822141

research-article2019
JOP0010.1177/0269881118822141Journal of PsychopharmacologyMillard et al.

Original Article

Perinatal exposure to fluoxetine increases

anxiety- and depressive-like behaviours and
alters glutamatergic markers in the prefrontal Journal of Psychopharmacology

cortex and hippocampus of male adolescent

1­–14
© The Author(s) 2019
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DOI: 10.1177/0269881118822141
https://doi.org/10.1177/0269881118822141

rats and the Wistar-Kyoto rat model of journals.sagepub.com/home/jop

depression

Samuel J Millard1,2, Jeremy S Lum1,2, Francesca Fernandez3, Katrina

Weston-Green1,2 and Kelly A Newell1,2

Abstract
Background: With approximately 10% of pregnant women prescribed antidepressant drugs for the treatment of depressive disorders, there is growing
concern regarding the potential long-term effects of this exposure on offspring. Research is needed in clinically relevant models to determine the
effects on offspring behaviour and associated neurobiological systems.
Aim: The aim of this study was to determine the effects of maternal fluoxetine treatment on anxiety-like and depressive-like behaviours in adolescent
offspring as well as associated glutamatergic markers, using a clinically relevant rodent model of depression.
Methods: Wistar-Kyoto (model of innate depression) and Sprague-Dawley rats were treated with fluoxetine (10 mg/kg) from gestational day 0
to postnatal day 14. Male offspring underwent behavioural testing (open field, elevated plus maze, forced swim test) at adolescence followed by
quantitative immuno-detection of glutamatergic markers in the prefrontal cortex and ventral hippocampus.
Results: Perinatal fluoxetine exposure exacerbated the anxiety-like and depressive-like phenotype in Wistar-Kyoto offspring and induced an anxiety-
like and depressive-like phenotype in Sprague-Dawley offspring. Wistar-Kyoto offspring showed reductions in NMDA receptor NR1, NR2A and NR2B
subunits, as well as post-synaptic density 95 (PSD-95) and metabotropic glutamate receptor subtype 1 (mGluR1) in the prefrontal cortex; perinatal
fluoxetine exposure further reduced NR1, NR2A, PSD-95 and mGluR1 expression in Wistar-Kyoto as well as Sprague-Dawley offspring. In the ventral
hippocampus perinatal fluoxetine exposure reduced PSD-95 and increased metabotropic glutamate receptor subtype 5 (mGluR5) and Homer1b/c in
both Sprague-Dawley and Wistar-Kyoto strains.
Conclusion: These findings suggest that maternal fluoxetine treatment exacerbates effects of underlying maternal depression on offspring behaviour,
which may be mediated through alterations in the glutamatergic system. Further research investigating how to minimise these effects, whilst ensuring
optimal treatment for mothers, is essential to move the field forward.

Keywords
Maternal depression, offspring, perinatal selective serotonin reuptake inhibitor, fluoxetine, antidepressant, Wistar-Kyoto, glutamate, N-methyl-D-
aspartate receptor, metabotropic glutamate receptor, post-synaptic density 95

