Brain, Behavior, and Immunity 70 (2018) 131–140

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Brain, Behavior, and Immunity

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Full-length Article

Impact of maternal immune activation on maternal care behavior,

offspring emotionality and intergenerational transmission in C3H/He
mice
Stefanie Berger a, Marianne Ronovsky a, Orsolya Horvath a, Angelika Berger b, Daniela D. Pollak a,⇑
a
Department of Neurophysiology and Neuropharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Austria
b
Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Austria

a r t i c l e i n f o a b s t r a c t

Article history: Maternal immune activation (MIA) is a well-established model for the investigation of the deleterious
Received 23 November 2017 effects of gestational infection on offspring mental health later in life. Hence, MIA represents a critical
Received in revised form 13 February 2018 environmental variable determining brain development and the depending neural and behavioral func-
Accepted 13 February 2018
tions in the progeny. Transgenerational transmission of some of the effects of MIA has been recently
Available online 1 March 2018
reported using the Polyinosinic:polycytidylic acid (Poly (I:C)) MIA model in C57BL/6 (C57) inbred mice.
However, little is known about the underlying molecular mechanisms and the possible relevance of
Keywords:
the specific genetic make-up of the inbred mouse strain used. Here we set out to characterize the effects
C3H/HeNCrl
Maternal behavior
of gestational Poly (I:C) treatment in C3H/HeNCrl mice (C3H), focusing on maternal care and offspring
Depression-like behavior depression-like behavior and its intergenerational potential. miRNA expression in the offspring hip-
Intergenerational transmission pocampus in the F1 and F2 generations was examined as possible mechanism contributing to the
Maternal immune activation observed behavioral effects.
miRNA The impact of MIA on maternal care and its transmission to F1 females was previously observed in C57
mice was also found in C3H mice. Depression-like behavior in the adult offspring in C3H F1 and F2
females differed from reports of the C57 strain in the literature, suggesting a potential modulating role
of the genetic background in the Poly(I:C) MIA mouse model. As the pattern of expression of selected can-
didate miRNAs in the F1 and F2 offspring hippocampus was not conserved between the two generations,
it is unlikely to be a direct consequence of altered maternal care, or to be an immediate determinant of
offspring emotionality.
Ó 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction tions underlying these multifaceted pathologies (Flint and

Psychopathologies are one of the most prevalent illnesses
worldwide (Whiteford, 2013). Besides the impact on the affected
individuals’ quality of life and the high mortality (90% of the 800
000 suicide cases every year worldwide are associated with psychi-
atric disorders (WHO, 2017) the burden on patients’ families and
the socioeconomic relevance of these severe conditions are signif-
icant. Yet, etiologies and pathomechanisms underlying psychiatric
disorders remain incompletely understood. This is largely due to
our still incomplete knowledge about the individual components
forming part of the intricate web of gene x environment interac-
⇑ Corresponding author at: Department of Neurophysiology and Neuropharma-
cology, Center for Physiology and Pharmacology, Medical University of Vienna,
Schwarzspanierstrasse 17, A-1090 Vienna, Austria.
E-mail address: daniela.pollak@meduniwien.ac.at (D.D. Pollak).
Kendler, 2014).
Increased risk factors for developing psychiatric disorders are
early adverse life events, including childhood trauma and exposure
to infections during pregnancy (Cowan, 2016; Groger, 2016;
McEwen, 2003; Meyer, 2008). Mounting evidence suggests an
association between maternal immune activation (MIA), as a result
of gestational infection, and the development of affective and per-
sonality disorders, as well as autism spectrum disorders later in life
of the offspring (Knuesel, 2014; Reisinger, 2015; Ronovsky, 2016).
Using a MIA mouse model we and others have shown a possible
relevance of altered maternal care behavior (MB) for derangements
in offspring emotional behavior (Meek, 2001; Meaney, 2001;
Champagne, 2008; Ronovsky, 2017). Specifically, the known
importance of MB for the regulation of epigenetic processes
(Champagne, 2013; Herman and Cullinan, 1997; Weaver, 2004)
suggests an involvement of the recently delineated transgenera-

