Neurobiology of Stress 23 (2023) 100526

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Neurobiology of Stress
journal homepage: www.elsevier.com/locate/ynstr

Social adversity during juvenile age but not adulthood increases

susceptibility to an immune challenge later in life
Cyprien G.J. Guerrin a, Janine Doorduin a, *, Kavya Prasad a, Daniel A. Vazquez-Matias a,
Lara Barazzuol b, c, Erik F.J. de Vries a
a
Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713, GZ, Groningen, the
Netherlands
b
Department of Radiation Oncology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713 GZ, Groningen, the Netherlands
c
Department of Biomedical Sciences of Cells and Systems, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713 GZ, Groningen, the
Netherlands

A R T I C L E I N F O A B S T R A C T

Handling Editor: Dr. Tallie Z Baram Adverse experiences in early life can increase mental vulnerability to immune challenges experienced later in
life, which may induce the development of stress-related psychopathologies. Here, we investigated whether the
Keywords: combined effect of both events is higher if the first adverse experience occurs when the brain is still in devel­
Social stress opment. Therefore, male Wistar rats were exposed to repeated social defeat (RSD, first hit) during juvenile age or
Psychopathologies
adulthood and to an immune challenge consisting of a single injection of lipopolysaccharide (LPS, second hit) in
Microglia priming
adulthood. Control animals were not exposed to RSD, but only to the LPS challenge. Translocator protein density,
Dual hit model
Adolescent social defeat a marker for reactive microglia, microglia cell density and plasma corticosterone levels were measured using in
LPS challenge vivo [11C]PBR28 positron emission tomography, iba1 immunostaining, and corticosterone ELISA, respectively.
Anhedonia, social behavior and anxiety were measured with the sucrose preference, social interaction, and open
field tests, respectively. Rats exposed to RSD during juvenile age exhibited enhanced anhedonia and social
interaction dysfunction after an immune challenge in adulthood. This enhanced susceptibility was not observed
in rats exposed to RSD during adulthood. In addition, exposure to RSD synergistically increased microglia cell
density and glial reactivity to the LPS challenge. This increase in microglia cell density and reactivity to the LPS
challenge was more pronounced in rats exposed to RSD during juvenile age than in adulthood. Exposure to RSD
alone in juvenile age or adulthood induced similar short-term anhedonia, a long-lasting increase in plasma
corticosterone and microglial activity, but no change in anxiety and social behavior. Our findings indicate that
exposure to social stress during juvenile age, but not adulthood, primes the immune system and increases the
sensitivity to an immune challenge experienced later in life. This suggests that juvenile social stress can have
more deleterious effects in the long term than similar stress in adulthood.

1. Introduction to the pathological effects of a second postnatal stimulus, such as

Traumatic social events can place individuals at risk for developing
stress-related psychopathologies and psychiatric disorders later in life
(Varchmin et al., 2021; Varese et al., 2012). Psychological trauma,
despite its relatively frequent occurrence (Comijs et al., 2007; Dong
et al., 2004), is usually not sufficient for disease induction (Stilo and
Murray, 2010) and seems to have a rather modest effect in large pop­
ulations (Varese et al., 2012). To explain this modest effect, it has been
proposed that social adversity can render an individual more vulnerable
infection or sepsis (Bayer et al., 1999; Guerrin et al., 2021; Hjorthøj
et al., 2020).
Social adversity activates the hypothalamus-pituitary-adrenal axis
(HPA) and can chronically increase glucocorticoids levels. A higher level
of glucocorticoids is an indicator of increased stress sensitivity, which is
commonly observed in individuals exposed to childhood adversity and
at risk of psychosis (Reininghaus et al., 2016; Walder et al., 2000).
Furthermore, higher glucocorticoid level is an important physiological
process in the development of psychotic experiences in the early stages

* Corresponding author.
E-mail address: j.doorduin@umcg.nl (J. Doorduin).