Introduction the most frequently prescribed antidepressants during pregnancy

Major depressive disorder (MDD) is one of the most common
mental illnesses affecting an estimated 300 m people worldwide
(World Health Organization, 2017). The overall incidence of
MDD has risen significantly over the last several decades
(Huybrechts et al., 2013; Kessler, 2003). Women display twice
the risk ratio compared to men, with the greatest risk of develop-
ing the disorder being at childbearing age (Huybrechts et al.,
2013; Marcus and Heringhausen, 2009). Untreated maternal
MDD and stress-related disorders are associated with adverse
outcomes for mother and child (Deave et al., 2008; Ronald et al.,
2011). Approximately 10% of pregnant women are prescribed
antidepressant drugs for the treatment of depressive and stress-
related disorders (Cooper et al., 2007; Huybrechts et al., 2013);
(in 80% of cases) are the selective serotonin reuptake inhibitors
(SSRIs) (Mitchell et al., 2011; Oberlander et al., 2009). SSRIs
such as fluoxetine (FLX), bind to the serotonin transporter,
1Molecular Horizons and School of Medicine, University of Wollongong,
Wollongong, NSW, Australia
2Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia
3School of Science, Australian Catholic University, Brisbane, QLD, Australia
Corresponding author:
Kelly A Newell, School of Medicine, Faculty of Science, Medicine and
Health, University of Wollongong, Northfields Ave, Wollongong, NSW
2522, Australia.
Email: knewell@uow.edu.au
2 Journal of Psychopharmacology 00(0)
blocking the reabsorption of serotonin by the presynaptic neuron
resulting in increased serotonin levels in the synaptic cleft
(Hiemke and Härtter, 2000). It has been shown that SSRIs admin-
istered during pregnancy cross the placenta (by as much as ~45–
70% of maternal levels) (Heikkinen et al., 2002) and are excreted
in breast milk (Hendrick et al., 2001; Kim et al., 2006), exposing
the developing foetus and newborn.
The serotonergic system plays an important role in early brain
development facilitating a number of neurodevelopmental pro-
cesses such as neuronal proliferation, migration and differentia-
tion (Vitalis and Parnavelas, 2003); of particular interest, the
serotonergic system regulates the development and maturation of
the brain’s primary excitatory neurotransmitter system, the gluta-
matergic system (Bonnin et al., 2007; Edgar and Price, 2001;
Lavdas et al., 1997; Vitalis and Parnavelas, 2003). Dysfunction of
the glutamatergic system, in particular the N-methyl-D-aspartate
(NMDA) ionotropic receptors and Group I metabotropic recep-
tors, has long been linked to neurodevelopmental and psychiatric
disorders including autism, attention deficit hyperactivity disorder
(ADHD), depression and schizophrenia (Deschwanden et al.,
2011; Hashimoto et al., 2007; Lakhan et al., 2013; Matosin et al.,
2013; Pérez de la Mora et al., 2006; Yuan et al., 2015). In line with
this, SSRI use during pregnancy has been associated with
increased risk of autism (Boukhris et al., 2016; Croen et al., 2011;
Gidaya et al., 2014; Harrington et al., 2014), ADHD (Clements
et al., 2015; Figueroa, 2010), depression (Malm et al., 2016) and
anxiety (Brandlistuen et al., 2015) in exposed children. However,
current evidence is conflicting and a number of studies have
reported no significant association between maternal SSRI expo-
sure and risk of neurodevelopmental or neuropsychiatric disor-
ders in offspring (for review see Millard et al., 2017). Additionally,
there is an inherent difficulty in discerning the effects of SSRI
exposure from that of the innate maternal depression in clinical
studies, especially given that maternal depression and adversity
are themselves associated with an increased risk for altered neu-
rodevelopment and associated disorders in offspring (Deave et al.,
2008; Glover, 2014; Ronald et al., 2011).
Preclinical studies investigating the effects of developmental
SSRI exposure on offspring behaviour in rodents have reported
alterations in social interaction, cognitive function, anxiety- and
depressive-like behaviours in adolescent and adult offspring (for
review see Millard et al., 2017). However, there has been no
investigation of the effects of SSRI exposure in rodent models of
innate depression that closely model MDD. Determining the
effects of maternal SSRI treatment on offspring behaviour in
rodent models of innate depression and understanding the neuro-
biological changes will be key to determining the clinical risk
associated with maternal SSRI use in context of the underlying
maternal psychopathology.
The aim of this study was to investigate the effects of maternal
SSRI (FLX) treatment on offspring behaviour and neurobiology
relevant to neurodevelopmental and psychiatric disorders.
Specifically, given the key role of serotonin in the development of
the glutamatergic system and the importance of the glutamatergic
system in neurodevelopmental disorders, we sought to investigate
the potential influence that maternal SSRI treatment has on key
glutamatergic markers at adolescence, particularly associated with
the NMDA and Group I metabotropic glutamate receptors and
associated scaffolding proteins. Furthermore, given the signifi-
cance of the prefrontal cortex (PFC) and ventral hippocampus
(vHPC) in neurodevelopmental and psychiatric disorders and their
biological sensitivity to the action of SSRIs (Brandon and McKay,
2015; Dusi et al., 2015), these brain areas were investigated. By
comparing the effects of maternal SSRI treatment in a rodent
model of innate depression (Wistar-Kyoto (WKY)) to that of a
standard laboratory rat strain (Sprague-Dawley (SD)), we aimed to
test the hypothesis that maternal depressive phenotypes (which
model depressive disorders) could influence the effects of SSRI
exposure on offspring neurobiology and behaviour.
Materials and methods
Animals
All experiments were approved by the University of Wollongong’s
Animal Ethics Committee in accordance with the Australian Code for
the Care and Use of Animals for Scientific Purposes (AE14/31). To
separate the effects of maternal FLX treatment from that of innate
maternal depression, SD (control strain) and WKY (model of innate
depression) rats were used in the study. The WKY rat strain has long
been established as a model of innate depression, displaying exagger-
ated stress responses, increased anhedonia, learned helplessness,
decreased locomotor activity and increased anxiety- and depressive-
like behaviours (López-Rubalcava and Lucki, 2000; Nam et al.,
2014; Pare and Tejani-Butt, 1996; Rittenhouse et al., 2002; Will et al.,
2003). The WKY strain is also characterised by alterations in stress
hormones and serotonergic alterations similar to those previously
reported in depressed individuals (Arango et al., 2001; Lemos et al.,
2011; López-Figueroa et al., 2004; Pardon et al., 2002). Studies have
reported increases in stress hormones such as basal corticosterone
and ACTH (Pardon et al., 2002; Will et al., 2003) and increased levels
of pro-opiomelanocortin in response to acute stress in comparison to
other strains (Redei et al., 1994; Will et al., 2003). The WKY strain
also shows decreased basal serotonin (5HT) concentrations,
decreased 5-hydroxyindoleacetic acid (5HIAA; metabolite of 5HT),
decreased serotonin transporter (SERT) density in hippocampus and
cortical regions, decreased 5HT1A receptor and a reduction in the rate-
limiting serotonin-producing enzyme, tryptophan hydroxylase
(Lemos et al., 2011; Pare and Tejani-Butt, 1996; Scholl et al., 2010).
Male and female SD and WKY rats (12 weeks of age) were
obtained from the Animal Resource Centre, Western Australia. Rats
were housed at the University of Wollongong Animal Housing
Facility in a temperature-controlled environment (20±20C) under a
12:12-hour light-dark cycle (lights on at 07:00). The rats were pro-
vided with ad libitum access to water and laboratory chow. Females
were housed one per cage (cage size 38×25×16 cm) to allow mon-
itoring of drug intake; males were housed two per cage of the same
strain. Following a minimum of one-week acclimatisation, male
and female SD or WKY rats were paired to elicit mating. At the
conclusion of the mating period, pregnant dams were randomly
assigned to a FLX or vehicle (VEH) (water) treatment group.
Following the birth of pups, which was designated as post-natal day
(PN) 0, pups were housed with their dam and litter mates until
PN21–28 days of age, at which time they were weaned and housed
2–3 per cage with their littermates of the same sex. Perinatal FLX
treatment did not influence litter sizes (VEH: 10.3±0.88 pups/litter;
FLX: 9.3±0.98 pups/litter; p>0.05), however dams of the WKY
strain did have significantly smaller litters compared to SD dams
(SD: 12.4±0.66 pups/litter; WKY: 7.6±0.63 pups/litter; p<0.001).
Pup body weights were determined at PN7, PN14 and PN42 as
Millard et al. 3
Figure 1.  Treatment paradigm and experimental timeline.
EPM: elevated plus maze; FLX: fluoxetine; FST: forced swim test; GD: gestational day; OFT: open field test; PFC: prefrontal cortex; PN: postnatal day; SD: Sprague-Dawley;
VEH: vehicle; vHPC: ventral hippocampus; WKY: Wistar-Kyoto.
measures of potential SSRI teratogenicity. Behavioural tests and
immunoblot analyses were performed at adolescence. Male off-
spring were the initial focus of this study, given the increased preva-
lence in males for neurodevelopmental disorders such as autism
(4:1) and ADHD (2.3:1), (Ramtekkar et al., 2010; Werling and
Geschwind, 2013), and the reported association between maternal
FLX treatment and the risk for these disorders (Millard et al., 2017).
However, it will be important to investigate the potential effects of
perinatal FLX exposure on female offspring in future studies.
Maternal SSRI treatment
Pregnant SD and WKY dams were dosed with FLX hydrochloride
(LKT Laboratories, St Paul, Minnesota, USA) (10 mg/kg dis-
solved in tap water) or VEH (tap water), for the full gestational
term and a further two weeks of the lactation period (i.e. five
weeks in total), with the last day of treatment corresponding to
PN14 of the offspring, equivalent to birth in humans (Semple
et al., 2013). In total, 27 dams (and litters) were used in this study
comprising 12 FLX-treated dams (6×SD, 6×WKY) and 15
VEH-treated dams (7×SD, 8×WKY) (Figure 1). FLX was
administered in light-resistant gravity-fed water bottles to mini-
mise drug degradation via photolysis (Lam et al., 2005). Dam
water consumption and body weight were recorded daily through-
out the treatment period to ensure the correct dosage. While WKY
dams weighed less than SD dams (~25%), there were no differ-
ences between the strains or the treatment groups in percentage of
body weight change during gestation (p=0.818) or lactation
(p=0.599) periods. The average daily water consumption over the
treatment period was lower in WKY dams compared to SD dams
(42.4±3.7 vs 53.8±2.1 ml/day; p=0.002) and lower in the FLX
treated dams compared to the VEH treated dams (43.3±2.6 vs
57.6±2.6 ml/day; p=0.001). However, when accounting for differ-
ences in body weight between the dams there was no significant
difference in average water intake in WKY vs SD dams over the
treatment period (177.4±9.5 vs 202.7±8.7 g water/kg bodyweight;
p=0.068); there was however a 16% lower water intake in the
FLX treated dams compared to VEH treated dams (173.5±7.7 vs
206.6±10.3 g water / kg body weight; p=0.021).
Bottles were replaced with fresh FLX or vehicle solution
daily to minimise drug degradation while also allowing dosage to
be adjusted based on dams body weight and water intake. The
dose of 10 mg/kg was chosen based on previous animal models
that have shown this dose to produce antidepressant-like effects
in rodents (Dulawa et al., 2004; Kiryanova et al., 2016; Thompson
et al., 2004). Moreover, human neuroimaging studies have dem-
onstrated that clinically efficacious doses of SSRIs have resulted
in 80% or greater SERT occupancy; a paradigm that has been
achieved previously in rodents with dosages of 8–12 mg/kg/day
of FLX (Capello et al., 2011). The rationale behind the selected
drug administration method was based on the desire to minimise
unwarranted stress to the animals that may occur using subcuta-
neous or intraperitoneal injections as well as to model a more
clinically relevant scenario in which FLX is primarily adminis-
tered orally (Koran et al., 1996). Finally, this method and dosage
is generally well accepted within the current literature (Ansorge
et al., 2008, 2004; Bairy et al., 2006; Cabrera-Vera and Battaglia,
1998; Karpova et al., 2009; Kiryanova et al., 2013; Olivier et al.,
2011; Rodriguez-Porcel et al., 2011; Thompson et al., 2004).
Behavioural testing
Male offspring underwent a series of behavioural tests to meas-
ure primarily anxiety- and depressive-like behaviours using the
open field test (OFT), elevated plus maze (EPM) and forced
swim test (FST), (n=8–14/group) (Figure 1). Behavioural testing
began at PN35, corresponding to the adolescent period in rodents
(Semple et al., 2013) and was conducted between 12:00–1700.
The procedures for behavioural testing in the OFT, EPM and FST
were based on protocols previously implemented by du Bois
et al. (2008), Foldi et al. (2011) and Turner and Burne (2014).
Testing was conducted from least stressful to most stressful in the
order of OFT<EPM<FST, to limit any behavioural influences
that may result from prior testing (McIlwain et al., 2001).
4 Journal of Psychopharmacology 00(0)
Additionally, rat offspring were given one day of rest in between
behavioural tests as suggested by Paylor et al. (2006).
OFT
The OFT was used as a measure of locomotion, however time
spent in different zones of the arena also provides information on
anxiety-like behaviours (Prut and Belzung, 2003). Parameters
analysed included total distance travelled (as a measure of loco-
motor activity), time spent in the centre of the arena and time
spent in corners (as measures of anxiety-like behaviour). The
OFT apparatus was made up of a 60 cm×60 cm square arena
with 40 cm high walls. Lighting remained constant throughout
the procedure with light intensity set at ~20 lux. Upon com-
mencement of the test a single rat was placed within the centre
arena of the OFT, and remained within the apparatus for a testing
duration of 10 min.
EPM
The EPM was used as a measure of anxiety-like behaviour, explo-
ration and locomotor activity (Walf and Frye, 2007). The main
determining parameter of anxiety-like behaviour was the amount
of activity in the open arm of the apparatus versus that of the
closed arm, corresponding to increased or decreased anxiety-like
behaviours as the animal engages in or refrains from exploratory
behaviour (Walf and Frye, 2007). The EPM apparatus comprised
of one pair of closed, and one pair of open arms, with respective
dimensions 50 cm×7 cm×30 cm (length×width×height) and 50
cm×7 cm×1 cm. There was an open roof and the apparatus was
arranged around a centre platform, with the arms perpendicular to
each other. The maze was elevated 60 cm above the ground and
the light intensity set at a constant of ~100 lux across the open
arms. Rats were tested individually for five minutes, beginning
with the placement of the subject within the centre of the appara-
tus facing an open arm.
FST
The FST is widely implemented to assess escape-driven behav-
iour in the face of an inescapable aversive situation (Commons
et al., 2017; Slattery and Cryan, 2012). Passive behaviour (i.e.
immobility) is typically measured, and was the focus of the FST
given that it can be clearly defined and is distinguishable from
active behaviour and coping measures (such as swimming and
climbing), which can be difficult to reliably measure across exper-
imental observers (Slattery and Cryan, 2012). Increased immobil-
ity has previously been associated with ‘depressive-like’ behaviour
due to the observation that antidepressants increase the duration
of time the rodent spends engaged in active and escape-driven
behaviour (Castagné et al., 2011). Rats were tested under ceiling
lighting (500–750 lux) in an opaque cylinder (30 cm diameter
×50 cm depth) filled to a level 1.25×length of the rat (snout to
tail) with (25±1oC) water (Slattery and Cryan, 2012). The rats
were tested over two days, the first day of which involved famil-
iarising the rat to the test environment by placing the subject in the
water for 15 min. On the second day (24-hour post-habituation),
the subject was placed in the test apparatus filled to a predeter-
mined level with water and observed for 5 min; water was changed
between subjects and the procedure repeated. Data was quantified
based on the duration of time that the rat spent unengaged in
escape-driven activity (floating in water without active move-
ments of forepaws) represented by total immobility time (passive
behaviour).
Behaviour from all three tests was video-recorded. For the OFT
and EPM, behaviour was analysed using video tracking software
(Ethovision, Noldus Information Technology). For the FST, immo-
bility time was measured manually by two assessors blinded to
experimental conditions, inter-rater reliability between the two
assessors was determined using SPSS (Intraclass correlation coef-
ficient=0.998, p<0.001). Baseline mean immobility scores for SD
and WKY strains were similar to those previously reported (Abe
et al., 2007; Calvo et al., 2011; Jama et al., 2008; Morley-Fletcher
et al., 2011; Nam et al., 2014).
Immunoblotting
Rats were euthanised 48 h after their final behavioural test
(PN42) via carbon dioxide (CO2) asphyxiation and the PFC
(Bregma +3.70 mm) and vHPC (Bregma −2.80 mm) were dis-
sected according to a standard rat brain atlas (Paxinos and
Watson, 2007). Equal amounts of protein (5 ug/well) were sepa-
rated in 4–20% Tris-Glycine-eXtended (TGX) precast gels (Bio-
Rad, Australia) under non-reducing conditions (Lum et al.,
2016). All protein-loading concentrations were in the linear
range of detection for each antibody. Following electrophoresis,
proteins were transferred onto polyvinylidene difluoride mem-
branes (Bio-Rad) and membranes blocked with 5% bovine
serum albumin or skim milk (w/v) for 60 min at room tempera-
ture. The membranes were incubated overnight in primary poly-
clonal antibodies (NR1, NR2A, NR2B, post-synaptic density 95
(PSD-95), mGluR1, mGluR5, Homer1b/c and β-Actin;
Supplementary Material Table 1). Membranes were subse-
quently incubated with horseradish peroxidase conjugated sec-
ondary antibodies. Bands were visualised using Amersham ECL
Western blotting detection reagent (GE Healthcare, Australia)
and membranes exposed to Hyperfilm (GE Healthcare,
Australia). Films were scanned using a GS-800 scanner (Bio-
Rad) and densitometry values were quantified. Relative densi-
tometry values for each protein were normalised to their
respective β-actin levels and an internal control value, to account
for protein loading and gel-gel variability, respectively. Each
sample (n=7/group) was run in triplicate.
Statistical analysis
Two-way analyses of variance (ANOVAs) were used to deter-
mine any effects of treatment (FLX or VEH) or strain (WKY or
SD) on body weight (PN42), behavioural parameters and relative
protein densities. Where significant interactions were found, one-
way ANOVAs were used to identify differences between groups.
Kruskal–Wallis tests were used to determine the effects of treat-
ment and strain on offspring body weight (PN7+PN14) and
behavioural data (OFT: Time spent in centre arena), where data
showed non-normal distribution (Kolmogorov–Smirnov
p<0.05). Where significant interactions were found, a post hoc
pairwise comparison test was performed to detect significant dif-
ferences between groups. Significance was set at an alpha level
of p=0.05.
Millard et al. 5

