cells

Article
Increasing Adult Hippocampal Neurogenesis Promotes
Resilience in a Mouse Model of Depression
Barbara Planchez 1 , Natalia Lagunas 1 , Anne-Marie Le Guisquet 1 , Marc Legrand 1 , Alexandre Surget 1 ,
René Hen 2,3,4 and Catherine Belzung 1, *

1 UMR 1253, iBrain, Université de Tours, Inserm, CEDEX 1, 37032 Tours, France;
[email protected] (B.P.); [email protected] (N.L.); [email protected] (A.-M.L.G.);
[email protected] (M.L.); [email protected] (A.S.)
2 Departments of Neuroscience, Psychiatry & Pharmacology, Columbia University, New York, NY 10027, USA;
[email protected]
3 Division of Integrative Neuroscience, Department of Psychiatry, New York State Psychiatric Institute,
New York, NY 10032, USA
4 Kavli Institute for Brain Sciences, Columbia University, New York, NY 10027, USA
* Correspondence: [email protected]

Abstract: Many studies evaluated the functional role of adult hippocampal neurogenesis (AHN)
and its key role in cognitive functions and mood regulation. The effects of promoting AHN on
the recovery of stress-induced symptoms have been well studied, but its involvement in stress
resilience remains elusive. We used a mouse model enabling us to foster AHN before the exposure to
unpredictable chronic mild stress (UCMS) to evaluate the potential protective effects of AHN on stress,



assessing the depressive-like phenotype and executive functions. For this purpose, an inducible


transgenic mouse model was used to delete the pro-apoptotic gene Bax from neural progenitors
Citation: Planchez, B.; Lagunas, N.; four weeks before UCMS, whereby increasing the survival of adult-generated neurons. Our results
Le Guisquet, A.-M.; Legrand, M.; showed that UCMS elicited a depressive-like phenotype, highlighted by a deteriorated coat state,
Surget, A.; Hen, R.; Belzung, C.
a higher immobility duration in the tail suspension test (TST), and a delayed reversal learning in a
Increasing Adult Hippocampal
water maze procedure. Promoting AHN before UCMS was sufficient to prevent the development of
Neurogenesis Promotes Resilience in
stressed-induced behavioral changes in the TST and the water maze, reflecting an effect of AHN on
a Mouse Model of Depression. Cells
2021, 10, 972. https://doi.org/
stress resilience. Taken together, our data suggest that increasing AHN promotes stress resilience on
10.3390/cells10050972 some depressive-like symptoms but also in cognitive symptoms, which are often observed in MD.

Academic Editor: Boldizsár Czéh Keywords: depression; adult hippocampal neurogenesis; chronic stress; inhibition; flexibility;
depressive-like behaviors; hippocampus; stress resilience
Received: 25 March 2021
Accepted: 19 April 2021
Published: 21 April 2021
1. Introduction
Publisher’s Note: MDPI stays neutral Major depression (MD) is a highly debilitating disorder that affects millions of people
with regard to jurisdictional claims in
worldwide and is thus a main contributor to the global burden of healthcare and the
published maps and institutional affil-
economy. Although many pharmacological approaches have been developed to alleviate
iations.
symptoms, almost a third of patients still exhibit symptoms after receiving a treatment [1,2],
and only 30% go through remission. Even though MD is not a homogenous pathology, it
exhibits core symptoms such as anhedonia and depressive mood, often associated with
sleep and eating disturbances, psychomotor alterations, and anxiety.
Copyright: © 2021 by the authors. Antidepressants currently in use are mainly based on active molecules modulating
Licensee MDPI, Basel, Switzerland. monoamine systems; among classic drugs are the selective serotonin reuptake inhibitors
This article is an open access article
(SSRIs). The efficacy of these drugs appears after a delay, leading to the idea that their
distributed under the terms and
antidepressant effects are not solely due to the direct modulation of monoamines but are
conditions of the Creative Commons
also the consequence of long-term downstream changes. As a matter of fact, antidepressants
Attribution (CC BY) license (https://
induce therapeutic effects along with an increase in hippocampal neurogenesis [3–9].
creativecommons.org/licenses/by/
Interestingly, rather than being a concomitant process, the increase in adult hippocampal
4.0/).

Cells 2021, 10, 972. https://doi.org/10.3390/cells10050972 https://www.mdpi.com/journal/cells

Cells 2021, 10, 972 2 of 18

neurogenesis (AHN) would be causal because its inhibition or depletion blocks some effects
induced by antidepressants [10–15].
AHN is a complex process by which adult-born neurons are continuously generated
in the hippocampus during adulthood in mammals. Neuronal stem cells, located in the
subgranular zone of the dentate gyrus (DG) of the hippocampus, go through successive
steps of proliferation, migration, and maturation in order to develop into neurons fully
incorporated into pre-existing circuits. The role of adult-born neurons has been exten-
sively studied since their discovery by Altman and colleagues [16]. Interestingly, AHN
is implicated in cognitive functions such as memory and learning, as well as in mood
and stress regulation [11,17–24]. While precise mechanisms remain elusive, recent studies
support the idea that adult-born neurons could act as key regulators of global hippocam-
pal activity. Indeed, while inhibition of AHN can increase the overall excitability of the
hippocampus, stimulation or promotion of AHN lowers mature granule cells’ activity
in the DG [25–27]. Thereby, AHN could participate in functions supported directly by
the hippocampus. Additionally, very recent studies showed that enhancement of AHN
could not only affect neuronal activity in trisynaptic hippocampal networks but also in
downstream circuits [28–30].
Many factors can act on AHN, such as enrichment and antidepressants, which posi-
tively regulate it [31,32], while stress negatively regulates it by decreasing the proliferation,
maturation, and survival of adult-born neurons [33–35]. Because stress is considered as a
trigger for MD [36] and that antidepressants induce behavioral responses supported by
AHN [11–14,37], it is supposed that a stress-induced impairment of AHN could be a key
factor in the pathophysiology of stress-related disorders such as MD. Accordingly, many
studies focused on the antidepressant effects of AHN on stress-induced impairments once
the symptoms were present, mainly reflecting the effects of AHN on the recovery, while
the protective role of AHN remains less studied. However, the involvement of AHN in
stress resilience is supported by the fact that factors that positively regulate AHN tend to
also promote resilience [38]. Additionally, because AHN buffers stress responses [39], it is
likely that increasing AHN could prevent the deleterious effects of stress on cognition and
mood. Indeed, some recent papers highlighted a potential role of AHN in the resilience to
stress: while the inhibition of the activity of adult-born neurons can lead to a vulnerability
to social defeat, increasing AHN promotes resilience [27]. Similarly, in an animal model
of chronic stress based on chronic corticosterone injections, increasing AHN was able to
protect preventively from stress-induced depressive-like behaviors [18].
Given the functional role of adult-born neurons, promoting AHN before stress could
prevent stress-induced impairments on hippocampus-dependent functions, which are
usually impaired in MD. Furthermore, if cognitive alterations are not the core symptoms of
MD, it is currently well known that patients suffering from MD exhibit memory deficits,
decreased flexibility, and impaired inhibitory control [40], which could then be prevented
by increasing AHN. Nonetheless, studies that tried to highlight a link between AHN and
stress resilience focused on anxiety and depressive-like behaviors while the effects on
cognitive functions are still poorly assessed. Additionally, because the hippocampus is part
of the corticolimbic system, normalizing its activity through AHN enhancement could also
affect interconnected regions involved in stress and mood regulation [41,42] and thereby
prevent the development of depressive and anxiety-like behaviors.
In an attempt to shed more light on the potential link between AHN and stress
resilience, we stimulated AHN in an animal model of unpredictable chronic mild stress
(UCMS), which is a naturalistic model of MD [43,44]. We used an inducible transgenic
mouse model in which it is possible to remove the pro-apoptotic Bax gene from neural stem
cells at the time of tamoxifen injection, which in turn promotes adult-generated neuron
survival and increases AHN [17]. In order to quantify the increase in AHN, survival and
maturation indexes were assessed by using doublecortin immunochemistry, a marker of
immature neurons. We first analyzed the effect of chronic stress and AHN on anxiety and
depressive-like behaviors; then, because cognitive deficits are often observed in MD [40],
Cells 2021, 10, 972 3 of 18