https://doi.org/10.1016/j.ynstr.2023.100526
Received 7 November 2022; Received in revised form 6 February 2023; Accepted 7 February 2023
Available online 8 February 2023
2352-2895/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
C.G.J. Guerrin et al.
of schizophrenia (Reininghaus et al., 2016; Walder et al., 2000).
Increased levels of glucocorticoids play a complex modulating role on
the immune system by having anti-and pro-inflammatory properties
(Yeager et al., 2011). Higher number of reactive microglia and other
neuroinflammatory changes, including higher brain levels of
pro-inflammatory cytokines, are often observed in patients with neu­
rodevelopmental disorders, such as schizophrenia and autism, or mood
disorders, such as bipolar disorders and depression (Hughes and Ash­
wood, 2020; Meltzer and Van de Water, 2017). Altogether, the stress
associated with social adversity could dysregulate the HPA axis and
prime the immune system to an aberrant response to a second postnatal
stimulus. Yet, vulnerability to social adversity seems to depend on age.
Social trauma experienced during juvenile age, a critical phase during
which the brain undergoes many neurodevelopmental changes, is
believed to have more detrimental effects than trauma experienced in
adulthood when the brain is fully developed (Varchmin et al., 2021).
However, this hypothesis still awaits direct verification.
In this study, we tested whether exposure to social adversity during
juvenile age or adulthood affects glial reactivity, glucocorticoid levels,
and behavior differently and whether such adversity alters the response
to infection later in life. To achieve social adversity, repeated social
defeat (RSD) was performed in juvenile and adult rats. Rats exposed to
RSD were previously found to display altered behavioral phenotypes
relevant to stress-related psychopathologies, including increased anxi­
ety, social withdrawal, and anhedonia (Iñiguez et al., 2014; Vidal et al.,
2007). Infection later in life was performed by injecting lipopolysac­
charide (LPS), the major component of the outer membrane of
gram-negative bacteria, a factor shown to induce sickness behavior in
humans and rodents (Bassi et al., 2012; Fullerton et al., 2016). To
measure glial reactivity to the stressors and serum glucocorticoid levels,
we performed non-invasive Positron Emission Tomography (PET) im­
aging using the PET tracer [11C]PBR28 to measure translocator protein
(TSPO) levels, protein associated with reactive microglia, and took
blood samples, at different time points. PET imaging allows to measure
TSPO levels in vivo non-invasively and, therefore, to conduct longitu­
dinal within-group comparisons. Additionally, it offers translational
value as TSPO tracers are also used and validated clinically to measure
neuroinflammation. Potential differences in microglia density were
examined by quantification of cells immunoreactive for the ionized
calcium-binding adaptor molecule 1 (Iba1) on day 93. To measure
anxiety-like behavior, social behavior, and anhedonia, symptoms often
observed in psychopathologies, we longitudinally performed the open
field test (OFT), social interaction test (SIT), and sucrose preference test
(SPT), respectively.
2. Materials and methods
2.1. Animals
All experiments were performed in accordance with European
Directive 2010/63/EU and the Law on Animal experiments in the
Netherlands. Twenty-four male outbred Wistar Unilever rats (HsdCpd:
WU, age 21–23 days) were purchased from Envigo (Horst, The
Netherlands), housed in pairs with food and water available ad libitum.
Rats were allowed to acclimatize for seven days before the start of the
experiments. Housing rooms were humidity-controlled and thermo-
regulated (21±2 ◦ C), with a 12:12-h light:dark cycle (lights on at 7 a.
m.). Day 35–39 and day 63–67 were considered as juvenile age and
adulthood, respectively (Babikian et al., 2010; Semple et al., 2013). Only
males were included in this study because the RSD model of social stress
is well validated in males but not females due to the males innate ter­
ritorial aggression toward other males intruding their territory, behavior
rarely observed in female rats.
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Neurobiology of Stress 23 (2023) 100526
2.2. Experimental design
Male rats were randomly divided into three groups: (1) rats not
exposed to social defeat (control, n = 8), (2) rats exposed to social defeat
at a juvenile age (day 35–39, RSDjuv, n = 8) but not adulthood, and (3)
rats exposed to social defeat during adulthood (day 63–67, RSDadu, n =
8) but not juvenile age. All animals were injected with LPS on day 90.
PET scans to measure glial activity, blood samples to measure plasma
corticosterone and behavioral tests to measure anhedonia, anxiety and
sociability were performed as shown in Fig. 1.
2.3. Repeated social defeat
12–16 week old male Long Evans rats (HsdBlu:LE – Envigo, USA)
were used as residents. To encourage territoriality, each male resident
was housed in a large cage (80*50*40 cm) with an ovariectomized fe­
male Long Evans rat that underwent tubal ligation for at least one week
before the screening. The screening consisted of measuring attack la­
tency and submission time in RSD sessions on five consecutive days to
select rats displaying the desired aggressive behavior and exclude rats
showing signs of violence (attack latency <10 s and attempts to kill the
intruder by attacking vital zones), or non-aggressive behavior (attack
latency>60s, absence of submission or submission time >120s, or the
resident being submitted) (Moraga-Amaro et al., 2022).
The RSD protocol was performed between 1 and 4 p.m. Female Long
Evans rats were removed from the resident cages 1 h before the exposure
of the experimental rat. The session lasted 1 h and started with the
introduction of the experimental rat in the cage of the resident, after
which they were allowed to interact until the experimental rat showed a
submissive posture for at least 5 s or after 10 min of interaction. Sub­
sequently, the experimental rat was placed inside a wire mesh cage and
put back in the cage of the resident to allow for visual, auditory, and
olfactory interactions for the remainder of the 1 h. The experimental rat
was exposed to five different residents for five consecutive days. Control
rats were placed in a wire mesh cage inside a clean cage for a duration of
1 h.
RSDjuv and RSDadu rats were individually housed during the RSD
protocol and the period thereafter during which the behavioral tests and
