups in chronically stressed and control birds and b) Time (in
seconds) spent in different quadrants of the arena containing the white cup, the spatial black cup, other
black cups in chronically stressed and control birds. * p≤0.05; ** p≤0.01; *** p≤0.001, significant
difference between the white cup, black spatial cup, and other black cups. Mean ± SEM are given.
The main objective of the present study was to address, in birds, whether chronic stress can
differentially impact two forms of long-term memory, the declarative spatial and the non-declarative
cue-based memories, according to the level of training. To this end, control and chronically stressed
Japanese quail were trained in a task that could be solved using spatial and cue-based memory, the dual
spatial/cued task, and tested for their performances on each type of memory after 5 and 15 days of
training (initial training and overtraining, respectively). Our results showed that the spatial memory of
quail was sensitive to the deleterious effect of chronic stress. Performances of chronically stressed birds
during the spatial test after initial training were impaired compared to that of control birds. During the
test after overtraining, however, these differences were lowered. Interestingly, during the test after an
emotional challenge, when birds were exposed to an open field, the negative impact of chronic stress on
spatial memory performances was fully reinstated. These findings indicate that the relationships between
chronic stress and spatial memory are complex and dynamic. By contrast, cue-based memory was not
affected in chronically stressed birds compared to control birds on any test occasion. Thus, this form of
memory is relatively resistant to the negative effect of chronic stress. Finally, a test conducted to better
qualify birds' cue-based memory performances, the colour test, provided support that chronically
stressed birds were still able to efficiently remember the spatial location of the target cup when this
location was signalled by a colour similar to that used during training. This suggests that chronically
stressed birds cannot efficiently recall stored spatial information in a context different from the one
experienced during training, which highlights that chronic stress reduces the ability to use spatial
Our results from habituation showed that the number of mealworms eaten increased over days
without any significant effect of chronic stress. Moreover, the latency to reach the target cup decreased
over days of training in both groups. These findings indicate that both control and chronically stressed
birds similarly habituated to the arena and the cups and similarly learned the dual spatial/cued task. They
also indicate differences between groups observed during the different tests performed could not be
attributed to evident motor or motivational differences and instead reflect stress-related changes in
During the spatial test, the number of errors before reaching the spatial location of the target cup
was higher in all test occasions (initial training, overtraining, after an emotional challenge) for
chronically stressed birds compared to control birds, evidencing the negative impact of chronic stress
on spatial memory performances. Although not significant (p=0.07), the interaction between test
occasion and stress suggests that the performance of both groups during overtraining tests was similar,
compared to initial training and after emotional challenge tests, when their performances differed more.
Confirming this tendency, the time spent in different quadrants revealed some specificities of the
animals' performance over the different test occasions. During spatial tests performed after initial
training, chronically stressed birds spent their time evenly between the quadrants, while control birds
spent significantly more time in the spatial quadrant. The same did not occur after overtraining. Indeed,
performances of both control and chronically stressed birds were relatively low during the spatial test
performed after overtraining. Contrasting to the spatial tests, chronic stress did not affect cue-based
memory performances in all displacement tests performed. In all test occasions, chronically stressed
birds and control had the same performance and followed the displaced black cup preferentially. These
findings indicate that chronic stress differentially impacts memory depending on the memory system,
with the spatial memory being much more sensitive to the adverse effects of chronic stress than the cue-
based memory [1,3,6,36].
Our results suggest that chronic stress has a stronger detrimental effect on spatial memory at
early stages compared to the late stages of training. They confirm previous studies in birds and mammals
reporting chronic stress that can negatively impact this memory system [1,3,6,7,15]. Moreover, the
lowering of chronic stress effects on spatial memory performances after overtraining may be explained
by a progressive acquisition of a predominant cue-based strategy to solve the spatial test over days of
training. In mammals, it is well documented that the involvement of memory systems is a dynamic
process that can vary over time of training. In particular, overtraining in mammals favours cue-based
memory [17,18,20,21]. In line with this, the number of errors to reach the black target cup during the
displacement tests decreased with training, evidencing an improvement of this type of memory over
time. These results confirm the idea that the protective effect of an extended period of training on the
negative consequences of chronic stress on memory may be due to a progressive shift toward a non-
declarative, cue-based memory system.
An alternative explanation for these results could be that extensive exposure to the task was
perceived, by the control animals, as stressful on its own, worsening their performance. For rats, while
a 4-day training did not impact brain plasticity, a 14-day training schedule drastically reduced
hippocampal neurogenesis [23,37]. If that is the case, it is noteworthy to state that the stress from
overtraining compared to the CUS procedure was perceived differently by the animals since the negative
effect of chronic stress on spatial memory performances were specifically restored after an emotional
challenge. Previous studies highlight that acute stress or stress hormone injections experienced just
before testing can induce the re-emergence of an extinguished memory [24,25]. These results suggest
that the progressive shift toward a cue-based memory system by control quail after an extending period
of training is not due to the elimination of the spatial memory, which can re-emerge depending on the
more immediate psychological state of the quail. Similarly, studies on mammals have shown that the
shift from spatial memory to cue-based memory with increased training is not due to the elimination of
the spatial memory [18,21]. Moreover, even after overtraining, the reappearance of chronic stress
adverse effects on the spatial memory of chronically stressed quail could indicate a long-lasting
sensibility of this memory system.
Finally, our study highlighted that chronic stress affected specific characteristics of spatial
memory but did not erase this form of memory. Indeed, in the last test, the colour test, control, and
chronically stressed birds made fewer visits before reaching the black cup located in the same spatial
location used during training. Chronically stressed birds seemed thus able to remember the location of
the target cup when this cup was black, as during training. It suggests that chronic stress specifically
impaired the capacity of birds to remember a previously learned location when this location was no
more signalled by the cue (black colour). This capacity is crucial to perform the spatial test efficiently.
Numerous studies conducted in birds and mammals evidenced that spatial memory critically requires
the hippocampus [11,38–42]. In mammals, the hippocampus was shown to be critically involved in
forming distinct memories from events with a high level of similarities (pattern separation) and to re-
establish previously acquired memory by using incomplete information as recall cues (pattern
completion) [43,44]. During spatial tests, the spatial location was no more signalled by the cue (all cups
were white). Birds should re-establish the complete spatial-cued information learned during training
(one rewarded black cup among seven non-rewarded white cups) from this incomplete information.
Under this framework, chronic stress, which negatively affects the hippocampus's functioning in quail
, may impair hippocampus-dependent processing of pattern completion.
In conclusion, the present study presents evidence that chronic stress can negatively impact the
birds' declarative spatial memory. It highlights that chronic stress specifically alters the ability to use
spatial memory flexibly. Also, we show for the first time in birds that an extended period of training can
lower the spatial memory differences between control and chronically stressed individuals, either by a
progressive shift to cue-based memory or by the fact that overtraining can be in itself perceived as
stressful by control animals, hindering their performance. These hypotheses should be tested further. An
emotional challenge, before testing, was capable of reinstating the differences in performance between
chronically stressed and control quail showing the long-lasting sensibility of the spatial memory system.
Altogether these findings reveal an original and dynamic dialogue between stress and memory systems
This work was supported by the Institut National de Recherche pour l'Agriculture,
l'Alimentation et l'Environnement (INRAE). FL and VHBF were supported by a grant from the French
Region Centre Val de Loire and from JUNIA ISA, respectively. We are grateful to all the members of
the experimental unit PEAT of INRAE (UE PEAT, INRAE, 2018. Experimental Poultry Facility, DOI:
10.15454/1.5572326250887292E12) and particularly to I. Grimaud for providing care to the birds.
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