Figure 2.  Average offspring body weight at (a) postnatal day (PN)7, (b) PN14 and (c) PN42. Data expressed as mean grams (g)+standard error of
the mean (SEM); **p<0.010, ***p<0.001. SD: Sprague-Dawley; WKY: Wistar-Kyoto.

Figure 3.  (a) Distance travelled, (b) time spent in the centre arena and (c) time spent in corners of the open field test (OFT). Data expressed as
mean centimetres (cm) or seconds (sec)+standard error of the mean (SEM); ^p=0.056, *p<0.05, **p<0.010, ***p<0.001; ###p<0.001 refers to
significant difference in Wistar-Kyoto (WKY) compared to Sprague-Dawley (SD) (overall strain effect).

Results
Offspring body weight
Kruskal-Wallis tests revealed significant effects of
TREATMENT (PN7: χ2=15.553; p<0.001; PN14: χ2=9.579;
p<0.05), STRAIN (PN7: χ2=77.046; p<0.001; PN14:
χ2=41.175; p<0.001) and significant TREATMENT×STRAIN
interactions (PN7: χ2=96.702; p<0.001; PN14: χ2=69.244;
p<0.001) on body weight of offspring at PN7 and PN14. FLX-
exposed offspring of both strains weighed significantly less
compared to VEH offspring at PN7 (SD: –12%, p<0.05; WKY:
–10%, p<0.001) and PN14 (SD: –8%, p<0.05; WKY: –17%,
p<0.01). This effect extended to PN42 in male SD offspring,
but not WKY offspring, with SD FLX offspring weighing sig-
nificantly less compared to SD VEH offspring (–15%, p<0.05).
WKY VEH offspring also weighed less than SD VEH offspring
at PN7 (–19%, p<0.001), PN14 (–11.1%, p<0.001) and PN42
(–42%, p<0.001) (Figure 2).
Adolescent behaviour
OFT.  Locomotor activity in the OFT was influenced by TREAT-
MENT (F1,47=13.526; p<0.001), STRAIN (F1,47=22.344;
p<0.001) and a significant interaction between
TREATMENT×STRAIN (F3,47=6.463; p<0.05). WKY-VEH
rats travelled less than SD-VEH rats (–82%; p<0.001). FLX
exposure reduced distance travelled in the OFT in SD offspring
(–61%; p<0.01) but this did not reach significance in WKY off-
spring. Similarly, there was a significant effect of TREATMENT
(χ2=162.0; p<0.05), STRAIN (χ2=93.0; p<0.05) and a
TREATMENT×STRAIN interaction (χ2=18.824; p<0.001) on
time spent in the centre of the OFT arena; WKY-VEH offspring
spent a substantially decreased amount of time in the centre
arena compared to SD-VEH offspring (-95%; p<0.05) while
FLX-exposed SD, but not WKY, rats spent less time in the cen-
tre of the OFT area compared to SD-VEH rats (–90%; p=0.056).
There were also significant effects of TREATMENT and
STRAIN on time spent in the corners of the OFT, with WKY
offspring spending increased time in the corners compared to
SD offspring (+10%; F1,47 =14.448; p<0.001) and FLX-
exposed offspring of both strains overall spending increased
time in the corners compared to VEH (+10%; F1,47=12.781;
p<0.05). There was no TREATMENT×STRAIN interaction on
time spent in corners (Figure 3).
EPM.  Adolescent WKY offspring travelled less in the EPM com-
pared to SD offspring (–27%; F1,48=8.974; p<0.004) and FLX-
exposed offspring travelled less in the EPM compared to VEH
(–54%; F1,48=34.618; p<0.001). There were no significant
TREATMENT×STRAIN interactions on distance travelled in
the EPM. WKY offspring spent less time in the open arms of the
EPM compared to SD rats (–52%; F1,47=4.781; p<0.05); simi-
larly, FLX-exposed offspring of both strains overall spent a
reduced amount of time exploring the open arms compared to
6 Journal of Psychopharmacology 00(0)
Figure 4.  (a) Distance travelled, (b) time spent in the open arms and (c) time spent in closed arms of the elevated plus maze (EPM). Data expressed
as mean centimetres (cm) or seconds (sec)+standard error of the mean (SEM); *p<0.05, ***p<0.001, #p<0.05; ##p<0.010 Wistar-Kyoto (WKY)
compared to Sprague-Dawley (SD) (overall strain effect).
Figure 5.  Time spent immobile in the forced swim test. Data
expressed as mean seconds (sec)+standard error of the mean (SEM);
***p<0.001; #p<0.05 Wistar-Kyoto (WKY) compared to Sprague-
Dawley (SD) (overall strain effect).
VEH offspring (–81%; F1,47=18.025; p<0.001). There was no
STRAIN×TREATMENT interaction on time spent in open arms.
There were significant TREATMENT (F1,48=55.379, p<0.001)
and STRAIN (F1,48=22.994, p<0.001) effects on time spent in
the closed arms and a significant STRAIN×TREATMENT inter-
action (F3,48=7.69, p=0.008). Post hoc analysis showed that WKY
VEH offspring spent an increased amount of time in the closed
arms compared to SD VEH offspring (+26%, p<0.001) and that
FLX-exposed SD and WKY offspring spent significantly
increased time in the closed arms of the EPM compared to their
respective SD (+91%, p<0.001) and WKY (+25%, p<0.05)
VEH control groups (Figure 4).
FST. Overall, offspring of the WKY strain spent more time
immobile in the FST compared to SD offspring (+308%;
F1,44=45.2; p<0.05). FLX-exposed offspring of both strains also
showed an increased amount of time spent immobile compared
to VEH (+27%; F1,44=6.404; p<0.001). There were no signifi-
cant TREATMENT × STRAIN interactions (Figure 5).
Immunoblot. Adolescent offspring of the WKY strain had
reduced levels of NR1 (–15%; F1,25=8.359; p<0.05), NR2A
(–28%; F1,26=22.028; p<0.001) and NR2B (–29%;
F1,23=15.113; p<0.001) in the PFC compared to offspring of
the SD strain, as well as reductions in the post-synaptic density
marker, PSD-95, a protein that anchors NMDA receptors
(–17%; F1,27=10.741; p<0.05). Furthermore, offspring of both
SD and WKY strains that were exposed to FLX expressed
reduced relative protein levels of NR1 (–12%; F1,25=4.515;
p<0.05), NR2A (–13%; F1,25=5.063; p<0.05) and PSD-95
(–17%; F1,27=8.389; p<0.01) compared to VEH offspring. In
the vHPC there were similar reductions in PSD-95 in WKY
offspring compared to SD offspring (–23%; F1,27=8.098;
p<0.01) as well as reductions following FLX exposure (–18%;
F1,27=5.213; p<0.05). There were no significant differences
between groups in any NMDA receptor subunits in the vHPC,
and no significant TREATMENT×STRAIN interactions
detected in either brain region (Figure 6).
Overall, the WKY strain showed reduced mGluR1 mono-
mer levels in the PFC compared to SD offspring (–41%;
F1,27=17.826; p<0.01) and FLX-exposed offspring of both
strains collectively showed reductions in relative levels of
mGluR1 monomer in the PFC compared to VEH (–37%;
F1,27=6.886; p<0.05). There were no TREATMENT×STRAIN
interactions. In the vHPC mGluR1 monomer expression was
influenced by TREATMENT (F1,26= 19.932, p<0.001),
STRAIN (F1,26=5.839, p<0.05) and a significant
TREATMENT×STRAIN interaction (F3,26=6.933; p<0.05);
relative mGluR1 monomer levels in the vHPC were signifi-
cantly reduced in WKY VEH offspring compared to SD VEH
(–51%, p<0.05) and FLX exposure reduced mGluR1 mono-
mer expression in SD offspring (–70%, p<0.001) but not
WKY offspring. There were no significant effects of treat-
ment, strain or interaction between the factors, on mGluR1
dimer levels in the PFC or vHPC (Figure 7).
In both the PFC and vHPC, there were significant effects of
STRAIN (PFC: F1,25=16.908, p<0.001; vHPC: F1,27=11.440,
p<0.01) and TREATMENT (PFC: F1,25=11.116, p<0.01;
vHPC: F1,27=40.567, p<0.001) on mGluR5 monomer levels
and significant interactions (PFC: F3,25=5.608, p<0.05; vHPC:
F3,27 =11.693, p<0.01). Post hoc analyses revealed that WKY
VEH offspring express reduced mGluR5 monomer levels com-
pared to SD VEH (PFC: p<0.01; –58%; vHPC: p<0.001;
–52%) and that FLX-exposed SD offspring express reduced
mGluR5 monomer levels compared to SD VEH in both brain
regions (PFC: p<0.01; –50%; vHPC: p<0.001; –75%), an
 Millard et al.