PET scan took place, i.e. on day 35–46 and day 63–74, respectively, as
social interaction during group housing can counteract the effect of
social defeat. The control group remained housed in pairs during the
RSD protocols. Hereafter the rats were group housed again.
2.4. LPS administration
On day 90, all the rats were intraperitoneally injected with a solution
of 1 mL of 2 mg/kg of E. coli lipopolysaccharide (Sigma Aldrich, L2630)
dissolved in Dulbecco’s phosphate-buffered saline (PBS).
2.5. Sucrose preference test
Sucrose preference tests were performed on days 34, 40, 62, 68, 89,
and 91 to measure anhedonia. SPT training consisted of placing a bottle
with 1% sucrose in water in the cage for 1 h on four consecutive days
(day 30–33), followed by overnight exposure to two bottles, one filled
with 1% sucrose solution and the other with normal drinking water. The
SPT consisted of overnight exposure to two bottles, one filled with a 1%
sucrose solution, and one filled with drinking water. The sucrose pref­
erence was measured as the outcome parameter and was calculated
according to the following formula: sucrose preference (%) = [Sucrose
intake (mL)/(Sucrose intake (mL)+ Water intake (mL))] *100%.
2.6. Open field test
Open field tests (OFT) were performed on day 40, 68 and 91 to
measure anxiety-like behavior and motility. Rats were allowed to
C.G.J. Guerrin et al.
Fig. 1. Study design. Behavior consists of sucrose preference test (day 40, 62, 68, 89 and 91), open field and social interaction tests (day 40,74, and 91). Blood
samples were collected before each PET scan. Rats were sacrificed following the last PET scan on day 93.
acclimatize to the experimental room for 1 h before being placed in a
circular area (100 cm diameter) for 5 min. Time spent in the center (>10
cm from the wall) was measured to determine the level of anxiety-like
behavior. Behavior was video recorded and analyzed offline using
Ethovision XT14 (Noldus Information Technology, Wageningen, The
Netherlands). A 10% ethanol solution was used to clean the arena after
each session.
2.7. Social interaction test
Social interaction tests (SIT) were performed on day 40, 68 and 91 to
measure social behavior. The test was performed following the OFT. To
account for the size differences and to allow for closer interactions be­
tween the rats, the first SIT was performed in a 50*50 cm squared arena
and the second and third SIT in a circular arena with a diameter of 100
cm. The total duration of the test was 10 min and consisted of two
phases: the habituation and the test phase. During the habituation
phase, the experimental rat was permitted to freely explore the arena
containing two empty wire mesh cages placed on opposite sides of the
arena for 5 min. Then, the rat was returned to its home cage and the
researcher placed an object and a test rat (age-matched; maximum of 14
days older) in the wire mesh cages. During test phase the experimental
rat was put in the arena and allowed to freely explore for 5 min.
Behavior was video recorded and analyzed offline using Ethovision
XT14 (Noldus Information Technology, Wageningen, The Netherlands).
A 10% ethanol solution was used to clean the arena after each test. The
time spent with the rat and the time spent with the object were recorded
and used as an indicator of social behavior. Rats spending less than 3 s
interacting with any of the wire mesh cages were excluded (day 68: 1
RSDadu, day 91: 1 RSDjuv + 1 RSDadu).
2.8. PET imaging
PET was used to measure TSPO levels as a marker of microglia
reactive to the stressors, on day 46, 74, 89, and 93. Rats were anaes­
thetized with isoflurane (induction 5% and maintenance 2%, in oxygen)
for an intravenous injection of [11C]PBR28 in the tail vein. The average
injected tracer dose was similar between groups. The average radioac­
tivity dose at the time of injection was 39.6 ± 6.2 MBq. After tracer
injection, rats were placed back in their home cage. About 30 min after
tracer injection, rats were anaesthetized with isoflurane and positioned
into the PET camera (microPET Focus 220, Siemens) for a transmission
scan of 10 min followed by an emission scan of 30 min, starting at 45
min after tracer injection. Heating pads and eye salve were used to
maintain body temperature and prevent dehydration of the eyes,
respectively. Heart rate and blood oxygen levels were monitored. After
the scan, rats were placed back into their home cage. After the final scan
on day 93, rats were terminated under deep anesthesia by cardiac
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Neurobiology of Stress 23 (2023) 100526
perfusion.
PET scans were iteratively reconstructed (OSEM2D, 4 iterations, 16
subsets) into a single frame, resulting in images with a 128 × 128 × 95
matrix, a pixel width of 0.632 mm, and a slice thickness of 0.762 mm.
PET images were automatically co-registered to a functional [11C]
PBR28 template [17], which was spatially aligned with a stereotaxic T2-
weighted MRI template in Paxinos space [18]. Predefined volumes of
interest (VOI) representing specific brain regions were copied to the
realigned PET images and used to calculate the tracer uptake, expressed
as percentage of the injected dose per gram of tissue. 1 control and 2
RSDjuv rats died before the first PET scan and 1 RSDjuv rat died before
the last scan. Due to tracer production failure, 1 RSDadu rat and 2
controls rats could not be scanned on day 68 and day 89, respectively.
2.9. Brain collection and immunohistochemistry
To collect brains for immunostaining, rats were perfused with PBS
and 4% paraformaldehyde (PFA) under deep anesthesia. Brains were
fixed for 48 h in 4% PFA at room temperature. After dehydration in 25%
sucrose at 4 ◦ C, the brains were embedded with optimal cutting tem­
perature OCT compound and stored at − 80 ◦ C.
For immunohistochemistry, 15 μm sections were prepared by cry­
osection. After washing three times with PBS, antigen retrieval was
performed by pressure cooking for 10 min in 10 mM sodium citrate, pH
6.0. Subsequently, the sections were washed and incubated in PBS with
0.3% hydrogen peroxide (H2O2) for 30 min to block endogenous per­
oxidases. The sections were then washed and blocked for 30 min with
2% normal donkey serum (NDS; Jackson Immuno Research, 017-000-
121) in PBS with 1% Triton X-100 (PBS+) and 2% bovine serum albu­
min (BSA). The sections were then incubated with the primary rabbit-
α-ionized calcium-binding adapter molecule 1 (Iba1) antibody (1:2000;
Wako, 01–19,741) with PBS+ and 1% BSA overnight at 4 ◦ C. The
