DOI: 10.1126/science.1228261
, 1095 (2013);339 Science
et al.Sandra Giovanoli
of Prenatal Immune Activation in Mice
Stress in Puberty Unmasks Latent Neuropathological Consequences
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and downstream inflammatory cascade play a
role in trigeminal activation. The effect of CBX
was not due to a direct vascular action or blockade
of gap junctions because CSD-induced CBF
changes, early MMA dilation, and Ca
+2
rise in
astrocytic syncytium were not altered by CBX
(Fig. 3, E and F, and fig. S5) (19). Interrupting
this signaling cascade with another Panx1 in-
hibitor , probenecid or HMGB1-shRNA, or NF-kB
activation inh ibitor 4-methyl-N1-(3-p henylpropyl)
benzene-1,2-diamine suppressed the late MMA
dilation (Fig. 3, G and H). Naproxen (40 mg/kg
intraperitoneally), a prostaglandin synthase in-
hibitor , also suppress ed the MMA response.
Moreover , CSD-induced dural mast cell degran-
ulation, another manifestation of the trigeminal
activation (21, 22), was significantly reduced by
CBX and HMGB1-shRNA (Fig. 3, I to L). Last,
we assessed the headache-like behavior induced
by repeated CSDs (which also induced PI influx
and nuclear NF-kB translocation) (19) with a meth-
od based on scoring facial grimace (23). Placement
of a KCl-pellet over dura in freely moving mice
caused pain-related mimics unlike saline-applied,
sham-operated mice. CSD-induced pain was re-
versed by means of CBX treatment (Fig. 3M).
These data show that CSD opens neuronal
Panx1 channels as reported during in vitro ische-
mia, NMDA over-activation, and aberrant bursting
(17). Activation of Panx1 by cellular stressors such
as excess potassium or glutamate stimulates the
inflammasome complex, subsequent caspase-1 ac-
tivation, and IL-1b production (17, 18), suggesting
that Panx1 megachannels may play a role as a
reporter linking neuronal stress to inflammatory
response. Similarly , CSD induced caspase-1 acti-
vation and HMGB1 release from neurons whose
Panx1 channe ls were activated.
HMGB1 is a member of the alarmin family,
which mediates the communication between in-
jured and surrounding cells (24). HMGB1 is
passively released from necrotic cells and ac-
tively secreted by cells under distress (25, 26).
HMGB1 behaves like a cytokine and promotes
inflammation when released (27, 28). Therefore,
HMGB1 and IL-1b released during CSD may take
part in initiation of the inflammatory response.
Subsequent NF-kB activation in astrocytes may
induce formation of cytokines, prostanoids, and
inducible NO synthase-derived NO (as suggested
by inhibition of MMA response by naproxen and
CSD-induced COX2 and iNOS expression in glia
limitans), which may be released to the sub-
arachnoid space via glia limitans and, hence,
stimulate trigeminal nerve endings around pial
vessels (fig. S6). By promoting sustained head-
ache, HMGB1 may thus serve to alarm the orga-
nism that the brain parenchyma has been stressed
by CSD or CSD-like events. HMGB1 is most
likely not the only mediator playing this role; other
cytokines as well as cells (such as microglia) may
also take part along the course of inflammatory
response (29, 30). In contrast to mediator s such as
potassium and proton s that are transiently released
during CSD, activation of the parenchymal in-
flammatory pathways may provide the sustained
stimulus required for sensitization of trigeminal
nerve endings and lasting pain as suggested by
suppression of long-lasting MMA vasodilatation,
mast cell degranulation, and importantly, headache-
like behavior by interrupting the inflammatory
cascade at one of the steps (11 ).
We propose a previously unknown link be-
tween a noxious intrinsic brain event and activa-
tion of the trigeminal pain fibers, involving the
opening of Panx1 megachannels on stressed neu-
rons, subsequent activation of the inflammatory
pathways, and transduction of this signal to the
trigeminal nerves around pial vessels (fig. S6).
References and Notes
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19. Materials and methods are available as supplementary
materials on Science Online.
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Brain Res. 477, 157 (1989).
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A. M. Strassman, Pain 130, 166 (2007).
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25. P. Scaffidi, T. Misteli, M. E. Bianchi, Nature 418, 191 (2002).
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332 (2004).