https://doi.org/10.1016/j.bbi.2018.02.008
0889-1591/Ó 2018 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
132 S. Berger et al. / Brain, Behavior, and Immunity 70 (2018) 131–140
tional effects of MIA in the Polyinosinic:polycytidylic acid (Poly (I:
C)) mouse model (Ronovsky, 2017; Weber-Stadlbauer, 2017).
Indeed, gestational psychosocial and infectious stress with impact
on MB and offspring behavior has been shown to affect essential
epigenetic mechanisms, including posttranslational modifications
of histones (Reisinger, 2016), DNA methylation (Weaver, 2004)
and microRNA (miRNA) expression (Zucchi, 2013) in the offspring
brain. However, our own and a previous study describing altered
MB in the Poly (I:C) MIA model (Ronovsky, 2016; Schwendener
et al., 2009), as well as the reports on the transmission of patholog-
ical traits in offspring after MIA (Ronovsky, 2017; Weber-
Stadlbauer, 2017) have been carried out in the C57BL/6 (C57)
mouse strain. Hence, little is known on the relevance of the genetic
background in this context, although high degrees of variability in
MB between different inbred strains of laboratory mice have been
reported (Koehl, 2012; van der Veen, 2008). This led us to explore
the consequences of MIA in C3H/He (C3H) mice, an inbred mouse
strain with demonstrated high levels of MB (Koehl, 2012; Carlier
et al., 1982), focusing on offspring emotional behavior, its intergen-
erational transmission and the role of miRNA expression in the off-
spring brain as potential molecular correlate.
2. Material and methods
2.1. Animals
C3H/HeNCrl (C3H) mice were used for all experiments. Initial
breeding animals were purchased from Charles River (Sulzfeld,
Germany) at 8 weeks of age. All animals were housed under stan-
dard conditions in a temperature controlled room (22 ± 1 °C) with
a 12 h light/dark cycle and food and water ad libitum unless other-
wise stated. The light intensity was 10 l inside the cages. All ani-
mal experiments were carried out in accordance with the ARRIVE
guidelines and the U.K. Animals (Scientific Procedures Act, 1986
and associated guidelines, EU Directive 2010/63/EU for animal
experiments). Animal experiments described in this study were
approved by the national ethical committee on animal care and
use (BMWF-66.009/0015-II/3b/2012; Bundesministerium für Wis-
senschaft und Forschung).
2.2. Breeding and maternal immune activation (MIA)
A timed mating procedure for breeding of the F1 and F2 genera-
tions was applied as previously described (Khan, 2014). Briefly, after
overnight mating for 14 h, the presence of vaginal plug and body
weight of females were recorded the following morning and this
day was considered as E0.5. Body weight was further determined
every 6 days and females with over 16% weight gain on E12.5 (Hau
and Skovgaard, 1987) were assumed to be pregnant and treated with
Poly(I:C) (Sigma Aldrich, Vienna, Austria) or 0.9% NaCl (control
group). Poly (I:C) phosphate salt was dissolved in 0.9% physiological
NaCl and 20 mg/kg of Poly(I:C) or 0.9% NaCl were applied intraperi-
toneal at 10 ml/ kg injection volume. The dosage regime and time
point were chosen based upon previous studies demonstrating
enhanced depression-like behavior in adult offspring after MIA on
E12.5 (Ronovsky, 2017; Reisinger, 2016; Khan, 2014).
Upon delivery (PD 0) the collective weight of each litter and the
number of pups were recorded.
MB was recorded and evaluated from postnatal day (PD) 1–6
according to a standardized protocol (Champagne and Variations,
2011). All pups were weaned on PD 21, separated by sex and
single-housed for behavioral experiments at the age of 8 weeks.
Adult F1 offspring were separated in several cohorts and used for
breeding of F2 offspring, hippocampal gene expression and behav-
ior experiments. 2–3 mice per litter have been used for behavioral
and 2 animals per litter for gene expression studies.
A mating scheme for generation of F1 and F2 offspring is illus-
trated in Fig. 1. For offspring behavioral characterization each
mouse underwent a battery of behavioral tests as depicted in Fig. 2.
2.3. Behavior analysis
2.3.1. Maternal care behavior (MB)
MB was recorded in F0 and F1 dams on PD 1–6 between 11am
to 1pm and 3pm to 5pm using commercially available webcams
(Logitech C525 HD Webcam, Microsoft LifeCam HD-3000, Trust
Widescreen HD-Webcam, Creative LIVE! Cam Chat HD USB-
Webcam). Behavioral observation was conducted by video scoring
every 3 min by an experimenter blinded to the experimental con-
ditions and following a previously described protocol (Champagne
and Variations, 2011). Parameters evaluated included pup licking/-
grooming, nest-building, nursing and non-pup related parameters
(eating/sleeping, self-grooming).
2.3.2. Sucrose preference test (SPT)
The SPT assessing anhedonic behavior was carried out accord-
ing to a standard procedure (Monje, 2011; Savalli, 2015). Briefly,
during a 3 h testing period mice were given the choice to drink
from 2 equal bottles placed in their home cage. One bottle con-
tained a 2% sucrose solution (Sucrose from Sigma Aldrich) while
the other one was filled with regular drinking water. The weight
change of the bottles before and after testing was evaluated in
order to calculate the volume of liquid consumption and percent-
age of sucrose preference.
2.3.4. Forced swim test (FST)
The FST, which measures depression-related behavior by
assessing active versus passive responses in an acute stress situa-
tion, was carried out according to a standardized protocol (Khan,
2014; Pollak, 2008). Mice were placed in glass beakers 19 cm in
diameter and 23 cm deep filled with water at 22–24 °C for the total
test period of 6 min. The movements of the mice were automati-
cally recorded using a tracking software (VIDEOTRACK (PORSOLT),
Viewpoint, Champagne au mont d’Or, France). Immobility (exclud-
ing movements to prevent drowning) was evaluated for the last 4
min of the test.
2.3.5. Open Field test (OF)
The OF procedure was adapted from a standard protocol (Khan,
2014) and recorded for 60 min by computational tracking system
(Activity monitor, Med Associates St Albans, VT, USA) in an 27.5 c
m  27.5 cm arena using horizontal infrared beams. Standard
parameters for locomotor activity and exploratory behavior were
automatically recorded using the software.
2.3.6. Rota rod test (RR)
The rota rod and a computational monitoring system (USB Rota
Rod ‘‘SOF-ENV-57X”, MedAssociates Inc., St. Albans USA) were
used to evaluate motor coordination according to a previously used
protocol (Khan, 2014; Rogers, 1999). The experiment lasted for a
maximum of 5 min and the latency for the animal to fall from
the rod and the speed of rotation was recorded by the software.
The experiment was repeated for 3 times within a 15 min break
between trials and the average of the time of an animal balancing
on the rod was calculated (Rogers, 1999).
2.3.7. Light/dark box test (LD)
In LD experiments the anxiety-like behavior was analyzed by
placing a designated insert into the OF test chambers (Activity
monitor, MedAssociates Inc., St. Albans USA). At the beginning of
 S. Berger et al. / Brain, Behavior, and Immunity 70 (2018) 131–140
Fig. 1. Breeding Scheme from F0 MIA dams to F2 offspring. Study design employed for the analysis of the effects of Poly (I:C)-induced maternal immune activation on
maternal care in the F0 and F1 generations and depression-like behavior and hippocampal miRNA expression in F1 and F2 female offspring.
Fig. 2. Battery of behavioral tests used for the phenotypical characterization of F1
