Fetal Programming of Brain Development - Role of

Intrauterine Stress and Stress Biology in

Susceptibility for Psychopathology
Claudia Buss, PhD, Sonja Entringer, PhD, & Pathik D Wadhwa, MD,
PhD.
UC Irvine Development, Health and Disease Research Program
Departments of Pediatrics, Psychiatry & Human Behavior, Obstetrics & Gynecology, and
Epidemiology
University of California, Irvine, School of Medicine

Responseand
NICHe Conference on ‘Stress Response and Child
ChildHealth’
Health’ ASWIN
May 18-20, 2012, Heraklio, Crete, Greece

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 Outline

 Conceptual framework- Fetal programming of

brain development
 Issues and considerations
 brain development
Empirical evidenceinfor
humans
developmental programming
of
 Future directions

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Conceptual Framework:Traditional view

Psychopathology/
Developmental Disorders

• Autism
• Brainin
Alterations Anatomy
the brain
Connectivity • Schizophrenia
• Structural and Functional • ADHD
Genotype / Risk Alleles
• Depression
• Anxiety Disorders
• Dementia

Vulnerability Hypothesis

It is unlikely that 30,000 genes can determine 240 trillion

synapses of the cerebral cortex.

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 Fetal Programming of Brain Development

Psychopathology/
Developmental Disorders
Prenatal Perturbations Alterations in the brain
• Autism
• Maternal Stress • Brain Anatomy
• Structural and Functional • ADHD
• Glucocorticoids
Connectivity • Schizophrenia
• Exogenous CRH
• Depression
• Infection/ Inflammation
• Anxiety Disorders
• Prenatal Drug Exposure
Maternal and Fetal Genotype • Dementia

Vulnerability Hypothesis

Vulnerability= f [Genes x Early Environment]

“Brain development is guided by genes but sculpted by the
environment.” (Lenroot et al. 2008)
The concept of programming describes the process by which the early
environment interacts with genetic and other factors to produce an
individual human constitution.
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 Issues and questions
1) Which organ systems are particularly amenable to
environmental influences during development?
2) During which periods of development is the influence
of environmental conditions particularly pronounced?
3) Which particular environmental conditions exert the largest
influence on development (or, represent the most potent
programming cues)?
4) How, or through which pathways, do stress-related
environmental conditions influence brain development?
5) What are the approaches to study fetal programming of brain
development?

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 Issue # 1.Which organ systems are particularly
amenable to environmental influences during
development?

Obstet d
ri
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 Issue # 2. During which periods of development is the
influence of environmental conditions particularly
pronounced?

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Issue # 3.Which particular environmental conditions exert the
largest influence on development (or, represent the most potent
programming cues)?

Rationale for the study of the effects of prenatal stress

 Concepts in evolutionary biology, developmental plasticity and life
history theory
 development – plastic process – range of possible phenotypes from same genotype
 unfolding of developmental process from genotype to phenotype – CONTEXT DEPENDENT – series
of conditional probabilities (interactions) over time
 Key environmental conditions that shape evolutionary selection and developmental
plasticity – variation in energy substrate (NUTRITION) and challenges that have the
potential to impact structural or functional integrity and survival till reproductive age
(STRESS)

Hypothesis: Prenatal stress would be expected to

influence many, if not all, aspects of
developmental outcomes.

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 Issue # 4. How, or through which pathways, do stress-related
environmental conditions influence brain development?
Early Environment
• Stress
• Obesity
Endocrine: Cortisol, CRH Mother • Undernutrition
Immune: Il-6,TNF-a Placenta • Obstetric
complications (e.g.
Maternal and Fetal Genotype

Fetus infection)

• Transcription factors
• Neurotrophic factors
• Neurotransmitters
• Growth hormones
• Thyroid hormones