Figure 6.  Relative levels of N-methyl-D-aspartate (NMDA) receptor subunits NR1, NR2A, NR2B and scaffolding protein post-synaptic density 95 (PSD-95) in the prefrontal cortex (PFC) ((a)–(d)) and
ventral hippocampus (vHPC) ((e)–(h)) at PN42. Data expressed as mean values+standard error of the mean (SEM). *p<0.05, ***p<0.001, #p<0.05; ###p<0.001 Wistar-Kyoto (WKY) compared to
Sprague-Dawley (SD) (overall strain effect).
7
8 Journal of Psychopharmacology 00(0)

Figure 7.  Relative levels of metabotropic glutamate receptor subtype 1 (mGluR1) monomer and dimeric forms in the prefrontal cortex (PFC) ((a) and
(b)) and ventral hippocampus (vHPC) ((c) and (d)) at post-natal day (PN) 42. Data expressed as mean values+standard error of the mean (SEM).
*p<0.05, ***p<0.001; ##p<0.01 Wistar-Kyoto (WKY) compared to Sprague-Dawley (SD) (overall strain effect). KDa: kilodalton.

Figure 8.  Relative levels of metabotropic glutamate receptor subtype 5 (mGluR5) monomer and dimeric forms, and scaffolding protein Homer1bc in the prefrontal
cortex (PFC) ((a)–(c)) and ventral hippocampus (vHPC) ((f)–(h)) at post-natal day (PN) 42. Data expressed as mean values+standard error of the mean (SEM).
*p<0.05, ***p<0.001; #p<0.05, ##p<0.010 Wistar-Kyoto (WKY) compared to Sprague-Dawley (SD) (overall strain effect). KDa: kilodalton.