following day, the sections were washed and incubated with the bio­
tinylated secondary donkey-α-rabbit IgG antibody (1:400; Jackson
Immuno Research, 711-065-152) for 2 h. After washing with PBS, the
section were incubated with Avidin/Biotinylated enzyme Complex ABC
solution (VECTASTAIN® ABC Kit, Vector Laboratories, PK-6100) for 30
min. The sections were washed and stained using 0.04% 3,3′ -Dia­
minobenzidine and 0.03% hydrogen peroxide for 10 min and subse­
quently dehydrated using a sequence of increasing ethanol
concentrations. After being air-dried, the slides were mounted with
coverslips using DePex (Serva) and stored at room temperature.
2.10. Microglial density and spatial distribution analysis
Microglia densities in the parietal and temporal cortices were
determined by counting all Iba1 positive cells in a specified cortical
region of interest (ROI) of known dimensions (0.28 mm2) at a 20X
C.G.J. Guerrin et al.
magnification using ImageJ software (http://rsb.info.nih.gov/ij/). The
spatial distribution of microglia in the cortex and the distance of the
microglia to their nearest neighbor, that is, the average Euclidian dis­
tances between the nearest cells, were determined using the NND plugin
for ImageJ. Between 8 and 12 ROIs per animal were selected for the
analysis. Five rats per group were used.
2.11. Plasma corticosterone with ELISA
PET acquisition was performed in the morning or in the afternoon.
We observed no difference in corticosterone between morning and af­
ternoon. At the time of each PET acquisition, about 0.50 mL blood
samples were collected from the tail vein, prior to injection of the PET
tracer, and immediately centrifuged at 5000 g for 3 min. Plasma was
collected, frozen in liquid nitrogen and stored at − 80 ◦ C. Plasma corti­
costerone measurement was performed using an enzyme-linked immu­
noassay (ELISA) using a commercially available kit (Arbor Assays,
DetectX Corticosterone Immunoassay kit) according to the manufac­
turer’s recommendations.
2.12. Statistical analysis
Statistical analysis of body weight, behavior, plasma corticosterone
levels and PET data was performed using SPSS (IBM SPSS Statistics,
Version 22.0). A generalized estimating equation (GEE) analysis was
performed, using ‘social defeat’ and ‘time’ as factors for longitudinal
data statistical analyses, as this analysis can account for missing data. A
paired t-test was used to assess differences in RSD severity and within
groups in the social interaction test. A one-way ANOVA was performed
to assess differences between groups in Iba1 staining. The box represents
the interquartile range, the whiskers the min to max values, and the
center line indicates the median.
3. Results
3.1. Juvenile rats were exposed to less severe social defeat than adult rats
Average attack latency (Supplementary figure 1A) was significantly
lower in rats exposed to RSD in adulthood than during juvenile age
(RSDjuv = 102 ± 112, RSDadu = 23 ± 21, p < 0.001). The number of
times the intruder was not submitted (RSDjuv = 88%, RSDadu = 30%, p
< 0.001) were significantly different between rats exposed to social
defeat during juvenile age and adulthood (Supplementary figure 1B).
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Neurobiology of Stress 23 (2023) 100526
3.2. Juvenile but not adulthood RSD reduced body weight
Body weight of all groups increased with time (p < 0.001, Fig. 2A.).
We observed a main effect of RSDjuv (p = 0.003) but not RSDadu (p =
0.634) on body weight. On day 40, the body weight of RSDjuv rats was
significantly lower compared to control and RSDadu rats (control: − 7%,
p = 0.004, RSDadu: − 9%, p = 0.010). This difference was still observed
on day 54 (control: − 6%, p = 0.050, RSDadu: − 10%, p = 0.029),
whereas only the difference compared to the control group remained
statistically significant on day 67 (control: − 5%, p = 0.050), and day 89
(control: − 6%, p = 0.001). The body weight of RSDadu rats was not
significantly different from that of controls at any time point. On day 93,
2 days after LPS injection, the body weight of the RSDjuv group (− 12%,
p < 0.0001), but not of the RSDadu group (− 6%, p = 0.115), was
significantly lower than that of controls. Within group comparison be­
tween day 89 and day 93, the period in which LPS was injected, showed
a reduction in bodyweight in control (− 8%, p < 0.0001), RSDjuv
(− 15%, p < 0.0001), and RSDadu (− 8%, p = 0.031) rats.
3.3. Juvenile but not adulthood RSD induced a synergistic decrease in
sucrose preference following LPS injection
The sucrose preference test was used to assess anhedonia (Fig. 2. B).
Overall, we observed a main effect of time (p < 0.001) but not of RSDjuv
(p = 0.217) or RSDadu (p = 0.181). On day 40, rats submitted to the RSD
protocol at juvenile age showed a reduction in sucrose intake compared
to controls (− 7%, p = 0.023). On day 67, rats submitted to the RSD
protocol during adulthood showed a reduction in sucrose uptake
compared to controls (− 15%, p = 0.024). On day 91, following the LPS
injection, RSDjuv rats, but not RSDadu rats, showed a significant
reduction in sucrose intake compared to controls (RSDjuv: − 18%, p =
0.027, RSDadu: − 15% p = 0.664). Within-group comparison showed a
consistent decrease in sucrose preference between day 89 and day 91
(period during which LPS was injected) in the control (− 16%, p =
0.018), RSDjuv (− 29%, p < 0.0001) and RSDadu (− 23%, p = 0.003)
group.
3.4. RSD did not significantly affect anxiety-like behavior nor locomotion
The percentage of time spent in the center of the arena and the total
distance travelled in the OFT were used to assess anxiety-like behavior
(Fig. 2C.) and locomotion (Fig. 2D.), respectively. We observed a main
effect of time (p < 0.001) and RSDadu (p = 0 < 001) but not of RSDjuv
Fig. 2. Bodyweight, anhedonia, locomotion and
anxiety changes. A. Bodyweight (n = 5 to 8 per
group). B. Anhedonia (n = 5 to 8 per group). C.
Anxiety-like behavior (n = 5 to 8 per group). D.
Locomotion (n = 5 to 8 per group). Day 40 is one day
after juvenile RSD, day 68 is one day after adulthood
RSD, and day 91 is one day after LPS injection. Box
represents the interquartile range, the whiskers the
min to max values and the center line indicates the
median. Statistically significant differences between
groups are indicated by asterisks: *p < 0.05, **p <
0.01, ***p < 0.001. Significant differences between
time points are not shown.
C.G.J. Guerrin et al.
(p = 0.271) on locomotion. RSDadu travelled significantly more than
controls on day 40 (p < 0.001) and day 91 (p < 0.0001). There was no
