Adverse neuropsychiatric development following perinatal brain injury: from a preclinical perspective

Abstract

Perinatal brain injury is a leading cause of death and disability in young children. Recent advances in obstetrics, reproductive medicine and neonatal intensive care have resulted in significantly higher survival rates of preterm or sick born neonates, at the price of increased prevalence of neurological, behavioural and psychiatric problems in later life. Therefore, the current focus of experimental research shifts from immediate injury processes to the consequences for brain function in later life. The aetiology of perinatal brain injury is multi-factorial involving maternal and also labour-associated factors, including not only placental insufficiency and hypoxia–ischaemia but also exposure to high oxygen concentrations, maternal infection yielding excess inflammation, genetic factors and stress as important players, all of them associated with adverse long-term neurological outcome. Several animal models addressing these noxious stimuli have been established in the past to unravel the underlying molecular and cellular mechanisms of altered brain development. In spite of substantial efforts to investigate short-term consequences, preclinical evaluation of the long-term sequelae for the development of cognitive and neuropsychiatric disorders have rarely been addressed. This review will summarise and discuss not only current evidence but also requirements for experimental research providing a causal link between insults to the developing brain and long-lasting neurodevelopmental disorders.

Full text

REVIEW ARTICLE
Adverse neuropsychiatric development following perinatal
brain injury: from a preclinical perspective
Ivo Bendix
1
, Martin Hadamitzky
2
, Josephine Herz
1
and Ursula Felderhoff-Müser
1
Perinatal brain injury is a leading cause of death and disability in young children. Recent advances in obstetrics, reproductive
medicine and neonatal intensive care have resulted in significantly higher survival rates of preterm or sick born neonates, at the
price of increased prevalence of neurological, behavioural and psychiatric problems in later life. Therefore, the current focus of
experimental research shifts from immediate injury processes to the consequences for brain function in later life. The aetiology of
perinatal brain injury is multi-factorial involving maternal and also labour-associated factors, including not only placental
insufficiency and hypoxia–ischaemia but also exposure to high oxygen concentrations, maternal infection yielding excess
inflammation, genetic factors and stress as important players, all of them associated with adverse long-term neurological outcome.
Several animal models addressing these noxious stimuli have been established in the past to unravel the underlying molecular and
cellular mechanisms of altered brain development. In spite of substantial efforts to investigate short-term consequences, preclinical
evaluation of the long-term sequelae for the development of cognitive and neuropsychiatric disorders have rarely been addressed.
This review will summarise and discuss not only current evidence but also requirements for experimental research providing a
causal link between insults to the developing brain and long-lasting neurodevelopmental disorders.
Pediatric Research (2019) 85:198–215; https://doi.org/10.1038/s41390-018-0222-6
INTRODUCTION
Over the past decades, it has become clear that the perinatal
environment in conjunction with genetic factors shapes an
individuals’predisposition to increased risk of disease
1
. Brain
development is influenced by various intrauterine, prenatal and
postnatal events interacting with the genotype to affect the neural
systems related to emotions, cognitive and language abilities,
which may also increase the risk for psychopathology later in life.
Noxious stimuli include maternal infection yielding excess
inflammation, maternal stress, obesity and metabolic disorders,
many of them associated with intrauterine growth restriction
(IUGR) and/or preterm birth. In addition to these foetal elements,
the immature brain is at risk to be exposed to additional injurious
stimuli during or early after birth, e.g. infection, foetal hypoxia,
intrapartum oxygen deprivation, hyperoxia, stress from maternal
separation and medical procedures, exposure to medication or
anaesthesia further increasing the risk for the development of
behavioural and neuropsychiatric brain diseases in later life.
Perinatal brain injury affects infants born at all gestational ages
with a higher incidence in the most vulnerable very immature
group of patients. Improved obstetric and neonatal care in
conjunction with the use of preventive and/or therapeutic
strategies such as antenatal steroids prior to preterm delivery
and the introduction of hypothermia treatment for
hypoxic–ischaemic encephalopathy (HIE) has led to increased
survival rates and also reduction of severe neurological impair-
ment. For instance, a recent meta-analysis of world-wide cohorts
born after 2006 revealed that the pooled prevalence of cerebral
palsy in infants born extremely preterm was with a rate of 6.8%
(95% confidence interval 5.5–8.4) reduced compared to previous
studies
2
. In the majority of cases suffering from an adverse
perinatal environment, physiological brain maturational processes
are affected leading to a variety of neurodevelopmental disorders
in later life such as cognitive impairment, autism spectrum
disorders (ASD), attention deficit (hyperactivity) disorder (ADHD)
and psychiatric disease like schizophrenia (SZ)
3
.
While these sequelae are frequently related to perinatal brain
injury of various origins, investigations of the underlying
anatomical and pathophysiological processes remain a major
goal for research in neonatal neurology. In this review, we give an
overview on multiple risk factors for the development of
neuropsychiatric diseases focussing on recent findings derived
from experimental models (Table 1). We further summarise shared
cellular and molecular mechanisms across a variety of neurode-
velopmental disorders. Finally, we discuss challenges for pre-
clinical research focussing on the assessment of neuropsychiatric
symptoms in experimental models.
RISK FACTORS FOR THE DEVELOPMENT OF
NEUROPSYCHIATRIC DISEASES
Maternal and early postnatal stress
Human studies suggest a positive association between maternal
stress and the risk of preterm delivery, foetal growth restriction,
altered social behaviour, ADHD symptoms and impaired cognitive
performance
4,5
. Furthermore, analysis in siblings demonstrated
that exposure to high levels of cortisol in utero are associated with
lower adult educational attainment and verbal cognition
6
. The
Received: 17 July 2018 Revised: 11 October 2018 Accepted: 15 October 2018
Published online: 26 October 2018
1
Department of Paediatrics I, Neonatology and Experimental Perinatal Neuroscience, University Hospital Essen, University Duisburg-Essen, Essen, Germany and
2
Institute of
Medical Psychology and Behavioural Immunobiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Correspondence: Ivo Bendix (ivo.bendix@uk-essen.de) or Ursula Felderhoff-Müser (ursula.felderhoff@uk-essen.de)
www.nature.com/pr
©International Pediatric Research Foundation, Inc 2018

Table 1. Modelling adverse neuropsychiatric development following perinatal insults
Models (Neuropatho)physiology Neuropsychiatric outcome/behavioural deficits References
Maternal and early postnatal stress
Prenatal restraint stress (pregnant
Sprague–Dawley rats 45 min, 3×/day from
embryonic day 11 (E11) until birth)
Male offspring, postnatal day 14 (PND14) and
PND22:
- reduced serum leptin levels
- reduced expression of γ2 subunit of gamma-
aminobutyric acid A receptors in amygdala
- altered expression of metabotropic glutamate
receptor 2/3/5 receptors in the amygdala and
hippocampus
PND30–300:
- hypertrophic adrenal glands
PND90–660:
- reduced hippocampal cell proliferation
PND22:
- increased anxiety (reduced time in open arms of
elevated plus maze (EPM))
- more ultrasonic vocalization in response to
isolation from their mothers and later suppression
of ultrasonic vocalization after exposure to an
unfamiliar male odour
PND120:
- delayed learning in a spatial memory task (Water
maze)
9,10
Maternal separation (rat pups separated for 3 h/
day between PND2–14
PND60:
- basal adrenocorticotropic hormone levels
increased
- reduced adrenocorticotropic hormone levels after
restraint stress
- decreased noradrenaline in limbic regions and
increased 5-hydroxyindoleacetic acid levels in the
frontal cortex and hippocampus
PND60:
- increased anxiety (reduced number of entries into
open arms and increased time in closed arms of
EPM)
21
Maternal social behaviour deficits (Long–Evans rat
dams had decreased nesting/bedding material
between PND8 and 12)
PND20–22 and PND42–48:
- reduced neural activation (cFos) in amygdala,
medial prefrontal cortex and nucleus accumbens
PND20–22 and PND42–48:
- social behaviour deficits (less time in the social
chamber)
PND75:
- depressive-like behaviour in forced-swim test
22
Maternal separation (Sprague–Dawley rat pups, 3
h/day, PND3–21)
PND21–60:
- hypomyelination and ultrastructural changes in
medial prefrontal cortex in males and females
- impaired oligodendrocyte precursor cell
differentiation in medial prefrontal cortex via
decreased the expression of histone deacetylases
1/2 and activation of Wnt signalling
PND21 and PND60:
- increased anxiety (decreased time in centre of
open field (OF))
- increased grooming and circling behaviour
- cognitive deficits: (decreased response in
nonmatching-to-place task in T maze)
- social behaviour deficits (reduced social
interaction with stranger rats)
23
Maternal obesity
Maternal obesity (12-week-old C57BL/6 dams,
high-fat diet, 60% calories from fat 6 weeks prior to
mating, control diet: 10% calories from fat)
PND42:
- increased microglia activation in amygdala in
female but not in male offspring
- increased cerebral pro-inflammatory cytokine
levels (IL-1beta and TNFalpha) in female but not
male offspring
PND32–35:
- hyperactivity (increased distance travelled in
open field in male offspring)
- increased anxiety in female offspring (decreased
distance in centre over total distance travelled in
OF)
PND39–42:
- social behaviour deficits in female but not male
offspring (reduced interest in stranger mice in
three-chamber sociability test after prolongation of
maternal high-fat diet into the lactation period)
31
Maternal high-fat diet (Long–Evans rats, 60% fat
for 4 weeks prior mating until weaning, controls:
standard chow (13.5% fat))
PND110:
- heightened response to restraint stress and a
slower return to baseline corticosterone levels
- increased mineralocorticoid receptor mRNA
expression in amygdala
- increased glucocorticoid receptor mRNA
expression in amygdala of females
- higher levels of nuclear factor kappa B transcript
in female offspring
- increased abundance of IL-6
PND90:
- increased anxiety in males (less time in centre of
OF and decreased preference for open arms of
EPM)
33
Maternal high fat diet (C57BL/6 dams, high-fat diet
(32% fat) 6 weeks before mating and throughout
pregnancy and lactation, controls: normal diet (4%
fat))
PND21:
- increased lipid peroxidation
- decreased mRNA and protein levels of BDNF in
the hippocampus
PND35:
- decreased dendritic length, number of branches
and dendrite formation
PND21–28:
- decreased number of readings in OF
- cognitive deficits (increased distance to reach the
target hole of the Barnes maze)
35
Maternal high-fat diet (C57BL/6 dams, 60% from
fat, 3 weeks before mating prolonged until
weaning, control: normal chow)
PND90:
- reduced amino acid levels (glutamate, aspartate,
GABA, taurine and tyrosine) in the medial
prefrontal cortex and hippocampus
PND90 and PND540:
- cognitive disabilities evident in a reference
memory ( T maze) and in an associative learning
(two-way active avoidance) paradigm
- potentiation of prepulse inhibition
36
Placental insufficiency/IUGR
Intrauterine artery ligation (Dunkin–Hartley guinea
pigs, E28–30)
PND56:
- enlargement of lateral ventricles
- atrophy of basal ganglia and septal region
PND84:
- reduced prepulse inhibition
53
Maternal caloric undernutrition (ICR mice, 50%
food restriction, E12–15 and PND0–21)
PND45, male offspring:
- increased brain insulin receptor expression
- reduced PSD95 expression
PND25–35
- learning disabilities (Morris water maze)
56
Meso‐ovarian vessel cauterisation (Wistar rats, E17) PND35:
- reduced levels of PSD95 and synaptophysin in the
hippocampus
PND25–35:
- spatial memory deficits (water maze)
55
Adverse neuropsychiatric development following perinatal brain injury:. . .