effect that was not seen in FLX exposed WKY offspring.
mGluR5 dimers in the PFC were not affected by treatment,
strain or interaction effects (p>0.05); in the vHPC however,
there was a significant effect of TREATMENT, with FLX-
exposed offspring overall showing increased levels of mGluR5
dimers in the vHPC (+53%; F1,27=5.102; p<0.05) (Figure 8).
In line with this increase, FLX-exposed offspring had signifi-
cantly increased vHPC levels of Homer1b/c, a scaffolding pro-
tein that increases cell surface expression of mGluR5 (Mao
et al., 2005), compared to VEH offspring (+9.6%: F1,25=5.233;
p<0.05). No other significant effects of TREATMENT,
STRAIN or interactions were detected for Homer1b/c levels in
the PFC or vHPC (Figure 8).
Discussion
There is growing concern regarding the consequences of maternal
SSRI treatment on offspring neurodevelopment. Animal models,
such as that used in the present study design, allow us to distin-
guish the effects of maternal SSRI treatment on offspring com-
pared to, or in conjunction with, the underlying effects of maternal
depression. Overall, our findings show that male adolescent off-
spring of the WKY strain display increased anxiety- and depres-
sive-like behaviours compared to offspring of the SD strain.
Furthermore, this study showed for the first time that maternal
FLX treatment exacerbated the anxiety- and depressive-like phe-
notype observed in the WKY offspring and induced these behav-
iours in the SD offspring. These FLX-induced behavioural changes
Millard et al. 9
were accompanied by altered protein expression of glutamatergic
receptors and associated structural proteins in the PFC and vHPC
of both SD and WKY offspring. These novel findings suggest that
maternal FLX treatment exacerbates effects of the underlying
maternal depression on offspring behaviour, which may be medi-
ated through alterations in the glutamatergic system.
Maternal FLX treatment reduces offspring
body weight
Maternal FLX treatment was associated with reduced bodyweight
of juvenile offspring at PN7 and PN14 in both SD and WKY rats.
While this finding is consistent with several previous studies in
FLX treated healthy rodents (for review see Hutchison et al.,
2018), this is the first report of offspring body weight followed
throughout early developmental time periods in a rodent model of
innate depression. In the clinic it is well reported that maternal
FLX exposure can cause low birth weight (Olivier et al., 2013),
however these studies are often complicated by confounders such
as maternal depression and anxiety, which are also risk factors for
perinatal complications such as low birth weight (Bansil et al.,
2010; Davalos et al., 2012; Field et al., 2004). In the clinic, there
are no investigations into offspring body weight beyond the early
childhood years (Hutchison et al., 2018); we report in the present
study that the effect of FLX exposure on body weight extends to
adolescence in male offspring, but only in the SD strain, suggest-
ing no long-term effects of perinatal FLX exposure in the depres-
sion model. In support of these findings, Gemmel et al. (2018)
recently reported that maternal FLX treatment in a model of pre-
gestational stress reduces offspring body weight at pre-adoles-
cence but this does not extend to adulthood, similar to our findings
which indicate no long-term effect on body weight.
Perinatal FLX exposure exacerbates the
anxiety-like and depressive-like phenotype in
male adolescent WKY offspring and induces
an anxiety-like and depressive-like phenotype
in male SD offspring
Maternal FLX treatment caused significant increases in anxiety-
like and depressive-like behaviour in male adolescent offspring of
both SD and WKY strains in the OFT, EPM and FST. While dis-
tance travelled and time spent in the centre arena during the OFT
did not reach statistical significance in the FLX exposed WKY off-
spring, it should be noted that the WKY offspring were already
showing low measures in these parameters making it difficult to
detect further reductions. These findings suggest that perinatal FLX
exposure in a model of depression exacerbates the anxiety-like and
depressive-like phenotype at adolescence. To our knowledge there
is only one other study modelling maternal SSRI treatment in a
rodent model showing an innate depressive phenotype. Glover et al.
(2015) showed that paroxetine-exposed adult (PN75) male off-
spring born of SD dams exhibiting increased anxiety-like pheno-
types (termed bred low responders (bLRs)) had significantly greater
immobility times in the FST, similar to our finding, but report no
effect of paroxetine exposure on time spent in the open arms of the
EPM in the bLR offspring, in contrast to our finding. This contrast
may be related to the difference in rat strains used, the different drug
treatment between the two studies (paroxetine vs fluoxetine) or the
ages of offspring examined (PN75 vs PN37). Several studies have
utilised models of maternal stress (intense light exposure, restraint,
confinement and other stressors) during various periods of gestation
coupled with FLX exposure (Boulle et al., 2016a, 2016b; Kiryanova
et al., 2016; Rayen et al., 2011). Consistent with our findings in
adolescent offspring, it has been reported that prenatal stress (gesta-
tional day (GD)15–21) coupled with postnatal FLX treatment
(post-partum day 1 to weaning) increases immobility time in the
FST in male, as well as female, adolescent offspring, however does
not change OFT measures (Rayen et al., 2011). While there is a lack
of further investigation of adolescent offspring, one study in adult
offspring reported increased immobility time during the FST in
female (but not male) offspring born to FLX-treated (post-partum
day 1 to weaning) and maternally stressed dams (GD15–21), com-
pared to offspring of untreated, stressed dams (Boulle et al., 2016a,
2016b), suggesting possible sex effects; they however reported no
significant effects for either sex on anxiety-like measures in the
OFT or Elevated Zero Maze in the adult offspring. Collectively,
these findings suggest that maternal SSRI treatment in models of
maternal depression or stress can influence offspring anxiety-like
and depressive-like behaviour, especially in adolescent offspring
and that the sex and age of offspring can influence these effects.
Indeed, given these observations, an emphasis on the differences
between innate and environmental models of depression, and the
influence of offspring sex and age should be taken into considera-
tion for further studies investigating the effects of maternal SSRI
treatment on offspring.
The findings of increased immobility in the FST observed in
the present study need to be taken with consideration given the
absence of additional behavioural tests assessing other facets of
depressive-like phenotypes (e.g. anhedonia in the sucrose prefer-
ence test). It has been argued that immobility in the FST repre-
sents a switch from active to passive behaviour in the face of an
aversive stressor, and represents a behavioural adaption rather
than depressive-like behaviours (Molendijk and de Kloet, 2015).
Given that a decrease in locomotor activity as the result of mater-
nal FLX exposure was noted in the OFT and EPM, it is possible
that the increased immobility time in FST could represent a
‘false-positive’ (Slattery and Cryan, 2012). Despite this, other
studies investigating developmental SSRI exposure on offspring
behaviour have noted increased immobility times in the FST in
the presence and absence of changes in locomotor activity in the
OFT/EPM (for review see, Millard et al., 2017), suggesting that
developmental SSRI exposure induces changes in immobility
time in the FST, independent of changes in locomotor activity. To
further define the behavioural phenotype, future studies should
assess behaviours related to neuropsychiatric and neurodevelop-
mental disorders in a wider battery of tests. It must also be
acknowledged that the effects of maternal FLX treatment on dam
behaviour were not assessed in the present study. Given that
maternal behaviour and care are known to influence offspring
development and behaviour (Ahmadiyeh et al., 2004; Curley and
Champagne, 2016), this should be considered in future investiga-
tions. Previous work in models of maternal stress have shown
that treatment with FLX at the same dose as the present study,
does not influence maternal care behaviours (Gemmel et al.,
2018) but does improve anxiety-like behaviours in the dam
(Pawluski et al., 2012). There is no evidence to suggest that FLX
treatment in models of depression further exacerbate maternal
10 Journal of Psychopharmacology 00(0)
depressive- or anxiety-like behaviours, thus it is unlikely that the
behaviours observed in the FLX exposed offspring in the present
study are a result of changes in maternal behaviour.
Perinatal FLX exposure alters NMDA and group I
metabotropic glutamate receptors, as well as the
key scaffolding proteins, PSD-95 and Homer1b/c,
in the cortex and ventral hippocampus of male
adolescent SD and WKY rats
Whilst preclinical studies clearly implicate the NMDA receptor
and associated scaffolding protein PSD-95 in the mechanism of
SSRI action (and to a lesser extent group 1 mGluRs) (Boyer
et al., 1998; Musazzi et al., 2013; Park et al., 2014), the effects of
SSRI exposure during neurodevelopment on these proteins have
not previously been investigated. We report that maternal FLX
exposure reduced the scaffolding protein, PSD-95, in both the
PFC and vHPC of both rat strains. PSD-95 regulates NMDA
receptor cell-surface expression and synaptic activity (Sheng,
2001) by directly anchoring the NR2 subunits, and indirectly
coupling with mGluR1/5 receptor complexes (Tu et al., 1999).
Accordingly, NMDA receptor NR1 and NR2A subunits were also
reduced, but only in the PFC. In line with this, previous work
investigating the effects of chronic treatment with the SSRI, cit-
alopram, in adult rodents, has reported reductions in mRNA lev-
els encoding the NR1 (–7%) and NR2A (–35%) subunits in the
frontal cortex (Boyer et al., 1998). Interestingly these findings
suggest that the mechanism through which the effects of perinatal
SSRI exposure influence NMDA receptor subunit expression in
the PFC at adolescence, parallel those that occur in chronic use in
adults. FLX exposure also reduced mGluR1 and mGluR5 mono-
mers in both the PFC and vHPC, however these changes were
primarily in the SD offspring only, highlighting the potential con-
sequences of maternal SSRI treatment in a healthy model and the
importance of using relevant models of maternal psychopathol-
ogy. In light of these findings, it is tempting to speculate that the
paralleled reductions of PSD-95 and NMDA receptor subunits in