Brain Development
Proliferation, Migration
Differentiation
Neurogenesis Cognitive
and Affective
Growth of axons and dendrites Processes
Synapse formation Mental Health Apoptosis / Pruning
Myelination Program Studi Obstetri Ginekologi Peminatan Kedokteran Fetomaternal Program
d
Issue # 4 (continued).What are the key signaling pathways by which
stress- related endocrine and immune/inflammatory pathways
influence brain development?
Key signaling pathways in brain development:
• Mammalian Target of Rapamycin (mTOR) signaling
• serine/threonine protein kinase that integrates input from upstream pathways and
regulates cell growth and survival, protein synthesis and transcription.
• Wnt signaling
• Wnt proteins are mophogens involved in cell fate decisions and tissue patterning by
regulating cell to cell interactions.
• Sonic Hedgehog (Shh) signaling
• Shh proteins play crucial role in dorso-ventral patterning of cell type
differentiation.
• Reelin signaling
• extracellular matrix glycoprotein involved in regulating processes of
neuronal migration and positioning in the developing brain.

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Issue # 4 (continued).What are the key signaling pathways by
which stress-related endocrine and immune/inflammatory
pathways influence brain development? Direct and indirect
effects.

Biological stress mediators may influence key signaling pathways…

1) Directly
2) Indirectly via altering availability of
• Neurotrophic factors
• Neurotransmitters
• Growth hormones
• Thyroid hormones

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Issue # 4 (continued).What are the key signaling pathways by
which stress-related endocrine and immune/inflammatory
pathways influence brain development? Glucocorticoids and
mTOR,Wnt, Shh and Reelin signaling.

High levels of glucocorticoids can change the brain developmental

trajectory by altering key signaling pathways:
• Mammalian Target of Rapamycin (mTOR) signaling
• Wnt signaling
• Sonic Hedgehog signaling
• Reelin signaling

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Issue # 4 (continued).What are the key signaling pathways by
which stress-related endocrine and immune/inflammatory
pathways influence brain development? Actions on neurotrophic
factors.

Neurotrophic Factor signaling Immune: Il-6,TNF-a Placenta

Endocrine: Cortisol, CRH Mother

Fetus
mitogen-activated (MAP) kinase cascade • Neurotrophic
• promotion of neuronal differentiation via
• promotion of neuronal survival or growth via • Neurotransmitters
• Transcription factors

Ras or GAB1 Brain Development

factors

• Growth hormones
• Thyroid hormones
Differentiation

Neurogenesis
• promotion of synaptic plasticity via activation of Proliferation, Migration
Synapse formation

phopholipase (PL)-C-y1 activating CA2+ and Myelination

Growth of axons and dendrites

protein kinase Apoptosis / Pruning

 High levels of glucocorticoids can alter the availibility of

neurotrophic factors and may interfere with these pathways with
consequences for neuronal survival, growth and synaptic plasticity.
Obstet d
ri
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Issue # 4 (continued).What are the key signaling pathways by
which stress-related endocrine and immune/inflammatory
pathways influence brain development? Actions on 5-HT.

Immune: Il-6,TNF-a
Endocrine: Cortisol, CRH Placenta
Mother
5-HT signaling
• impact on cell migration by altering reelin Fetus
• Neurotrophic factors

signaling • Transcription factors

• Growth hormones

and subsequent opening of cAMP-gated sodium

• impact on axon growth via increases in cAMP • Neurotransmitters

• Thyroid hormones
Proliferation, Migration

• impact on synaptogenesis via activation of the Neurogenesis

small Rho- family GTPase Cdc42 by

channels Brain Development Synapse formation

phospholipase C (PLC) and phosphoinositide 3- Differentiation

Myelination

Growth of axons and dendrites

Apoptosis / Pruning

kinase (PI3 kinase) pathways

Reduced serotonin availability in association with prenatal endocrine and

immune stress may impair fundamental brain developmental processing like
cell migration, axon development and
Program synaptogenesis.
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 Issue # 4 (continued).What are the key signaling pathways by
which
stress-related endocrine and immune/inflammatory pathways
influence brain development? Actions on growth hormones.
Immune: Il-6,TNF-a Placenta