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199
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1234567890();,:

Table 1 continued
Models (Neuropatho)physiology Neuropsychiatric outcome/behavioural deficits References
Hypoxia–ischaemia
Neonatal hypoxia–ischaemia (PND7
Sprague–Dawley rats, right common carotid artery
ligation, 3 h hypoxia at 8% oxygen )
PND95:
- macroscopic injury to the hippocampus, ranging
from hippocampal atrophy and neuronal loss to
severe reduction of the ipsilateral hemisphere with
the loss of cerebral cortex and hippocampu s
PND21:
- increased motor activity
PND36:
- cognitive deficits (difference in spontaneous and
forced alternation in T maze)
PND50:
- spatial memory deficits (increased error rates in
water maze)
PND90:
- increased apomorphine-induced stereotypy in
open field
74
Neonatal hypoxia–ischaemia (PND4 Spraque-
Dawley rats, bilateral carotid artery occlusion
followed by to hypoxia, 8% oxygen for 15 min)
PND21:
- reduced white matter size, decrease in thickness
of corpus callosum, reduction in numbers of
mature oligodendrocytes and hypomyelination
- enlarged ventricles, decrease in hippocampal
axon dendrite length, reduced numbers of tyrosine
hydroxylase-positive neurons
- enhanced microgliosis
PND21:
- hyperactivity (increased distance travelled in OF)
- reduced anxiety/increased impulsivity (increased
entries and time in open arms of the EPM)
69
Neonatal hypoxia–ischaemia (PND10
129T2xC57BL/6 F1 hybrid mice, right common
carotid artery ligation followed by 1 h hypoxia at
10% oxygen)
PND94:
- decreased brain weight and hippocampal injury
PND49:
-deficits in novel object recognition PND56-91:
- spatial memory deficits (water maze)
- increased circling following apomorphine
challenge
72
Neonatal hypoxia–ischaemia (PND9 C57BL/6 mice,
occlusion of the right common carotid artery
followed by 1 h hypoxia (8 or 10% oxygen)
PND16:
- neuronal and axonal loss
- hypomyelination and astrogliosis
- microglia and endothelial activation
- peripheral leukocyte
infiltration
- increased pro-inflammatory cytokine levels (IL-6,
IL-1beta)
PND51:
- brain atrophy mainly affecting the cortex,
hippocampus and striatum
PND28–49:
- reduced anxiety (increased time in open arms in
EPM)
- cognitive deficits (reduced recognition memory in
object recognition)
67,71
Neonatal hypoxic–ischaemia (left common carotid
artery ligation in male PND9 C57BL/6 mice
followed by 30 min hypoxia at 10% oxygen)
PND55:
- increased reactive gliosis (GFAP, Iba1) in the
hippocampus
- hippocampal volume reduction by >50%
- reduced neuronal density
PND50–54:
- cognitive deficits (reduced recognition memory in
object recognition)
70
Neonatal hypoxia–ischaemia (PND10 Wistar rats,
occlusion of the right common carotid artery
followed by 65 min hypoxia at 8% oxygen)
PND11, 24 and 94
- brain atrophy mainly affecting hippocampal and
thalamo-striatal regions
PND50–70:
- cognitive deficits (recognition memory in object
recognition)
75
Prenatal transient systemic hypoxia–ischaemia,
(Sprague–Dawley dams, E18, transient occlusion of
uterine arteries for 60 min, shams: laparotomy for
60 min)
PND35–40:
- diffusion tensor imaging abnormalities
- impaired axonal integrity and reduced
myelination
PND25–32:
- hyperactivity (distance and velocity in OF)
- social behaviour deficits (reduced social
interaction)
77
Hyperoxia
24 h 80% oxygen ( Wistar rats, PND6) PND7:
- increased lipid peroxidation
- increased microglia activation and pro-
inflammatory cytokine expression
- oligodendrocyte degeneration and impaired
maturation
- reduced mRNA expression of synaptic plasticity-
related molecules
PND11:
- hypomyelination
PND125:
- microstructural white matter abnormalities in
corpus callosum and external capsule
PND30 and PND90:
- cognitive deficits (increased time to find escape
hole, probe trial in Barnes maze, and recognition
memory deficits in object recognition)
88,95
48 h 80% oxygen (C 57BL/6 mice, PND6) PND30 and PND53:
- disturbed white matter diffusivity in corpus
callosum
PND44–53:
- hyperactivity (higher maximum velocity in wheel
running)
- impaired capacity to compensate complex motor
challenge (irregularly spaced crossbars in running
wheel)
96
Inflammation
Maternal immune activation (MIA, YFP-H C57BL/6
mice, E12.5, 20 mg/kg poly I:C, i.p.)
PND17 and PND90:
- reduced cortical spine density, dynamics
PND17–19:
- altered excitatory and inhibitory synaptic
connectivity in offspring
PND60:
- repetitive behaviour (increased marble burying)
104
MIA (C57BL6/N mice, E9, 5 mg/kg poly I:C, i.v.) PND40 and PND90:
- impaired expression of presynaptic and
postsynaptic proteins in the hippocampus
PND90:
- increase in hippocampal IL-1βexpression
PND90:
- prepulse inhibition deficits
105
Adverse neuropsychiatric development following perinatal brain injury:. . .
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200
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Table 1 continued
Models (Neuropatho)physiology Neuropsychiatric outcome/behavioural deficits References
MIA (C57BL/6, E15, 5 mg/kg poly I:C on E15, i.p.) PND120, male offspring:
- decreased cell proliferation and reduced dendritic
intersections in dentate gyrus
PND120, male offspring:
- prepulse inhibition deficits of the acoustic startle
response
- cognitive deficits (reduced spatial memory in the
T and Morris water maze)
- increased depressive like behaviour (tail-
suspension test)
106
MIA (mice, E12.5, 20 mg/kg poly I:C, i.p) E14.5:
elevated IL-17Ra mRNA expression in the brain
PND9:
- abnormal ultrasonic vocalisation
PND56–360:
- social interaction deficits in male mice
- increased repetitive behaviour (marble burying)
108
Cytomegalovirus infection (BALB/c mice, PND0,
750 plaque-forming units, i.p.)
PND30:
- brain atrophy and cerebellar hypoplasia
PND 30–40:
- social behaviour deficits (lack of preference for
social novelty)
109
MIA (rhesus monkeys; E43, 44, 46, 47, 49 and 50;
0.25, 0.5 and 1 mg/kg poly I:C, i.v.)
PND1280:
- smaller apical dendrites and greater number of
oblique dendrites in dorsolateral prefrontal cortex
PND180:
increased whole-body stereotypies (pacing)
112
MIA (C57BL/6 mice, 5 mg/kg poly I:C, i.v. either at
E9 or E17)
PND24 and PND180:
MIA E9:
- reduction of dopamine D1-receptors, Reelin and
Parvalbumin in medial prefrontal cortex
- reduced Reelin-positive cells in hippocampal and
reduced doublecortin expression in dentate gyrus
MIA E17:
- reduction in NMDA-receptor subunit NR1 in
dorsal hippocampus
- decrease in postnatal Reelin- and Parvalbumin-
positive cells in the medial prefrontal cortex
- reduced doublecortin expression and enhanced
apoptosis (caspase-3) in dentate gyrus
PND98–112:
MIA E9:
- reduced prepulse inhibition of acoustic startle
response
MIA E17:
- impaired working memory
- enhanced locomotor reaction to dizocilpine (MK-
801)
- perseverative behaviour (reversal learning in the T
maze)
114,117
MIA (Sprague–Dawley rats, 50 μg/kg LPS, i.p., on
E12 or E16)
E18:
- decreased dopaminergic neurons in the midbrain
(MIA at E16)
PND30
- reduced anxiety (more time in centre of the open
field, MIA E16)
- altered amphetamine response in reward
processing (MIA E12)
115
Postnatal inflammation (Wistar rats, PND3, 0.25
mg/kg LPS)
PND5:
- cellular degeneration, reactive gliosis
- increased IL-1beta levels
PND11:
- hypomyelination
PND120:
- microstructural deficits in the white matter
assessed by diffusion tensor imaging
P30 and P90:
- cognitive deficits (latency in probe trial in the
Barnes maze, recognition memory in object
recognition)
120
Postnatal inflammation (Swiss mice, 10 µg/kg IL-
1beta twice a day from PND1 to PND4 and once on
PND5, i.p.)
