Maternal care and Gene - Environment Interactions Defining ... Meaney, Maternal... · Maternal care...

Maternal care and Gene - Environment

Interactions Defining Development

Michael J Meaney

James McGill Professor

Dept. Psychiatry

McGill University

Douglas Hospital Research Centre

The development of an individual is an active process

of adaptation that occurs within a social and economic

context:

• To resource (food, shelter, safety) availability.

• To social interactions.

• To independence from the parent.

Early experience

Abuse

Family strife

Emotional neglect

Harsh discipline

Health Risks

Depression

Drug abuse

Anxiety

Diabetes

Heart disease

Obesity

Mechanism?

Developmental Origins of Adult Disease

Early experience

Abuse

Family strife

Emotional neglect

Harsh discipline

Health Risks

Depression

Drug abuse

Anxiety

Diabetes

Heart disease

Obesity

Individual differences

in neural and endocrine

responses to stress (defensive responses)

Stress Diathesis Models

Early experience

Abuse

Family strife

Emotional neglect

Harsh discipline

Health Risks

Depression

Drug abuse

Anxiety

Diabetes

Heart disease

Obesity

Individual differences

in neural and endocrine

responses to stress

Poverty

Early experience

Abuse

Family strife

Emotional neglect

Harsh discipline

Depression

Drug Abuse

Anxiety

Obesity

Diabetes

Heart Disease

Poverty

Effects of poverty on emotional and cognitive development are

mediated by parental factors (Conger, McLloyd, Eisenberg).

Poverty

Maternal

emotional

distressParenting

Child

behavioural

problems

Linver, Brooks-Gunn & Kohen Dev Psychol 2002; same model predicts child cortisol

levels (Lupien, McEwen, Meaney Biol Psychiatry 2000)

Poverty

Home

environment

Child

cognitive

development

Environmental

adversity

Parenting/

Home

resources

Child

development

Nutrient Supply

Mate Quality

Violence

Infection

Population density

Parental

investment

Defensive Strategies

Foraging/Metabolism

Reproductive Strategies

Environmental Parental Developmental

signal mediation outcome

Robert Hinde: Evolution has shaped the young to use parental

signals as a „forecast‟ of the quality of the environment

into which they have been born. For most species, there is

no single, optimal phenotype.

Evolutionary biology - Maternal effects

Defense to snake predation in skink lizards

Most frequent prey

• smaller

• shorter tails

• less reactive to

snake cues

If mother has been exposed to the scent of a

predatory snake then offspring are larger, with

longer tails and ….

Control Perfume Snake0

1

2

3

4

Response to snake odours

To

ng

ue

-Flic

k

Offspring

are significantly

larger and with

longer tails

Nutrient Supply

Mate Quality

Violence

Infection

Population density

Parental

investment

Defensive Strategies

Foraging/Metabolism

Reproductive Strategies

Environmental Parental Developmental

signal mediation outcome

Robert Hinde: Evolution has shaped the young to use parental

signals as a „forecast‟ of the quality of the environment

into which they have been born. For most species, there is

no single, optimal phenotype.

Evolutionary biology - Maternal effects

How might the parent-child relations that define early family

life influence the development of individual differences in

brain development and function?

Summary

• Parental care affects the activity of

genes in the brain that regulate stress

responses, neural development and

reproduction.

• This parental effect involves a form

a “plasticity” at the level of the DNA.

Epigenetics: Any functional change in the genome that

does not involve an alteration of DNA sequence.

Every cell in your body has the same nuclear genes, but…?

Multiple phenotypes from a common genotype

If they ask you anything you don‟t know,

just say its due to epigenetics.

Epigenetics

Any functional change to the genome that

does not involve an alteration of DNA sequence.

Epigenetics is the biological basis for gene x

environment interactions.

Warning!!!

Incomprehensible scientific jargon

will follow…

C T A C G T A C T C G G A A T C T C GCH

3CH

3CH

3

C T A C G T A C T C G G A A T C T C G

Genetic code is defined by the sequence of four nucleotides that

produce proteins and other molecules that serve cell function.

RNAs, Protein

Epigenetic effects refer to modifications of the chemistry of the DNA,

but not to a change of sequence. Epigenetics alters the activity of the

gene, but not its function.