we evaluated through a water maze paradigm the functions of flexibility and inhibition in
UCMS mice and the effects of increasing AHN on them.

2. Method
2.1. Animals
Male iBax mice aged 11 weeks at the start of the experiments were used (N = 83).
The generation and characterization of the Nes-CreERT2 transgenic mouse line are already
described in detail [17]. To induce CreERT2 -mediated recombination of Bax in neural
stem cells in the adult brain, mice of 11 weeks of age were given 55 mg/kg tamoxifen
(20 mg mL−1 , T-5648, Sigma, St-Louis, MO, USA) intraperitoneally, once a day for 5 con-
secutive days, or 10 mL/kg body weight of corn oil for vehicle-treated mice. Animals
were group-housed and kept under standard laboratory conditions (12/12 h light-dark
cycle with lights on at 8:30 p.m. and room temperature at 22 ± 2 ◦ C), in enriched cages
(46 × 29 × 25 cm, PAULA Ferplast, Castelgomberto, Italy) for 4 weeks starting from the
first tamoxifen injection. Access to food and water was ad libitum. Mice were then divided
into two groups depending on whether they had received the unpredictable chronic mild
stress regimen (UCMS) or not (non-stressed: NS). Mice subjected to the UCMS regimen
were housed in 24 × 11 × 12 cm cages without any environmental enrichment from week 5
to week 9, while NS mice remained in enriched cages. All procedures were compliant with
Directive 2010/63/EU guidelines on animal ethics (referral 2019092017334223, approved
by the ethical committee CEEvdl).

2.2. Experimental Design

Mice of 11 weeks of age received 5 consecutive injections of tamoxifen and were
housed in an enriched environment for 4 weeks, and then half of the mice underwent the
UCMS protocol for 4 additional weeks, while the other half stayed in enriched cages and
were not stressed. Based on stress application and treatment, mice were divided into four
groups, whether they underwent the UCMS regimen or not (UCMS: N = 43; NS: N = 40) and
whether they received tamoxifen or vehicle treatment (tamoxifen: N = 43; vehicle: N = 40).
After the UCMS regimen, all mice were tested for behaviors; however, we separated mice
into two cohorts depending on which behavior was assessed. The first cohort (N = 49) was
tested for anxiety-like behaviors as well as anhedonic and goal-motivated behaviors, and
at the end of the experiment, mice were sacrificed to quantify hippocampal neurogenesis.
Additionally, a second cohort (N = 34) was tested for stress-coping behaviors and cognition
only (Figure 2a). We tested mice for cognitive impairments with a water maze learning
task. Although learning tasks are known to be affected by hippocampal neurogenesis, it
is also frequently observed that hippocampal neurogenesis is modified by learning; for
this reason, we chose to test mice in the water maze paradigm in a second cohort where
only the stress-coping behaviors were assessed before in order to avoid any side effect of a
learning-induced increase in hippocampal neurogenesis on depressive-like and anxiety
behaviors and/or on the quantification of hippocampal neurogenesis.

2.3. UCMS
The UCMS regimen was used as previously described [10,45]. Briefly, UCMS mice
were isolated in individual cages and subjected to various socio-environmental stressors
of mild intensity on a daily basis according to an unpredictable schedule for 4 weeks.
Stressors included removal of sawdust, damping the sawdust, replacing the sawdust with
water at 21 ◦ C, repeated sawdust changes, tilting the cages at 45◦ , placing a mouse into a
cage that had been occupied by another mouse, contention in small tubes, and alterations
of the light/dark cycle.
Cells 2021, 10, 972 4 of 18

2.4. Coat State

The coat state of each animal was assessed at the beginning of each week from week 5
to week 9 of the experiment in order to monitor the behavioral degradation induced by the
UCMS regimen [10].

2.5. Nest-Building Test

Animals were isolated in bigger individual cages (Makrolon Type III, Tecniplast,
Buguggiate, Italy) 12 h before the start of the test for habituation. One square piece of
pressed cotton (5 × 5 cm) was placed in each cage at 7:30 a.m., and the quality of the nest
was assessed at two time points: after 5 h and after 24 h according to an already described
1–5 rating protocol (Deacon, 2006). The mice were then put back in their home cages.

2.6. Light/Dark Box

The apparatus consisted of a lightbox with transparent sides (20 × 20 × 15 cm) and
a dark box with the same dimensions and opaque sides. The boxes were connected by a
small opaque plastic tunnel (5.5 × 6.5 × 10 cm). Animals were placed in the lightbox, and
once the animal entered the tunnel, the test started and lasted for 5 min. The number of
entries and the total time spent in the dark box were recorded. An entry into a box was
recorded when the animal placed all four paws in the box. This procedure is based on
the innate avoidance of mice for well-lit areas and is used as an indicator of anxiety-like
behaviors [46].

2.7. Novelty-Suppressed Feeding Test (NSF)

As previously described [10], the apparatus consisted of a square (30 × 30 × 30 cm)
with the floor covered with litter and illuminated by red light; a small food pellet was
placed in the middle of the apparatus on a small piece of white paper (2 × 2 cm). This test
is based on the natural aversion of mice for open areas, and they face a conflict between
the drive to eat and the aversion for the open space. Once the mice were positioned in
the apparatus (head facing the wall), the latency to explore and the latency to eat the
pellet were measured. Once the mice started to bite the pellet, they were gently put back
in their home cages with the pellet and allowed to eat for 5 additional minutes, and the
consumption of the pellet was then recorded.

2.8. Splash Test

The test was conducted as previously described [47]. Mice were sprayed on their
dorsal coats with a 10% sucrose solution in their home cages and under red light. Grooming
behaviors were stimulated by the palatability of the solution. The duration of grooming
was recorded during a period of 5 min after the spray.