PND15 and PND30:
- increase of oligodendrocyte progenitors
associated with a reduction of mature
PND30:
- reduced expression of myelin proteins
PND35:
- microstructural abnormalities in the white matter
oligodendrocytes
PND29:
- cognitive deficits (recognition memory in novel
object recognition and in object location memory)
121
Postnatal inflammation (Sprague–Dawley rats,
PND14, 100 µg/kg LPS)
PND14:
increased cFos positive neurons in central
amygdala
PND40:
- increased anxiety (less time in open arms of EPM
for females)
PND65:
- altered fear behaviour (increased freezing rate in
auditory cued fear conditioning)
122
Multiple hit
MIA (Lewis rats, 200 µg/kg LPS, i.p., every 12 h from
E17 until end of gestation)+hypoxia–ischaemia at
PND1 (occlusion of the right common carotid
artery followed by 210 min hypoxia at 8% oxygen)
PND25:
- enlarged right lateral ventricles
- decreased thickness of corpus callosum
- reactive astrogliosis and microgliosis
- decreased number of dopaminergic neurons in
substantia nigra
PND15, 20, 25:
- reduced exploratory behaviour and increased
immobility (decreased total distance travelled in
OF, increased freezing)
124
transient systemic hypoxia–ischaemia
(Sprague–Dawley rats, 60 min ute rine artery
occlusion at E18)+perinatal inflammation (intra-
amniotic injection of 4 µg LPS after occlusion)
PND2:
- ventricular enlargement
- increased microglial and astroglial
immunoreactivity
PND15:
- reduced myelin basic protein expression
PND28:
- motor impairment and gait abnormalities in
forelimbs and hindlimbs (DigiGait analysis)
125
MIA (1 mg/kg poly I:C i.v. E9, C57BL/6 mice)+stress
in puberty (electric foot shock, restraint stress,
swimming stress, water deprivation, repeated
home cage changes, PND30–40)
PND41:
- increased microglia activation and pro-
inflammatory cytokine levels in the hippocampus
PND70:
- increased dopamine in the nucleus accumbens
independent of stress
- decreased serotonin in medial prefrontal cortex
independent of MIA
PND70-PND100:
- increased anxiety independent of MIA (increased
time in open arms of EPM)
- disruption of selective associative learning
independent of MIA and stress (impaired latent
inhibition in conditioned active avoidance
paradigm)
- synergistic effects in sensorimotor gating
129
Adverse neuropsychiatric development following perinatal brain injury:. . .
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201
Pediatric Research (2019) 85:198 – 215

underlying reasons for these effects are assumed to be stress-
related alterations in brain structure. As such, it was shown that
children born to mothers who experienced high levels of anxiety
in the early second trimester of pregnancy or chronic perceived
stress had region-specific reductions in grey matter volume in the
cortex, hippocampus and cerebellum
7,8
. Nevertheless, information
about the detailed cellular and molecular mechanisms is mainly
limited to animal studies
4
. In experimental models, prenatal stress
can be induced either by restraint under bright light, nest
manipulation or predator stimuli, which alters the offsprings’
anxiety-related behaviour, learning and memory and stress
regulation
4,9–12
. Neuropathology includes alterations in hippo-
campal gene expression, cortical inhibitory neuron subtypes and
hypothalamic–pituitary–adrenal axis responses or cortisol reactiv-
ity
13–15
. Furthermore, basic neurodevelopmental processes like
cellular proliferation, differentiation and migration are
delayed
16,17
. Importantly, maternal stress is supposed to interact
with multiple genes involved in neurogenesis, serotonergic genes
and genes involved in GABAergic transmission, implicated in both
SZ and ASD
18
. For example, interaction between the serotonergic
gene 5-hydroxytryptamine transporter (5-HTT aka. sodium-
dependent serotonin transporter and solute carrier family 6 member
4(SLC6A4)) and stress was observed for ultrasonic vocalisation
19
and ASD-like stereotypic repetitive grooming behaviour
20
.In
addition to maternal stress, early postnatal stress models have
been developed with maternal separation being one of the most
prominent causes leading to long-lasting increases in anxiety,
depressive behaviour and disruption of stress responses. These
changes are accompanied by reduced neural activation in the
amygdala and other areas implicated in social functioning,
including the medial prefrontal cortex and nucleus accum-
bens
21,22
. Recently, it was shown that long-lasting stress (i.e. 3 h
maternal separation daily for the first 3 postnatal weeks) results in
increased anxiety and reduced social interaction accompanied by
impaired oligodendrocyte differentiation and myelination in the
prefrontal cortex
23
. Nevertheless, often only single regions and
single cell types were analysed. Other molecular events such as
alteration not only in synaptic strength and neuron connections
but also inflammatory processes may contribute to early stress-
induced behavioural alterations in later life. The relationship
between synaptogenesis, synaptic pruning and inflammation has
been demonstrated in a variety of studies
24–26
. However, whether
these mechanisms are associated with early-life stress and later life
changes remains to be investigated.
Maternal obesity
Causal links between maternal obesity and the development of
severe perinatal brain injury, e.g. periventricular leukomalacia,
HIEorintraventricularhaemorrhage are rather scarce
27,28
.In
contrast, high amount of human studies indicate that it is
associated with an increased risk for various mental health
disorders, such as not only ADHD, ASD, depression and SZ but
also cognitive impairments (reviewed elsewhere
29
). This is
supported by animal research
30
.Forinstance,inarodentmodel
of maternal high-fat diet (HFD) it was shown that female
offspring revealed decreased sociability and males presented
with increased hyperactivity
31
.However,thisseemstocontra-
dict other reports demonstrating that HFD leads to signs of
anxiety and depression assessed in the elevated plus maze
(EPM), open field (OF) and forced swim test in male off-
spring
32,33
.Inadditiontorodents,anon-humanprimatemodel
was used demonstrating that adiposity in pregnancy increases
the risk of impulsive and disruptive behaviour
34
.Besides
alteration of emotions, cognitive function is reduced with
delayed spatial learning, deficits in reference memory and
associative learning accompanied by attenuated levels of amino
acids in the medial prefrontal cortex and hippocampus
35,36
.
Preclinical studies in rodents and primates suggest abnormal
development of the serotonergic and dopaminergic system and
obesity-induced inflammation as underlying mechanisms
29,34
.
This is supported by human studies demonstrating that
maternal obesity is associated with changes in cytokines,
hormones and acute-phase proteins in the second trimester
and with an increased risk of maternal infection or childhood
asthma
37,38
.
Placental insufficiency
IUGR often caused by placental insufficiency is a major contributor
to neurodevelopmental deficits involving problems in motor skills,
cognition and memory in school children
39
. It is assumed that
deviant brain development and altered maturational processes
may lead to increased chances of later cognitive, emotional and
behavioural problems
40–42
. Neuroimaging studies revealed struc-
tural brain alterations and disturbed white matter organisation
leading to impaired neurodevelopmental outcomes, which persist
into young adulthood
43–45
.
Growth-restricted foetuses due to placental insufficiency are
chronically hypoxemic and hypoglycaemic. In rodents, placental
dysfunction can be caused by reduced utero placental perfusion
leading to hypertension and placental ischaemia
46,47
or genetic
knockdown of the insulin receptor reflecting maternal diabetes
48
.
Despite initial epidemiological evidence indicating a relationship
between these maternal metabolic disorders (i.e. hypertension
and diabetes) and the risk for the development of SZ
49,50
, animal
research in terms of long-term neuropsychiatric outcomes in
above described experimental models is still lacking.
Most commonly applied models for evaluation of neurodeve-
lopment in IUGR offspring include umbilical/uterine artery ligation
or chronic hypoxia in sheep, piglets or rodents
39
. Neuropatholo-
gical consequences include reductions in total brain volume,
myelination deficits and impaired brain connectivity
39
. Further-
more, unilateral ligation of the uterine artery at mid-gestation was
associated with ventriculomegaly, reduced basal ganglia volume,
impaired synaptogenesis and altered cerebellar Purkinje cell
development in the guinea-pig brain at adolescence, resembling
anatomical changes found in some individuals with SZ
18,51–54
.Of
note, prepulse inhibition (PPI) of the acoustic startle response
(ASR) was reduced
53
. In addition to the PPI deficit that is supposed
to parallel sensorimotor gating deficits observed in SZ, cognitive
deficits have been observed in rodent models of IUGR induced
Table 1 continued
Models (Neuropatho)physiology Neuropsychiatric outcome/behavioural deficits References
- synergistic effect for dopamine in the
hippocampus
deficiency (reduced PPI of the acoustic startle
reflex)
MIA (100 µg/kg LPS i.p., E18 and E19, Wistar rats)
+systemic hypoxia (PND4, 8% oxygen, 140 min)
PND18 and PND30:
- hypomyelination
PND18 and PND69:
- impaired oligodendrocyte maturation
PND18:
- microglia activation and astrogliosis
PND28:
- reduced social play behaviour in males (pinning)
PND35:
- ASD-like symptoms (repetitive grooming)
PND49:
- increased anxiety (reduced transition inner–outer
zone in open field)
128
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either by maternal caloric undernutrition or by meso-ovarian
vessel cauterisation leading to learning disabilities and spatial
memory deficits in adolescent offspring
55,56
.
Hypoxia–ischaemia (HI)
Another frequent insult in the perinatal period is term asphyxia
with HIE affecting approximately 1.5–3 in 1000 live births
57
.