main effect in the percentage of time the animal spent in the center or
any significant difference between groups at any timepoint.
3.5. Juvenile but not adulthood RSD altered social preference following
LPS injection
To test social behavior, we quantified the preference of a rat for
interacting with another rat versus an object. During the familiarization
phase, we observed no difference in time spent with the empty wire
mesh cages and in distance travelled between groups (data not shown).
There was no significant difference in percentage of time spent with the
rat between groups at any timepoints (data not shown). On day 40 (p <
0.001) and day 68 (p < 0.01), all groups showed social preference for a
rat over an object (Fig. 3. A. B.). Unlike control (p < 0.001) and RSDadu
(p < 0.01) rats, RSDjuv (p = 0.89) rats did not show social preference for
a rat over an object anymore, one day after the exposure to LPS (day 91)
(Fig. 3C.).
3.6. RSD increased corticosterone levels in adulthood
Serum concentrations of corticosterone were measured 6 days after
RSD and 1 day before and 3 days after LPS injection (Fig. 4.). Six days
after exposure to RSD during juvenile age (day 46) or adulthood (day
74), the corticosterone levels were not different from controls. On day
89, the corticosterone levels were significantly higher in RSDjuv (+61%,
p = 0.048), when compared to controls. In RSDadu rats, the cortico­
sterone levels were also higher than in controls, but this was not sta­
tistically significant (+56%, p = 0.083). We observed a main effect of
time on corticosterone (p < 0.050). On day 91, following the LPS in­
jection, corticosterone levels were not different anymore between
groups. Within-group comparison between day 89 and day 93, the
period during which LPS was injected, showed that serum corticosterone
levels were significantly reduced in the RSDjuv group (− 53%, p =
0.002). A reduction was also observed in the RSDadu group (− 43%, p =
0.068), but this was not statistically significant. No change was observed
for the control group (− 11%, p = 0.169).
3.7. RSD induced a long-lasting increase in reactive microglia,
synergistically enhancing the effect of LPS injection
To determine the effect of RSD and LPS on glial activity, [11C]PBR28
PET was performed on day 46, 74, 89, and 93 (Fig. 5A. B.C.D.; Table 1).
A main effect of time and group was observed for the whole brain
tracer uptake (p < 0.0001). RSD induced a significant increase in tracer
uptake in RSDjuv rats on day 46 in the frontal (+18%, p < 0.001),
frontal association (+23%, p < 0.001), occipital (+21%, p < 0.0001),
and parietal cortices (+18%, p < 0.001). In RSDadu rats, significantly
enhanced tracer uptake in the whole brain (+13%, p = 0.011), cere­
bellum (+21%, p = 0.012), corpus callosum (+20%, p < 0.01), nucleus
accumbens (+28%, p = 0.01), hippocampus (+16%, p = 0.011) and
frontal (+16%, p = 0.026), parietal (+17%, p < 0.01) and occipital
cortices (+21%, p < 0.01) was observed on day 74, when compared to
control animals. RSD also induced a long-lasting significant increase in
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Neurobiology of Stress 23 (2023) 100526
Fig. 4. Plasma corticosterone levels. n = 5 to 8 rats per group. Day 46 is 6
days after juvenile RSD, day 74 is 6 days after adulthood RSD, day 89 precedes
LPS injection, day 93 is 3 days after LPS injection. Box represents the inter­
quartile range, the whiskers the min to max values and the center line indicates
the median. Statistically significant differences between groups are indicated by
asterisks: *p < 0.05. Significant differences between time points are not shown.
tracer uptake in the frontal association (+17%, p < 0.01) and temporal
cortices (+9%, p = 0.050), bed nucleus of the stria terminalis (BNST)
(+32%, p = 0.013), cerebellum (+18%, p < 0.01), striatum (+21%, p <
0.001), and hippocampus (+14%, p = 0.021) of RSDjuv rats on day 89
(before LPS injection), and in the whole brain (+15%, p < 0.01), cere­
bellum (+37%, p < 0.01), corpus callosum (+19%, p < 0.01), striatum
(+18%, p = 0.001), hippocampus (+22%, p = 0.001), midbrain (+15%,
p = 0.042), and brainstem (+22%, p < 0.01) of RSDadu rats, when
compared to control animals. On day 89, RSDadu rats had a significantly
higher tracer uptake in the midbrain (+13%, p = 0.042) and cerebellum
(+16%, p = 0.01) than RSDjuv rats.
Three days after LPS injection (day 93), tracer uptake was signifi­
cantly higher in the entorhinal (+13%, p = 0.017), insular (+14%, p <
0.01), and temporal cortices (+13%, p = 0.023), amygdala (+20%, p =
0.010), nucleus accumbens (+17%, p < 0.01), hippocampus (+14%, p
= 0.042), and basal ganglia (+16%, p < 0.01) of RSDjuv rats, and in the
entorhinal (+19%, p = 0.047), and temporal cortices (+19%, p =
0.037), cerebellum (+18%, p = 0.0001), hippocampus (+23%, p <
0.01), and brainstem (+17%, p = 0.019) of RSDadu rats, when
compared to control animals. On day 93, RSDadu rats had a significantly
higher tracer uptake in the occipital cortex (+25%, p < 0.01) and cer­
ebellum (+20%, p = 0.0001) than RSDjuv rats.
Within group comparison between day 89 and day 93, the period
during which LPS was injected, showed that tracer uptake in control rats
had significantly increased in the BNST (+34%, p = 0.026), cerebellum
(+15%, p = 0.021), frontal association (+11%, p = 0.007), and medial
prefrontal cortices (+15%, p = 0.050), striatum (+18%, p = 0.016),
midbrain (+21%, p = 0.020), brainstem (+13%, p = 0.45). RSDjuv rats
showed a significant increase in tracer uptake in the amygdala (+17%, p
= 0.001), entorhinal (+17%, p < 0.001), insular (+13%, p < 0.001), and
temporal cortices (+10%, p = 0.023), midbrain (+18%, p = 0.003),
brainstem (+13%, p = 0.44), and basal ganglia (+14%, p < 0.0001), but
a significantly lower tracer uptake in the frontal association (− 13%, p =
0.008) and frontal cortices (− 12%, p = 0.018) over this period. RSDadu
rats had a significantly higher tracer uptake in the entorhinal (+12%, p
Fig. 3. Social behavioral changes. Social prefer­
ence on A. Day 40 (following juvenile RSD), B. Day
68 (following adulthood RSD during), C. Day 91
(following LPS injection). N = 4 to 8 per group. Box
represents the interquartile range, the whiskers the
min to max values and the center line indicates the
median. Statistically significant differences between
the time spent interacting with the rat and the object
are indicated by asterisks: *p < 0.05. Significant dif­
ferences between time points are not shown.
C.G.J. Guerrin et al. Neurobiology of Stress 23 (2023) 100526