2.9. Cookie Test

To test for anhedonic traits, mice were subjected to a reward maze test [11,48]. The
apparatus was composed of three consecutive chambers (20 × 20 × 20 cm), which com-
municate through openings. During the test, mice were placed in the first chamber, and
the palatable reward (butter cookie, Saint-Michel) was placed in the center of the third
chamber. Once the mice entered the second chamber, the communication between the first
and the second chamber was closed. The latency to eat the reward and the consumption of
the reward was measured for up to 5 min from the moment the mice were placed in the
first chamber.
On the testing day, common food pellets were removed from the cage lid 1 h before the
test. Moreover, to minimize environmental neophobia, mice were habituated three times
to the device, and the test was performed under red light. Mice were familiarized with
the cookie by giving a sample every 2 days 1 week before the test (for more details, see
supplementary material and methods).
Cells 2021, 10, 972 5 of 18

2.10. Tail Suspension Test

Stress-coping behaviors were assessed by the tail suspension test: mice were sus-
pended above the ground by their tails with tape for 6 min without any possibility of
escape or hold onto any surface [49]. This test allows measuring the immobilization time,
which reflects the resignation of the mice and then can illustrate depressive-like behaviors.

2.11. Flexibility/Inhibition in the Water Maze

This test aims to evaluate two aspects of executive functions: cognitive flexibility and
inhibitory control. Executive functions represent a set of high-level cognitive processes
that support the elaborations and control of complex and adaptive behavioral responses.
Among these processes, cognitive flexibility represents the ability to switch between dif-
ferent strategies or behavioral responses, while inhibitory control represents the ability
to inhibit or override a behavioral response previously learned that became inefficient or
irrelevant in order to implement adapted goal-oriented strategies.
We used a cross-shaped water maze with 4 arms (N, E, S, W) placed in a circular pool
(diameter: 90 cm). The water was maintained at a temperature of 22 ± 2 ◦ C, and the light
intensity at the center of the device was approximately 100 lux. Mice were trained to find a
hidden platform placed in one of the maze’s arms: the platform (5 × 5 cm2 ) was placed at
its extremity slightly below the water surface (between 1 and 1.5 cm). The arm N contained
a visual cue (a card with a strong black-and-white contrast) and, just above its extremity, a
small lamp lit the visual cue at 500 lux. Sensory cues were placed in the water: small plastic
lenses to support contextual learning; whether the water was clean or filled with lenses
indicated a different localization strategy to reach the platform. The departure occurred
at the extremity of one of the 3 other arms (E, S, W), the mouse’s head angled toward the
center of the maze, and the departure position varying from a trial to another.
Mice had to learn different strategies in order to find the platform depending on the
context (with or without lenses). A specific context was then associated with a specific task:
mice had to find the platform by allocentric navigation strategy depending on the location
of the cue (“cue training”), or by egocentric navigation strategy using a specific sequence
of directions, independently from the arm of departure (“direction training”). Accordingly,
each mouse during the experiment was subjected to 2 different contexts, and each of these
contexts was associated with a different task.
For the training paradigm, on days 1–2, mice had to learn one of the two strategies
in the presence of the first tactile cue. They had to associate a specific context to a task
(Context A-Task 1). To do so, each mouse was given 4 blocks composed of 5 trials in a day,
with a 30-min inter-block delay and a 30-s inter-trials delay on the platform.
On days 3–4, mice had to learn the second strategy in the presence of the other tactile
cue (Context B-Task 2). Similarly to the first 2 days, mice underwent a learning phase of 4
blocks of 5 trials each day.
For each trial, mice were released from different starting positions; if the mouse
had not reached the platform within 1 min, the experimenter gently led the mouse to
the platform.
Subsequently, 1 week after the training phase, mice underwent the flexibility test. Each
mouse was given 6 blocks in a day, but this time we alternated the cue task and the direction
task between blocks. Then, blocks 1-3-5 were in context A while blocks 2-4-6 were in context
B; accordingly, mice had to alternate their strategies to find the platform (Figure 3b). For
all trials, the latency to reach the platform and the total number of perseverative errors
(perseverative/interfering errors were defined as entries in the arm where the platform
should be located in the other learned task) were recorded.
Thereafter, to test for inhibition (reversal learning), each mouse was given 6 blocks in
a day, and contexts that were previously associated with a specific task, “Context A-Task 1”
and “Context B-Task 2” were switched into “Context A-Task 2” or Context B-Task 1”.
Accordingly, mice had to inhibit the behavioral response previously learned to find the
platform. For all trials, the latency to reach the platform and the total number of persistent
Cells 2021, 10, 972 6 of 18

errors (when mice enter the arm where the platform was located in the previously learned
task) were recorded (for more details, see Supplementary Material and Methods).

2.12. Immunohistochemistry
At the end of the experiments, mice were injected with an overdose of pentobarbital so-
lution (100 mg/kg, Dolethal® , Vetoquinol, Lure, France), then transcardially perfused with
50 mL of heparin saline solution to remove blood, followed by 100 mL of 4% paraformalde-
hyde (PFA) in phosphate buffer 0.1 M solution to fix the brain. Brains were then extracted
and placed overnight in PFA 4% solution, then cryoprotected in sucrose solution (20%) and
stored at 4 ◦ C. For immunochemistry, brains were cut into 30-µm coronal sections with a
cooled microtome (−20 ◦ C, Leica CM 3050 S, Paris, France). In order to quantify the AHN, a
free-floating immunochemistry against doublecortin (DCX), a marker of immature neurons,
was performed. Briefly, a heat antigen retrieval in citrate buffer (10 mM, pH = 6) was per-
formed on brain slices for 10 min at 95 ◦ C followed by incubation with primary antibodies
at 4 ◦ C for 48 h (DCX antibody 1/750 dilution, ab18723; Abcam, Cambridge, UK) and
incubation with secondary antibodies (Donkey anti-mouse Alexa Fluor555, 1/500 dilution,
ab150106; Abcam) for 2 h at room temperature. Finally, slices were mounted onto slides,
covered with Vectashield® mounting medium (Vector Laboratories, Burlingame, CA, USA),
and stored at 4 ◦ C (for more details, see supplementary material and methods).
The immunolabeled sections were observed under a Zeiss Z.2 Imager microscope in
emitted-light mode, and DCX-labeled cells were counted in the DG at X20 magnificence.
An unbiased and blinded protocol was used to count the DCX-labeled cells in the granule
cell layer of the DG along its septotemporal axis. For quantification, 7 matched sections
were selected for each mouse (4 sections for the dorsal hippocampus from bregma −1.3 to
−1.8 mm, and 3 sections for the ventral hippocampus from bregma −3.3 to −3.6 mm) and
DCX cells were expressed as normalized cellular densities (DCX cells+/mm2 ). Additionally,
to evaluate the maturation, DCX cells with at least tertiary dendrites were counted, the
maturation index was then expressed as the ratio of DCX cells with at least tertiary dendrites
over the total number of DCX cells.

2.13. Statistical Analysis

Data were analyzed using ordinary two-way ANOVAs or mixed-factor two-way
ANOVAs (repeated measures over time) followed by a Fisher post hoc test when needed
for multiple comparisons (only when p < 0.05). For behavioral tests and DCX quantification,
we evaluated the effect of treatment (tamoxifen or vehicle) and stress (UCMS or NS); thus,
we had two categorical factors. For the coat state, we had the stress and treatment as
categorical factors, as well as the weeks. For the water maze task, we had stress, treatment,
and blocks as categorical factors. All results are presented as mean ± SEM. Detailed
statistical analyses are provided in the supplementary statistics.