Endocrine: Cortisol, CRH Mother

Growth hormone signaling •Fetus

Neurotrophic factors

• Growth hormones
• axon development
• antiapoptotic via activation
effects of MAPK)
via PI3K/Akt or signaling • Transcription factors

pathway 3
MAPK/Erk
• Neurotransmitters

Proliferation, Migration • Thyroid hormones

• cell proliferation via mitogen-activated Neurogenesis

protein(MAPK) signaling pathway 2

kinase Brain Development
Synapse formation
signaling pathway 3
Differentiation

Growth of axons and dendrites

Apoptosis / Pruning
Myelination

Inhibition of growth hormones by cortisol, Il-6 and TNF-alpha may

interfere with these pathways with consequences for cell proliferation and
survival and axon development.
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Issue # 4 (continued).What are the key signaling pathways by
which stress-related endocrine and immune/inflammatory
pathways influence brain development? Actions on thyroid
hormones.

Immune: Il-6,TNF-a Placenta

Thyroid hormone (TH) signaling
Endocrine: Cortisol, CRH Mother

• cell migration via reelin and downstream signaling Fetus

• Neurotrophic factors

• Growth hormones
cascade • Transcription factors • Thyroid hormones

• differentiation of embryonic neural stem cells • Neurotransmitters

Proliferation, Migration
(promotion
through TRa1of neural proliferation and inhibition of Neurogenesis
astrocyte differentiation)
• cell proliferation by inhibiting via
and differentiation STAT3 signaling Brain Development
Synapse formation
Differentiation

sonic hedgehog signaling Myelination

Growth of axons and dendrites

Apoptosis / Pruning

 Inhibition of thyroid hormones by cortisol, Il-6 and TNF-alpha

may interfere with these pathways with consequences for neuronal
migration, proliferation and differentiation.

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Issue # 5.What are the approaches to study fetal programming of
brain development?

Species Differences in Brain Anatomy and Developmental Programming

of the
Brain
 The complexity of brain anatomy and connectivity largely differs by
species.
 Due to the accelerated trajectory of brain development in smaller species, the
impact the environment has on sculpting the brain differs by species.

 Animal models are necessary to study molecular mechanisms that cannot

be studied in humans but testing hypotheses in the human context is
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 Issue # 5.What are the approaches to study fetal programming of
brain development?

Brain Structure Brain Function

Brain Anatomy Behavior
Structural Connectivity Neurocognitive Performance

Functional Connectivity Psychiatric Symptoms

Considerations:
Temporal Sequence
Importance of prospective longitudinal brain imaging
studies with serial assessments
Limitations of MRI-based methods
 Importance of accompanying mechanistic studies in
animal models
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 Empirical evidence for fetal programming
of human brain development

Programming Cues
I. Cortisol as a biological ligand
II. Obesity as a clinical condition associated with
high inflammation

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Study Overview

PC Gestation Birth Brain Development

Infancy Childho od P Adulthood

Prospective longitudinal study with up to five assessments during pregnancy and


neurocognitive assessments of their children
 Cognitive assessments
 Brain MRI

15, 19, 25, 31 and 37 weeks gestation

Neuroendocrine & Obstetric Child:
assessments
psychosocial complications
Birth outcomes MRI
Neurocognitive &

Covariates: obstetric complications, gestational age at birth, birth weight, maternal

postnatal depression and IQ, race/ ethnicity, age and sex of the child

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Study I. Maternal cortisol and child brain development

 Cortisol is known to exert a wide array of metabolic, endocrine and immune

effects on most if not all cells and is known to play a key role in events
underlying the development of the brain.
 Cortisol is one of the primary biomarkers of the physiologically-stressed state
because its production, bioavailability and activity is altered by all adverse
conditions that have been shown to program the developing brain.
 While glucocorticoids play an essential role in normal brain development,
abnormal or inappropriate levels particularly during sensitive periods may induce
neurotoxicity with detrimental long-term consequences.