Despite improvement of outcomes after introduction of hypother-
mia treatment mortality remains significant and severe motor and
cognitive impairment may lead to cerebral palsy evident in the
first 2 years of life. Beside the severe forms of brain injury children
may also suffer from attention, memory and behaviour problems,
requiring developmental surveillance beyond 2 years of age.
Diversity of outcome across grades of HIE is reported but only few
studies have addressed the milder consequences of HIE at school
age both in the pre- and post-cooling period
58–60
. Furthermore,
children with diagnosed stroke in the neonatal period, a disease
with an incidence between 7.1 in 100.000 and 16 in 100.000 live
births, may also present with poor neurological and psychosocial
outcome, which is largely dependent on the lesion size
61–64
. Those
deficits can be attributed to the pattern of injury and affected
brain structures, such as hippocampus, cortex and striatum
65
.
The most commonly used neonatal asphyxia model has been
established in rodents by Rice–Vannucci in 1981 with unilateral
common carotid artery (CCA) occlusion followed by systemic
hypoxia (HI) leading to excitotoxicity, apoptosis, inflammation and
subacute white and grey matter injury resulting in long-term brain
atrophy mainly affecting cortex, hippocampus and striatum
66–68
.
This type of injury leads to long-term sensorimotor deficits while
general locomotor activity is only transiently increased resolving
at 3 weeks of age
67,69,70
. This transient increase in locomotor
activity may reflect hyperactivity, a major hallmark of ADHD.
Furthermore, adolescent and young adult animals reveal reduced
anxiety, an increased risk-taking behaviour, deficits in sustained
attention and increases in impulsivity and compulsivity
67,69,71–73
.
Together, these results suggest that HI contributes to the
development of characteristics related to ADHD. In addition to
alterations in impulsive behaviour, several groups also demon-
strated deficits in cognitive abilities either in recognition or
working memory or learning capability
67,69–71,74,75
. With regard to
symptoms associated with SZ, discrepant results have been
described. While unilateral HI in 10-day-old mice did not modulate
sensorimotor gating
72
, bilateral CCA ligation in 12-day-old rats
revealed a significant deficit in this behavioural domain
76
. The
variability of outcomes can be most likely explained by differences
in experimental models, age and species.
In addition to postnatal HI in term-born equivalent rodents a
prenatal hypoxic–ischaemic brain injury model has been recently
developed. Offspring of dams exposed to transient intrauterine
systemic HI have been shown to develop symptoms of ASD
revealed by poor social interaction
77
. Subplate neuronal loss and
impaired cortical maturation resulting in impaired thalamocortical
connections have been discussed as potential underlying
mechanisms
77
.
Hyperoxia
While HI causes an acute disruption of oxygen supply reflecting
asphyxia, preterm birth is frequently associated with exposure to
non-physiological high oxygen concentrations. This is caused by a
sudden rise in oxygen tension compared to intrauterine condi-
tions and is further increased by intensive care
78,79
. Although our
understanding of neuropathological hallmarks of hyperoxia-
triggered brain injury has increased within the past decade
80
,
involving inflammatory responses and induction of reactive
oxygen species associated with oligodendroglial cell death and
hypomyelination triggering ultrastructural changes of the devel-
oping white matter
81–94
, the evaluation of long-lasting functional
deficits has only recently begun
88,95,96
. Neonatal hyperoxia is
associated with reduced motor-skill learning and memory deficits
assessed in the complex running wheel, Barnes maze and novel
object recognition that persist into late adulthood
88,95,96
. These
findings resemble the difficulties observed in neurodevelopmental
follow-up studies in ex-preterm infants, emphasising the impor-
tance of experimental preclinical research.
Inflammation
Early perinatal immune activation is associated with long-lasting
consequences on cognitive and emotional health throughout life.
Clinical studies have linked early- and late-onset infections to
adverse outcome with development of cerebral palsy
97
.In
addition, pattern of inflammatory markers have been related to
neurological outcome in very immature infants as investigated up
to 10 years of age
98,99
. Different models of immune activation
have been established. The most common immunogens are the
viral mimetic polyinosinic–polycytidylic acid (poly I:C) and the
bacterial mimetic lipopolysaccharide (LPS). Poly I:C induces several
symptoms of ASD and SZ, such as impaired cerebral communica-
tion, abnormal social and repetitive behaviour, decreased
sensorimotor gating, working memory deficits and increased
anxiety
100–102
. These behavioural abnormalities are associated
with neuropathological alterations, e.g. not only reduced cortical
thickness and decreased volumes of hippocampus, amygdala and
striatum
103
but also ASD-associated aberrations in Purkinje cells
and altered levels of synaptic proteins, as well as deficits, in
synaptic transmission
102,104–106
. Furthermore, neurochemical
alterations involving serotonergic and dopaminergic signalling
have been linked to SZ and ASD pathology
101,107
. While LPS and
poly I:C induce a general rise in pro-inflammatory cytokines,
prenatal administration of selective cytokines such as interleukin-6
(IL-6) or IL-17 are sufficient to elicit ASD- and SZ-like beha-
viours
102,108
. Besides these artificial immune stimuli, models of
congenital viral infections have been used, such as cytomegalo-
virus infection leading to delayed acquisition of neurological
milestones (e.g. righting and grasping reflexes, cliff aversion and
negative geotaxis) and disturbances in social behaviour and
exploratory activity in adolescent animals
109
. Clinical signs were
accompanied by brain atrophy, cerebellar hypoplasia and
neuronal loss
109
. In spite of the huge number of available models
altogether underpinning the detrimental impact of perinatal
inflammation on long-term neurodevelopment, it needs to be
critically emphasised that most outcome measures focus on
distinct and selective behavioural domains, single brain regions
and/or single analysis time points in a single animal model.
Comprehensive systematic analyses identifying common neuro-
biological principles underlying distinct pathologies should be a
future goal in this research field.
In addition to immune stimuli, several different species have
been studied with various outcomes, i.e. while maternal treatment
with poly I:C in mice leads to long-lasting deficits in sensorimotor
gating in both male and female
102
, Wistar rats seem to be less
sensitive to maternal immune activation as sensorimotor deficits
were only observed in males
110
. To overcome limitations
regarding translation, non-human primate models of maternal
immune activation have been developed
111,112
also recapitulating
symptoms of neurodevelopmental disorders, e.g. abnormal
repetitive behaviour.
Timing of immune challenge seems to be critical not only for
foetal brain pathology
113
but also for long-term behavioural
outcomes. For instance, maternal immune activation with poly I:C
at late gestation is supposed to be more effective to induce
working memory deficits compared to early gestation
110,114
.
Similarly, maternal LPS injection at early gestation (i.e. embryonic
day 12 (E12)) leads to impaired reward seeking behaviour while at
late gestation (i.e. E16) impaired motor behaviour is induced
115
.
Comparative analysis in the poly I:C model further demonstrated
that prenatal immune activation at E9 but not at E17 reduces OF
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exploration, impairs the latent inhibition effect of associative
learning and alters sensorimotor gating
82,114,116,117
. On the other
hand, E17 but not E9 immune activation led to perseverative
behaviour
117
. Considering the delay in rodent brain development
compared to humans, with the first postnatal week in rodent brain
development corresponding to the third trimester in
humans
118,119
, several postnatal models of inflammation have
been developed. A single low-dose LPS injection at postnatal day
3 (PND3) or repetitive injections of IL-1beta over the first 5 PNDs
impairs recognition memory, which is accompanied by long-
lasting alterations of the white matter microstructure
120,121
.
Interestingly, locomotor and anxiety-related behaviour were not
modulated
120
. In addition to cognitive dysfunction, pain and
conditioned fear responses can be modulated by neonatal
inflammation as previously shown after immune challenge with
LPS at PND14
122
.
MULTIPLE HIT HYPOTHESIS
Epidemiological studies suggest that the probability to develop a
neuropsychiatric disorder does not only rely on a single risk factor
but also requires exposure to multiple “hits”. For instance maternal
immune activation is supposed to be a “disease primer”increasing
susceptibility to effects of genetic mutations and postnatal risk
factors that synergise to trigger mental diseases in later life
102,123
.
This is supported by clinical and experimental data, e.g. the effect
of maternal infection in increasing the risk for SZ is higher in
families with SZ history
101
. In preclinical models, it was shown that
low-dose poly I:C synergises with SZ- and ASD-related genes like
DISC1, NRG1, NR4A2 and TSC2 inducing increased ASD- and SZ-
related abnormalities compared to single insults
101,102,107
.
In addition to the combination with genetic variations, animal
models of prenatal inflammation have been combined with
intrauterine or early postnatal HI
124–127
. However, the majority of
studies mainly focussed either on analysis of acute inflammatory
responses and/or pathological alterations involving for example
myelination deficits
126,127
. Furthermore, correlations to beha-
vioural changes have rarely been assessed and if so these
analyses focussed on motor deficits that were similar in single
intrauterine-induced hypoxic–ischaemic injury and in the com-
bined setting
125
. Analysis of behaviours relevant to mental
disorders have only recently been explored in more subtle models
of white matter injury, e.g. by combination of foetal inflammation
with postnatal hypoxia not involving severe lesions or gross
anatomical changes. This combination led to long-term changes in
oligodendrocyte maturation, which was associated with increased
anxiety and signs of autism-like behaviour, i.e. reduced social play
behaviour and increased repetitive grooming
128
. Besides episodes
of hypoxia, exposure of preterm babies to high oxygen
concentrations is critical in terms of brain development (see
above). Combining an inflammatory stimulus (i.e. LPS) with
hyperoxia (80% oxygen) results in long-lasting impairment of
white matter microstructural integrity, which did not significantly
differ compared to single insults. However, underlying mechan-
isms were different in so far that hyperoxia but not LPS
predominantly induced apoptotic cell death while LPS rather
impaired oligodendrocyte maturation
82
. Even though these
pathophysiological processes are important to understand,
analyses of behaviours potentially relevant to cognitive and
neuropsychiatric disorders are lacking.