and F2 offspring. F1 and F2 MIA offspring and control animals were behaviorally
characterized using the depicted behavioral tests in the displayed order. An inter-
test interval of 24 h was observed in all instances. SPT: Sucrose Preference Test, OF:
Open Field, LD: Light Dark Box, RR: Rota Rod, FST: Forced Swim Test. The green
boxes represent depression-related behavior, yellow boxes are general behavioral
assays and orange shows tests for anxiety-like behavior.
the 10 min testing period, animals were placed into the dark com-
partment and allowed to move freely between the two sides of the
chamber. The locomotor activity and the amount of time the ani-
mals spent in the light, dark or transition area and the zone entries
were automatically recorded by the software (Activity monitor,
Med Associates Inc., SOF 811).
2.4. Molecular analysis
2.4.1. Brain dissection
Animals were sacrificed by neck dislocation, brains were rapidly
dissected out and hippocampi were separated from the rest of the
brain tissue. Samples for subsequent mRNA analysis were stored in
RNase-free Eppendorf tubes containing RNAlaterÒ (Ambion, Aus-
tin, TX, USA).
133
2.4.2. RNA Extraction, cDNA synthesis and quantitative real-time PCR
(qRT-PCR)
Total RNA (including miRNAs) was extracted from hippocampal
tissue using a commercial kit (miRNeasy microKit, Qiagen) accord-
ing to the provided protocol. RNA concentration and purity were
determined using a Nanodrop photometer (NanoPhotometerTM,
IMPLEN, 7122 V2.3.1).
500 ng of total RNA was used for the synthesis of cDNA from
mature miRNAs using miScript II RT Kit (Qiagen) according to the
instructions provided.
qRT-PCR was carried out using the miScript SYBR Green PCR Kit
(Qiagen) and a BioRad (Life Science Group, Vienna, Austria) PCR
machine (CFX384 Touch Real-Time PCR Detection System). All
miRNA primers were obtained from Qiagen (miScript Primer Assay,
Qiagen; see Supplementary Table 1 for primer sequences). Each
cDNA sample was analyzed in duplicate. dCT was determined by
normalization of the Ct value for each miRNA to Ct value of the ref-
erence small nucleolar RNA SNORD61 as housekeeping control
(Qiagen). Data were transformed into relative values 2-DDCT
(ddCT), with ddCT constituting the difference between the mean
dCT of each group and the mean dCT of the control group, as pre-
viously described (Savalli, 2015).
2.5. Statistical analysis
All data were tested for normality using the Kolmogorov-
Smirnov test prior to further statistical evaluation. Statistical anal-
ysis of MB in F0 generation was based upon using unpaired two-
tailed Student’s t test. Two-way analysis of variance (ANOVA) (tr
eatment  sex) or (mother  father) was employed for statistical
evaluation of the MB in the F1 generation as well as SPT, FST, LD,
OF and RR in F2 offspring and gene expression in F1 and F2 off-
spring. For evaluation of between-group differences in F1 maternal
behavior, F2 depression-like behavior and F2 gene expression, pair-
wise comparisons were calculated. All statistical analyses were
134 S. Berger et al. / Brain, Behavior, and Immunity 70 (2018) 131–140
performed using SPSS software, for windows, Version 23 (IBM cor-
poration, Chicago, IL, USA).
3. Results
3.1. Maternal immune activation affects maternal behavior of F0 dams
Following a previous report in C57Bl/6N mice (Ronovsky, 2017),
analysis of MB from PD 1–6 was conducted in C3H dams after ges-
tational treatment with Poly (I:C) (PIC) at E12.5. Statistical analysis
revealed a significant reduction in the time PIC mothers spent in
licking/grooming (LG) behavior (p < 0.01) while more time spent
in nest-building behavior was observed (p < 0.05) (Fig. 3a and b).
No differences in nursing and non pup-oriented behaviors (eating/-
drinking, sleeping, self-grooming) were observed (Fig. 3c and d).
No effect of MIA on growth in pregnancy, litter size and composi-
tion was observed (Supplementary Table 3).
3.2. Augmentation of anhedonic behavior in F1 female offspring
In order to evaluate the effects of MIA on depression-like behav-
ior in F1 offspring (Ronovsky, 2017; Reisinger, 2016; Khan, 2014),
the behavioral performance of C3H F1 female MIA and control off-
spring were evaluated in the SPT and the FST, respectively. A signif-
icant reduction in sucrose preference in the SPT was observed in
MIA as compared to saline control offspring (Fig. 4a). No group dif-
ferences in immobility in the FST, locomotor activity in the OF,
anxiety-like behavior in the OF (center entries) and LD and motor
coordination in the RR were detected in F1 female offspring
(Fig. 4b–f).
3.3. Alterations in maternal behavior in F1 MIA offspring
In order to determine a potential impact of MIA on offspring
maternal care behavior, MB was evaluated in F1 dams of PIC and
Saline (SAL) exposed females mated in a 2  2 design in order to
evaluate possible differential contributions of the effects of MIA
Fig. 3. Maternal care behavior in F0 dams after MIA induced by Poly (I:C) stimulation at E12.5. Presentation of a.) licking/grooming, b.) nest-building, c.) nursing and d.) non
pup-oriented behaviors in Poly (I:C) stimulated (PIC) mothers relative to the percentage of total time spent by saline control (SAL) mothers (n = 14 per group). Data are
depicted as mean ± SEM; *: p < 0.05, **: p < 0.01.
on MB along the maternal or paternal lineages (Fig. 1). 2-way
ANOVA analysis of F1 MB revealed a significant main effect of
the mother (F(3,21) = 6.5, p < 0.05) and a significant mother  father
interaction (F(3,21) = 6.3, p < 0.05) on the time mothers spent in LG.
For LG behavior, post-hoc comparisons identified a significant dif-
ference (p < 0.05) between $SAL  #SAL and $SAL  #PIC and a sig-
nificant difference (p < 0.01) between $SAL  #PIC and $PIC  #PIC
(Fig. 5a). For the time F1 mothers engaged in nest-building behav-
ior, a significant mother x father interaction (F(3,21) = 10.3, p < 0.01)
was observed. Post-hoc comparisons determined a significant dif-
ference (p < 0.01) between $PIC  #SAL and $PIC  #PIC and a sig-
nificant difference (p < 0.01) between $SAL  #PIC and $PIC  #PIC
(Fig. 5b). A significant interaction mother  father (F(3,22) = 6.1, p <
0.05) was also detected for the time dams spent nursing the pups.
Here, post-hoc comparisons revealed a significant difference (p <
0.05) between $PIC  #SAL and $PIC  #PIC and a significant dif-
ference (p < 0.05) between $SAL  #PIC and $PIC  #PIC (Fig. 5c).
No statistically significant effects on the time a mother engaged
in non pup-oriented behavior were found (Fig. 5d). F1 breedings
of all groups showed comparable litter size and composition (Sup-
plementary Table 3).
3.4. Intergenerational transmission of depression-like behavior to F2
offspring
We next went on to examine the behavioral phenotype of F2
offspring originating from one of the four different pedigrees. For
anhedonic behavior in the SPT, two-way ANOVA revealed a signif-
icant mother x father interaction (F(3,34) = 43.3, p < 0.0001); post-
hoc pairwise comparisons determined a significant difference (p
< 0.01) between $SAL  #SAL and $SAL  #PIC, a significant differ-
ence (p < 0.001) between $SAL  #SAL and $PIC  #SAL, a signifi-
cant difference (p < 0.001) between $SAL  #PIC and $PIC  #PIC
and a significant difference (p < 0.001) between $PIC  #SAL and
$PIC  #PIC revealing augmented sucrose consumption in these
animals (Fig. 6a). For the FST a significant main effect of the mother
(F(3,29) = 7.0, p < 0.05) and significant main effect of the father
 S. Berger et al. / Brain, Behavior, and Immunity 70 (2018) 131–140 135

