α as a measure of the pro-inflammatory response. a b pg ... · the prenatal maternal response to...
TRANSCRIPT
The prenatal maternal response to immune activation predicts
offspring cognitive dysfunction in a rat model of schizophreniaHarry G. Potter†a, Grace Revillb, Rebecca M. Woodsb, Hager M. Kowashc, Jocelyn D. Glazierac, Joanna C. Neillb, and Reinmar Hagera
aDivision of Evolution & Genomic Sciences, School of Biological Sciences; bDivision of Pharmacy & Optometry, School of Health Sciences; cDivision of Developmental Biology & Medicine, School of Medical Sciences.
Faculty of Biology, Medicine, & Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, M13 9PL
20 female Wistar rats were timed-mated and the presence of a vaginal plug was designated gestational day 1 (GD1).
On GD15 pregnant rats were treated with a single intraperitoneal injection of 10 mg/kg bodyweight poly I:C (low molecular weight form, InvivoGen) or vehicle (endotoxin-free 0.9 % saline). A tail vein blood sample was taken at 3 hours post-treatment to measure maternal interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) as a measure of the pro-inflammatory response.
On postnatal day 1 (PD1) offspring (n=212) were cross-fosteredusing a split litter design – half of each litter remained with their biological mother whilst the other half were crossed to a dam of the opposite treatment group.
Offspring ultrasonic vocalisations (USVs) were recorded on PD6, 10, and 14 for 3 minutes and analysed using the open-source MATLAB script MUPET5.
Offspring were tested on the attentional set-shifting task (ASST)on PD100 as a measure of cognitive flexibility (Box 2).
In a second cohort, DNA was extracted from fetal (GD21) or offspring (PD21-175) brain tissue and used to measure global DNA 5-methylcytosine by ELISA (Enzo Life Sciences, UK).
GD1
GD15
PD1
PD6-14
PD100
Schizophrenia is a neurodevelopmental disorder (NDD) whichis characterised by symptoms including impaired workingmemory, executive function, and attentional deficits.
Maternal immune activation (MIA) is recognised as a riskfactor for NDDs such as schizophrenia in the adult offspring(Box 1)1,2.
The double-stranded viral RNA mimetic polyinosinic-polycytidylic acid (poly I:C) is used to model MIA in animalmodels1.
The mechanism by which MIA increases this risk is poorlyunderstood, perhaps accounted for by issues associated withthe reproducibility of animal models1,3.
It is known that the pre- and postnatal maternal environmentsare critical influencers of offspring neurodevelopment4.
Here, we used cross-fostering in the poly I:C model of MIA tounderstand the role of the pre- and postnatal maternalenvironments in predicting offspring developmental deficits.
How does maternal immune
activation increase risk for NDDs?
Box 1: The maternal immune activation hypothesis of schizophrenia and other NDDs.Immunogens such as the viral mimetic poly I:C are used to study the pathogenesis ofNDDs such as schizophrenia, based on epidemiological evidence of increased risk forschizophrenia in offspring of mothers exposed to the influenza virus (top panel).Activation of the maternal immune system during pregnancy involves maternal andplacental production of pro-inflammatory cytokines such as IL-6 and TNF-α which mayalter developmental programming of the fetus (middle panel) or lead to alterations inthe early postnatal maternal environment (such as maternal care behaviours; bottompanel). These factors may contribute to developmental and behavioural deficits in theadult offspring relevant to schizophrenia and other NDDs.
References1Kowash et al. (2019). Brain Behav. Immun. (in press).
2Brown & Meyer (2018). Am. J. Psychiatry 175:1730-1830.
3Kentner et al. (2019). Neuropsychopharm. 44:245-258.
4Potter et al. (2019). Front. Neuroendocrinol. 52:195-205.
5Van Segbroeck et al. (2017). Neuron 94:465-485.
Molecular analysis will be carried out on the brains of offspring exposed to poly I:C:
qPCR will look at whether MIA affects expression of candidate genes (e.g. Pvalb, Gad67, Dnmt1, Reln) in brain regions that are affected in schizophrenia.
Protein expression of parvalbumin and Gad67 will be analysed by high-throughout Western blot (WES) to identify functional changes (Box 3).
Neuronal populations (immunohistochemistry for Pvalb, Iba-1) and morphology (Golgi-Cox stain) will be carried out to further assess the deficit caused by MIA.
@HPNeuro
Imm
un
oge
nP
ren
atal
en
viro
nm
ent
Post
nat
al
envi
ron
men
t
Maternal infection during pregnancye.g. Influenza
Models of MIAe.g. poly I:C
Prenatal effectse.g. placental transport
Maternal and placental production of pro-inflammatory
cytokinese.g. IL-6 and TNF-α
Postnatal effectse.g. maternal care
Developmental and behavioural deficits in the adult offspring
MethodsIntroduction
Results (1)MIA increases USV production and
duration regardless of cross status.