the PFC, could be indicative of overall reductions in post-synap-
tic densities, the specialised sites of glutamatergic synapses that
harbour key proteins responsible for glutamatergic signalling and
transduction (Sheng, 2001). However, in the vHPC, whilst we
also report reduced PSD-95 in FLX-exposed offspring, we
observed no change in NMDA receptor subunits and increased
levels of mGluR5 dimers and the associated scaffolding protein,
Homer1b/c (which positively regulates mGluR5 signalling (Lum
et al., 2018)), two proteins also indirectly coupled to PSD-95 via
complexes deep in the post-synaptic density (Sheng and Kim,
2011) suggesting that SSRI exposure induces brain region-spe-
cific effects.
While to our knowledge there have been no prior investiga-
tions of the glutamatergic system in models of perinatal SSRI
exposure, one recent study reported no effect of maternal FLX
treatment (from GD10–PN21) in SD rats on PSD-95 immunore-
activity in the PFC of pre-adolescent and adult offspring (Gemmel
et al., 2018); they did however show reduced PSD-95 in male
offspring of dams exposed to pre-gestational stress only (Gemmel
et al., 2018). SSRIs are well known to mediate their effects in
adults, at least in part, by downstream effects on brain derived
neurotrophic factor (BDNF) signalling (Martinowich and Lu,
2007), with glutamate signalling and BDNF production intri-
cately linked (Mattson, 2008). The BDNF signalling system is
crucial for normal neurodevelopment and ongoing regulation of
synaptic plasticity (Martinowich and Lu, 2007). Studies have
shown that mRNA expression of BDNF and its receptor,
Tropomyosin receptor kinase B (TrkB), are differentially altered
in models of prenatal stress±postnatal exposure to FLX (Boulle
et al., 2016a, 2016b), and that negative correlations exist between
immobility time in the FST and hippocampal BDNF mRNA
(Boulle et al., 2016b). Given our current findings of reduced
NMDA receptor subunits and PSD-95 in exposed offspring,
increased immobility time in the FST and the link between SSRIs
and BDNF, it would be of interest for future investigations of
perinatal SSRI±maternal depression, to determine whether glu-
tamatergic changes in the present study are associated with alter-
ations in the BDNF/TrkB signalling system.
Perturbations in glutamatergic signalling have repeatedly
been associated with anxiety- and depressive-like phenotypes.
Specifically, preclinical studies have shown reduced relative
levels of NR1 and NR2A in the PFC and hippocampus (HPC) of
prenatally stressed offspring and concomitant increases in anxi-
ety- and depressive-like behaviours (Sun et al., 2013a, 2013b).
Changes in PSD-95 and related increases in anxiety-like behav-
iours have also previously been reported; anxiety-like behaviour
in the novelty-induced hypophagia test and the OFT, induced by
social isolation, has been shown to be accompanied by decreases
in PSD-95, as well as NR1, in the frontal cortex and increases in
PSD-95 in the HPC and amygdala (Hermes et al., 2011; Zhang
et al., 2012). Furthermore, in the present study we showed that
the WKY strain, which has a prominent anxiety- and depressive-
like phenotype, displayed reduced glutamatergic markers
including NR1, NR2A, NR2B and PSD-95, predominately in the
PFC. Similar observations have been reported in postmortem
brains of those suffering from depression (Feyissa et al., 2009).
Despite the evidence for altered glutamatergic markers in clini-
cal cases of MDD, these had not been previously measured in
the WKY strain. These findings add further support to the clini-
cal relevance and validity of the WKY strain as an innate depres-
sion model. Interestingly, others have shown that functional
antagonists of the NMDA receptor cause an antidepressant
behavioural response in a model of inescapable stress (tail sus-
pension test) (Trullas and Skolnick, 1990), suggesting that
NMDA hyperactivity could underpin depressive-like behav-
iours. Indeed, NMDA antagonists such as ketamine, have rapid
and profound antidepressant properties (Iadarola et al., 2015).
The observation that the WKY strain shows reduced NMDA
subunit levels in the PFC suggests that they could be resistant to
the effects of NMDA antagonists. Interestingly, however, studies
have found the contrary, with WKY rats being extremely respon-
sive to the effects of ketamine on immobility time in the FST
(Browne and Lucki, 2013; Tizabi et al., 2012). In the PFC, it is
the NMDA receptors located on gamma-aminobutyric acid
(GABA)-ergic interneurons that are reportedly more susceptible
to the effects of ketamine (Moghaddam and Javitt, 2012); it may
be that the reduction in NMDA receptor subunits observed in the
present study is predominantly occurring on other neuronal pop-
ulations, such as pyramidal cells, in which case antagonism of
NMDA receptors on GABA-ergic interneurons would help
restore excitatory/inhibitory balance. Further investigation into
how the changes in the present study reflect specific neuronal
Millard et al. 11
populations, or whether they extend to other brain regions will
be important to further understand the potential and mechanisms
of novel antidepressant treatments.
Beyond the behavioural parameters regarding anxiety- and
depressive-like phenotypes, others have also reported that altera-
tions in glutamatergic signalling are associated with changes in
cognitive ability. NR1Hypo mice (expressing ~5% of normal NR1
levels) (Mohn et al., 1999) have been shown to display alterations
in cognition, including impairments in short- and long-term mem-
ory in spatial and non-spatial tasks (Barkus et al., 2012).
Furthermore, abnormalities in PSD-95 have consistently been
associated with altered cognition (VanGuilder et al., 2011). Indeed,
two studies investigating the effects of developmental SSRI expo-
sure on offspring behaviour have noted alterations in cognition
evident through changes in time taken to reach the platform in the
Morris water maze: however, these have been in standard labora-
tory rodents not modelling maternal depression (Bairy et al., 2006;
Sprowles et al., 2016). Our findings of reduced NMDA receptor
subunits and PSD-95 warrant further investigation of cognitive
behaviours using appropriate models of maternal depression.
In addition to changes observed in NMDA receptor subunits
and PSD-95, our findings also suggest that mGluR5 signalling is
fundamentally altered in the vHPC following FLX exposure.
Although the exact mechanism underlying this finding is
unknown, the relationship between anxiety-like behaviours and
mGluR5 have been well documented (Terbeck et al., 2015).
Evidence from a large number of preclinical studies has estab-
lished profound anxiolytic effects of mGluR5 antagonists
(Swanson et al., 2005). For example, following 2-methyl-
6(phenylethynyl)pyridine (MPEP) treatment, rodents display
reduced exploratory inhibition by spending more time in the
open arms of the EPM (Pérez de la Mora et al., 2006;
Tatarczyńska et al., 2001). Previous studies have also noted
robust anxiolytic effects of selective intra-amygdaloid (a region
known to play a critical role in anxiety and fear) injections of
MPEP in different rodent models of anxiety (Pérez de la Mora
et al., 2006). Considering the vHPC is highly interrelated both
structurally and physiologically with the amygdala and this cir-
cuit mediates anxiety-like behaviour (Fanselow and Dong,
2010; Sah et al., 2003), it is not surprising to see changes in
mGluR5 in the vHPC of FLX-exposed offspring displaying
increased anxiety-like behaviour. Collectively our findings sug-
gest that perinatal SSRI exposure is associated with increased
anxiety-like and depressive-like behaviours which may be medi-
ated by altered glutamatergic signalling.
Conclusion
Overall, our findings show that maternal FLX treatment in a clin-
ically relevant rodent model of depression induces changes in
body weight, behaviour and neurochemical phenotypes in
exposed offspring. Specifically, we report reduced postnatal bod-
yweight as well as increased anxiety- and depressive-like behav-
iours and alterations in cortical and hippocampal glutamatergic
markers in male adolescent offspring. How the changes observed
in the present study reflect glutamatergic synapses and whether
these changes are transient in nature or persist into adulthood will
be key to understanding the clinical implications of maternal
SSRI exposure. Given that dysfunction of key glutamatergic
receptors and associated proteins has been linked to several
neurodevelopmental disorders including autism, ADHD and
mood disorders, our findings warrant further investigation into
the clinical effects of maternal SSRI exposure. Future studies
assessing the influence of sex and maternal SSRI exposure are
also needed to provide deeper insight into the potential differ-
ences of exposure between the sexes. Incidence of maternal
depression is on the rise, and untreated maternal depression can
have significant negative effects on the health of both the mother
and child. Antidepressants play a critical role in minimising the
risks associated with untreated maternal depression. However,
we are just beginning to understand the potential effects that
maternal SSRIs may cause, and future studies investigating ways
to minimise these effects will be critical to ensuring healthy preg-
nancies and offspring development.
Acknowledgements
The authors wish to thank Rebecca Webby, Marta Ramos and Connor
Mackay for assistance with the rodent studies.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the
research, authorship and/or publication of this article: This study has been
conducted with the support of a University of Wollongong, Faculty of
Science, Medicine and Health Target Grant to KAN and the Australian
Government Research Training Program Scholarship awarded to SJM
and JSL. JSL is further supported by Australian Rotary Health, in the
form of an Ian Scott Scholarship.
ORCID iD
Kelly A Newell https://orcid.org/0000-0002-1245-5585
Supplementary material
Supplementary material for this article is available online.
References
Abe H, Hidaka N, Kawagoe C, et al. (2007) Prenatal psychological stress
causes higher emotionality, depression-like behavior, and elevated
activity in the hypothalamo-pituitary-adrenal axis. Neurosci Res 59:
145–151.
Ahmadiyeh N, Slone-Wilcoxon JL, Takahashi JS, et al. (2004) Maternal
behavior modulates X-linked inheritance of behavioral coping in the
defensive burying test. Biol Psychiatry 55: 1069–1074.
Ansorge MS, Morelli E and Gingrich JA (2008) Inhibition of serotonin
but not norepinephrine transport during development produces
delayed, persistent perturbations of emotional behaviors in mice. J
Neurosci Off J Soc Neurosci 28: 199–207.