In addition to early prenatal and postnatal factors, events in
later in life may also increase the risk for neurodevelopmental
diseases. This was elegantly demonstrated in a translational
mouse model that combined exposure to prenatal immune
challenge and peripubertal stress resulting in synergistic effects on
adult behavioural alterations associated with neuropsychiatric
diseases
129
.
In spite of a whole body of clinical and experimental evidence
(with only a few selected here) confirming the multiple hit
hypothesis for the development of mental illness, the underlying
mechanisms remain obscure. Further research is necessary
combining diverse prenatal and postnatal risk factors to char-
acterise phenotypes and illuminate the cellular and molecular
pathways for each factor.
SEXUAL DIMORPHISM
Compelling clinical and preclinical evidence demonstrates a
pronounced sexual dimorphism across a huge variety of cognitive
deficits and neuropsychiatric disorders. This is supported by
experimental work since males display deficits in executive
function and attention while females presented increased
immobility in the forced swim test and reduced saccharin intake
after immune activation in lactating dams
130
. These findings
recapitulate the clinical situation where men have an increased
risk for SZ and women for depression
131,132
. Of note, hippocampal
and striatal volume reductions were similar in males and females.
However, the inflammatory response, i.e. pro-inflammatory
cytokine levels (i.e. IL-6, interferon-gamma) in the offsprings’brain
were increased in males supporting the concept of cytokine-
mediated perinatal programming of neuropsychiatric disorders.
Similar sex differences were observed in a model of intrauterine
inflammation induced by LPS injection at E15 demonstrating
increased cytokine responses in males
133
. However, behavioural
phenotypes were similar in that study contradicting the results by
Arad et al., which might be explained by different behaviour tests
applied but more likely by the different timing of perinatal
immune activation
133
. This is supported by a recent report where
postnatal LPS challenge between P5 and P7 resulted in differential
behavioural responses in males and females suggesting that the
sexual dimorphism in behavioural domains is more prominent
after late immune challenges
134
. Even though divergent beha-
vioural responses dependent on sex were confirmed, the kind of
behavioural deficits differed comparing studies of Custodio et al.
and Arad et al., i.e. depressive-like behaviour was observed in
female but not in male mice after immune challenge by lactating
dams
130
while direct immune challenge via intraperitoneal
injection to neonatal animals at a similar time point resulted in
the opposite with males but not females presenting depressive-
like behaviour
134
. These different outcomes suggest that not only
timing but also the mode of immune challenge may be decisive
for final outcome and sexual dimorphism in adulthood. On the
other hand, these differences might have been caused by
alteration of maternal behaviour after injection of LPS to the
lactating dam. Similar low levels of maternal care have been
described when immune activation occurs in mid-gestation
135,136
.
Besides long-lasting divergent outcomes in behaviour, emerging
evidence suggests that inflammatory responses also differ
between adult males and females. As such, males demonstrate
increased myeloperoxidase activity in the hippocampus at P35
and P70
134
. Furthermore, cytokine responses to adult LPS
challenge are increased in males at adulthood after intrauterine
immune challenge
133
.
In addition to perinatal immune challenges, neonatal HI induces
long-lasting immune responses, which differ between genders.
For instance, in adolescent male mice circulating levels of tumour
necrosis factor (TNF)alpha were increased and males revealed
more lymphocyte infiltration in injured brain hemispheres
accompanied by reduced neurogenesis
137
. Regarding HI-induced
atrophy and neurobehavioural deficits, gender differences were
observed after neonatal HI in P9 mice with reduced brain atrophy
in females while deficits in OF behaviour were increased, likely
representing injury not detectable by volume measurements
alone
138
.
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Sex differences have also been observed in models of maternal
obesity with female offspring of HFD-fed dams revealing increased
anxiety in the OF but males being more hyperactive
31
. Further-
more, latency in initial and reverse Barnes maze trials were
decreased in male but not in female offspring of HFD-fed dams
139
.
The most obvious mechanism underlying sexually dimorphic
outcomes seem to be sex hormones that is supported by
epidemiological data revealing a higher incidence and earlier
onset of SZ in males compared to females while incidence in
females increases with age and outnumbers that of males after
the age of 50 years
140
. The sex hormone hypothesis is in line with
numerous reports on the neuroprotective effects of oestro-
gen
84,141–143
. Major target mechanisms of sex hormones are
supposed to be regulation of neurotransmitter dysfunction and
synaptic plasticity
144,145
. Nevertheless, preclinical reports demon-
strating no sex differences in hormone levels but differences in
outcome regarding early and late inflammatory responses and
histological outcomes after neonatal HI
137,146
imply that additional
intrinsic mechanisms may play a role in divergent susceptibility to
the development of neurological deficits. This was demonstrated
in in vitro cultures of isolated cells or brain slices revealing
differences in cell death pathways and differentiation of specific
hippocampal neuron subpopulations independent of the pre-
sence of hormones
147,148
. In view of the huge amount of
epidemiological evidences for more than two decades, preclinical
in vivo research is now beginning to include stratified analyses for
sex across a huge variety of perinatal injury models.
MECHANISMS POTENTIALLY RELEVANT FOR THE
DEVELOPMENT OF NEURODEVELOPMENTAL DISORDERS
In spite of the well-known heterogeneity of cognitive and
neuropsychiatric symptoms, only some of them overlapping
between different diseases and despite the obvious diversity of
risk factors, several common molecular and cellular mechanisms
have been proposed to underlie the pathology of neuropsychiatric
disorders. Here we will focus on the most prominent molecular
and cellular components.
Cytokines
The major hypothesis how maternal immune activation impairs
neurodevelopment and potential consecutive development of
neuropsychiatric symptoms is an alteration of the balance
between pro- and anti-inflammatory cytokines. As such, blockade
of IL-6 and IL-17 during maternal immune activation via poly I:C
reverses behavioural abnormalities resembling ASD-like behaviour
while an increase of IL-10 is protective
101,108
. Whether maternal
increases in pro-inflammatory cytokines mediate direct effects on
the foetal brains or rather indirectly through induction of placental
inflammation leading to downstream effects such as oxidative
stress and nutrient deficiency is still not entirely clear
149,150
.Of
note, elevated cerebral cytokines levels may persist into adulthood
in a region- and age-specific manner
114,117,151,152
. Changes in
cerebral major histocompatibility complex-I expression and
associated intracellular mammalian target of rapamycin signalling
are proposed mechanisms underlying altered synaptic plasticity
and function associated with neuropsychiatric disorders
100
.
In addition to prenatal immune activation, hyperoxia also
generates inflammatory responses in the developing brain, as
demonstrated by a marked increase in mRNA and protein levels of
caspase-1 and its downstream effectors IL-1beta, IL-18, and IL-18
receptor alpha (IL-18Ralpha) in PND6 rodents exposed to
hyperoxia for 2–48 h
88,153
. Of note, both intraperitoneal injection
of a specific inhibitor of IL-18 and genetic deficiency of IL-1
receptor-associated kinase 4, which is pivotal in both IL-1beta and
IL-18 signal transduction, attenuated hyperoxic brain injury
153
.
Even though a direct correlation to cognitive development and
neuropsychiatric symptoms remains to be determined, these
findings causally link inflammation triggered by pro-inflammatory
cytokines such as IL-1beta and IL-18 to early hyperoxia-induced
cell death resulting in long-lasting white matter structural
abnormalities
86–89,95
.
Similarly to hyperoxia, maternal obesity and neonatal HI
increase cytokine levels in the offsprings’brain. This was
demonstrated in rodent and non-human primate models of
obesity
34,154,155
. Moreover, after neonatal hypoxic–ischaemic brain
injury in rodents, cerebral levels pro-inflammatory proteins (e.g. IL-
1beta and TNFalpha) were increased compared to sham-controls
while anti-inflammatory cytokines (e.g. IL-10) were decreased
4 days after injury that was associated with long-term alteration of
emotional and impulsive behaviour assessed in the EPM
71
.In
addition to short-term regulation of brain cytokines, recent work
also demonstrated a long-lasting increase in the pro-inflammatory
cytokine TNFalpha up to 30 days after injury at least in males
137
.
Taken together, these findings strongly suggest that cytokines
are a contributing mechanism underlying the pathology of
developmental neuropsychiatric disorders. Nevertheless, determi-
nation of molecular targets and dynamic changes of a larger
number of cytokines across brain regions, developmental stages
and animal models to identify immune signatures associated with
specific neuropsychiatric symptoms will be a major goal for future.
Microglia
Microglia as resident immune cells of the brain play an important
role during physiological brain development. Microglia functions
during development include not only phagocytosis of synaptic
elements and living and dying cells but also stimulation of
myelination, neurogenesis and cell survival
24
. The majority of
studies in early brain injury models propose a detrimental
activation of microglia mainly based on their amoeboid morphol-
ogy associated with increased pro-inflammatory cytokine release
similar as in adult neurodegenerative diseases. However, these
simple conclusions might be misleading because immature
microglia differ from adult microglia for example in gene
expression in response to LPS activation
156
. Microglia develop-
ment proceeds through three distinct stages—early, pre-adult and
adult—with characteristic gene expression and functional states.
Therefore, it is not surprising that perturbations of this matura-
tional process during early brain development modulate micro-
glia. For instance, maternal immune activation results in a
transcriptional shift of pre-microglia towards the more advanced
developmental stage
157
. Thus analysis by morphology alone
might provide limited information. Assessment of microglia
function by gene expression, phagocytic activity or altered
ultrastructure is suggested to be more accurate
24
.