Fig. 4. Behavioral phenotypes of F1 female MIA offspring. a.) Sucrose Preference in the sucrose preference test (SPT) and b.) immobility in the forced swim test (FST) as
indicators of depression-like behavior in F1 female MIA and saline control offspring. c.) exploratory and locomotor activity in the Open Field (total distance travelled), d-e.)
anxiety – like behavior in the Open Field and light/dark box (with center zone entries and percentage of time in the light area) and f.) motor coordination in the Rota Rod
(latency to fall of the rod) in F1 female MIA and control offspring. Data are displayed as mean ± SEM (n = 13–15 per group); *: p < 0.05.

Fig. 5. Maternal care behavior in F1 MIA dams. a.) Licking/grooming, b.) nest-building, c.) nursing and d.) non pup-oriented behaviors of F1 dams of PIC or SAL mothers mated
with male offspring of PIC or SAL mothers relative to the percentage of total time spent by SAL controls (mother ($) saline and father (#) saline). Data are depicted as mean ±
SEM. *p < 0.05, **p < 0.01 indicates results of post hoc pairwise comparisons.

(F(3,29) = 4.1, p < 0.05) was observed with highest immobility in the
$PIC  #PIC group (Fig. 6b). In the OF we found a significant mot
her  father interaction (F(3,40) = 5.7, p < 0.05) for the total distance
travelled in F2 offspring, revealing a pattern paralleling the obser-
vation in the SPT (Fig. 6c). Furthermore a significant main effect of
father (F(3,43) = 5.4, p < 0.05) was detected for the entries into the
center zone (Fig. 6d). No significant differences in the performance
between F2 offspring groups for anxiety-like behavior in the LD
and motor coordination in the RR were observed (Fig. 6e and f).
3.5. Hippocampal miRNA expression in F1 MIA offspring
In the search for a neurobiological mechanism possible mediat-
ing the consequences of F0 MIA on offspring emotionality in the F1
and the F2 generations we focused on specific miRNA (miR) species
with potential relevance for the neuro-immune system and
depressive disorders, according to a literature search (Supplemen-
tary Table 2). The hippocampus was selected as target region due
to its prominent involvement in the neural circuitry of depression
(Pandya, 2012; Krishnan and Nestler, 2008; Nestler, 2002) and ear-
lier reports of MIA induced molecular changes in F1 and F2 off-
spring hippocampus (Ronovsky, 2017; Reisinger, 2016; Khan,
2014).
Hippocampal levels of 13 miRNAs were analyzed by qRT-PCR in
behaviorally naïve MIA and control offspring of the F1 generation.
For the expression of miR-15a, no differences between groups were
observed (Fig. 7a). Levels of miR-15b-2 were significantly higher in
female PIC offspring (p < 0.05; Fig. 7b). A similar pattern with sig-
nificantly higher levels in the F1 generation of mice after MIA as
compared to control were revealed for miR-98-1 (Fig. 7c), miR-
136 S. Berger et al. / Brain, Behavior, and Immunity 70 (2018) 131–140

Fig. 6. Behavioral phenotypes of F2 female MIA offspring. a.) Percentage of sucrose preference in the sucrose preference test (SPT) and b.) immobility in the forced swim test
(FST) as indicators of depression-like behavior in F2 female offspring with heritage of PolyI:C stimulation along the maternal ($), paternal (#) or both ($  #) lineages. c.)
exploratory and locomotor activity in the Open Field (total distance traveled) d and e.) anxiety-like behavior in the Open Field and light/dark box (center zone entries and
percentage of time in the light area) and assessment of f.) motor coordination in the Rota Rod (latency to fall off the rod) in the same animals.and Data are depicted as mean ±
SEM. *p < 0.05, **p < 0.01, ***p < 0.001 indicates results of post hoc pairwise comparisons.