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PC Gestation Birth Brain Development
Infancy Childhood P Adulthood

Independent of adverse

Prenatal cortisol concentrations, limbic structures and

affective problems in the child

Methods (N=65):
• Maternal saliva collection during pregnancy
for cortisol analyses
• Structural MRI (amygdala and hippocampus
volume) at 6-9 years
• Child affective problems (maternal report,
CBCL) at 6-9 years

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“Communication” between Mother and Fetus

Fetus PLACENTA MOTHER

CRH
CRH

ACTH

ACTH
CRH

CORTISOL 11-bHSD 1 and 2 CORTISOL

Mechanism: - stimulation of placental CRH

- transplacental passage of cortisol

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Ginekologi
PC Gestation Birth Brain Development
Infancy Childho od P Adulthood

Independent of adverse
Right amygdala volume (cm3)

Girls
Maternal Cortisol (µg/dl) during Pregnancy

Maternal Cortisol (µg/dl) during Pregnancy

Boys
1.0 1.6 1.0
1.4

1.2

0.0
1.0

0.8 0.8 0.8

0.0 0.3 0.4 0.5 0.6

Cortisol (µg/dl) at 15 weeks gestation

0.6 0.6

0.4 0.4

0.0 0.0
0.2 0.2

0 15 20 25 30 35 0 15 20 25 30 35
Weeks Gestation Weeks Gestation

Small Child Amygdala (lowest tertile)

Large Child Amygdala (highest tertile)

Age: 6-9 years

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PC Gestation Birth Brain Development
Infancy Childho od P Adulthood

Independent of adverse

The association between high prenatal cortisol and affective

symptoms is mediated by the size of the amygdala .

Girls
Right
Amygdala

P=0.02 P=0.02

Prenatal P=0.04 Affective Symptoms

Maternal in the Offspring
Cortisol P=0.10

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PC Gestation Birth Brain Development
Infancy Childho od P Adulthood

Independent of adverse

Maternal prenatal cortisol and child limbic

structures

 Higher susceptibility in females

 Higher susceptibility of the amygdala than the
hippocampus
 Laterality: higher susceptibility of the right amygdala

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Study II. Maternal pre-pregnancy obesity and child ADHD symptoms

 Prenatal inflammatory milieu as a risk factor for various

neurodevelopmental disorders.
 Maternal obesity as one particularly potent condition that produces an increased
inflammatory milieu during gestation.
 Maternal obesity before and during pregnancy has been associated with
deficits in neurodevelopmental outcomes in the offspring including attention-
deficit/
hyperactivity disorder (ADHD).

What are the neurocognitive alterations that underlie the association between
maternal obesity and increased risk for child ADHD?

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 PC
Maternal BMI Gestation Birth Brain Development
Infancy Childho od P Adulthood

Independent of weight gain Independent of adverse

ADHD risk in children of preconceptionally

obese mothers

 ADHD symptoms in the child (maternal report, CBCL,

N=174)
 Neurocognitive Assessment (executive function, N=174)

Gestational Child
pregnancy Executive
Pre- Child

weight gain Behavior

BMI Function

Preconception Pregnancy
7 years age

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 PC
Maternal BMI Gestation Birth Brain Development
Infancy Childho od P Adulthood

Independent of weight gain Independent of adverse

ADHD risk in children of preconceptionally obese mothers

*
**
 Pre-pregnancy BMI but not
ADHD symptoms (CBCL)

0.8
weight gain during pregnancy is
associated with ADHD symptoms in
0.6 the child at 7 years age (F(1,158)=4.80,
p=0.03).
0.4

 suggested 2.8-fold increase in the

0.2 prevalence of ADHD among
children of obese compared to
0.0
those of non-obese mothers.
Normal Overweight Obese
weight

Age: 6-9 years

Buss/ Entringer et al. 2012, PLoS ONE

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 PC
Maternal BMI Gestation Birth Brain Development
Infancy Childho od P Adulthood

Independent of weight gain Independent of adverse

ADHD risk in children of preconceptionally obese mothers

**
18 *
 Pre-pregnancy BMI but not
16 weight gain during pregnancy is
Executive Function

correct responses)
(Reaction time/

associated with child performance

on the Go/No-Go Task at 7 years
14 age (F(1,157)=8.38, p<0.01).