Ansorge MS, Zhou M, Lira A, et al. (2004) Early-life blockade of the
5-HT transporter alters emotional behavior in adult mice. Science
306: 879–881.
Arango V, Underwood MD, Boldrini M, et al. (2001) Serotonin 1A
receptors, serotonin transporter binding and serotonin transporter
mRNA expression in the brainstem of depressed suicide victims.
Neuropsychopharmacology 25: 892–903.
Bairy KL, Madhyastha S, Ashok KP, et al. (2006) Developmental and behav-
ioral consequences of prenatal fluoxetine. Pharmacology 79: 1–11.
12 Journal of Psychopharmacology 00(0)
Bansil P, Kuklina EV, Meikle SF, et al. (2010) Maternal and fetal outcomes
among women with depression. J Womens Health 19: 329–334.
Barkus C, Dawson LA, Sharp T, et al. (2012) GluN1 hypomorph mice
exhibit wide-ranging behavioral alterations. Genes Brain Behav 11:
342–351.
Bonnin A, Torii M, Wang L, et al. (2007) Serotonin modulates the
response of embryonic thalamocortical axons to netrin-1. Nat Neu-
rosci 10: 588–597.
Boukhris T, Sheehy O, Mottron L, et al. (2016) Antidepressant use dur-
ing pregnancy and the risk of autism spectrum disorder in children.
JAMA Pediatr 170: 117–124.
Boulle F, Pawluski JL, Homberg JR, et al. (2016a) Prenatal stress and
early-life exposure to fluoxetine have enduring effects on anxiety
and hippocampal BDNF gene expression in adult male offspring.
Dev Psychobiol 58: 427–438.
Boulle F, Pawluski JL, Homberg JR, et al. (2016b) Developmental fluox-
etine exposure increases behavioral despair and alters epigenetic
regulation of the hippocampal BDNF gene in adult female offspring.
Horm Behav 80: 47–57.
Boyer P-A, Skolnick P and Fossom LH (1998) Chronic administration of
imipramine and citalopram alters the expression of NMDA receptor
subunit mRNAs in mouse brain. J Mol Neurosci 10: 219–233.
Brandlistuen RE, Ystrom E, Eberhard-Gran M, et al. (2015) Behavioural
effects of fetal antidepressant exposure in a Norwegian cohort of dis-
cordant siblings. Int J Epidemiol 44: 1397–1407.
Brandon NJ and McKay R (2015) The cellular target of antidepressants.
Nat Neurosci 18: 1537–1538.
Browne CA and Lucki I (2013) Antidepressant effects of ketamine: Mecha-
nisms underlying fast-acting novel antidepressants. Front Pharmacol 4:
161.
Cabrera-Vera TM and Battaglia G (1998) Prenatal exposure to fluox-
etine (Prozac) produces site-specific and age-dependent alterations
in brain serotonin transporters in rat progeny: Evidence from autora-
diographic studies. J Pharmacol Exp Ther 286: 1474–1481.
Calvo N, Cecchi M, Kabbaj M, et al. (2011) Differential effects of social
defeat in rats with high and low locomotor response to novelty. Neu-
roscience 183: 81–89.
Capello CF, Bourke CH, Ritchie JC, et al. (2011) Serotonin transporter
occupancy in rats exposed to serotonin reuptake inhibitors in utero or
via breast milk. J Pharmacol Exp Ther 339: 275–285.
Castagné V, Moser P, Roux S, et al. (2011) Rodent models of depression:
Forced swim and tail suspension behavioral despair tests in rats and
mice. Curr Protoc Neurosci, 55: 8.10A.1-8.10A.14.
Clements CC, Castro VM, Blumenthal SR, et al. (2015) Prenatal antide-
pressant exposure is associated with risk for attention deficit-hyper-
activity disorder but not autism spectrum disorder in a large health
system. Mol Psychiatry 20: 727–734.
Commons KG, Cholanians AB, Babb JA, et al. (2017) The rodent forced
swim test measures stress-coping strategy, not depression-like
behavior. ACS Chem Neurosci 8: 955–960.
Cooper WO, Willy ME, Pont SJ, et al. (2007) Increasing use of antide-
pressants in pregnancy. Am J Obstet Gynecol 196: 544.e1–544.e5.
Croen LA, Grether JK, Yoshida CK, et al. (2011) Antidepressant use dur-
ing pregnancy and childhood autism spectrum disorders. Arch Gen
Psychiatry 68: 1104–1112.
Curley JP and Champagne FA (2016) Influence of maternal care on the
developing brain: Mechanisms, temporal dynamics and sensitive
periods. Front Neuroendocrinol 40: 52–66.
Davalos DB, Yadon CA and Tregellas HC. (2012) Untreated prenatal
maternal depression and the potential risks to offspring: A review.
Arch Womens Ment Health 15: 1–14.
Deave T, Heron J, Evans J, et al. (2008) The impact of maternal depres-
sion in pregnancy on early child development. Int J Obstet Gynaecol
115: 1043–1051.
Deschwanden A, Karolewicz B, Feyissa AM, et al. (2011) Reduced
metabotropic glutamate receptor 5 density in major depression
determined by [(11)C]ABP688 PET and postmortem study. Am J
Psychiatry 168: 727–734.
du Bois TM, Huang X-F and Deng C (2008) Perinatal administration of
PCP alters adult behaviour in female Sprague–Dawley rats. Behav
Brain Res 188: 416–419.
Dulawa SC, Holick KA, Gundersen B, et al. (2004) Effects of chronic
fluoxetine in animal models in anxiety and depression. Neuropsy-
chopharmacology 29: 1321–1330.
Dusi N, Barlati S, Vita A, et al. (2015) Brain structural effects of antide-
pressant treatment in major depression. Curr Neuropharmacol 13:
458–465.
Edgar JM and Price DJ (2001) Radial migration in the cerebral cortex is
enhanced by signals from thalamus. Eur J Neurosci 13: 1745–1754.
Fanselow MS and Dong H-W (2010) Are the dorsal and ventral hippo-
campus functionally distinct structures? Neuron 65: 7–19.
Feyissa AM, Chandran A, Stockmeier CA, et al. (2009) Reduced levels
of NR2A and NR2B subunits of NMDA receptor and PSD-95 in the
prefrontal cortex in major depression. Prog Neuropsychopharmacol
Biol Psychiatry 33: 70–75.
Field T, Diego M, Hernandez-Reif M, et al. (2004) Prenatal maternal bio-
chemsitry predicts neonatal biochemistry. Int J Neurosci 114: 933–945.
Figueroa R (2010) Use of antidepressants during pregnancy and risk of
attention-deficit/hyperactivity disorder in the offspring. J Dev Behav
Pediatr 31: 641–648.
Foldi CJ, Eyles DW, McGrath JJ, et al. (2011) The effects of breeding
protocol in C57BL/6J mice on adult offspring behaviour. PLoS One
6: e18152.
Gemmel M, Kokras N, Dalla C, et al. (2018) Perinatal fluoxetine prevents
the effect of pre-gestational maternal stress on 5-HT in the PFC, but
maternal stress has enduring effects on mPFC synaptic structure in
offspring. Neuropharmacology 128: 168–180.
Gidaya NB, Lee BK, Burstyn I, et al. (2014) In utero exposure to selec-
tive serotonin reuptake inhibitors and risk for autism spectrum disor-
der. J Autism Dev Disord 44: 2558–2567.
Glover ME, Pugh PC, Jackson NL, et al. (2015) Early-life exposure to
the SSRI paroxetine exacerbates depression-like behavior in anxiety/
depression-prone rats. Neuroscience 284: 775–797.
Glover V (2014) Maternal depression, anxiety and stress during preg-
nancy and child outcome; what needs to be done. Best Pract Res Clin
Obstet Gynaecol 28: 25–35.
Harrington RA, Lee L-C, Crum RM, et al. (2014) Prenatal SSRI use and
offspring with autism spectrum disorder or developmental delay.
Pediatrics 2013–3406.
Hashimoto K, Sawa A and Iyo M (2007) Increased levels of glutamate in
brains from patients with mood disorders. Biol Psychiatry Depress
62: 1310–1316.
Heikkinen T, Ekblad U and Laine K (2002) Transplacental transfer of
citalopram, fluoxetine and their primary demethylated metabolites
in isolated perfused human placenta. Int J Obstet Gynaecol 109:
1003–1008.
Hendrick V, Stowe ZN, Altshuler LL, et al. (2001) Fluoxetine and nor-
fluoxetine concentrations in nursing infants and breast milk. Biol
Psychiatry 50: 775–782.
Hermes G, Li N, Duman C, et al. (2011) Post-weaning chronic social
isolation produces profound behavioral dysregulation with decreases
in prefrontal cortex synaptic-associated protein expression in female
rats. Physiol Behav 104: 354–359.
Hiemke C and Härtter S (2000) Pharmacokinetics of selective serotonin
reuptake inhibitors. Pharmacol Ther 85: 11–28.
Hutchison SM, Mâsse LC, Pawluski JL, et al. (2018) Perinatal selective
serotonin reuptake inhibitor (SSRI) effects on body weight at birth
and beyond: A review of animal and human studies. Reprod Toxicol
77: 109–121.
Huybrechts KF, Palmsten K, Mogun H, et al. (2013) National trends in
antidepressant medication treatment among publicly insured preg-
nant women. Gen Hosp Psychiatry 35: 265–271.
Millard et al. 13
Iadarola ND, Niciu MJ, Richards EM, et al. (2015) Ketamine and other
N-methyl-D-aspartate receptor antagonists in the treatment of
depression: A perspective review. Ther Adv Chronic Dis 6: 97–114.
Jama A, Cecchi M, Calvo N, et al. (2008) Inter-individual differences
in novelty-seeking behavior in rats predict differential responses to
desipramine in the forced swim test. Psychopharmacology (Berl.)
198: 333–340.
Karpova NN, Lindholm J, Pruunsild P, et al. (2009) Long-lasting behav-
ioural and molecular alterations induced by early postnatal fluox-
etine exposure are restored by chronic fluoxetine treatment in adult
mice. Eur Neuropsychopharmacol 19: 97–108.
Kessler R (2003) Epidemiology of women and depression. J Affect Dis-
ord 74: 5–13.
Kim J, Riggs KW, Misri S, et al. (2006) Stereoselective disposition of
fluoxetine and norfluoxetine during pregnancy and breast-feeding.
Br J Clin Pharmacol 61: 155–163.
Kiryanova V, McAllister BB and Dyck RH (2013) Long-term outcomes
of developmental exposure to fluoxetine: A review of the animal lit-
erature. Dev Neurosci 35: 437–439.
Kiryanova V, Meunier SJ, Vecchiarelli HA, et al. (2016) Effects of
maternal stress and perinatal fluoxetine exposure on behavioral out-
comes of adult male offspring. Neuroscience 320: 281–296.
Koran LM, Cain JW, Dominguez RA, et al. (1996) Are fluoxetine plasma
levels related to outcome in obsessive-compulsive disorder? Am J
Psychiatry 153: 1450–1454.
Lakhan SE, Caro M and Hadzimichalis N (2013) NMDA receptor activ-
ity in neuropsychiatric disorders. Front Psychiatry 4: 52.
Lam MW, Young CJ and Mabury SA (2005) Aqueous photochemical
reaction kinetics and transformations of fluoxetine. Environ Sci
Technol 39: 513–522.
Lavdas AA, Blue ME, Lincoln J, et al. (1997) Serotonin promotes the differ-
entiation of glutamate neurons in organotypic slice cultures of the devel-
oping cerebral cortex. J Neurosci Off J Soc Neurosci 17: 7872–7880.
Lemos JC, Zhang G, Walsh T, et al. (2011) Stress-hyperresponsive WKY
rats demonstrate depressed dorsal raphe neuronal excitability and
dysregulated CRF-mediated responses. Neuropsychopharmacology
36: 721–734.
López-Figueroa AL, Norton CS, López-Figueroa MO, et al. (2004) Sero-
tonin 5-HT1A, 5-HT1B, and 5-HT2A receptor mRNA expression in
subjects with major depression, bipolar disorder, and schizophrenia.
Biol Psychiatry 55: 225–233.
López-Rubalcava C and Lucki I (2000) Strain differences in the behav-
ioral effects of antidepressant drugs in the rat forced swimming test.
Neuropsychopharmacology 22: 191–199.
Lum JS, Fernandez F, Matosin N, et al. (2016) Neurodevelopmental expres-
sion profile of dimeric and monomeric group 1 mGluRs: Relevance to