103-2 (Fig. 7d) and miR-124-1 (Fig. 7e). No significant group differ-
ences for any of the other miRNAs examined was detectable
(Fig. 7f–m).
3.6. Hippocampal miRNA expression in F2 MIA offspring
We further continued to test whether the detected changes in
the hippocampal expression of selected miR in the F1 generation
were also reflected in the F2 generation employing qRT-PCR anal-
ysis of hippocampal tissue of behaviorally naïve adult female F2
offspring with ancestral exposure to MIA and controls (see
Fig. 1). For miR-16, statistical analysis revealed a significant main
effect of the father (F(3,26) = 5.5, p < 0.05; Fig. 8a) with F2 offspring
of PIC along the paternal line showing reduced expression. A reflec-
tion of this pattern was also observed for miR-212-5p (F(3,24) = 7.6,
p < 0.05; Fig. 8b) and miR-132, albeit not reaching statistical signif-
icance in the latter (Fig. 8c). No expressional changes between
groups were found for any of the other miRNAs examined
(Fig. 8d–m).
4. Discussion
In the search for the origins of highly complex and severely dis-
abling mental illnesses, the prenatal period has emerged as a crit-
ically determining phase. The developing brain is particularly
vulnerable to the impact of environmental adversities which can
directly impinge on the wiring of the fetal neurons and induce
long-lasting structural, cellular and molecular alterations which
may form the basis for behavioral and cognitive dysfunctions later
in life. Infections during pregnancy have been long suspected to
contribute to the development of offspring psychiatric disorders
(Cowan, 2016; McEwen, 2003; Meyer, 2008) and excellent animal
models exists which have corroborated and extended epidemio-
logical findings and provided valuable tools for the examination
of the neural underpinnings (Reisinger, 2015; Ronovsky, 2016).
We and others have recently employed the Poly(I:C) mouse model
to demonstrate transgenerational effects of MIA on offspring emo-
tionality (Ronovsky, 2017; Weber-Stadlbauer, 2017) in C57 mice.
Here we built upon these findings to extend previous observations
in the MIA model to the C3H strain and explore the involvement of
miRNAs as possible molecular mediators.
We first focused on MB which was previously found to be
impacted by MIA in the C57 and BALB/cAnNCr1 strains
(Schwendener et al., 2009; Lucchina, 2010). In agreement with
our own study in C57 mice (Ronovsky, 2017) we here found that
also in C3H mice the amount of licking/grooming was reduced in
PIC dams which had experienced stimulation during pregnancy.
The other evaluated parameters, i.e. nest-building behavior, nurs-
 S. Berger et al. / Brain, Behavior, and Immunity 70 (2018) 131–140 137

Fig. 7. Hippocampal miRNA expression F1 female MIA offspring. Relative gene expression of a.) miR-15a-1, b.) miR-15b-2, c.) miR-98-1, d.) miR-103-2, e.) miR-124-1, f.) miR-
132-1, g.) miR-212-3p, h.) miR-212-5p, i.) miR-16-2, j.) miR-190b-1, k.) miR-144-3, l.) miR-219-1, m.) miR-219-2-3p determined by qRT-PCR in hippocampal tissue of F1
female MIA (PIC) and control (SAL) offspring. Results were normalized to SNORD61 as housekeeping gene and plotted relative to the mean of controls (n = 6–8 per group).
Data are displayed as mean ± SEM, *: p < 0.05.

ing and non pup-oriented behavior also followed the pattern ear-
lier described in C57 PIC dams (Ronovsky, 2017). Although it has
been reported that C3H mice display endogenously higher levels
of MB (Koehl, 2012; van der Veen, 2008), the current results sug-
gest that the genetically determined variation in MB does not mod-
ulate the impact of MIA. This is of particular relevance as LG is the
most significant form of tactile stimulation neonatal mouse pups
experience and constitutes a major influencing factor for emotional
behavior and gene expression in later in life (Champagne, 2003;
Pauline, 2014; Claessens, 2011; Pena et al., 2013; Fish, 2004;
Kaffman and Meaney, 2007; Ruthschilling, 2012; Hellstrom,
2012; Pedersen, 2011). However, in the absence of a direct statis-
tical comparison on the effects of MIA on MB in the same study,
it is not possible to draw final conclusions on strain-dependency
and the role of the genetic background.
Turning towards the consequences of MIA for offspring behav-
ior, augmented depression-related anhedonia was revealed in
female C3H MIA offspring in the SPT, along the lines of earlier
demonstrations in C57 mice (Reisinger, 2015). Interestingly, no
alterations in performance in the FST in C3H MIA offspring as com-
pared to controls was observed herein, which is in contrast to the
effects of PIC MIA reported in the C57 strain (Reisinger, 2015). We
138 S. Berger et al. / Brain, Behavior, and Immunity 70 (2018) 131–140