12  Maternal pre-pregnancy
obesity is associated with
inattentive/ less efficient
processing.
0 weight
N

l Program Studi Obstetri Ginekologi Peminatan Kedokteran Fetomaternal Program

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Impaired Child Attention Mediates the Association between Maternal
Pre-pregnancy Obesity and Child ADHD Symptoms

Executive
function (child)
0.123** 0.047***

BMI (Mother) β =0.18* ADHD

symptoms
β =0.12
(child)
Sobel test: P<0.05

Age: 6-9 years Buss/ Entringer et al. 2012, PLoS ONE

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 PC Gestation Birth Brain Development
Infancy Childhood P Adulthood

Conclusions

• Preconceptional and prenatal factors affect brain development

• Cognitive and affective function
• Size of limbic structures
• Implications for neuropsychiatric illness?

 Critical / sensitive developmental periods

 Effects independent of birth outcomes (birth weight, length of gestation)
 Other determinants that influence vulnerability (e.g. sex, genotype)

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PC Gestation Birth Brain Development
Infancy Childhood P Adulthood

R01 MH-091351, R01 HD-06028, R01 HD-065825

10-12 20-22 30-32
Weeks Weeks Weeks Birth 6 12
Gestation Gestation Gestation Months Months

▪ Baseline Blood/ Hair Sample ▪ Neonatal Brain ▪Infant Cognitive, ▪Infant Brain MRI
▪ Fasting Blood sample MRI (structural Motor & Emotional (structural MRI,
▪ Psychosocial Stress Assessment MRI, DTI, resting Behavior (Bayley’s) DTI, resting state
▪ Neuropsychological Battery state fMRI) ▪Infant Home fMRI)
▪ Fetal Ultrasound ▪ Neonatal Motor Environment ▪Infant Cognitive,
▪Cervicovaginal
▪Anthropometric Swabs
Measures Performance
(TIMP) ▪ Mother/InfantInteractions
Motor & Emotional Behavior (Bayley’s)

▪ 4 Day Ambulatory Period: ▪Infant StressReactivity▪ Mother/Infant

▪Diurnal Salivary Cortisol (7 per day)
Interactions
▪Actiheart Device
▪Electronic Diary (Smart Phone)
▪ 2 Random Dietary Recall Assessments
▪24 hr Urine
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 Future Directions
 Maintenance of Cohort for Future Follow-Up
 MRI as well as cognitive, motor and temperament assessments at older ages
 Gene* Environment Interactions
 Variation in genes associated with risk for developmental disorders and
psychopathology: serotonin transporter gene, dopamine D4 receptor gene,
BDNF Val66Met gene
 Additional Assays of Biological Specimen
 Effects of other prenatal conditions on brain development: iron
deficiency, nutrition, omega 3 fatty acids, oxidative stress, insulin
resistance
 Stem cell differentiation studies in tissue culture

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University of California Irvine Universität Trier
Claudia Buss Dirk H. Hellhammer
Elysia P. Davis
Sonja Entringer McGill University
Nora Sonia J. Lupien
Steven Moog
G. Potkin Michaeal J.
Jerod Rasmussen Meaney
Curt A. Sandman Jens C. Pruessner
James M.
University of North Carolina
Swanson Pathik
J H. Gilmore
D.Wadhwa
ohn Styner
Martin
Feizal W. affarn

Oregon Health & Science University

Damien Fair

Funding Agencies:
Deutsche Forschungsgemeinschaft (DFG )
Deutsches Cusanuswerk
Canadian Institute of Health Research (C IHR)
National Institutes of Healthof California
The University (NIH)
Irvine
MH-091351, PerinatologyHD-065825,
HD-06028, Research HD-51852, HD-28413
Program
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