Fig. 5. Microglial reactivity in the whole brain

(A.), amygdala (B.), hippocampus (C.) and ento­
rhinal cortex (D.), as measured with [11C]PBR28
PET (n = 5 to 8 per group). Day 46 is 6 days after
juvenile RSD, day 74 is 6 days after adulthood RSD,
day 89 precedes LPS injection, day 93 is 3 days after
LPS injection. Box represents the interquartile range,
the whiskers the min to max values and the center
line indicates the median. Statistically significant
differences between groups are indicated by asterisks:
*p < 0.05, **p < 0.01, ***p < 0.001. Significant
differences between time points are not shown.

= 0.019), insular (+14%, p = 0.11), and orbitofrontal cortices (+9%, p dysfunction. Such effects were not observed when RSD occurred during
= 0.003), striatum (+14%, p = 0.038), hippocampus (+13%, p = adulthood, indicating that the susceptibility to RSD is age dependent.
0.001), thalamus (+11%, p = 0.010), midbrain (+13%, p = 0.021), RSD is a validated model of social stress that mimics some of the
brainstem (+9%, p = 0.26) and forebrain (+12%, p = 0.008) on day 93 effects of bullying in humans (Björkqvist, 2001). In accordance with
than on day 89. other studies (Iñiguez et al., 2014; Kopschina Feltes et al., 2017), we
found that juvenile and adult rats exposed to five days of social defeat
developed short, but not long term, anhedonia-like behavior as indi­
3.8. Previous exposure to RSD synergistically increased microglial density
cated by a reduced sucrose preference in the SPT. However, our RSD
following LPS exposure at adulthood
model did not induce anxiety-like behavior, altered locomotion, or so­
cial dysfunction, as the time spent in the center of the open field, the
To determine the effect of RSD and LPS on microglia, the effect on
distance travelled, and the social interaction were not affected. Previous
microglia cell density was determined in the parietal (Fig. 6 A.B.C.) and
studies using the RSD protocol during juvenile age or adulthood have
temporal (Fig. 6 D.E.F.) cortices by counting the number of Iba1-positive
shown contradictory results, as either an increase in anxiety and social
cells.
dysfunction (MacKay et al., 2017; Mancha-Gutiérrez et al., 2021; Man­
On day 93, 3 days after the injection of LPS, the density of microglia
cini et al., 2021), or no effect (Moraga-Amaro et al., 2022; Mouri et al.,
cells in the parietal (+50%, p < 0.01) and temporal cortices (+43%, p <
2018) have been reported. Many factors may explain such differences,
0.001) was significantly higher in the rats previously exposed to juvenile
including housing conditions, differences in the RSD procedure, its
RSD than in control rats injected with LPS (Fig. 6G. H.). The density of
duration and timing, and the interval between the RSD and the behav­
microglia cells in the parietal cortex (+31%, p < 0.05) of rats previously
ioral tests.
exposed to RSD during juvenile age was also significantly higher than in
Infection by injection of LPS is a well-validated model to challenge
rats previously exposed to RSD at adulthood. Rats previously exposed to
the immune system. In the present study, LPS injected in adulthood (day
RSD at adulthood had a higher microglial density in the temporal
90) induced sickness behavior and reduced body weight and sucrose
(+31%, p < 0.05) but not parietal cortex, when compared to control rats
preference in SPT. This is consistent with findings in animal studies
injected with LPS.
demonstrating sickness behavior following LPS injection (Bassi et al.,
In line with the increased microglial density, a significant decrease in
2012) and clinical data indicating similar behavior in response to an LPS
the nearest neighbor distance between microglia in the parietal (− 16%,
injection or an infection (Fullerton et al., 2016). The observed LPS ef­
p < 0.05) and temporal (− 14%, p < 0.01) cortices of rats exposed to
fects on behavior were more pronounced when rats were previously
juvenile RSD and LPS was observed, as compared to control rats injected
exposed to RSD during juvenile age. Sucrose preference dropped
with LPS (Fig. 6. I.J.). Rats previously exposed to RSD at adulthood also
significantly more in the rats exposed to juvenile RSD. In addition, only
had a significant decrease in the nearest neighbor distance between
the combination of RSD and LPS induced dysfunction of social interac­
microglia in the temporal (− 10%, p < 0.05), but not parietal cortex,
tion characterized by a lack of social preference towards a rat and an
compared to control rats exposed to LPS only.
object, whereas RDS or LPS alone had no effect. These results indicate
that social stress experienced during juvenile age affects the response to
4. Discussion
immune challenges in adulthood.
The synergistic effect of an immune challenge during adulthood and
The present findings indicate that exposure to RSD at juvenile age or
RSD during juvenile age, but not during adulthood, is an interesting
adulthood induced similar short-term anhedonia-like behavior, a long-
observation as resident rats were less aggressive towards juvenile than
lasting increase in plasma corticosterone and microglial reactivity, but
adult intruder rats. Residents took more time to attack and spent less
no change in anxiety or social behavior. In addition, iba1 staining and
time attacking juvenile rats than adult intruder rats. Furthermore, ju­
within-group TSPO PET imaging revealed that exposure to RSD syner­
venile intruder rats showed less avoidance behavior and were submitted
gistically increased microglia cell density and reactive microglia
less often than adult intruder rats. Therefore, the level of stress experi­
following an immune challenge at adulthood. This effect was less pro­
enced by the juvenile rats during the social encounter is expected to be
nounced in rats previously exposed to RSD during adulthood. Rats
weaker than that experienced by adult rats. Another study made similar
exposed to juvenile RSD in combination with an immune challenge at
observations in mice (Montagud-Romero et al., 2017). However, despite
adulthood exhibited enhanced anhedonia and social interaction

6
C.G.J. Guerrin et al. Neurobiology of Stress 23 (2023) 100526

Table 1
[11C]PBR28 PET: tracer uptake in the brain of control animals (control), and animals exposed to social defeat at juvenile age (RSD-juv) or adulthood (RSD-adu).
Tracer uptake (% injected dose/g) is presented for different brain areas. Data are shown as mean ± SD. Statistically significant differences compared to control animals
on the same day are indicated with an asterisk: *p < 0.05; **p < 0.01, ***p < 0.001. Statistically significant differences between RSDjuv and RSDadu on the same day
are indicated with a $: $p < 0.05; $$p < 0.01, $$$p < 0.001.
Brain regions Day 46 (after RSDjuv) Day 74 (after RSDadu) Day 89 (before LPS) Day 93 (after LPS)