3. Results
3.1. Genetic Deletion of Bax Gene Tends to Enhance Survival and Promotes Maturation in
Adult-Born Neurons
We first analyzed the DCX-labeled cells in the total hippocampus (Figure 1a). We
observed that UCMS did not impact the total DCX cells (F1,12 = 0.089, p = 0.771) or the
maturation index (F1,12 = 1.180, p = 0.299) regardless of the treatment. Tamoxifen induced a
main increase in the DCX-labeled cells (F1,12 = 4.755, p = 0.050) compared to the vehicle
groups. Multiple comparisons showed no significant difference between non-stressed
vehicle mice and non-stressed tamoxifen mice (Fisher: p = 0.236), but UCMS tamoxifen
mice tended to have an increase in the DCX-labeled cells compared to UCMS vehicle mice
(Fisher: p = 0,091). The effect of tamoxifen on the maturation index was more important
(F1,12 = 19.322, p = 0.001), revealing an increase in the maturation in the non-stressed mice
tamoxifen compared to vehicle mice (Fisher: p = 0.009) as well as in UCMS tamoxifen mice
 maturation index (F1,12 = 1.180, p = 0.299) regardless of the treatment. Tamoxifen induced
a main increase in the DCX-labeled cells (F1,12 = 4.755, p = 0.050) compared to the vehicle
groups. Multiple comparisons showed no significant difference between non-stressed
vehicle mice and non-stressed tamoxifen mice (Fisher: p = 0.236), but UCMS tamoxifen
Cells 2021, 10, 972 mice tended to have an increase in the DCX-labeled cells compared to UCMS vehicle 7 of 18
mice (Fisher: p = 0,091). The effect of tamoxifen on the maturation index was more im-
portant (F1,12 = 19.322, p = 0.001), revealing an increase in the maturation in the non-
stressed mice tamoxifen compared to vehicle mice (Fisher: p = 0.009) as well as in UCMS
compared mice
tamoxifen to UCMS vehicle
compared to mice
UCMS vehiclep mice
(Fisher: = 0.009). Additionally,
(Fisher: we analyzed the
p = 0.009). Additionally, we DG
separately along its dorso-ventral axis (Figure 1b–c).
analyzed the DG separately along its dorso-ventral axis (Figure 1b–c).

Figure 1.
Figure 1. Modifications
Modificationsinduced
inducedbyby UCMS
UCMSand tamoxifen
and tamoxifenon hippocampal
on hippocampal neurogenesis. (a) DCX-labeled
neurogenesis. cells and
(a) DCX-labeled cellsmatu-
and
ration index in the total hippocampus. UCMS did not affect the number of adult-born neurons or the ratio of DCX-labeled
maturation index in the total hippocampus. UCMS did not affect the number of adult-born neurons or the ratio of DCX-
cells with at least tertiary dendrites on the total number of DCX-labeled cells, defined as the maturation index in the total
labeled cells with at least tertiary dendrites on the total number of DCX-labeled cells, defined as the maturation index
hippocampus; however, tamoxifen increased both. (b) DCX-labeled cells and maturation index in the dorsal hippocampus.
in the total
UCMS hippocampus;
decreased the numberhowever, tamoxifen
of total increased
DCX-labeled cells, both. (b) DCX-labeled
and tamoxifen inducedcells and maturation
an increase index in the
in the maturation dorsal
index. (c)
hippocampus. UCMS decreased the number of total DCX-labeled cells, and tamoxifen induced an increase
DCX-labeled cells and maturation index in the ventral hippocampus. No effect of UCMS or tamoxifen was observed in in the maturation
index. (c) DCX-labeled
the total DCX-labeled cells
cells;and maturation
however, index in
tamoxifen the ventral increased
significantly hippocampus. No effect ofindex.
the maturation UCMS(d) or tamoxifen was
Representative
observed in the total DCX-labeled cells; however, tamoxifen significantly increased the maturation index. (d) Representative
hippocampal sections immunostained for DCX from NS vehicle and tamoxifen mice, as well as UCMS vehicle and tamoxifen
mice. ANOVA followed by post hoc multiple comparisons when necessary. *: p < 0.05 UCMS vs. NS; #: p < 0.05; ##: p < 0.01;
###: p < 0.001 vehicle vs. tamoxifen. UCMS: unpredictable chronic mild stress; NS: non-stressed.

Results showed that in the dorsal hippocampus, UCMS had the main effect by de-
creasing the total number of DCX-labeled cells (F1,12 = 4.968, p = 0.046), and we observed
a trend toward a decrease in the DCX-labeled cells in UCMS vehicle mice compared to
non-stressed vehicle (Fisher: p = 0.091) but no significant difference between non-stressed
and UCMS tamoxifen mice (Fisher: p = 0.213). However, UCMS did not affect the matura-
tion index (F1,12 = 2.260, p = 0.159). Tamoxifen only tended to increase the total number of
DCX-labeled cells (F1,12 = 3.922, p = 0.071) but significantly increased the maturation index
(F1,12 = 7.186, p = 0.020) in the dorsal hippocampus. Multiple comparisons revealed a trend
Cells 2021, 10, 972 8 of 18

toward an increase in the maturation index in the non-stressed tamoxifen mice compared
to non-stressed vehicle mice (Fisher: p = 0.072) and in the UCMS tamoxifen mice compared
to UCMS vehicle mice (Fisher: p = 0.094; Figure 1b).
Concerning the ventral hippocampus, no main effect of UCMS was observed in
the total number of DCX-labeled cells (F1,12 = 2.942, p = 0.112) or the maturation index
(F1,12 = 0.005, p = 0.946) compared to non-stressed group. However, we observed that
tamoxifen tended to increase the total number of DCX cells (F1,12 = 3.948, p = 0.070) and
significantly increased the maturation index (F1,12 = 40.715, p < 0.0001) in non-stressed
tamoxifen mice (Fisher: p < 0.001) and UCMS tamoxifen mice (Fisher: p < 0.001; Figure 1c)
compared to non-stressed vehicle mice and UCMS vehicle mice, respectively.