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Acknowledgments: We are grateful to M. A. Moskowitz for
helpful discussions, K. Kilic and E. Lule for their expert help
with the figures, and to A. Can for his help with confocal
microscopy. This work was supported by the Turkish Academy
of Sciences (T.D.), Hacettepe University Research Fund
08-D07-101-011 (Y.G.-O.), and the Brain Research
Association (H.K.).
Supplementary Materials
www.sciencemag.org/cgi/content/full/339/6123/1092/DC1
Materials and Methods
Supplementary Text
Figs. S1 to S5
Table S1
References (31–42)
23 October 2012; accepted 30 January 2013
10.1126/science.1231897
Stress in Puberty Unmasks Latent
Neuropathological Consequences of
Prenatal Immune Activation in Mice
Sandra Giovanoli,
1
Harald Engler,
2
Andrea Engler,
2
Juliet Richetto,
3,4
Mareike Voget,
5,6
Roman Willi,
7
Christine Winter,
6
Marco A. Riva,
3,4
Preben B. Mortensen,
8,9
Manfred Schedlowski,
2
Urs Meyer
1
*
Prenatal infection and exposure to traumatizing experiences during peripuberty have each been
associated with increased risk for neuropsychiatric disorders. Evidence is lacking for the cumulative
impact of such prenatal and postnatal environmental challenges on brain functions and
vulnerability to psychiatric disease. Here, we show in a translational mouse model that combined
exposure to prenatal immune challenge and peripubertal stress induces synergistic pathological
effects on adult behavioral functions and neurochemistry. We further demonstrate that the
prenatal insult markedly increases the vulnerability of the pubescent offspring to brain immune
changes in response to stress. Our findings reveal interactions between two adverse environmental
factors that have individually been associated with neuropsychiatric disease and support theories
that mental illnesses with delayed onsets involve multiple environmental hits.
P
renatal maternal infection and postnatal ex-
posure to psychological trauma are two
environmental risk factors for developmen-
tal psychiatric disorders, including autism, schizo-
phrenia, and bipolar disorder (1–4). In spite of
their relatively frequent occurrence (5–7), both
factors seem to have rather modest effect sizes
in large populations (4, 8, 9). For example, the
global incidence of schizophrenia after influenza
pandemics only increases marginally (relative risk
ratios of 1 to 2.5) even though 20 to 50% of the
general population is typically infected during
influenza pand emics (9, 10). It has therefore been
proposed that developmental stressors, such as
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infection or traumatizing experiences, may unfold
their neuropathological impact primarily in genet-
ically predisposed subjects (11). Another feasible
scenario is that initial exposure to a prenatal en-
vironmental insult, such as infection, can render
the offspring more vulnerable to the pathological
effects of a second postnatal stimulus, such as
stress (12, 13). However , this hypothesis still
awaits direct verification. We therefore tested if
stress in puberty has the potential to unmask latent
psychopatholog y in neurodevelopmenta lly vulne r-
able subjects with prenatal infectious histories.
We compared the consequences of prenatal
immune activation with or without additional post-
natal stress challenge in mice (fig. S1 and sup-
plementary methods). Prenatal immune activation
was induced by the viral mimetic polyriboinosinic-
polyribocytidilic acid [poly(I:C)], a synthetic analog
of double-stranded RNA that induces a cytokine-
associated, viral-like acute-phase response (14).
We used a low dose of poly(I:C) [1 mg/kg, admin-
istered intravenously on gestation day 9 (GD9)]
to mimic physiologically relevant and transient
cytokine elevations ( fig. S2) (14). Of fspring born
to poly(I:C)-exposed or control mothers were
then left undisturbed or exposed to variable and
unpredictable stress during peripubertal devel-
opment, a maturational period known to be highly
sensitive to the disrupting effects of traumatiz-
ing events relevant to psychosis-related disease
(15, 16). The stress protocol included five dis-
tinct stressors: (i) electric foot shock, (ii) restraint
stress, (iii) swimming stress, (iv) water depriva-
tion, or (v) repeated home cage changes, applied
on alternate days between postnatal days (PNDs)
30 and 40 (fig. S1 and supplementary methods).