Control RSDjuv Control RSDadu Control RSDjuv RSDadu Control RSDjuv RSDadu

Amygdala 0.11 ± 0.12 ± 0.018 0.086 ± 0.092 ± 0.067 ± 0.078 ± 0.077 ± 0.015 0.076 ± 0.091 ± 0.089 ± 0.022
0.017 0.008 0.008 0.014 0.011 0.010 0.012*
BNST 0.084 ± 0.087 ± 0.073 ± 0.083 ± 0.059 ± 0.078 ± 0.071 ± 0.011 0.079 ± 0.074 ± 0.076 ± 0.007
0.018 0.027 0.012 0.011 0.017 0.009* 0.015 0.015
Basal Ganglia 0.087 ± 0.082 ± 0.067 ± 0.071 ± 0.058 ± 0.065 ± 0.063 ± 0.008 0.064 ± 0.074 ± 0.071 ± 0.018
0.010 0.020 0.007 0.012 0.008 0.010 0.009 0.004**
Brainstem 0.123 ± 0.123 ± 0.102 ± 0.105 ± 0.083 ± 0.092 ± 0.101 ± 0.094 ± 0.104 ± 0.110 ±
0.013 0.012 0.009 0.015 0.010 0.006 0.015* 0.009 0.015 0.017*
Cerebellum 0.173 ± 0.174 ± 0.133 ± 0.161 ± 0.116 ± 0.137 ± 0.159 ± 0.133 ± 0.131 ± 0.157 ±
0.022 0.024 0.017 0.027* 0.013 0.014** 0.019***$$ 0.015 0.014 0.012***$$$
Corpus callosum 0.094 ± 0.102 ± 0.076 ± 0.091 ± 0.072 ± 0.082 ± 0.086 ± 0.081 ± 0.078 ± 0.089 ± 0.012
0.012 0.016 0.005 0.013** 0.010 0.015 0.009** 0.013 0.013
Cortex Entorhinal 0.138 ± 0.149 ± 0.099 ± 0.109 ± 0.076 ± 0.082 ± 0.090 ± 0.018 0.085 ± 0.096 ± 0.101 ±
0.023 0.025 0.013 0.013 0.011 0.009 0.011 0.007* 0.021*
Cortex Frontal 0.195 ± 0.230 ± 0.130 ± 0.151 ± 0.129 ± 0.137 ± 0.131 ± 0.013 0.135 ± 0.120 ± 0.135 ± 0.014
0.021 0.019** 0.012 0.024* 0.017 0.017 0.021 0.022
Cortex Frontal 0.310 ± 0.380 ± 0.200 ± 0.220 ± 0.175 ± 0.205 ± 0.192 ± 0.030 0.194 ± 0.178 ± 0.205 ± 0.027
Association 0.032 0.034** 0.026 0.041 0.018 0.024** 0.021 0.029
Cortex Insular 0.166 ± 0.177 ± 0.099 ± 0.110 ± 0.082 ± 0.088 ± 0.088 ± 0.016 0.087 ± 0.099 ± 0.100 ± 0.022
0.023 0.018 0.007 0.015 0.009 0.011 0.011 0.006**
Cortex Medial 0.154 ± 0.166 ± 0.104 ± 0.125 ± 0.094 ± 0.098 ± 0.104 ± 0.019 0.108 ± 0.096 ± 0.107 ± 0.023
Prefrontal 0.023 0.023 0.013 0.034 0.018 0.015 0.013 0.021
Cortex Occipital 0.136 ± 0.165 ± 0.111 ± 0.134 ± 0.117 ± 0.130 ± 0.135 ± 0.013 0.120 ± 0.100 ± 0.125 ± 0.013
0.012 0.013*** 0.011 0.019** 0.022 0.026 0.023 0.018 $$
Cortex 0.220 ± 0.240 ± 0.126 ± 0.145 ± 0.121 ± 0.130 ± 0.126 ± 0.025 0.120 ± 0.122 ± 0.137 ± 0.022
Orbitofrontal 0.033 0.028 0.019 0.026 0.018 0.013 0.014 0.013
Cortex Parietal 0.149 ± 0.175 ± 0.110 ± 0.129 ± 0.117 ± 0.123 ± 0.120 ± 0.010 0.117 ± 0.101 ± 0.113 ± 0.008
0.017 0.014** 0.006 0.016** 0.020 0.021 0.021 0.019
Cortex Temporal 0.158 ± 0.171 ± 0.106 ± 0.115 ± 0.086 ± 0.094 ± 0.098 ± 0.017 0.091 ± 0.103 ± 0.108 ±
0.022 0.021 0.008 0.013 0.007 0.008* 0.012 0.008* 0.020*
Forebrain 0.099 ± 0.105 ± 0.081 ± 0.085 ± 0.072 ± 0.080 ± 0.078 ± 0.013 0.082 ± 0.086 ± 0.087 ± 0.015
0.017 0.019 0.010 0.012 0.012 0.010 0.012 0.012
Hippocampus 0.093 ± 0.096 ± 0.074 ± 0.086 ± 0.063 ± 0.072 ± 0.077 ± 0.071 ± 0.081 ± 0.087 ±
0.013 0.013 0.008 0.010* 0.008 0.007* 0.009** 0.009 0.010* 0.013**
Midbrain 0.098 ± 0.087 ± 0.089 ± 0.090 ± 0.078 ± 0.080 ± 0.090 ± 0.094 ± 0.094 ± 0.102 ± 0.017
0.011 0.016 0.010 0.013 0.011 0.006 0.012*$ 0.015 0.011
Nucleus 0.120 ± 0.120 ± 0.075 ± 0.096 ± 0.062 ± 0.075 ± 0.079 ± 0.022 0.077 ± 0.090 ± 0.092 ± 0.029
Accumbens 0.015 0.029 0.010 0.021* 0.015 0.009 0.013 0.007**
Striatum 0.086 ± 0.086 ± 0.067 ± 0.076 ± 0.056 ± 0.068 ± 0.066 ± 0.066 ± 0.071 ± 0.075 ± 0.016
0.011 0.019 0.007 0.017 0.005 0.008** 0.011** 0.010 0.007
Thalamus 0.101 ± 0.106 ± 0.083 ± 0.085 ± 0.075 ± 0.082 ± 0.079 ± 0.013 0.083 ± 0.088 ± 0.088 ± 0.015
0.019 0.019 0.010 0.012 0.013 0.011 0.012 0.013
Whole Brain 0.136 ± 0.147 ± 0.102 ± 0.115 ± 0.092 ± 0.103 ± 0.106 ± 0.101 ± 0.100 ± 0.110 ± 0.013
0.015 0.011 0.007 0.014* 0.009 0.010 0.010* 0.012 0.011

this apparent weaker threat, juvenile social defeat has induced long term
consequences, characterized by a reduction in body weight, higher
plasma corticosterone levels in adulthood and enhanced susceptibility to
an immune challenge, which were not observed if the social defeat
occurred during adulthood. These results suggest that the timing of the
insult is important, and that juvenile age is a period of higher suscep­
tibility to stressors than adulthood. During juvenile age many neuro­
developmental changes still take place, such as neurogenesis,
myelination, and synaptic pruning, which could explain increased
vulnerability to stress at juvenile age (Hefner and Holmes, 2007; Spear,
2013; Yoo et al., 2013).
In the present work, we found that juvenile RSD increased cortico­
sterone levels in the plasma in adulthood relative to control animals.
This increase in plasma corticosterone levels suggests an altered activity
of the hypothalamus-pituitary (HPA) axis, believed to be an indicator of
heightened stress sensitivity (Holtzman et al., 2013). Such heightened
stress sensitivity characterized by higher corticosterone levels, is com­
mon in individuals exposed to childhood adversity and at risk for psy­
chosis and is an important psychological process in the development of
psychotic experiences in the early stages of schizophrenia (Reininghaus
et al., 2016; Walder et al., 2000). It has been observed that juvenile
social stress in mice increased corticosterone serum levels in juvenile age
and adulthood and induced long term memory impairments (Man­
cha-Gutiérrez et al., 2021). Conversely, another study observed reduced
corticosterone levels in adulthood in rats exposed to juvenile social
stress (Mancini et al., 2021). Interestingly, they also observed that ju­
venile social stress confers resilience against emotional behavior and
spatial memory dysfunction induced by exposure to stressors later in
life. Many factors may explain such differences with their and our study,
including the rat species used, the timing of the RSD protocol as they
performed it between day 28 and 34 while we did it between day 35 and
40, differences in RSD procedure, and in the adulthood stressor as they
performed a single prolonged stress (2 h restraint stress followed by
15min forced swim and isoflurane until loss of consciousness) while we
conducted an immune challenge by injecting LPS (Mancini et al., 2021).
In our study, enhanced glial reactivity to the LPS injection was
apparent in several brain regions after the combination of social stress
and the LPS immune challenge, as compared to only an LPS challenge.
Non-invasive [11C]PBR28 PET imaging revealed that LPS injection alone
increased TSPO levels, a protein associated with microglial reactivity in