Fig. 8. Hippocampal expression of miRNA in F2 female MIA offspring. Relative gene expression a.) miR-16-2, b.) miR-212-5p, c.) miR-132-1, d.) miR-15a-1, e.) miR-15b-2, f.)
miR-98-1, g.) miR-103-2, h.) miR-212-3p, i.) miR-124-1, j.) miR-190b-1, k.) miR-144-3, l.) miR-219-1, m.) miR-219-2-3p determined by qRT-PCR in hippocampal tissue of F2
female offspring with heritage of Poly (I:C) stimulation along the maternal ($), paternal (#) or both ($  #) lineages. Results were normalized to SNORD61 as reference gene
and plotted relative to the mean of the controls (mother ($) saline and father (#) saline) (n = 4–9 per group). Data are depicted as mean ± SEM. *p < 0.05, **p < 0.01 indicates
results of post hoc pairwise comparisons.
 S. Berger et al. / Brain, Behavior, and Immunity 70 (2018) 131–140
focused on consequences of MIA on female offspring brain and
behavior in the present study for several reasons: First, the inves-
tigation of depression-related behaviors in animal models and
their neural underpinnings is of particular interest in female ani-
mals, considering the significantly higher prevalence of depression
in women compared to men (Kessler, 1994; Nolen-Hoeksema,
1987). Second, despite this relevance of the investigation of female
subjects in preclinical as well as in clinical studies, an overwhelm-
ingly larger part of experiments are currently being conducted
exclusively in males, although an emphasis on the characterization
of female animals has been declared a priority in depression-
related research (Krishnan and Nestler, 2011). Third, in light of
the investigation of maternal care behavior and its intergenera-
tional transmission in the present study, which naturally can only
be studied in dams, we have opted to also focus on females for the
analysis of offspring behavior and its molecular correlates. Addi-
tionally, when examining potential consequences of MIA of male
C3H offspring, sex-effects have been noted, with the effects on
depression-like behavior being more pronounced in dams than in
sires (data not shown).
The effects of MIA on offspring immobility in the FST observed
herein in C3H mice differ from the reports in C57 mice which may
in part relate to the described baseline differences between the
strains in the FST (Uz and Manev, 2001). For anxiety-like behavior,
exploratory activity and motor coordination, no effect of MIA was
observed in adult offspring which is in line with published results
in C57 mice (Reisinger, 2015; Khan, 2014).
We next went on to explore the potential intergenerational
transmission of the effects of PIC MIA in C3H mice by examining
MB in F1 offspring. The overall pattern of MB in F1 C3H mothers
was comparable to the earlier observations in C57 mice, confirm-
ing that LG was reduced when one parent had PIC origins, but
enhanced in $PIC  #PIC matings, possibly constituting compen-
satory mechanisms (Ronovsky, 2017). Interestingly, the amount
spent nest-building was lowest in $PIC  #PIC dams while in con-
trast to the previous report in C57 mice, no differences in non pup-
oriented behavior was found in F1 dams. Thus, while the individual
results may depend on the genetic background, results of the pre-
sent study in CH3 F1 mothers confirm the central finding that the
impact of gestational PIC treatment on MB can be transmitted to
the following generation.
This conclusion let us to further examine the possible impact of
MIA on emotional behavior in the F2 generation, which has been
earlier described in C57 F2 offspring (Ronovsky, 2017; Weber-
Stadlbauer, 2017). For depression-related behaviors which were
investigated in the SPT and FST, an interesting arrangement of
varying degrees of intergenerational transmission and modifica-
tion of individual behavioral traits was revealed: sucrose consump-
tion in the SPT was augmented in F2 offspring with either F1
parent having gestational PIC heritage, suggesting a ‘‘protective”
effect of MIA on anhedonic behavior in the F2 generation in the
C3H strain which differs from earlier findings in C57 mice
(Ronovsky, 2017). In contrast, performance in the FST was found
to follow the pattern previously described in C57 mice with F2 off-
spring from the $PIC  #PIC group presenting with the highest
degree of immobility (Ronovsky, 2017). Interestingly, no differ-
ences in MIA versus control offspring were found in performance
in the FST in F1 generation of C3H mice. The novel emergence of
augmented depression-like behavior in the FST in the F2 genera-
tion is reminiscent of earlier findings in C57 mice (Weber-
Stadlbauer, 2017) where – using a PIC treatment regime differing
from the employed herein and in our previous report (Ronovsky,
2017) – enhanced depression-like behavior in the FST was found
to emerge de-novo in the F2 MIA offspring and remained stable
until the F3 generation (Weber-Stadlbauer, 2017). Overall, these
observations further highlight the complexity of endogenous and
139
exogenous factors contributing to the development of emotional
disturbances during an individuaĺs lifespan.
Searching for molecular correlates we turned towards epige-
netic mechanisms as known mediators of intergenerational effects,
focusing on miRNAs which are suggested as predictors of these
outcomes (Champagne, 2016). In a nested screening approach we
analyzed the expression of selected miRNAs in the hippocampus,
a brain region central to the neural circuitry in F1 and F2 MIA
and control offspring. Of the 13 miRNA species examined, which
have been included in the present study due to their reported
involvement in (1) depression-like behavior (Beveridge, 2010;
Hollins and Cairns, 2016); (2) prenatal stress (Zucchi, 2013) and
(3) neuroimmune mechanisms (Soreq and Wolf, 2011), only miR-
15b-2 (1), miR-98-1 and miR-103-2 (2) and miR-124-1 (3) were
found to be differentially expressed in hippocampal tissue of F1
MIA as compared to control offspring. MiR-219-2-3p (2) as well
as miR-132-1 and miR-212-5p-1 (3) showed increased levels in
MIA offspring, but fell short of statistical significance. This analysis
provides evidence for a specific pattern of miRNA expression
induced by gestational exposure to immune activation in the adult
offspring which differs from previous observations after prenatal
stress and changes observed in other animal models of
depression-like behavior. While other, herein not examined miR-
NAs might also be involved, all differentially expressed miRNAs
in the present study were found to present with higher levels in
the MIA offspring, suggesting a possible common regulatory mech-
anism (Pandya, 2012; Krishnan and Nestler, 2008). Interestingly,
when the same set of miRNAs was examined in the F2 generation
offspring, an overall pattern different to the F1 generation
emerged. No changes in the miRNAs differentially expressed in
the F1 generation was observed in the F2 progeny descending from
the any of the three different F1 MIA breeding groups. However,
the expression of miR-16-2 and mir-212-5p which was unaltered
in the F1 MIA hippocampus was determined by a main effect of
the father in F2 offspring. Albeit small, these changes were statis-
tically significant and indicate an intergenerational modification
in the effects of MIA on miRNA expression in the F1 and F2 off-
spring hippocampus. Overall, the rather modest magnitude of
change together with the different patterns in F1 and F2 genera-
tions suggest that the observed miRNAs are most likely not to be
considered as relevant epigenetic mediators of the behavioral con-
sequences of intrauterine exposure to immune activation and its
effect across generations.
5. Conclusion
The present study in C3H mice suggests a possible role for the
genetic background in the long-term consequences of MIA on off-
spring emotionality which remains to be further substantiated by
direct strain-wise comparisons. An impact of MIA on maternal care
and its transmission to F1 females was supported by the current
findings which are generally consistent with previous observations
in C57 mice. However, in the absence of cross-fostering experi-
ments, we are currently not in a position to discriminate prenatal
from postnatal influences or to determine the amount of the phe-
notype being caused by alterations in maternal care. Furthermore,
the precise nature of the contribution of the paternal lineage to
brain and behavior in the progeny is dependent on future experi-
ments which will, by the means of assisted reproduction technolo-
gies, aid to discriminate germline from non-germline determined
inheritance mechanisms, which are delineated in detail in excel-
lent reviews on this topic (Bale, 2015; Weber-Stadlbauer, 2017).
Considering that the pattern of hippocampal miRNA expression
is not conserved between the two generations, it is unlikely to be a
direct consequence of altered MB or to be an immediate determi-
140 S. Berger et al. / Brain, Behavior, and Immunity 70 (2018) 131–140