A major mechanism potentially relevant for the pathophysiol-
ogy of developmental brain disorders is an alteration of microglia-
mediated synaptic pruning. This is supported by a recent report
using a model of prenatal inflammation by a single LPS stimulus at
E15 resulting in symptoms reminiscent of ASD (e.g. increased
stereotypic movements and reduced ultrasonic vocalisation,
reduced social preference)
158
. Of note, these long-term beha-
vioural alterations were accompanied by reduced hippocampal
expression of the fractalkine receptor CX3CR1 at P15
158
, the time
point and structure where effects would be most readily detected
for microglia-mediated synaptic pruning
159
. Even though
microglia-specific CX3CR1 deletion studies are still missing, a
causal relationship between CX3CR1 signalling and impaired
social behaviour was recently demonstrated by Zhan et al.
160
.
Deficient synaptic pruning due to CX3CR1 deletion (CX3CXR1
knockout mice) coincided with weak synaptic transmission and
altered functional connectivity in the prefrontal cortex leading to
deficits in social interaction and increased grooming behaviour
160
.
Interestingly, CX3CR1 mRNA expression is only reduced in males
after prenatal immune activation
158
suggesting that different
microglia responses to perinatal insults might be a major cause for
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observed sex differences in behavioural outcomes as described
above.
While the importance of CX3CR1 is largely acknowledged by
previous reports
159,160
, recent work in a model of postnatal
inflammation by repetitive IL-1beta injection very elegantly
identified a potential new molecular player previously not linked
to microglia function but rather considered as a marker of
neuronal postsynaptic density
161
. Using complex gene network
analysis of the microglial transcriptomic response to injury, it was
demonstrated that discs large homologue 4 (DLG4, aka. post-
synaptic density protein-95) is expressed in microglia of the
immature mouse and human brain, which was developmentally
regulated and modulated by inflammation
161
. Since DLG4 knock-
out mice reveal increased repetitive behaviour, abnormal com-
munication and social behaviour
162
, this might be a new
important mechanism how microglia contribute neurodevelop-
mental disorders after perinatal infection. Even though the
underlying mechanism of DLG4 action in microglia remains
unclear, its involvement in crosstalk between oligodendrocytes,
astrocytes and microglia or regulation of glutamatergic and
GABAergic signalling was suggested
161
. Nevertheless, cell-specific
deletion would be important to dissect the functional role of DLG4
on microglia for long-term neurodevelopmental outcome.
In addition to immune activation, early-life stress has also been
implicated in perturbation of microglia function in the hippo-
campus
163
. Furthermore, neonatal HI and hyperoxia are known to
result in increased abundance of the microglial cell marker Iba-
1
71,88,164
. In-depth analysis in experimental neonatal HI revealed a
change in microglia phenotypes even though no consistent
classification could be made regarding the classical M1 and M2
polarisation states
165
. Furthermore, the classical view of detri-
mental microglia activation following perinatal insults is chal-
lenged by the growing number of evidence for a protective role of
microglia under certain conditions, such as neonatal ischaemic
brain injury
25
. It was shown that depletion of microglia enhances
local inflammation induced by ischaemia–reperfusion and
increases injury severity in the developing brain
166
. Taken
together, the functional relevance of phenotypic and functional
microglia differences associated with perinatal noxious insults for
the development of cognitive and neuropsychiatric disorders in
later life remains to be further investigated.
Oligodendrocytes
The development of the mammalian brain involves complex
cellular processes, like migration, glial cell proliferation, axonal and
dendritic outgrowth, synaptogenesis and myelination of axons
167
.
Although neurogenesis is mostly completed around 24 weeks of
gestation, i.e. at the border of viability of preterm infants,
outgrowth and the formation of long-range connectivity sup-
ported by glia maturation and axonal myelination are still in
progress
168,169
. Development of diffuse white matter alterations
and a reduction of cortical and hippocampal grey matter volume
are often associated with cognitive impairment, attention deficit
disorder, behavioural problems, autism and development of
psychiatric disease in later life
170–173
.
Experimental work detecting long-lasting subtle alterations of
white matter integrity and brain connectivity following perinatal
insults like HI, hyperoxia, inflammation or IUGR
80,164,174,175
confirm
reports about white matter abnormalities in psychiatric
patients
176–178
. Oligodendrocyte development is characterised
by different developmental stages, ranging from oligodendrocyte
precursor cells to fully mature, myelinating oligodendrocytes.
Whereas mature oligodendrocyte seem to be less sensitive to
noxious stimuli, oligodendrocyte precursor cells and pre-
myelinating oligodendrocytes, which are the most abundant
oligodendrocyte populations around birth, are particularly vulner-
able
174
. Hypoxia or hyperoxia-induced imbalances of the cellular
redox system of developing, immature oligodendrocytes have
been suggested to cause cell death
84,179,180
while inflammation-
triggered perinatal brain injury rather leads to maturational arrest
of oligodendrocytes
82,121
. In spite of different underlying causes,
both insults result in disturbed structural white matter develop-
ment
82,86–89,120,121
.
While most of these findings remain at a descriptive level, a
causal link between minor myelination deficits and psychiatric
symptoms has recently been reported. Using mice heterozygous
for the oligodendroglial gene MBP, Poggi et al. demonstrated that
very subtle hypomyelination without gross lesions and/or striking
differences in basic and cognitive behaviour results in persistent
defects of PPI of the startle response, as a surrogate marker of
gating defects in SZ patients
181
. A similar association between
myelin deficits and behaviour was demonstrated in CNP
−/−
mice
that show signs of catatonia, a psychomotor syndrome observed
across neuropsychiatric diseases
182
. Interestingly, these mice have
increased inflammatory responses predominantly in white matter
tracts. Furthermore, signs of catatonia were reversed after
microglia depletion. These data demonstrate that altered myelin
gene expression and minor structural abnormalities of central
nervous system myelin trigger white matter inflammation as
underlying cause of catatonic signs
182
. Even though experimental
data are sparse in the field of neonatology, these basic findings
strongly support the concept that perinatal injurious stimuli
inducing subtle myelination deficits, e.g. by inflammation and/or
oxygen disturbances, contribute to long-lasting neuropsychiatric
consequences.
Epigenetics
Epigenetic mechanisms, i.e. the enzymatic regulation of transcrip-
tion activity and gene expression without altering DNA sequence
via acetylation, methylation, ubiquitination, phosphorylation and
sumoylation on histones, DNA or via microRNA-mediated regula-
tion of translation plays a pivotal role in normal and disturbed
brain development and may be associated with neurodevelop-
mental and/or neuropsychiatric diseases later in life
183–185
.
Interestingly, perinatal brain injury either induced by inflammatory
triggers or by stress has been linked to sustained epigenetic
alterations associated with adverse neurodevelopmental out-
come
18,150
. Furthermore, maternal HFD-associated alteration of
cognitive performance and anxiety-related behaviour were
associated with increased brain-derived neurotrophic factor and
Grin2b methylation and increased expression of DNA methyl-
transferases
186
. Whereas the latter reviews and reports rather
focus on prenatal insults, several studies have also provided a
direct link between early postnatal insults, epigenetic modifica-
tions and neuropsychiatric outcome. For instance, early-life stress-
induced cognitive impairment and anxiety is attenuated by
silencing of miRNA124a in the hippocampus of adult male rats
187
.
Furthermore, maternal separation was associated with persistent
dysregulation of histone modifiers in the medial prefrontal cortex,
one of the key structures regulating stress responses and mood-
related behaviour
188
. For perinatal brain injury induced by
postnatal inflammation, alterations in miRNA expression and
histone acetylation have been described, which potentially
contribute to disturbed oligodendrocyte maturation
121,189,190
even
though this hypothesis needs to be proven in cell- and region-
specific analyses.
CHALLENGES FOR PRECLINICAL RESEARCH
Clinical and epidemiological studies are limited to establish a
causal relationship between perinatal insults and the develop-
ment of neuropsychiatric disorders. This may be attributed to
impossible randomisation of handling or behaviour. Humans are
ecologically and behaviourally heterogeneous limiting the asso-
ciative power and thus necessitating large studies. Furthermore,
symptoms often do not appear for many years after birth. The
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multiple hit scenario further impedes clear definitions of causal
relationships. Therefore, animal models are essential to identify
target mechanisms and therapeutics.
Challenges for experimental modelling and data analysis
Technical difficulties emerge from currently used animal models of
perinatal brain injury that often induce severe cerebral lesions and
associated motor deficits that may confound interpretation of
results from behavioural analyses assessing emotional, social and
cognitive tasks. Furthermore, owing to improvements in obstetric
and neonatal care the number of infants suffering from rather
subtle and late-onset cognitive and socio-emotional impairments
in later life requires the development of additional more subtle
injury models than the currently used severe injury models with
high mortality and morbidity. First attempts have been made by
combining prenatal immune challenge with postnatal hypoxia
only, instead of combination with the more severe model of HI
128
.
Nevertheless, even with refinement of experimental models,
which are indispensable to increase our abilities for the design of
therapies, most likely no single animal model can reflect all
neuropathological alterations and behavioural deficits of a
complex human disorder. For instance, while maternal immune
activation has been shown to induce behavioural abnormalities
resembling symptoms of ASD and SZ
100–102
, neuropathological
alterations do not always parallel observations in clinical studies.