A BMale Female
Mean number of syllables produced
** *
C DMale Female
Mean vocalisation duration
* *
Figure 1: Prenatal exposure to poly I:C, regardless of future cross-fostering status,programmes offspring to vocalise significantly more frequently and for longer duringearly postnatal life. Poly I:C treatment had a significant main effect to increase thenumber of USV syllables produced between PD6-14 in A) males (GLMM, F1,20=8.87,**p=0.008; n=25-31) and B) females (GLMM, F1,19=6.26, *p=0.022; n=24-28), as well asto increase the total vocalisation duration in C) males (GLMM, F1,21=5.75, *p=0.026;n=25-31) and D) females (GLMM, F1,20=5.41, *p=0.031; n=24-28). Postnatal day alsohad a significant main effect on all four dependent variables, whereas cross-fosteringstatus did not have a significant main effect (statistics not shown).
M a le F e m a le
0
1
2
3
ID/E
D s
hif
t
V e h ic le /H o m e
V e h ic le /C ro s s e d
P o ly I:C /H o m e
P o ly I :C /C ro s s e d
* * * *
M a le F e m a le
0
1
2
3
ID/E
D s
hif
t
V e h ic le /H o m e
V e h ic le /C ro s s e d
P o ly I:C /H o m e
P o ly I :C /C ro s s e d
* * * *
S D C D R 1 ID S R 2 E D S R 3
0
5
1 0
1 5
2 0
M a le n u m b e r o f t r ia ls
P h a s e
Nu
mb
er o
f tr
ials
M V H
M V X
M P H
M P XS D C D R 1 ID S R 2 E D S R 3
0
5
1 0
1 5
2 0
F e m a le n u m b e r o f t r ia ls
P h a s e
Nu
mb
er o
f tr
ials
F V H
F V X
F P H
F P X
A BMale Female
M a le F e m a le
0
1
2
3
ID/E
D s
hif
t
V e h ic le /H o m e
V e h ic le /C ro s s e d
P o ly I:C /H o m e
P o ly I :C /C ro s s e d
* * * *
MIA causes a deficit in cognitive
flexibility on PD100, regardless of cross-
fostering.
M a le F e m a le
0
1
2
3
ID/E
D s
hif
t
V e h ic le /H o m e
V e h ic le /C ro s s e d
P o ly I:C /H o m e
P o ly I :C /C ro s s e d
* * * *
M a le F e m a le
0
1
2
3
ID/E
D s
hif
t
V e h ic le /H o m e
V e h ic le /C ro s s e d
P o ly I:C /H o m e
P o ly I :C /C ro s s e d
* * * *
M a le F e m a le
0
1
2
3
ID/E
D s
hif
t
V e h ic le /H o m e
V e h ic le /C ro s s e d
P o ly I:C /H o m e
P o ly I :C /C ro s s e d
* * * *
M a le F e m a le
0
1
2
3
ID/E
D s
hif
t
V e h ic le /H o m e
V e h ic le /C ro s s e d
P o ly I:C /H o m e
P o ly I :C /C ro s s e d
* * * *
Figure 2: Prenatal exposure to poly I:C, regardless of futurecross-fostering status, programmes offspring for cognitivedeficits, specifically in cognitive flexibility as measured usingthe attentional set-shifting task (ASST) in adulthood. Poly I:Cexposure on GD15 causes an increase in the number of trialsneeded to complete extradimensional shift (EDS) phase of theASST for both crossed (dashed lines) and home-raised (solidlines) offspring in both A) males and B) females, resulting in C) asignificant main effect of treatment group on the intra-extradimensional (ID/ED) shift (ratio of scores betweenintradimensional shift (IDS) and EDS, represented by purplearrows; GLMM, F1,49=7.42, **p=0.009; n=5-7).
C
Future workConclusionsMIA by poly I:C:
‒ programmes offspring to produce more USVs and of a longer duration across early postnatal life (PD6-14).
‒ programmes offspring to have a significant and clinically-relevant deficit in the EDS phase of the ASST in adulthood.
These behavioural deficits were not sex-specific showing no differences between male and female offspring.
Furthermore, these deficits were all present regardless of whether offspring were cross-fostered on PD1.
This suggests that MIA by poly I:C induces robust behavioural deficits in offspring which are programmed by the prenatal maternal environment.
Finally, MIA appears to affect global DNA methylation in the offspring brain in a sex-, age-, and region-specific manner.
Results (3)
A B
Box 2: The attentional set-shifting task (ASST) is used tomeasure cognitive flexibility in rodents. A food reward is hiddenunder media (e.g. gravel or sand) in bowls anointed with odours(e.g. orange or coconut). Animals are trained to associate arelevant dimension (i.e. either media or odour) with thepresence of this food reward and ignore the distractor. Betweeneach phase of the ASST, the rule changes and animals must learnthe new rule and forget the outgoing rule. A clinical analogue ofthis task is used to identify cognitive symptoms in schizophreniapatients.