3.2. Depressive-Like and Anxiety-like Behaviors Induced by UCMS Were Partially Prevented by
Increasing AHN
The effects of UCMS were first evaluated on physical assessments such as the coat state
(Figure 2b) and assessed with the nest-building score, a measure of daily-living behaviors
(Figure 2c). Two-way ANOVA with repeated measures revealed significant differences
between groups for the coat state (week*UCMS*tamoxifen: F5,225 = 9.249, p < 0.0001).
UCMS induced a gradual deterioration of the coat state (week*UCMS: F5,225 = 68.254,
p < 0.0001), reaching statistical significance from the second week on (Figure 2b). Indeed,
UCMS vehicle mice had a significantly increased score compared to non-stressed vehicle
mice from week 2 to week 6 (week 2–week 6, Fisher: p < 0.00001), similarly, UCMS
tamoxifen mice had a significantly increased score compared to non-stressed tamoxifen
mice starting from the second week (week 2–week 6, Fisher: p < 0.001). Tamoxifen did
not have effect on the coat state (F1,45 = 0.028, p < 0.968). Additionally, evaluation of
the nest-building score highlighted a UCMS*tamoxifen interaction effect (F1,45 = 8.299,
p = 0.006), showing a UCMS effect in vehicle mice, as UCMS mice had higher scores than
non-stressed mice (Fisher: p < 0.0001). This UCMS effect was not present in tamoxifen mice:
the nest-building score was not different between UCMS and non-stressed tamoxifen mice
(Fisher: p = 0.905; Figure 2c). Moreover, non-stressed tamoxifen mice had a higher score
than vehicle non-stressed mice (Fisher: p = 0.005). Interestingly, increasing hippocampal
neurogenesis was not sufficient to prevent the coat state degradation induced by UCMS,
but it appeared that it could affect daily-life measures such as nest-building.
The effects of UCMS on goal-directed behaviors were assessed by the duration of
grooming following a splash of sucrose solution over the fur of the animal (Figure 2d).
Our results revealed no effect of UCMS (F1,45 = 1.604, p = 0.210) or tamoxifen (F1,45 = 0.792,
p = 0.378).
Additionally, the cookie test was used to evaluate anhedonia (Figure 2e); to this end,
we compared the latency to eat palatable food as well as the total consumption between
groups. No difference between groups was observed for the consumption following UCMS
(F1,41 = 0.856, p = 0.360) and tamoxifen injection (F1,41 = 0.043, p = 0.837). However, while
UCMS had no significant effect on the latency to eat (F1,41 = 1.867, p = 0.179), an effect
of tamoxifen was present (F1,41 = 5.593, p = 0.023), revealing a significant decrease in
the latency to eat the cookie in UCMS tamoxifen mice compared to UCMS vehicle mice
(Fisher: p = 0.008); this effect was not observed in non-stressed tamoxifen mice compared
to non-stressed vehicle mice (Fisher: p = 0.498). Accordingly, even if UCMS did not elicit
depressive-like behavior in the cookie test, it seems that tamoxifen-induced increased
hippocampal neurogenesis was sufficient to affect motivated behaviors in UCMS mice.
In order to measure anxiety, we used different behavioral paradigms such as the
novelty-suppressed feeding test (NSF) and the light/dark box (Figure 2f,g). We observed
no effect of UCMS and tamoxifen in the NSF on the latency to smell and the consumption
(UCMS: latency to smell F1,45 = 0.051, p = 0.823; consumption F1,45 = 0.293, p = 0.591;
tamoxifen: latency to smell F1,45 = 1.716, p = 0.197; consumption F1,45 = 1.429, p = 0.238).
Thus, because no effect of UCMS was observed, it renders the results non-conclusive on
how neurogenesis could affect stress-elicited anxiety-like behaviors in the NFS test.
Cells 2021, 10, 972 9 of 18
Cells 2021, 10, x FOR PEER REVIEW 9 of 19

Figure 2.2. The

Figure The effects
effects of
of UCMS
UCMS and and tamoxifen
tamoxifen were
were assessed
assessed in in physical
physical measures,
measures, motivational
motivational behaviors,
behaviors, anxiety-like
anxiety-like
and depressive-like behaviors in the first cohort. (a) Schematic representation of the experimental design. (b–g) UCMS
and depressive-like behaviors in the first cohort. (a) Schematic representation of the experimental design. (b–g) UCMS
elicited abnormal behaviors partially alleviated by tamoxifen. (b) UCMS induced deterioration of the coat state from the
elicited
second abnormal
week to the behaviors
end of thepartially alleviated
experiment, whichbywas
tamoxifen. (b) UCMS
not reversed induced deterioration
by tamoxifen. of the coat
(c) UCMS modified state from the
the nest-building
second week to the end of the experiment, which was not reversed by tamoxifen. (c) UCMS
score in vehicle mice but not in tamoxifen mice. (d) No effect of UCMS or tamoxifen was observed in the splash modified the nest-building
test. (e)
score in vehicle mice but not in tamoxifen mice. (d) No
No difference was found in the consumption of the cookie; however, UCMSeffect of UCMS or tamoxifen mice had a decreased splash
was observed in the latencytest.
to
eatNo
(e) thedifference
cookie compared
was found to the UCMS
in the vehicle mice.
consumption (f) No
of the effecthowever,
cookie; of UCMSUCMS or tamoxifen wasmice
tamoxifen observed
had aindecreased
the NSF test. (g)
latency
Ineat
to thethe
light/dark box, UCMS
cookie compared to mice had anvehicle
the UCMS increased
mice. number of entries
(f) No effect in theor
of UCMS dark box compared
tamoxifen to non-stressed
was observed in the NSF mice;
test.
nevertheless, no effect of UCMS and tamoxifen were seen for the time spent in the dark box. ANOVA,
(g) In the light/dark box, UCMS mice had an increased number of entries in the dark box compared to non-stressed mice; followed by post
hoc multipleno
nevertheless, comparisons
effect of UCMSwhen necessary.
and tamoxifen *: were
p < 0.05;
seen**:
forpthe
< 0.01;
time ***: p <in0.001
spent UCMS
the dark box.vs. NS; #: pfollowed
ANOVA, < 0.05; ##:
by ppost
< 0.01
hoc
tamoxifen vs. vehicle. UCMS: unpredictable chronic mild stress; NS: non-stressed; NSF: novelty-suppressed feeding test.
multiple comparisons when necessary. *: p < 0.05; **: p < 0.01; ***: p < 0.001 UCMS vs. NS; #: p < 0.05; ##: p < 0.01 tamoxifen
vs. vehicle. UCMS: unpredictable chronic mild stress; NS: non-stressed; NSF: novelty-suppressed feeding test.
The effects of UCMS on goal-directed behaviors were assessed by the duration of
grooming
Anxietyfollowing
was alsoa splash of sucrose
assessed solution over
in the light/dark box,theinfur of the
which animal
the (Figure
frequency and2d).
time
Our results revealed no effect of UCMS (F 1,45 = 1.604, p = 0.210) or tamoxifen (F1,45 = 0.792, p
spent in the dark box were measured (Figure 2g). An effect of UCMS was observed for
= 0.378).
the entries in the dark box while no effect was seen for the time spent in the dark box
Additionally,
(respectively: F1,45 the cookiep test
= 14.269, was Fused=to0.970,
= 0.000; evaluate anhedonia
p = 0.330). UCMS (Figure 2e);mice
vehicle to this
entered
1,45
end, we compared the latency to eat palatable food as well as the total consumption
the dark box more compared to the non-stressed vehicle mice (Fisher: p = 0.003); similarly, be-
tween groups. No difference between groups was observed for the consumption
Cells 2021, 10, 972 10 of 18

UCMS tamoxifen mice entered the dark box more than non-stressed tamoxifen mice (Fisher:
p = 0.037). Additionally, no effect of tamoxifen was observed in this test for the entries
(F1,45 = 0.505, p = 0.481) or the duration spent in the dark box (F1,45 = 0.605, p = 0.441). Taken
together, these results show that in our experiments, increasing hippocampal neurogenesis
was not sufficient to prevent UCMS-induced anxiety-like effects.
In the second cohort, which later underwent the water maze paradigm, stress-coping
behaviors were assessed in the tail suspension test (Figure 3a). Two-way ANOVA revealed
only a trend regarding tamoxifen injection (F1,30 = 3.302, p = 0.079) but a main effect of
UCMS (F1,30 = 7.039, p = 0.013). UCMS vehicle mice spent more time immobile compared to
the non-stressed vehicle mice (Fisher: p = 0.015), while no difference was observed between
UCMS tamoxifen mice and non-stressed tamoxifen mice (Fisher: p = 0.260). Moreover,
UCMS tamoxifen mice spent less time immobile than UCMS vehicle mice (Fisher: p = 0.040).
Our data revealed that while UCMS induced higher immobility, increasing hippocampal
neurogenesis prevented the effects of UCMS in this paradigm, thus decreasing deficits in
stress-coping behaviors.