nant of depression-like behavior in the adult offspring which man-
ifested distinctively in the F1 and F2 females.
Conflict of interest
The authors declare no conflict of interest.
Funding
This work was supported by the Austrian Science Fund (FWF):
P272520 (to DDP) and the ‘‘Verein zur Foerderung der Forschung
auf dem Gebiet der Neonatologie und paediatrischen Inten-
sivmedizin” (to AB).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bbi.2018.02.008.
References
Bale, T.L., 2015. Epigenetic and transgenerational reprogramming of brain
development. Nat. Rev. Neurosci. 16 (6), 332–344.
Beveridge, N.J. et al., 2010. Schizophrenia is associated with an increase in cortical
microRNA biogenesis. Mol. Psychiatry 15 (12), 1176–1189.
Carlier, M., Roubertoux, P., Cohen-Salmon, C., 1982. Differences in patterns of pup
care in Mus musculus domesticus l-comparisons between eleven inbred strains.
Behav. Neural. Biol. 35 (2), 205–210.
Champagne, F.A. et al., 2003. Variations in maternal care in the rat as a mediating
influence for the effects of environment on development. Physiol. Behav. 79 (3),
359–371.
Champagne, D.L. et al., 2008. Maternal care and hippocampal plasticity: evidence
for experience-dependent structural plasticity, altered synaptic functioning,
and differential responsiveness to glucocorticoids and stress. J. Neurosci. 28
(23), 6037–6045.
Champagne, F.A., 2013. Early environments, glucocorticoid receptors, and
behavioral epigenetics. Behav. Neurosci. 127 (5), 628–636.
Champagne, B.F.J.P.C.F.A., 2011. In: Measuring Variations in Maternal Behavior:
Relevance for Studies of Mood and Anxiety. Springer Link, pp. 209–224.
Champagne, F.A., 2016. Epigenetic legacy of parental experiences: Dynamic and
interactive pathways to inheritance. Dev. Psychopathol. 28 (4pt2), 1219–1228.
Claessens, S.E. et al., 2011. Development of individual differences in stress
responsiveness: an overview of factors mediating the outcome of early life
experiences. Psychopharmacology (Berl) 214 (1), 141–154.
Cowan, C.S. et al., 2016. The lasting impact of early-life adversity on individuals and
their descendants: potential mechanisms and hope for intervention. Genes
Brain Behav. 15 (1), 155–168.
Fish, E.W. et al., 2004. Epigenetic programming of stress responses through
variations in maternal care. Ann. N.Y. Acad. Sci. 1036, 167–180.
Flint, J., Kendler, K.S., 2014. The genetics of major depression. Neuron 81 (5), 1214.
Groger, N. et al., 2016. The transgenerational transmission of childhood adversity:
behavioral, cellular, and epigenetic correlates. J. Neural Transm. (Vienna) 123
(9), 1037–1052.
Hau, J., Skovgaard Jensen, H.J., 1987. Diagnosis and monitoring of pregnancy in
mice: correlations between maternal weight, fetal and placental mass and the
maternal serum levels of progesterone, pregnancy-associated murine protein-2
and alpha-fetoprotein. Lab. Anim. 21 (4), 306–310.
Hellstrom, I.C. et al., 2012. Maternal licking regulates hippocampal glucocorticoid
receptor transcription through a thyroid hormone-serotonin-NGFI-A signalling
cascade. Philos. Trans. R. Soc. Lond. B Biol. Sci. 367 (1601), 2495–2510.
Herman, J.P., Cullinan, W.E., 1997. Neurocircuitry of stress: central control of the
hypothalamo-pituitary-adrenocortical axis. Trends Neurosci. 20 (2), 78–84.
Hollins, S.L., Cairns, M.J., 2016. MicroRNA: small RNA mediators of the brains
genomic response to environmental stress. Prog. Neurobiol. 143, 61–81.
Kaffman, A., Meaney, M.J., 2007. Neurodevelopmental sequelae of postnatal
maternal care in rodents: clinical and research implications of molecular
insights. J. Child Psychol. Psychiatry 48 (3–4), 224–244.
Kessler, R.C. et al., 1994. Lifetime and 12-month prevalence of DSM-III-R psychiatric
disorders in the United States. results from the national comorbidity survey.
Arch. Gen. Psychiatry 51 (1), 8–19.
Khan, D. et al., 2014. Long-term effects of maternal immune activation on
depression-like behavior in the mouse. Trans. Psychiatry 4, e363.
Knuesel, I. et al., 2014. Maternal immune activation and abnormal brain
development across CNS disorders. Nat. Rev. Neurol. 10 (11), 643–660.
Koehl, M. et al., 2012. Interplay of maternal care and genetic influences in
programming adult hippocampal neurogenesis. Biol. Psychiatry 72 (4), 282–
289.
Krishnan, V., Nestler, E.J., 2008. The molecular neurobiology of depression. Nature
455 (7215), 894–902.
Krishnan, V., Nestler, E.J., 2011. Animal models of depression: molecular
perspectives. Curr. Top Behav. Neurosci. 7, 121–147.
Lucchina, L. et al., 2010. Evaluating the interaction between early postnatal