➢ For the first 5 phases (SD, CD, R1, IDS, R2), media is the relevant dimension.
➢ For EDS and R3, the relevant dimension switches to odour.
➢ Number of trials taken to complete (6 correct in a row), number of incorrect choices, and mean trial time are recorded.
Odour = or ?
Media = or ?
HO
W D
OES
TH
E TA
SK W
OR
K?
Vehicle
Poly I:C
Results (2)
6 1 0 1 4
0
2 0 0
4 0 0
6 0 0
M a le n u m b e r o f s y lla b le s
Nu
mb
er o
f s
yll
ab
les
6 1 0 1 4
0
2 0 0
4 0 0
6 0 0
F e m a le n u m b e r o f s y lla b le s
Nu
mb
er o
f s
yll
ab
les
6 1 0 1 4
0
1 0
2 0
3 0
4 0
5 0
F e m a le
Tim
e (
ms
)
6 1 0 1 4
0
1 0
2 0
3 0
4 0
5 0
M a le
Tim
e (
ms
)
Postnatal day Postnatal day
Postnatal day Postnatal day
MIA affects offspring brain global
DNA methylation in an age- and
region-specific manner.
PD35 PD175
Males Females
PD35 PD175
PD
35/m
ale
/veh
PD
35/m
ale
/pic
Gap
PD
175/m
ale
/veh
PD
175/m
ale
/pic
Gap
PD
35/f
em
ale
/veh
PD
35/f
em
ale
/pic
Gap
PD
175/f
em
ale
/veh
PD
175/f
em
ale
/pic
0
2 0
4 0
6 0
P re fro n ta l c o r te x
Glo
ba
l C
pG
me
thy
lati
on
(%
)
#
A
PD
35/m
ale
/veh
PD
35/m
ale
/pic
Gap
PD
175/m
ale
/veh
PD
175/m
ale
/pic
Gap
PD
35/f
em
ale
/veh
PD
35/f
em
ale
/pic
Gap
PD
175/f
em
ale
/veh
PD
175/f
em
ale
/pic
0
2 0
4 0
6 0
D o rs a l h ip p o c a m p u s
Glo
ba
l C
pG
me
thy
lati
on
(%
)*
#
Box 3: Planned molecular analysis will investigate the effect ofpoly I:C on GABAergic markers such as parvalbumin and Gad67 atthe gene, protein, and neuronal level in the offspring brain.
Co
nce
ntr
atio
n (
pg
/ml)
GD21 PD21 PD35 PD175 GD21 PD21 PD35 PD175
Males Females
GD21
/mal
e/ve
h
GD21
/mal
e/pic
Gap
PD21
/mal
e/ve
h
PD21
/mal
e/pic
Gap
PD35
/mal
e/ve
h
PD35
/mal
e/pic
Gap
PD17
5/m
ale/
veh
PD17
5/m
ale/
picGap
GD21
/fem
ale/
veh
GD21
/fem
ale/
picGap
PD21
/fem
ale/
veh
PD21
/fem
ale/
picGap
PD35
/fem
ale/
veh
PD35
/fem
ale/
picGap
PD17
5/fe
mal
e/ve
h
PD17
5/fe
mal
e/pic
0
20
40
60
Frontal cortex
Glo
ba
l C
pG
me
thyla
tio
n (
%)
**
*
E
Co
nc
en
trati
on
(p
g/m
l)
IL-6 TNFα
Co
nc
en
trati
on
(p
g/m
l)B
C D
PD35 PD175
Males Females
PD35 PD175
Poly I:C elicited an increase in maternal plasma concentration of the pro-inflammatory cytokines A)IL-6 (GLM, F1,15=4.12, +p=0.060) and B) TNF-α (GLM, F1,15=17.92, p=0.001) at 3 hours post-treatment. C) Both treatment (GLMM, F1,7=6.22, *p=0.041) and the magnitude of prenatal exposureto maternal IL-6 (GLMM, F1,7=8.10, #p=0.025) and TNF-α (GLMM, F1,7=9.30, #p=0.019) predictedglobal DNA methylation in the dorsal hippocampus in PD35 in females only. D) In the prefrontalcortex of PD35 female offspring it was the magnitude of the prenatal exposure to IL-6 (GLMM,F1,8=7.96, #p=0.022) and TNF-α (GLMM, F1,8=7.45, #p=0.026) that predicted global DNA methylationlevels, and not treatment group (GLMM, F1,10=0.77, p=0.402). E) An increase in frontal cortex globalDNA methylation of poly I:C-exposed offspring was predicted by the significant main effect oftreatment in male GD21 fetuses (GLMM, F1,8=18.97, **p=0.002). In contrast, on PD21 it was femalefetuses exposed to poly I:C who had an increase in global DNA methylation (GLMM, F1,8=6.39,*p=0.035).
Figure 3: Prenatal exposureto poly I:C affects globalDNA methylation in thefrontal cortex, prefrontalcortex, and dorsalhippocampus of offspring.
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