3.3. AHN Promotion Partially Promotes Resilience to Stress-Induced Cognitive Impairments

Although depressive mood and anhedonia are considered core symptoms of de-
pression, patients suffering from depressive episodes also exhibit important cognitive
impairments [40]. As such, memory deficits, as well as difficulties in executive functions,
are often observed. Because hippocampal neurogenesis has been widely linked to cogni-
tion [17,41,50,51], and that few studies have evaluated the protective effects of promoting
hippocampal neurogenesis on stress-elicited cognitive impairments in an animal model of
MD, we decided to evaluate the effect of increasing hippocampal neurogenesis on cognitive
impairments induced by UCMS. Therefore, we used a water maze protocol in order to
evaluate flexibility and inhibition (Figure 3b). Since associative learning can promote
hippocampal neurogenesis [52], this cognitive task was performed on the second cohort
after the TST in order to avoid any effect of learning-induced hippocampal neurogenesis
stimulation on previous measures.
Our results showed significant differences between sessions in the flexibility task
(Figure 3c), highlighting a progressive decrease in the latency to find the platform all
along the procedure (session: F5,135 = 16.753, p < 0.0001); nevertheless, no effect of UCMS
was observed (F1,27 = 0.001, p = 0.970). Moreover, UCMS elicited no difference for the
average latency to find the platform (F1,27 = 0.060, p = 0.809). Tamoxifen did not induce a
significant effect, but a trend was observed, showing a slight decrease in latency to reach
the platform all along the procedure compared to vehicle mice (F1,27 = 3.072, p = 0.088)
as well as a trend to decrease the average latency compared to vehicle mice (F1,27 = 3.267,
p = 0.082). Moreover, while perseverative failures were not affected by UCMS (F1,27 = 0.008,
p = 0.931), we observed a main effect of tamoxifen, which seemed to have induced a
decrease in perseverative failures (F1,27 = 5.183, p = 0.031; Figure 3c). Multiple comparisons
revealed no difference between non-stressed vehicle and non-stressed tamoxifen mice
(Fisher: p = 0.162), whereas tamoxifen tended to decrease the perseverative failures in
UCMS mice compared to UCMS vehicle mice (Fisher: p = 0.087).
For the inhibition task (Figure 3c), two-way ANOVA with repeated measures revealed
significant differences among groups (session*UCMS*tamoxifen: F5,135 = 3.586, p = 0.004),
showing a decrease in latency to find the platform all along the procedure for all groups
(Session: F5,135 = 51,808, p < 0.0001) and a UCMS*tamoxifen interaction effect, revealing
that UCMS vehicle mice spent more time to find the platform compared to all other groups
(F1,27 = 9.152, p = 0.005). Indeed, in the two first blocks, UCMS vehicle mice spent more time
to reach the platform compared to non-stressed vehicle mice (block 1, Fisher: p < 0,0001;
block 2, p = 0.006) and the UCMS tamoxifen mice (block 1, Fisher: p < 0,0001; block 2,
p = 0.014). As such, while UCMS seems to delay the learning of the new task in vehicle
mice, increasing hippocampal neurogenesis seems to prevent this effect.
Cells 2021, 10, 972 11 of 18
Cells 2021, 10, x FOR PEER REVIEW 11 of 19

Figure 3.
Figure 3. Promoting
Promotinghippocampal
hippocampalneurogenesis
neurogenesiscounteracts
counteracts UCMS-induced
UCMS-induced cognitive impairments
cognitive impairments in the
in water maze
the water and
maze
TST in the second cohort. (a) The TST revealed that UCMS vehicle mice spent more time immobile than non-stressed
and TST in the second cohort. (a) The TST revealed that UCMS vehicle mice spent more time immobile than non-stressed
vehicle mice, which was reversed by tamoxifen. (b) Schematic representation of the water maze protocol. While task 1 was
vehicle mice, which was reversed by tamoxifen. (b) Schematic representation of the water maze protocol. While task 1 was
based on an egocentric strategy (direction task), task 2 required an allocentric strategy (cue task). In context A (in gray),
based on an
the water wasegocentric
filled withstrategy
plastic(direction task), task
lenses, whereas 2 required
for context B (inan allocentric
white), strategy
the water was(cue
not. task). In context
(c) A session A (in
effect wasgray), the
present,
water
showing wasa filled
decrease withinplastic lenses,towhereas
the latency reach theforplatform
context B (in white),
during the watertest.
the flexibility wasNonot. (c) Aofsession
effect UCMSeffect was present,
or tamoxifen was
showing
observed afor decrease in the
flexibility, latency
either in thetoevolution
reach theof platform during
the latency theaverage
or the flexibility test. however,
latency; No effect ofweUCMS or tamoxifen
observed was
that tamoxifen
decreasedfor
observed theflexibility,
number ofeitherperseverative failures.of(d)
in the evolution theThe inhibition
latency or thetest revealed
average a decrease
latency; in the
however, wetime to reach
observed thetamoxifen
that platform
all along the
decreased thetest,
numberbut aofUCMS*tamoxifen interaction
perseverative failures. (d) Theeffect highlighted
inhibition the facta that
test revealed UCMS
decrease in vehicle
the timemice spent
to reach themore time
platform
to find
all alongthe
theplatform
test, butcompared to all other
a UCMS*tamoxifen groups ineffect
interaction blockhighlighted
1 and blockthe 2. fact
Additionally,
that UCMSthe comparison
vehicle of the
mice spent average
more time
latencies
to find therevealed
platform that while UCMS
compared to allelicited an increase
other groups in latency
in block 1 and to reach
block 2. the platform and
Additionally, thean increase inofperseverative
comparison the average
failures, tamoxifen totally reversed these effects. (c-d) ANOVA, followed by post hoc multiple comparisons when neces-
latencies revealed that while UCMS elicited an increase in latency to reach the platform and an increase in perseverative
sary. *: p < 0.05; **: p < 0.01 UCMS vs. NS; #: p < 0.05; ##: p < 0.01; ###: p < 0.001; ####: p < 0.0001 tamoxifen vs. vehicle. UCMS:
failures, tamoxifen totally reversed these effects. (c-d) ANOVA, followed by post hoc multiple comparisons when necessary.
unpredictable chronic mild stress; NS: non-stressed; TST: tail suspension test.
*: p < 0.05; **: p < 0.01 UCMS vs. NS; #: p < 0.05; ##: p < 0.01; ###: p < 0.001; ####: p < 0.0001 tamoxifen vs. vehicle. UCMS:
unpredictable chronic mild 3.3. stress;
AHNNS: Promotion
non-stressed; TST: tailPromotes
Partially suspension test.
Resilience to Stress-Induced Cognitive Impairments
Although depressive
Additionally, two-waymoodANOVA andrevealed
anhedonia are effect
a main considered
of UCMS core(Fsymptoms of de-
1,27 = 5.514, p = 0.026)
pression, patients
and tamoxifen suffering
(F1,27 = 6.432,from depressive
p = 0.017) for theepisodes
number also exhibit important
of perseverative cognitive
failures (Figure 3c)
impairments [40]. As such, memory
as well as a UCMS*tamoxifen deficits,
interaction asfor
effect well
theasaverage
difficulties in executive
latency to find thefunctions,
platform
are often
(F1,27 observed.
= 9.152, Because
p = 0.005). UCMS hippocampal
vehicle mice neurogenesis
had a higherhas been widely
number linked to failures
of perseverative cogni-
tion [17,41,50,51], and that few studies have evaluated the protective
compared to non-stressed vehicle mice (Fisher: p = 0.012); however, this UCMS effecteffects of promot-
ing
washippocampal neurogenesis
reversed in tamoxifen on as
mice, stress-elicited cognitive
UCMS tamoxifen miceimpairments
had a decreasedin an animal
number of
model of MD, failures
perseverative we decided to evaluate
compared the effect
to UCMS ofmice
vehicle increasing
(Fisher:hippocampal neurogenesis
p = 0.006). Overall, UCMS
on cognitive
vehicle mice impairments
spent more time induced
to findbythe
UCMS. Therefore,
platform compared we used a water maze
to non-stressed protocol
vehicle mice
in order to evaluate flexibility and inhibition (Figure 3b). Since associative learning can
Cells 2021, 10, 972 12 of 18