We assessed the effects of the double-hit pro-
tocol on adult (PND 70 to 100) brain functions
using behavioral tests relevant to translational
models of neuropsychiatric disease (14)(supple-
mentary methods and tables S1 to S19). Stress
exposure increased anxiety-like behavior in the
elevated plus maze test independently of the pre-
natal immunological manipulation (Fig. 1A),
which suggests that peripubertal offspring with
a prenatal infectious history do not differ from
prenatal controls in the development of stress-
induced anxiety-like abnormalities. We further
revealed independent effects of immune chal-
lenge and stress in the disruption of selective as-
sociative learning as measured by the paradigm
of latent inhibition (LI): Nonstressed control off-
spring displayed a robust LI effect in the condi-
tioned active avoidance paradigm (Fig. 1B). This
LI effect arising from repeated preexposures to
the conditioned stimulus before conditioning was
fully abolished in all other groups (Fig. 1B). Pre-
natal immune activation and peripubertal stress
caused synergistic effects in the development
of sensorimotor gating deficiency, as assessed
by the paradigm of prepulse inhibition (PPI) of
the acoustic startle reflex (Fig. 1C), as well as in
the precipitation of behavioral hypersensitivity
to the psychotomimetic drugs amphetamine
(AMPH) (Fig. 1D and fig. S3A) and dizocilpine
(MK-801) (Fig. 1E and fig. S3B). Neither im-
mune activation alone nor stress alone affected
sensorimotor gating and psychotomimetic drug
sensitivity. Abnormalities in these domains be-
came evident only after combined exposure to
the two environmental factors.
We further evaluated the postnatal onset of
the identified behavioral abnormalities in our
environmental double-hit model. With the ex-
ception of anxiety-related behavior, none of the
other behavioral functions were affected at peri-
pubertal age (PND 41 to 45) (Fig. 2 and fig. S4).
Hence, the emergence of multiple behavioral
dysfunctions such as LI deficiency, PPI atten-
tion, and psychotomimetic drug hypersensitivity
in singly or doubly ch allenged offspring are
dependent on postpubertal maturational processes ,
which, in turn, is consistent with the clinical
course of mental illnesses with delayed onsets,
including schizophrenia and bipolar disorder
(17). We also revealed that a later application
of stress in adolescence, at PND 50 to 60, did
not elicit the interaction with prenatal immune
activation (fig. S5). The findings emphasize that
the precise timing of postnatal stress is critical
for the interaction with the prenatal immune
challenge.
The adult behavioral abnormalities emerging
after prenatal immune activation and peripubertal
stress exposure are unlikely to be associated with
1
Physiology and Behavior Laboratory, Swiss Federal Institute
of Technology (ETH) Zurich, 8603 Schwerzenbach, Switzerland.
2
Institute of Medical Psychology and Behavioral Immunobiology,
University Hospital Essen, University of Duisburg-Essen, 45122
Essen, Germany.
3
Center of Neuropharmacology, Department
of Pharmacological Sciences, Università degli Studi di Milano,
20133 Milan, Italy.
4
Center of Excellence on Neurodegenerative
Diseases, Department of Pharmacological and Biomolecular
Sciences, Università degli Studi di Milano, 20133 Milan, Italy.
5
International Graduate Program Medical Neurosciences, Charité
Universitaetsmedizin Berlin, 10117 Berlin, Germany.
6
Department
of Psychiatry, Technical University Dresden, 01062 Dresden,
Germany.
7
Neuroscience Discovery, F. Hoffmann–La Roche Ltd.,
4070 Basel, Switzerland.
8
National Centre for Register-Based
Research, Aarhus University, 8000 Aarhus C, Denmark.
9
Lundbeck
Foundation Initiative for Integrative Psychiatric Research, iPSYCH,
8000 Aarhus, Denmark.
*To whom correspondence should be addressed. E-mail:
urmeyer@ethz.ch
Fig. 1. Prenatal immune activation and peripubertal stress cause independent and synergistic patho-
logical effects on adult behavioral functions. (A) Adult mice subjected to peripubertal stress (S+) display
enhanced anxiety-like behavior in the elevated plus maze test (as indexed by the reduced time spent on
the open arms) compared with nonstressed (S-) offspring regardless of the prenatal conditions [CON,
vehiclecontrol;POL,poly(I:C)];
+
P < 0.05, main effect of peripubertal stress. N =16to19pergroup.(B)
Response latencies in nonpreexposed (NPE) and CS-preexposed (PE) subjects as a function of successive
10-trial blocks in the LI test with a conditioned active avoidance procedure. The LI effect is present in
CON/S- subjects (**P < 0.01) but is completely disrupted by prenatal immune activation alone (POL/S-),
peripubertal stress alone (CON/S+), or their combination (POL/S+). NPE, N =7 to 9 per group; PE, N =7
to 10 per group. (C) Sensorimotor gating as assessed by PPI of the acoustic startle reflex. (Left) % PPI as
a function of increasing prepulse intensities (dB above background of 65 dB); (right) the mean % PPI
across all prepulse levels. *P < 0.05, reduction of % PPI in POL/S+ animals relative to all other groups.