schizophrenia pathogenesis and treatment. Sci Rep 6: srep34391.
Lum JS, Millard SJ, Frank E, et al. (2018) Chronic adolescent CDPPB
treatment alters short-term, but not long-term, glutamatergic receptor
expression. Neurochem Res 43: 1683–1691.
Malm H, Brown AS, Gissler M, et al. (2016) Gestational exposure to
selective serotonin reuptake inhibitors and offspring psychiatric dis-
orders: A National Register-Based Study. J Am Acad Child Adolesc
Psychiatry 55: 359–366.
Mao L, Yang L, Tang Q, et al. (2005) The scaffold protein Homer1b/c
links metabotropic glutamate receptor 5 to extracellular signal-regu-
lated protein kinase cascades in neurons. J Neurosci Off J Soc Neu-
rosci 25: 2741–2752.
Marcus SM and Heringhausen JE (2009) Depression in childbearing
women: When depression complicates pregnancy. Prim Care Clin
Off Pract 36: 151–165.
Martinowich K and Lu B (2007) Interaction between BDNF and serotonin:
Role in mood disorders. Neuropsychopharmacology 33: 73–83.
Matosin N, Frank E, Deng C, et al. (2013) Metabotropic glutamate recep-
tor 5 binding and protein expression in schizophrenia and following
antipsychotic drug treatment. Schizophr Res 146: 170–176.
Mattson MP (2008) Glutamate and neurotrophic factors in neuronal
plasticity and disease. Annals of the New York Academy of Sciences
1144: 97–112.
McIlwain KL, Merriweather MY, Yuva-Paylor LA, et al. (2001) The use
of behavioral test batteries: Effects of training history. Physiol Behav
73: 705–717.
Millard SJ, Weston-Green K and Newell KA (2017) The effects of
maternal antidepressant use on offspring behaviour and brain devel-
opment: Implications for risk of neurodevelopmental disorders. Neu-
rosci Biobehav Rev 80: 743–765.
Mitchell AA, Gilboa SM, Werler MM, et al.; National Birth Defects Pre-
vention Study (2011) Medication use during pregnancy, with par-
ticular focus on prescription drugs: 1976–2008. Am J Obstet Gynecol
205: 51.e1–8.
Moghaddam B and Javitt D (2012) From revolution to evolution: the
glutamate hypothesis of schizophrenia and its implication for treat-
ment. Neuropsychopharmacol Off Publ Am Coll Neuropsychophar-
macol 37: 4–15.
Mohn AR, Gainetdinov RR, Caron MG, et al. (1999) Mice with reduced
NMDA receptor expression display behaviors related to schizophre-
nia. Cell 98: 427–436.
Molendijk ML and de Kloet ER (2015) Immobility in the forced swim
test is adaptive and does not reflect depression. Psychoneuroendo-
crinology 62: 389–391.
Morley-Fletcher S, Mairesse J, Soumier A, et al. (2011) Chronic agomel-
atine treatment corrects behavioral, cellular, and biochemical abnor-
malities induced by prenatal stress in rats. Psychopharmacology
(Berl.) 217: 301.
Musazzi L, Treccani G, Mallei A, et al. (2013) The action of antidepres-
sants on the glutamate system: Regulation of glutamate release and
glutamate receptors. Biol Psychiatry 73: 1180–1188.
Nam H, Clinton SM, Jackson NL, et al. (2014) Learned helplessness and
social avoidance in the Wistar-Kyoto rat. Front Behav Neurosci 8: 109.
Oberlander TF, Gingrich JA and Ansorge MS (2009) Sustained neurobe-
havioral effects of exposure to SSRI antidepressants during devel-
opment: Molecular to clinical evidence. Clin Pharmacol Ther 86:
672–677.
Olivier JDA, Åkerud H, Kaihola H, et al. (2013) The effects of maternal
depression and maternal selective serotonin reuptake inhibitor expo-
sure on offspring. Front Cell Neurosci 7: 73.
Olivier JDA, Vallès A, Heesch F van, et al. (2011) Fluoxetine admin-
istration to pregnant rats increases anxiety-related behavior in the
offspring. Psychopharmacology (Berl.) 217: 419–432.
Pardon M-C, Gould GG, Garcia A, et al. (2002) Stress reactivity of the brain
noradrenergic system in three rat strains differing in their neuroendo-
crine and behavioral responses to stress: Implications for susceptibility
to stress-related neuropsychiatric disorders. Neuroscience 115: 229–242.
Pare WP and Tejani-Butt SM (1996) Effect of stress on the behavior and
5-HT system in Sprague-Dawley and Wistar Kyoto rat strains. Integr
Physiol Behav Sci 31: 112.
Park SW, Lee JG, Seo MK, et al. (2014) Differential effects of antide-
pressant drugs on mTOR signalling in rat hippocampal neurons. Int
J Neuropsychopharmacol 17: 1831–1846.
Pawluski JL, Brain UM, Underhill CM, et al. (2012) Prenatal SSRI expo-
sure alters neonatal corticosteroid binding globulin, infant cortisol
levels, and emerging HPA function. Psychoneuroendocrinology 37:
1019–1028.
Paxinos G and Watson C (2007) The rat brain in stereotaxic coordinates.
(6th Edition). San Diego: Academic Press.
Paylor R, Spencer CM, Yuva-Paylor LA, et al. (2006) The use of behav-
ioral test batteries, II: Effect of test interval. Physiol Behav 87: 95–102.
Pérez de la Mora M, Lara-García D, Jacobsen KX, et al. (2006) Anxio-
lytic-like effects of the selective metabotropic glutamate receptor 5
antagonist MPEP after its intra-amygdaloid microinjection in three
different non-conditioned rat models of anxiety. Eur J Neurosci 23:
2749–2759.
14 Journal of Psychopharmacology 00(0)
Prut L and Belzung C (2003) The open field as a paradigm to measure
the effects of drugs on anxiety-like behaviors: A review. Eur J Phar-
macol 463: 3–33.
Ramtekkar UP, Reiersen AM, Todorov AA, et al. (2010) Sex and age
differences in attention-deficit/hyperactivity disorder symptoms and
diagnoses: Implications for DSM-V and ICD-11. J Am Acad Child
Adolesc Psychiatry 49: 217–28.e1–3.
Rayen I, van den Hove DL, Prickaerts J, et al. (2011) Fluoxetine during
development reverses the effects of prenatal stress on depressive-like
behavior and hippocampal neurogenesis in adolescence. PLoS One 6:
e24003.
Redei E, Pare WP, Aird F, et al. (1994) Strain differences in hypotha-
lamic-pituitary-adrenal activity and stress ulcer. Am J Physiol Regul
Integr Comp Physiol 266: R353–R360.
Rittenhouse PA, López-Rubalcava C, Stanwood GD, et al. (2002) Ampli-
fied behavioral and endocrine responses to forced swim stress in the
Wistar–Kyoto rat. Psychoneuroendocrinology 27: 303–318.
Rodriguez-Porcel F, Green D, Khatri N, et al. (2011) Neonatal exposure
of rats to antidepressants affects behavioral reactions to novelty and
social interactions in a manner analogous to autistic spectrum disor-
ders. Anat Rec Adv Integr Anat Evol Biol 294: 1726–1735.
Ronald A, Pennell CE and Whitehouse AJO (2011) Prenatal maternal
stress associated with ADHD and autistic traits in early childhood.
Front Psychol 1: 223.
Sah P, Faber ESL, Lopez De Armentia M, et al. (2003) The amygdaloid
complex: Anatomy and physiology. Physiol Rev 83: 803–834.
Scholl JL, Renner KJ, Forster GL, et al. (2010) Central monoamine lev-
els differ between rat strains used in studies of depressive behavior.
Brain Res 1355: 41–51.
Semple BD, Blomgren K, Gimlin K, et al. (2013) Brain development
in rodents and humans: Identifying benchmarks of maturation and
vulnerability to injury across species. Prog Neurobiol 106–107:
1–16.
Sheng M (2001) The postsynaptic NMDA-receptor—PSD-95 signal-
ing complex in excitatory synapses of the brain. J Cell Sci 114:
1251.
Sheng M and Kim E (2011) The postsynaptic organization of synapses.
Cold Spring Harb Perspect Biol 3: a005678–a005678.
Slattery DA and Cryan JF (2012) Using the rat forced swim test to
assess antidepressant-like activity in rodents. Nat Protoc 7: 1009–
1014.
Sprowles JLN, Hufgard JR, Gutierrez A, et al. (2016) Perinatal exposure
to the selective serotonin reuptake inhibitor citalopram alters spatial
learning and memory, anxiety, depression, and startle in Sprague-
Dawley rats. Int J Dev Neurosci 54: 39–52.
Sun H, Guan L, Zhu Z, et al. (2013a) Reduced levels of NR1 and NR2A
with depression-like behavior in different brain regions in prenatally
stressed juvenile offspring. PLoS One 8: e81775.
Sun H, Jia N, Guan L, et al. (2013b) Involvement of NR1, NR2A differ-
ent expression in brain regions in anxiety-like behavior of prenatally
stressed offspring. Behav Brain Res 257: 1–7.
Swanson CJ, Bures M, Johnson MP, et al. (2005) Metabotropic glutamate
receptors as novel targets for anxiety and stress disorders. Nature
Reviews Drug Discovery 4: 131–144.
Tatarczyńska E, Kłodzińska A, Chojnacka-Wójcik E, et al. (2001) Poten-
tial anxiolytic- and antidepressant-like effects of MPEP, a potent,
selective and systemically active mGlu5 receptor antagonist. Br J
Pharmacol 132: 1423–1430.
Terbeck S, Akkus F, Chesterman LP, et al. (2015) The role of metabo-
tropic glutamate receptor 5 in the pathogenesis of mood disorders
and addiction: Combining preclinical evidence with human Positron
Emission Tomography (PET) studies. Front Neurosci 9.
Thompson MR, Li KM, Clemens KJ, et al. (2004) Chronic fluoxetine
treatment partly attenuates the long-term anxiety and depressive
symptoms induced by MDMA (‘Ecstasy’) in rats. Neuropsychophar-
macol Off Publ Am Coll Neuropsychopharmacol 29: 694–704.
Tizabi Y, Bhatti BH, Manaye KF, et al. (2012) Antidepressant-like
effects of low ketamine dose is associated with increased hippocam-
pal AMPA/NMDA receptor density ratio in female Wistar-Kyoto
rats. Neuroscience 213: 72–80.
Trullas R and Skolnick P (1990) Functional antagonists at the NMDA
receptor complex exhibit antidepressant actions. Eur J Pharmacol
185: 1–10.
Tu JC, Xiao B, Naisbitt S, et al. (1999) Coupling of mGluR/Homer and
PSD-95 complexes by the Shank family of postsynaptic density pro-
teins. Neuron 23: 583–592.
Turner KM and Burne THJ (2014) Comprehensive behavioural analysis
of Long Evans and Sprague-Dawley rats reveals differential effects
of housing conditions on tests relevant to neuropsychiatric disorders.
PLoS One 9: e93411.
VanGuilder HD, Farley JA, Yan H, et al. (2011) Hippocampal dysregula-
tion of synaptic plasticity-associated proteins with age-related cogni-
tive decline. Neurobiol Dis 43: 201–212.
Vitalis T and Parnavelas JG (2003) The role of serotonin in early cortical
development. Dev Neurosci 25: 245–256.
Walf AA and Frye CA (2007) The use of the elevated plus maze as
an assay of anxiety-related behavior in rodents. Nat Protoc 2:
322–328.
Werling DM and Geschwind DH (2013) Sex differences in autism spec-
trum disorders. Curr Opin Neurol 26: 146–153.
Will CC, Aird F and Redei EE (2003) Selectively bred Wistar-Kyoto rats:
An animal model of depression and hyper-responsiveness to antide-
pressants. Mol Psychiatry 8: 925–932.
World Health Organization (2017) Depression and Other Common
Mental Disorders: Global Health Estimates. Geneva: World Health
Organization.
Yuan H, Low C-M, Moody OA, et al. (2015) Ionotropic GABA and
glutamate receptor mutations and human neurologic diseases. Mol
Pharmacol 88: 203.
Zhang Y, Zu X, Luo W, et al. (2012) Social isolation produces anxiety-
like behaviors and changes PSD-95 levels in the forebrain. Neurosci
Lett 514: 27–30.