(Fisher: p < 0.001), an effect that was reversed in tamoxifen mice, as UCMS tamoxifen mice
presented a decreased average latency compared to UCMS vehicle mice (Fisher: p < 0.001).
Moreover, UCMS tamoxifen mice do not present any difference compared to non-stressed
tamoxifen mice (Fisher: p = 0.642), leading to the idea that promoting hippocampal neu-
rogenesis not only decreased the intensity of UCMS-induced cognitive deficits but was
sufficient to protect mice from them.
These results indicate that increasing hippocampal neurogenesis before UCMS re-
verses the UCMS-induced reversal-learning impairment in the inhibition task. Moreover,
while UCMS did not impact flexibility in our model, increased hippocampal neurogenesis
affected the number of perseverative failures but not the time to reach the platform in the
flexibility task.

4. Discussion
A majority of studies evaluated the impact of AHN on stress vulnerability through
its ablation or the antidepressant effects of its increase on stress-induced depressive-like
behaviors. However, it is still poorly described how promoting AHN could act as a factor
of resilience. Additionally, whereas the involvement of AHN in cognitive functions is
now well described, how promoting AHN could prevent stress-induced cognitive deficits
remains less studied. In the present study, to explore the contribution of AHN in the re-
silience to chronic stress, we used transgenic mice in which AHN was selectively promoted,
and we aimed to examine the impact of increasing AHN before the onset of stress in a
well-characterized animal model of MD, the UCMS model. Our results showed that the
UCMS regimen induced a depressive-like phenotype in vehicle mice, characterized by an
altered coat state and nest-building score, changes in stress-coping strategies in the TST,
and anxiety-like behaviors in the light/dark box as well as cognitive deficits in a water
maze task. However, increasing AHN before the stress was sufficient to prevent the effect
of stress in some depressive-like behaviors (TST, nest-building score) as well as in executive
functions (reversal learning).
Exposing mice to a 4-week UCMS regimen reduced hippocampal neurogenesis in
the dorsal hippocampus, and even if it did not reach significance, tamoxifen tended to
reverse this reduction. This stress effect on hippocampal neurogenesis is consistent with
previous results underlying the negative impact of stress on the survival of adult-born
neurons [45,53,54]. However, it seems that the ventral hippocampus was not affected by
UCMS in our study, possibly due to the fact that before the UCMS regimen, mice were
housed in an enriched environment, which could have partially prevented the effects of
stress [31]. Moreover, most studies that observed a UCMS-induced decrease in AHN used
protocols of 6 to 8 weeks; it is then highly probable that our 4-week protocol was not
sufficient to reach a significant decrease in AHN in the ventral hippocampus [11,45,55–57].
Nevertheless, tamoxifen injection significantly increased the number of adult-born
neurons in the total hippocampus and the maturation of these adult-born neurons in the
dorsal and the ventral hippocampus. Therefore, our results are in line with previous studies
using this transgenic model, which found increased survival and maturation, but no effect
on proliferation [17,45,57].
Adult-born neurons have distinct functions along the dorso-ventral axis of the hip-
pocampus: while the dorsal population is mainly involved in cognitive functions, the
ventral population is more important for mood and stress regulation [58–60]. Our model
was able to enhance AHN all along the dorso-ventral axis, therefore allowing us to inves-
tigate the effects of promoting AHN on the development of a depressive-like phenotype
but also on cognitive functions. However, because our transgenic model is based on the
selective ablation of the pro-apoptotic Bax gene in Nestin-expressing stem cells, one could
think that behavioral changes could not solely be the consequences of the increase in
adult-born neurons but also of changes in the glial cells ratio. Indeed, it has been repeatedly
found that MD was associated with glial loss [61], raising the possibility that affecting the
ratio of glial cells could alter behaviors in our model. It is nevertheless unlikely, as previous
Cells 2021, 10, 972 13 of 18

characterizations of this transgenic line showed no difference in the proportion of neurons

and glia [17,18]. This previous characterization led us to think that our results were mostly
explained by the increase in survival and maturation of adult-born neurons.

4.1. Increasing Adult Hippocampal Neurogenesis Partially Promotes Resilience to the

Depressive-Like Phenotype Induced by UCMS
Daily-life measures such as the coat state and the nest-building score revealed that
UCMS induced a significant deterioration of the coat state starting one week after the
beginning of the UCMS regimen, as well as changes in the nest-building score. These
UCMS-induced changes were prevented only for the nest-building test, while degradation
of the coat state was not reversed by increasing AHN.
Additionally, we observed UCMS-induced impairments of motivational behaviors
in the TST illustrated by an increase in immobility in vehicle mice that was not observed
in mice treated with tamoxifen. Thus, promoting AHN did not only decrease the effect
of stress on stress-coping behaviors but prevented their appearance. Even though we
did not observe any significant anhedonic effect of stress in the cookie test, increasing
AHN induced a decreased latency to eat. Therefore, even if our protocol did not induce
significant anhedonic traits, promoting AHN could potentially affect them. According to
these results, the stimulation of AHN seemed to have partially increased the resilience to
the effects of chronic stress, as observed on despair and nest-building in our behavioral
paradigms, whereas it did not affect the coat state. We previously observed a similar effect
of enhanced AHN on these stress-coping strategies with this transgenic strain under a
UCMS regimen; nonetheless, AHN was in this case promoted after the onset of stress, thus
counteracting the UCMS effects once they were established [45,57].
Increasing AHN did not induce any behavioral changes in the splash test and the
NSF test. However, in these tests, because chronic stress did not yield effects, it is difficult
to conclude about the protective effects of AHN. This lack of stress effect in some of our
behavioral paradigms could probably be explained by the fact that we used a shorter
UCMS protocol compared to previous studies that highlighted stress-induced behavioral
changes [11,45,57] and that our protocol did not elicit a decrease in AHN in the ventral
part of the hippocampus. Nonetheless, these findings point out the fact that AHN alone
may not be sufficient to induce antidepressant or anxiolytic effects in control non-stressed
mice, which is consistent with previous studies evaluating the effect of AHN enhancement
in animal models of chronic stress or MD [17,18].
Thus, increased AHN does not act as an antidepressant per se but rather plays a key
role in counteracting the disruption of neural circuits and the behavioral modifications
induced by chronic stress. AHN is involved in the negative feedback from the hippocampus
on the hypothalamo-pituitary-adrenal axis, which is impaired by chronic stress [11,39,62];
thus, promoting AHN could prevent the deleterious effects of stress, whereby protecting
from the development of symptoms. Therefore, our protocol had one limitation as we did
not evaluate the stress-induced modifications of the HPA axis activity. Indeed, measures of
glucocorticoid levels would have given us insights to understand the mechanisms through
which increasing AHN could prevent stress-induced behavioral changes. Even though
the present findings reveal only partial effects, it appears that increasing hippocampal
neurogenesis could at least partially prevent chronic stress-related effects on stress-coping
behaviors and anhedonia. However, it seems that increasing AHN hardly affects anxiety-
like behaviors.