7
C.G.J. Guerrin et al.
several brain regions, such as the frontal cortex, striatum, and brain­
stem. In accordance with other studies (Moraga-Amaro et al., 2022;
Rodríguez-Arias et al., 2018), social stress during juvenile age or
adulthood induced a long-lasting increase in reactive microglia in brain
regions related to stress-induced psychopathologies, such as the frontal
cortex, striatum, brainstem and hippocampus. This result is in line with
human studies that observed a link between early life stress character­
ized as childhood maltreatment and being raised in a ‘harsh family’ and
heightened peripheral and central markers of inflammation (Danese and
J Lewis, 2017; Miller and Chen, 2010). Furthermore, our TSPO PET
results have translational value as TSPO tracers are also used clinically
and imaging studies observed increased proinflammatory states and
TSPO upregulation in the hippocampus, amygdala, prefrontal cortex,
and the anterior cingulate cortex in people suffering from major
depressive disorder (Gritti et al., 2021; Kim and Won, 2017).
The longitudinal within-group comparisons using in vivo TSPO PET
imaging revealed that the increased microglial reactivity to the LPS
challenge was more pronounced when the rats were previously exposed
to social adversity during juvenile age or adulthood. The microglial
reactivity as indicated by TSPO upregulation was significantly higher in
the basal ganglia, amygdala, nucleus accumbens, temporal, entorhinal
and insular cortices of the rats previously exposed to juvenile RSD.
Microglial cell density, as measured with iba1 staining, was higher in the
temporal and parietal cortices of rats previously exposed to juvenile
RSD. This effect was less pronounced in rats previously exposed to
adulthood RSD. These results indicate the increased susceptibility
induced by the previous exposure to social stress may alter the respon­
sivity of the immune system. Another research group observed that
8
Neurobiology of Stress 23 (2023) 100526
Fig. 6. Density of microglia 3 days after the LPS
injection on day 93. Representative Iba1 staining of
microglia in the parietal cortex of control (A.),
RSDjuv (B.), and RSDadu (C.) rats and temporal
cortex of control (D.), RSDjuv (E.), and RSDadu (F.)
rats. Number of Iba1-positive cells/mm2 in the pari­
etal (G.) and temporal (H.) cortices. Nearest neighbor
distance analysis in the parietal (I.) and temporal (J.)
cortices. n = 5 rats per group. Box represents the
interquartile range, the whiskers the min to max
values and the center line indicates the median. Sta­
tistically significant differences between groups are
indicated by asterisks: *p < 0.05, **p < 0.01, ***p <
0.001.
previous exposure to social adversity enhanced the peripheral and
central inflammatory response to a subsequent acute social stress chal­
lenge one week later (Finnell et al., 2019). Furthermore, our results are
also in accordance with a similar study that observed that RSD enhanced
the microglial response in the hippocampus, ventricular nucleus and
PFC following an innate immune challenge consisting in injecting LPS 4
h after the last episode of social adversity (Wohleb et al., 2012). Our
studies are complementary as they observed short-term priming of the
immune system following the last episode of social stress, while we
observed that this priming effect remained for more than a month
following the last stressor.
Long-lasting microglial reactivity to the stressors was apparent in
several brain regions important for the regulations of emotions, stress,
memory, and cognition and known to be involved in psychopathologies,
such as schizophrenia. The frontal association cortex and hippocampus
are primary integrators of the stress response (McEwen et al., 2016).
These regions keep developing and maturing throughout that juvenile
age and until early adulthood (Andersen and Teicher, 2008; He and
Crews, 2007). Increased inflammation in these regions may compromise
their development and underlie the long-term behavioral alterations
induced by social stress and increased the susceptibility to stressors and
immune challenges later in life. For example, extended activation of
CD11b + cells in the hippocampus is associated with decreased neuro­
genesis and cognitive impairment (Wohleb et al., 2012).
Despite that the use of in vivo PET imaging proved useful to non-
invasively monitor the effects of social stress and LPS on microglial
states in vivo, our study includes several limitations. Limitations to this
study include the absence of direct measurement of brain and plasma
C.G.J. Guerrin et al.
pro- and anti-inflammatory cytokines such as interleukin 1 b, and
interleukin 10, respectively. Furthermore, our study design does not
allow to find causality between the changes in corticosterone levels,
microglia, and behavior. Future studies using drugs such as colony-
stimulating factor 1 receptor inhibitors to deplete microglia during the
stressors could verify causality.
5. Conclusion
Our results indicate that previous exposure to social adversity
increased sensitivity to an immune challenge later in life. The stress-
induced enhanced sensitivity was accompanied by a long-term in­
crease in plasma corticosterone levels and microglial reactivity to the
RSD. However, only the rats exposed to the social stress during juvenile
age, but not adulthood, exhibited increased microglial density in the
frontal cortex and enhanced susceptibility towards the development of
anhedonia and social interaction dysfunction after the immune chal­
lenge. This suggests that juvenile social stress can have more deleterious
effects in the long term than a similar stress in adulthood.
Formatting of funding sources
This research did not receive any specific grant funding agencies in
the public, commercial, or not-for-profit sectors.
CRediT authorship contribution statement
Cyprien G.J. Guerrin: Conceptualization, Methodology, Formal
analysis, Investigation, Writing – original draft. Janine Doorduin:
Conceptualization, Methodology, Writing – review & editing, Supervi­
sion. Kavya Prasad: Investigation, Writing – review & editing. Daniel
A. Vazquez-Matias: Investigation, Writing – review & editing. Lara
Barazzuol: Resources, Writing – review & editing. Erik F.J. de Vries:
Conceptualization, Methodology, Writing – review & editing,
Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors would like to thank Prof. Dr. Wia Baron and the mem­
bers of the Baron’s groups and Jurgen Sijbesma for the experimental
support and Ulysse Guerrin for the drawings of the graphical abstract.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ynstr.2023.100526.
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