As such, while cortical thickness is reduced in the rat model of
maternal immune activation, clinical studies demonstrated wide-
spread increased cortical thickness in ASD patients
191
. Another
example is neonatal encephalopathy related to HI that is modelled
by induction of unilateral brain damage and thus is unlikely to
fully model the global brain injury seen in humans. Nevertheless,
work in this model has shown suppression of electroencephalo-
gram power after unilateral HI, as is often seen in infants with
HIE
192
. Thus, despite the unilateral nature of the injury, the HI
model replicates at least some important aspects of the
encephalopathy observed clinically. With regard to preterm-
birth-related complications caused by high oxygen concentra-
tions, the hyperoxia model has been developed. It is a postnatal
model where experimental animals do not suffer from lung injury
when exposed for 24–48 h to high oxygen concentrations in
contrast to preterm infants. It also lacks the clinical fluctuation of
oxygen concentrations observed in clinical practice of neonatal
care
85,193
. However, it represents a very standardised and
reproducible type of injury that can be combined with additional
noxious stimuli, i.e. an inflammatory challenge can be easily added
to closer mimic the clinical situation
82
.
In addition to these difficulties, one has to keep in mind for all
models with maternal targeting that this involves whole-litter
manipulations where littermates share similar in utero and
postnatal environments, which can produce considerable litter
effects. Therefore, in experimental designs with maternal target-
ing, littermates may not be treated as fully independent in
statistical analyses that would artificially increase sample size. It is
rather suggested to randomly select only one subject from each
litter and to allocate animals of one litter to different outcome
measures to avoid ethical issues of wasting animals
194
.An
alternative would be the use of appropriate statistical models as
previously described
195
. Potential effects by altered maternal care
and/or pain due to injections/interventions can also be con-
founders in studies with maternal targeting.
Challenges for assessment of neuropsychiatric symptoms in
rodents
Symptoms like hallucination, delusions and major thought
disorders are difficult to measure in primates and impossible to
assess in small animals, such as rodents. Therefore, the aim should
not be to fully mimic the entire syndrome but rather focus on
distinct behavioural physiological and neuroanatomical
phenotypes. Nevertheless, multi-task approaches are needed to
model multi-symptomatic diseases and several studies have
shown that neuropsychopathological deficits and disorders can
be reliably modelled in animals with the help of a battery of
neurological and neuropsychological tests screening behavioural
abnormalities in rodents
196,197
. This chapter briefly presents and
describes paradigms that are commonly used in rodents to screen
for behavioural deficits frequently associated with certain
neuropsychiatric disorders or after experimentally induced devel-
opmental manipulations
198,199
. However, rodent behaviours have
limitations when compared to the complexity of human
behaviour. Thus any tests used for the assessment of behavioural
patterns in general are crucial to provide established face validity,
construct validity and predictive validity
200
.
Anxiety-related behaviours. The most commonly used uncondi-
tioned tests of anxiety comprise the OF test, the EPM, and the
light/dark test (LD). All of them reveal the conflict between
avoidance behaviour towards potentially dangerous spaces and
the motivation to explore novel, unfamiliar areas
201
.
Elevated plus maze: One prominent paradigm assessing anxiety-
related behaviour is the EPM test. The apparatus consists of a
centre platform with four branching arms, two open and two
opposing closed arms. By placing the animal on the centre
platform always facing an open arm, behaviour is assessed over a
certain time. The dependent measures are usually the number of
open arm entries, time spent in open/closed arms, the distance
covered on the open/closed arms and head dips (the frequency of
the animal protruding its head over the ledge of an open arm and
down towards the floor). An increase in anxiety-like behaviour is
characterised by reduced number of entries into the open arms
and the increased time spent on the closed arms of the maze
compared to controls
202–205
. Likewise, the reduction in head dips
is suggested to reflect deteriorated risk assessment-related
behaviour
206
and thus also supports the assumption that
anxiety-like behaviour is apparent.
Rodents’sickness behaviour is in part reflected by reduced
exploration and motor activity
207–212
. Thus observed changes in
EPM performance may be simply explainable due to impaired
locomotor activity induced by experimental interventions. Such an
assumption can, however, be ruled out easily by quantifying the
total distance covered on the maze between experimental groups.
Second, analysing the number of entries into the closed arms is
also considered as good indicator of locomotion rather than
anxiety
213
.
Open field: A simple rectangular acrylic box with black walls and
a frosted floor with infrared backlighting can be used as an OF
arena. For testing, animals are placed in the centre of the dimly lit
OF arena
214
and movements are assessed by a video tracking
system over 5, 10, 20 or more minutes. The dependent measures
are usually general motor activity, that is covered distance and
velocity, as well as straighten up-postures for vertical activity.
Anxiety-like behaviour in this test is reflected as less time that is
spent in the centre of the arena and vice versa more time spent at
the borders of the arena. Like in the EPM, quantifying the total
distance covered in this test may check simple effects on
locomotor activity.
LD test: Another simple but meaningful setting to investigate
decreased exploratory behaviour as an index for anxiogenic
effects in laboratory rodents is provided by the LD. This plexiglas-
made arena consists of two compartments, while two-thirds of the
arena is white coloured and illuminated, one-third was black
coloured and darkened. The two compartments are separated by
a black partition equipped with photocells across the opening, as
well as in the overall arena, to measure locomotor activity. Animals
Adverse neuropsychiatric development following perinatal brain injury:. . .
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Pediatric Research (2019) 85:198 – 215

are placed in the centre of the bright chamber facing the
separating wall. The total numbers of transitions between the two
compartments and total locomotor activity, as well as time spent
in the bright side are recorded
215
. In this test which is based on
rodents innate aversion to brightly illuminated areas
216
, less time
exploring the lit compartment (risk assessments) and low latency
to enter the lit compartment are usually interpreted as increased
levels of anxiety
217
.
While the aforementioned paradigms are frequently used to
assess anxiety in rodents, there are drawbacks that need to be
taken into consideration. First of all, the ideal animal model of
anxiety does not exist, and the tests available (EPM, OF and the
LD) are characterised by their originality. Second, using solely one
task to measure anxiety-like effects is biased, since rodent
behaviours have limitations when compared to the complexity
of human behaviour
200
. Thus, to gain broader understanding
about underlying mechanisms and to increase validity of data,
multiple behavioural tests should be used to characterise the
anxiogenic/anxiolytic impact of experimentally induced interven-
tions
218,219
, which also improve translation from animals to
humans. Moreover, it is proposed that short-term, intra-
individual variations in emotionality probably constitute an
important factor for investigating anxiety-related behaviour that
may differ between tests
220
.
Deficits in sensorimotor gating, break point and stereotypic
behaviour
Sensorimotor gating: The startle response is a fast twitch of eye-
lid-closure, facial, neck and skeletal muscles evoked by a sudden
and intense tactile, visual or acoustic stimulus
221,222
. This
characteristic response pattern, best described as ASR, is observed
in a variety of species such as rodents and humans and is thought
to be a protective function against injury from a predator or harm
and of the preparation of a flight-or-fight response
223
. However,
when a distinctive non-startling stimulus (prepulse) is presented
30–500 ms prior to the actual startling stimulus, the response
magnitude is naturally reduced. This mechanism of inhibited
contemporaneous sensory or motor events that would interfere
with the ongoing processing of the prepulse reflects a funda-
mental principle of the neuronal control of behaviour, which in
turn is necessary for stimulus recognition and sequential
organisation of the appropriate behaviour
224
. This phenomenon
also known as PPI is considered to be a pre-attentive filter
mechanism, reflecting the ability of an organism to gate out
irrelevant sensory information
221,222,225–227
. PPI can be measured
with almost identical procedures in humans (eye blink reflex) and
rodents (startle chamber)
228,229
. Since PPI is disrupted in schizo-
phrenic patients, animal models of sensorimotor gating may offer
the possibility to investigate the neural mechanisms related to
some characteristical impairment in SZ
230
. While lesioning the
medial prefrontal cortex
198
or the ventral thalamus
231
in adults are
known to induce deficits in sensorimotor gating, neonatal lesions
of the entorhinal cortex did not impact PPI measured during
adulthood
232
. However, multiple studies involving different forms
of maternal immune activation demonstrated reduced PPI
responses
105,108,117
.
Break point: The so-called “break point”or progressive ratio (PR)
test is an operant behaviour task that measures the instrumental
effort a rat is willing to invest in order to obtain a reward
233,234
.In
this paradigm, animals are initially trained to press a lever in order
to obtain a reward. At the level of stable performance, the number
of lever presses required for the reward is progressively
increased
235
, and at a certain point of instrumental effort, animals
cease to “work”for the reward and stop responding. The “break
point”is considered as operational measure for a shift in
motivation, indicating that the value of the reward is lower than
the effort the animal is willing to invest
236
. PR schedules have
been shown to provide a valuable method to measure the impact
of experimental manipulations that might affect the perceived
reinforcement value of gustatory stimuli
233
. Moreover, it was
assumed that a reduced break point and hence a lower
performance in the PR- test might serve as an animal model for
anhedonia, a core negative symptom in SZ
236
. When adult rats
were tested for motivation in the PR task following neonatal
lesions of the entorhinal cortex, a reduced break point in operant
responding was observed. These findings indicate that early-life
brain injury reduced motivation during operant behaviour
reflecting a state of anhedonia as also observed in SZ
232
.
Stereotypy: Stereotypy is not only a fundamental feature of the
behavioural syndrome induced by psychomotor stimulant drugs
but also a cardinal feature of a broad range of neuropsychiatric
disorders
237–240
. Behaviours of patients that typify stereotypy in
clinical disorders range from repetitions of single or multiple
movements (motor stereotypies) to repetitive, inflexible patterns
of attention, emotion, planning and cognition
241
. Most commonly,
video recordings of animals for a certain amount of time are
evaluated for specific patterns, such as focussed sniffing, repetitive
head and limb movements and oral movements (chewing, licking
and biting)
242,243
.
Cognitive deficits
Morris water maze: Neurodevelopmental disorders due to
perinatal insults are often associated with cognitive deficits
later in life
244
. A prominent task, which assesses spatial learning
in rodents is the Morris water maze test
245
.Thistestisbased
upon the premise that animals have evolved an optimal strategy
to explore their environment and escape from an unpleasant
environment (the water tank) with a minimum amount of
effort (swimming the shortest distance possible). The time the
animals need to discover a hidden platform in the tank after
previous exposure to the set-up with only external proximal and
distal cues available is considered as an index for spatial
memory
246,247
.