4.2. Increasing Adult Hippocampal Neurogenesis Promotes Resilience to Stress on

Executive Functions
In addition, we examined the effects of increasing AHN on cognitive performance
in a modified water maze paradigm, allowing us to measure inhibition and flexibility
capacities. During the experiment, mice went through a 4-day training stage in which they
had to associate two contexts with two different strategies to find the platform, according
to either the self-location (egocentric navigation strategy) or the spatial cues (allocentric
Cells 2021, 10, 972 14 of 18

navigation strategy), with no strategy alternation within each training day (Figure 3b).
This was followed by a flexibility task in which mice were exposed alternatively to each
context and therefore had to alternate the strategy to find the platform. Finally, during
an inhibition task, the previous association context/strategy was inverted, and the mice
had to inhibit their previous learning and learn the novel context/strategy association to
find the platform successfully. This protocol was designed to evaluate (1) the capacity to
switch between already learned rules (flexibility) and (2) the capacity of reversal learning
(inhibition) and examine if these cognitive functions are distinctly modulated by stress
and/or AHN.
We observed a significant effect of stress in the inhibition task. UCMS vehicle mice
spent more time to reach the platform compared to non-stressed mice and exhibited a
higher number of perseverative responses, showing a deficit in inhibiting previously
learned associative rules and persisted in searching the platform in the wrong arm. In
our model, UCMS seemed to partially alter cognitive functions, in line with what is
often observed in clinical studies [40,63]. Interestingly, these were reversed by tamoxifen
treatment: increasing AHN before chronic stress was thus sufficient to promote stress
resilience in the inhibition task. Surprisingly, we did not find any effect of UCMS for
the latency to find the platform in the flexibility task, and AHN increase only tended to
decrease latency without reaching significance. Nevertheless, increasing AHN seemed
to have partially improved the capacities to switch between rules, as was observed by a
decrease in perseverative errors in the flexibility task.
Interestingly, while spatial memory performance positively correlates with the level
of AHN [64–66], it appears that a learning task such as the water maze influences the
level of AHN [67,68]. Therefore, we separated our mice into two cohorts: mice that went
through the water maze were not used for other behavioral tests than the TST to avoid
any side effects of learning-induced increased neurogenesis on other behavioral tasks. For
the same reason, we did not measure the levels of AHN on these mice, as this learning
protocol could have induced augmentation of AHN even in stressed mice, thus rendering
the quantification non-conclusive. Previous findings highlighted a putative role of AHN in
reversal learning, while ablation induced cognitive deficits [50,69], stimulation of AHN was
able to restore allocentric navigation and contextual memory in aged mice in a water maze
task [29]. In addition, we showed that stimulation of AHN could improve performance in
an inhibition task impaired by chronic stress and promote the appropriate updating of the
learning strategy leading to a decrease in perseverative errors in the water maze task.
One of the main functions that have been proposed for AHN is pattern separation [17,24],
which is the capacity to disambiguate two similar inputs coming from the entorhinal cortex
into two distinct contextual representations. It is usually proposed that the sparse coding
of the hippocampus plays a crucial role in this function as it decreases the probability of
interferences between two similar encoded contexts. Since adult-born neurons seem to
play a critical role in the modulation of the overall activity of the hippocampus [26,70], it is
possible that they reduce memory overlapping through their inhibition control, thereby
improving cognitive flexibility and reversal learning [19,41]. Indeed, X-irradiation of
AHN increases the activity of mature granule cells in the DG along with a decreased
performance in a reversal-learning task [50]. Conversely, increasing AHN decreased the
DG excitability [25], improved contextual discrimination [17], and facilitated the encoding
of new memories [71]. Interestingly, whereas the effects of AHN on executive functions
were mainly observed in non-stressed conditions in previous studies, our data highlighted
that directly promoting AHN before the onset of stress was sufficient to prevent the deficits
of reversal learning. Taken together, these findings suggest that increasing AHN could play
an important role in resilience to stress by modulating the sparse hippocampal activity
and so promoting cognitive functions directly supported by the hippocampus. Moreover,
because the hippocampus projects to prefrontal regions involved in cognitive processes and
that stress disrupt this pathway [72], modulating hippocampal activity could act indirectly
on these projections, thereby positively shaping executive functions and stress resilience.
Cells 2021, 10, 972 15 of 18

4.3. Conclusions
Our findings add to accumulative evidence of the link between AHN and MD and
open a novel insight into the role of AHN in the pathophysiology of MD. Our study
highlights commonly accepted results such as a negative impact of chronic stress on hip-
pocampal neurogenesis and behaviors. However, we also point out a novel insight into the
neuronal mechanism underlying resilience to stress, showing that increasing hippocampal
neurogenesis before the onset of stress could partially alleviate the depressive-like pheno-
type as well as the cognitive impairments. However, from a perspective point of view, more
specific characterization of neuronal activity would be an asset to better understand the
mechanisms underpinning the behavioral changes that we observed. Because adult-born
neurons have been linked to neuronal control of the hippocampus [25–27,70], it would
be interesting to clarify their direct and indirect involvement in neuronal circuits during
behavioral tasks affected by stress.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/cells10050972/s1, Supplementary materials and methods.
Author Contributions: Investigation, N.L., A.-M.L.G., and B.P.; Formal analysis, B.P.; Writing—
original draft, B.P.; Methodology, A.S. and C.B.; Visualization, M.L. and B.P.; Writing—review and
editing, M.L., A.S., R.H. and C.B.; Conceptualization, C.B.; Funding acquisition, C.B. All authors
have read and agreed to the published version of the manuscript.
Funding: B.P. is supported by a grant from Fondation de la recherche Médicale (FRM).
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Directive 2010/63/EU, and approved by the Ethics Committee CEEvdl (n◦ 2019092017334223).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to privacy concerns.
Acknowledgments: B.P. is supported by a grant from Fondation de la recherche Médicale (FRM)
Conflicts of Interest: The authors declare no conflict of interest.

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