N =16to19pergroup.(D) The mean distance moved in a standard open-field arena during a 90-min
period after administration of AMPH [2.5 mg/kg, intraperitoneally (i.p.)]. *P <0.05and**P < 0.01, increase
in AMPH-induced activity displayed by POL/S+. N =8to10pergroup.(E) The mean distance moved in
the open field during a 90-min period after administration of MK-801 (0.15 mg/kg, i.p.). **P <0.01,
increase in MK-801–induced activity displayed by POL/S+ compared with all other groups, post hoc
group comparisons. N = 8 per group. All data are means T SEM.
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changes in the hypothalamus-pituitary-adrenal
(HPA) stress-response system: Neither single nor
combined exposure to the environmental adver-
sities affected basal plasma levels of corticoster-
one (COR T), the main effector hormone of the
HPA axis (fig. S6). The developmental stressors
also did not affect COR T secretion after acute
stress reexposure in adulthood (fig. S6). How-
ever , our high-performance liquid chromatogra-
phy analyses identified brain region–specific
neurochemical changes in adult mice exposed to
prenatal immune activation and/or peripubertal
stress: Prenatal immune activation was sufficient
to increase the levels of dopamine (DA) in the
nucleus accumbens (NAc) independently of post-
natal stress (Fig. 3A), and stress exposure de-
creased the content of serotonin (5-HT) in the
medial prefrontal cortex (PFC) regardless of the
prenatal history (Fig. 3B). It intrigued us that
enhanced DA levels in the hippocampus (HPC)
were only manifest after combined exposure
to prenatal immune challenge and peripubertal
stres s (Fig. 3A); this highlighted synergistic ef-
fects between the two adverse events in the pre-
cipitation of adult hippocampal DA imbalances.
Prenatal immune activation (at high intensity)
and chronic stress exposure have individually
been linked to the development of immune alter-
ations in the brain and periphery (18–20). Here,
we elucidated whether initial exposure to prenatal
immune challenge could change the offspring’s
neuroimmunological responses to peripubertal
stress. T w o brain areas of primary interest were
selected, namely, the HPC (in cluding CA1 to CA3
subregions and dentate gyrus) and the PFC (in-
cluding anterior cingulate, prelimbic, and infra-
limbic cortices). These brain regions are highly
sensitive to chronic stress exposure (21) and show
neuroanatomical abnormalities after intense pre-
natal immune challenge, including CNS immune
changes (19). We also included a cortical con-
trol region (secondary motor cortex, MC) that is
Fig. 3. Neurochemical parameters in adult offspring exposed to single
or combined prenatal immune activation and peripubertal stress. (A)DA
contents (nM/mg protein, square root–transformed) in the medial PFC,
NAc, and HPC of adult (PND 70) offspring under the conditions described
above.
+
P < 0.05, main effect of prenatal immune activation; *P < 0.05,
increase in HPC DA levels in POL/S+ mice relative to all other groups. N =
8 to 11 per group. (B) 5-HT contents (nM/mg protein, square root –
transformed) in the PFC, NAc, and HPC of groups of adult mice.
+
P <
0.05, main effect of stress exposure. N = 8 to 11 per group. All data are
means T SEM.
Fig. 2. Short-term effects of
single or combined exposure
to prenatal immune activation
and peripubertal stress on be-
havioral functions in pubescence.
(A) Peripubertal mice subjected
to stress in puberty (S+) displa y
enhanced anxiety-like behavior
in the elevated plus maze test
(as indexed by the reduced time
spentontheopenarms)com-
pared with nonstressed (S-) off-
spring regardless of the prenatal
conditions;
+
P < 0.05, main ef-
fect of peripubertal stress. N =
13 to 15 per group. (B)Response
latencies in NPE and PE sub-
jects as a function of successive
10-trial blocks in the LI test
with a conditioned active avoid-
ance procedure. *P < 0.05, main
effect of CS preexposure (LI) in
all groups. NPE, N =9pergroup;
PE, N = 9 to 10 per group. (C)
Sensorimotor gating as assessed
by PPI of the acoustic startle reflex.