inflammation and maternal care in the programming of adult anxiety and
depression-related behaviors. Behav. Brain. Res. 213 (1), 56–65.
McEwen, B.S., 2003. Early life influences on life-long patterns of behavior and
health. Ment Retard Dev. Disabil. Res. Rev. 9 (3), 149–154.
Meaney, M.J., 2001. Maternal care, gene expression, and the transmission of
individual differences in stress reactivity across generations. Annu. Rev.
Neurosci. 24, 1161–1192.
Meek, L.R. et al., 2001. Effects of stress during pregnancy on maternal behavior in
mice. Physiol. Behav. 72 (4), 473–479.
Meyer, U. et al., 2008. Adult brain and behavioral pathological markers of prenatal
immune challenge during early/middle and late fetal development in mice.
Brain Behav. Immun. 22 (4), 469–486.
Monje, F.J. et al., 2011. Constant darkness induces IL-6-dependent depression-like
behavior through the NF-kappaB signaling pathway. J. Neurosci. 31 (25), 9075–
9083.
Nestler, E.J. et al., 2002. Neurobiology of depression. Neuron 34 (1), 13–25.
Nolen-Hoeksema, S., 1987. Sex differences in unipolar depression: evidence and
theory. Psychol. Bull. 101 (2), 259–282.
Pandya, M. et al., 2012. Where in the brain is depression? Curr. Psychiatry Rep. 14
(6), 634–642.
Pan Pauline, F.A.S., Daeria, Lawson, Jenkins Jennifer, M., McGowan Patrick, O., 2014.
Within – and between-litter maternal care alter behavior and gene regulation in
female offspring. Behav. Neurosci. 128 (6), 736–748.
Pedersen, C.A. et al., 2011. Variations in maternal behavior in C57BL/6J mice:
behavioral comparisons between adult offspring of high and low pup-licking
mothers. Front. Psychiatry 2, 42.
Pena, C.J., Neugut, Y.D., Champagne, F.A., 2013. Developmental timing of the effects
of maternal care on gene expression and epigenetic regulation of hormone
receptor levels in female rats. Endocrinology 154 (11), 4340–4351.
Pollak, D.D. et al., 2008. An animal model of a behavioral intervention for
depression. Neuron 60 (1), 149–161.
Reisinger, S. et al., 2015. The poly(I:C)-induced maternal immune activation model
in preclinical neuropsychiatric drug discovery. Pharmacol. Ther. 149, 213–226.
Reisinger, S.N. et al., 2016. Maternal immune activation epigenetically regulates
hippocampal serotonin transporter levels. Neurobiol. Stress 4, 34–43.
Rogers, D.C. et al., 1999. Use of SHIRPA and discriminant analysis to characterise
marked differences in the behavioural phenotype of six inbred mouse strains.
Behav. Brain Res. 105 (2), 207–217.
Ronovsky, M. et al., 2016. Animal models of maternal immune activation in
depression research. Curr. Neuropharmacol. 14 (7), 688–704.
Ronovsky, M. et al., 2017. Maternal immune activation transgenerationally
modulates maternal care and offspring depression-like behavior. Brain Behav.
Immun. 63, 127–136.
Ruthschilling, C.A. et al., 2012. Analysis of transcriptional levels of the oxytocin
receptor in different areas of the central nervous system and behaviors in high
and low licking rats. Behav. Brain Res. 228 (1), 176–184.
Savalli, G. et al., 2015. Anhedonic behavior in cryptochrome 2-deficient mice is
paralleled by altered diurnal patterns of amygdala gene expression. Amino
Acids 47 (7), 1367–1377.
Schwendener, S., Meyer, U., Feldon, J., 2009. Deficient maternal care resulting from
immunological stress during pregnancy is associated with a sex-dependent
enhancement of conditioned fear in the offspring. J. Neurodev. Disord. 1 (1), 15–
32.
Soreq, H., Wolf, Y., 2011. NeurimmiRs: microRNAs in the neuroimmune interface.
Trends Mol. Med. 17 (10), 548–555.
Uz, T., Manev, H., 2001. Prolonged swim-test immobility of serotonin N-
acetyltransferase (AANAT)-mutant mice. J. Pineal Res. 30 (3), 166–170.
van der Veen, R. et al., 2008. Impact of intra- and interstrain cross-fostering on
mouse maternal care. Genes Brain Behav. 7 (2), 184–192.
Weaver, I.C. et al., 2004. Epigenetic programming by maternal behavior. Nat.
Neurosci. 7 (8), 847–854.
Weber-Stadlbauer, U., 2017. Epigenetic and transgenerational mechanisms in
infection-mediated neurodevelopmental disorders. Trans. Psychiatry 7 (5),
e1113.
Weber-Stadlbauer, U. et al., 2017. Transgenerational transmission and modification
of pathological traits induced by prenatal immune activation. Mol. Psychiatry
22 (1), 102–112.
Whiteford, H.A. et al., 2013. Global burden of disease attributable to mental and
substance use disorders: findings from the global burden of disease study 2010.
Lancet 382 (9904), 1575–1586.
WHO, Suicide (Fact Sheet), 2017.
Zucchi, F.C. et al., 2013. Maternal stress induces epigenetic signatures of psychiatric
and neurological diseases in the offspring. PLoS One 8 (2), e56967.