Barnes maze: Similarly, but not as aversive, however, is the
Barnes maze task, which also assesses spatio-temporal memory
248
.
Here animals are placed in the centre of a brightly illuminated
round board equipped with 20 holes at the border. Animals are
required to escape from the very unpleasant environment by
seeking an escape box that is attached to one of the 20 holes at
the border region. After training, the latency to find the escape
box is assessed after a couple of training days reflecting the
memory performance
249
.
Radial maze: Spatial memory abilities may also be assessed in
an eight-arm radial maze using a reinforced alternation task. The
maze consists of an octagonal central platform 48 cm in
diameter connected to eight equally spaced arms projecting
radially from the central platform with adjacent arms separated
by 45°. At the distal end of each arm, food cups are located (2.5
cm high ×4 cm in diameter) that prevents the rodent from
viewing the food reward. The test, usually in an experimental
room equipped with several extra-maze visual cues, is initiated
by placing the rodent into the centre of the maze with
orientation varying from trial to trial towards distinct arms
232
.
Four arms (alternating) of the maze are always baited with food
rewards.Theanimalsareplaced in the centre of the maze, and
arm choices are recorded when an animal collects the reward or
reaches the end of an arm. Entering an arm that had never been
baited reflects a reference memory error, while a re-entry into an
arm that was baited indicates a working memory error
250
.Since
a re-entry into a never-baited arm could be related to either
process of working or reference memory, it is scored as a
“perseveration error”
251
.
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Novel object recognition: Another method to test cognitive
functioning in rodents is provided by the novel object recognition
test
252–254
. To verify that performance is less dependent on spatial
information, an actual modified version of the task is performed in
a Y-maze. Here no external cues are visible for the animal from
inside the maze. Initially, three identical objects are placed at the
end of the maze arms for familiarisation purposes. On testing days,
one familiar object is replaced by a novel, differently shaped
object, which is consistent in height material. As most reliable
parameter for assessing recognition memory is the time spent in
the distal half-arms during the first 2 min of the test. In healthy
subjects, this should indeed be longer for the arm with the
unfamiliar object
255
.
Social behaviour
Social interaction: In humans, early prefrontal–cortical damage
has been shown to impair cooperative behaviour and social
interaction, as well as social cognition
256,257
. The so-called
“unsocial”behaviour is also a characteristic outcome of neurode-
velopmental psychiatric disorders, such as SZ and autism. Hence,
social withdrawal and social isolation are most commonly seen as
negative symptoms in SZ, while the core symptoms of autism
comprise specific impairments of reciprocal social relationships,
affected cooperative play with peers and atypical social
behaviour
258,259
.
Impairment of social behaviour in rodents can be measured
using the social interaction test. This paradigm is based on the fact
that active interaction reflects motivation to interact and does not
necessarily reflect the behaviour of both animals, while passive
interaction depends on the pair. Moreover, the evaluation of
active and passive interaction separately and for each animal of
the pair allow the investigation of social behaviours between
animals of different strains or drug treatments, increasing the
repertoire of information originated from this task
260–262
. Briefly,
pairs of unfamiliar rats or mice are simultaneously but placed
apart in an unfamiliar arena (e.g. OF). Over a certain time period,
the time spent in active (sniffing and following) or passive
behaviour (animals lie next to each other in distance) are
scored
263
. Based on its predictive and face validities, a decrease
in social interaction has been described in several animal models
of SZ as a behavioural parameter that mirrors the negative
symptoms of this disease
199,264–267
but need to be evaluated
following subtle perinatal insults in the future.
Three chamber sociability test: A different method to analyse
sociability in rodents is provided by the three chamber sociability
test. Here the animal is placed in a roofless apparatus comprising
three compartments, a small centre compartment and two equally
sized end compartments. During testing, the subject is placed in
the middle compartment. One end compartment hosts a strange,
unfamiliar rodent in an inverted grid cup, while the other end
compartment contains an identical but only an empty grid cup,
sometimes with an object
268,269
. Restraining the unfamiliar rodent
in a grid cup limits the mobility of this animal but still allows visual,
olfactory and tactile contact to the testing subject. Moreover,
possible aggressive responses between test and stranger animal
are prevented
270
. However, rodents normally prefer to spend
more time with an unfamiliar counterpart. Thus valid measures for
sociability in this test are the amount of time the test subject
spends in each compartment and the amount of exploration time
(sniffing), as well as the transitions between the compartments.
Besides sociability, social novelty may be assessed in a subsequent
test setting
271
. Here a second stranger animal is placed in the
former empty grid cup. In this phase, the test subject may choose
whether to explore the rodent that was already present during the
sociability phase (now a familiar animal) or the newly introduced
one. Measures for social novelty during this testing phase are the
same ones as described above.
Impulsive behaviour. Although not yet experimentally assessed
following insults to the developing brain, high levels of impulsive
behaviour are evident in a number of psychiatric disorders, such
as obsessive–compulsive disorder, ADHD, SZ, antisocial and
addictive behaviour
272,273
. Owing to the range of behaviours that
the term impulsivity encompasses, it is proposed that impulsivity
not as a unitary construct but rather as a set of diverse and
complex phenomena that may have independent underlying
biological mechanisms
274,275
. Common aspects of impulsivity
comprise decreased inhibitory control, intolerance of delay to
rewards and quick decision making due to lack of consideration,
as well as poor attention ability and hyperactivity
276,277
. Despite
this very broad range of symptomatology, it is possible to devise
different paradigms to measure the various forms of impulsive
behaviour in both humans and laboratory animals
278–280
.In
general, impulsivity includes two major categories: behaviour that
results from diminished ability to inhibit actions, often referred to
as impulsive action, and behaviour that reflects impulsive decision
making, for example intolerance to delay of gratification also
known as delay aversion
277,281
.
Impulsive action (inhibitory control): Impulse control is often
described as active inhibitory mechanism, modulating internally
and externally driven pre-potent desires for primary reinforcers,
such as food, sex or other highly desirable rewards. This inhibitory
control mechanism may provide lower cognitive mechanisms to
guide behaviour, while rapid conditioned responses and reflexes
are transiently suppressed
277,282
. The most popular clinical
measure of sustained attention, vigilance and response inhibition
in humans is the continuous performance task
281,283
. The
preclinical analogue of the continuous performance task designed
for rodents is the five-choice serial reaction time task, an operant-
based test originally developed to measure visuo-spatial attention.
In this 30–40 min task, animals are required to be attentive and
withhold responding (nose poke) while monitoring five apertures
for brief light stimuli (e.g. ≤1 s) presented randomly therein
284,285
.
Subsequently, at the beginning of a trial and prior to presentation
of a light stimulus, there is a 5-s inter-trial interval during which
the animals have to withhold a response at all. Any responses
made during this time are described as premature responses. Low
levels of premature responses require the ability to inhibit actions,
whereas high levels are seen as a measure of motoric impulsivity
reflected by disturbances in the inhibition of behaviour
279,284
.
Impulsive decision-making (delay aversion): Owing to the fact
that impulsive patients are not able to take time to carry out
appropriate evaluations of incoming information in order to
choose behavioural responses on a detailed analysis of a given
situation
286,287
, the inability to tolerate delays of gratification or
reward is also an important aspect of impulsive behaviour
288–290
.
A well-established paradigm for testing sensitivity to delays of
reward in laboratory animals is the delay-based decision-making
task, carried out in random operant chambers with two retractable
levers and a food pellet dispenser. Here the ability to wait for a
food reward is taken as an operational measure of impulsive-like
behaviour
275,291
. More specific, rats are given the choice between
a small (one food pellet) and a large reinforcer (five food pellets).
The programmed delay associated with the large reinforcer is
increased stepwise from 0 to 60 s during the session. Trained rats
begin each session by choosing the lever providing the larger
reinforcer but then switch the preference to the smaller one as the
delay increases. This switch from the large to the small reinforcer
indicates an increase in impulsive choice
275,292
.
CONCLUSION
Owing to the increasing amount of clinical neurodevelopmental
follow-up studies in large cohorts confirming the increased risk
Adverse neuropsychiatric development following perinatal brain injury:. . .
I Bendix et al.
209
Pediatric Research (2019) 85:198 – 215

for cognitive, neurobehavioural and neuropsychiatric problems
following perinatal insults, the focus of translational experi-
mental research has shifted from the sole analysis of the
underlying pathophysiology to assessment of long-lasting
neurobehavioural deficits reminiscent to typical neurodevelop-
mental disorders. However, several hurdles remain. While the
multiple hit hypothesis is increasingly implemented in experi-
mental research, underlying cellular and molecular mechanisms
of each factor and potential synergistic actions remain largely
unclear. Furthermore, stratified analyses for sex across a huge
variety of perinatal injury models needs to be included in future
studies. Major evidence for a direct link between perinatal
insults and long-term neurodevelopmental disorders is derived
from prenatally or postnatally induced inflammation while the
impact of prenatal stress, IUGR and oxygen imbalances on long-
lastingneuropsychologicalsymptomsislesswellexplored.
Moreover, most outcome measures focus on distinct and
selective behavioural domains, single brain regions and/or
single analysis time points in a single animal model. Thus
comprehensive systematic analyses including multi-task beha-
vioural tests and the identification of common neurobiological
principles underlying distinct pathologies should be a future
goal in this research field. This will need multidisciplinary
experimental approaches combining knowledge from the field
of basic neurobiology, neonatology, paediatrics, psychology and
psychiatry to explore the full spectrum of perinatal program-
ming of neurodevelopmental disorders.
ADDITIONAL INFORMATION
Competing interests: The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations.
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