(Left) % PPI a s a function of increasing prepulse intensities (dB above background of
65 dB); (right) the mean % PPI across all prepulse levels. N =13to15pergroup.(D)
The mean distance moved in a standard open-field arena during a 90-min period
after admini stration of AMPH (2.5 mg/kg, i.p.). N =12to13pergroup.(E)The
mean distance moved in the open field during a 90-min period after administration
of MK-80 1 (0.15 mg/kg, i.p.) . N = 10 to 11 per group. All data are means T SEM.
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largely insensitive to neuronal and immunolog-
ical adaptations after chronic stress.
We first used immunohistochemical techniques
to study the activation of microglia, a popula-
tion of immunocompetent cells i n the CNS (22).
Unbiased stereological estimations of microglia
immunoreactive for the calcium-binding protein
Iba1 revealed that peripubertal stress only led to
a ~5% increase in total microglia numbers in the
HPC at the adult stage (PND 70), without affect-
ing microglia morphology (fig. S7). Neither sin-
gle nor combined exposure to prenatal immune
activation and stress altered the expression of
CD68 (fig. S8), a cellular marker typically ex-
pressed by activated microglia in the CNS (22).
Likewise, the two environmental factors did not
change the hippocampal levels of the inflam-
matory molecules interleukin-1b (IL-1b), tumor
necrosis factor–a (TNF-a), and prostaglandin
E
2
(PGE
2
) in adulthood (fig. S8). Thus, single
or combined exposure to prenatal immune ac-
tivation and peripubertal stress exert only a min-
imal long-term impact on microglia cells and
induce no overt changes in the central and pe-
ripheral secretion of prototypical inflammatory
factors.
However , we revealed that the pr enatal insult
markedly increased the offspring’s vulnerability
to stress-induced neuroimmunological changes at
peripubertal age (PND 41): Combined immune
activationandstressledtoa2.5-to3-foldin-
crease in hippocampal (Fig. 4, B to D) and pre-
frontal (fig. S9, B to D) expression of markers
characteristic of activated microglia (CD68 and
CD1 1b) at PND 41. No such chan ges were found
in the MC c ontrol reg ion (fig. S10, B and C). The
hippocampal microglia response was further ac-
companied by the presence of elevated levels of
the proinflammatory cytokines IL-1b and TNF-a
(Fig. 4E) but not with plasma changes in inflam-
matory markers or the stress hormone COR T
(fig. S1 1). The neuroimmunological effects of
combined exposure to the two environmental in-
sults thus appear to be localized in stress-sensitive
brain areas, such as the HPC and PFC, and are
unlikely to be associated with functional changes
in the HPA axis. Single or combined exposure
to immune activation and stress also did not affect
the number or activation status of astrocytes in PND
41 mice (fig. S13), which suggests that the two
environmental challenges largely spared astroglial
functions at peripubertal age.
We performed additional molecular analyses
to expl ore whe ther th e transie nt micro glia changes
Fig. 4. Altered neuroimmune responses in the pubescent
brain after combined prenatal immune activation and peri-
pubertal stress. All measures were taken on postnatal day 41,
i.e., 1 day after exposure to the last peripubertal stressor.
(A) Stereological estimates and cell soma area of Iba1-positive
microglia cells in the HPC of mice treated as described above.
#
P <0.05and
§
P < 0.01, main effect of peripubertal stress.
N = 11 to 12 per group. The number of primary processes
and number of branch points of Iba1-positive microglia cells
intheHPCaregiveninfig.S12.(B) Stereological estimates
of CD68-positive cells in the HPC; **P < 0.01, post hoc
comparisons. N = 11 to 12 per group. (C)Therelative
optical density [arbitrary units (AU)] of CD11b immuno-
reactivity in the HPC; ***P < 0.001, post hoc compar-
isons. N = 11 to 12 per group. (D) Color-coded coronal brain sections of representative CON/S+ and POL/S+ offspring at the level of the HPC [CA1 to CA3
regions and dentate gyrus (DG) are highlighted] stained with antibodies against Iba1, CD68, or CD11b. Insets are at higher magnification. In color-coded
sections (CD68 and CD11b), the strongest staining intensities are yellow; the background is represented in dark purple (color scale bar). (E) Contents of
IL-1b,TNF-a,andPGE
2
in the HPC measured by using particle-based flow cytometry; **P <0.01and***P < 0.001, post hoc comparisons. N =10to12
per group. HPC levels of IL-6 and IL-10 protein were below detection limits. All data are means T SEM.
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