CH3

CH3CH

3

• Epigenetic effects refers to modifications of the

chemistry of the DNA, but not to a change of sequence.

• Epigenetics alters the activity of the gene, but not its function.


CH3

CH3CH

3




• DNA methylation: The addition of a methyl group onto a cytosine.


CH3

CH3CH

3





• DNA methylation is chemically very stable (potentially lasting for

the life of the organism).


CH3

CH3CH

3





• DNA methylation is chemically very stable (potentially lasting for

the life of the organism).

• DNA methylation silences gene expression.

Nucleosome core particle: ribbon traces for the 146-bp DNA phosphodiester backbones

(brown and turquoise) and eight histone protein chains (Luger et al. Nature 1997).

+ -

Prevents TF

binding to DNA

TF binding involves

alteration of

chromatin structure

Acetyl group

B. Turner. Chromatin structure and gene regulation. 2001.


CH3CH

3CH3


Genetic code is defined by the sequence of four nucleotides that

produce proteins and other molecules that serve cell function.

RNAs, proteins

Epigenetic effects refer to modifications of the chemistry of the DNA,

but not to a change of sequence. Epigenetics alters the activity of the

gene, but not its function.

SIN

SIN

CH3

CH3

CH3

CH3 MeCP2

HDACCH3

CH3

MeCP2

DNA Methylation can inhibit gene expression

by blocking transcription factors binding

HDAC HDAC: Histonedeacetylase

Methylated DNA

binding protein

HDAC

B. Turner. Chromatin structure and gene regulation. 2001.

Parental care Epigenetic mark PhenotypeGene expression

Naturally-occurring

variations in maternal care

Expression of specific genes

in brain regions

HPA function

Stable individual differences

in stress reactivity

Maternal licking/grooming

Source of tactile stimulation/nurturance: Enhances

activity of endocrine systems (e.g., GH/IGF) that

promote somatic growth, suppresses those

(glucocorticoids) that inhibit growth

Variations in maternal care

Fre

qu

en

cy c

ou

nt

% Licking/grooming

Variations in maternal care

Fre

qu

en

cy c

ou

nt

% Licking/grooming

Low LG

Mean - 1SD

High LG

Mean + 1SD

- - - - -

HighLow

Days of

Age

Lic

kin

g/g

roo

min

g

Variations in maternal care X Days

* No differences

in time with pups

864206

8

10

12

14

16

18

High/Stress

High/Ctl

Low/Stress

Low/Ctl

% L

ickin

g/g

room

ing

Day

Gestational stress/maternal behaviour

Popeski et al: Pictures of „own‟ infant increase activity

in ventral striatum (nucleus accumbens – dopamine target)

by comparison to other infant or neutral stimuli in fMRI

studies – this effect is abolished by chronic stress.

Comparable effects in humans

Are these naturally-occurring variations

in maternal behaviour associated with

the development of individual differences

in endocrine and behavioural responses

to stress?

* Effects hold for both males and females

fetal postnatal peripubertal adulthood

Maternal care Outcomes

GlucocorticoidReceptor

Hypothalamus

Pituitary

Adrenals

Hippocampus

Glucocorticoids

Stress

(-)

CRF

ACTH

CRF: corticotropin releasing factor. ACTH: adrenocorticotropin

Hypothalamus

Pituitary

Adrenals

Hippocampus

Glucocorticoids

(-)

CRF

ACTH

(-)

Hypothalamus

Pituitary

Adrenals

Hippocampus

Glucocorticoids

(-)

CRF

ACTH

(-)

Individual differences in glucocorticoid receptor

levels lead to altered pituitary-adrenal responses

to stress

Hypothalamus

Pituitary

Adrenals

High LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)

Hypothalamus

Pituitary

Adrenals

Low LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)



to stress

Adult offspring of High LG mothers show

more modest HPA responses to stress

Pre S10 S20 P20 P40 P60 P1200

200

400

600

800

1000High LGLow LG

Pla

sm

a A

CT

H (

pg

/ml)

Pre 10 20 40 60 80 1000

10

20

30

40

50

60

70

80High LGLow LG

Cort

icoste

rone (

µg/d

l)

Intra-hippocampal infusion of a GR antagonist completely

eliminates the maternal effect on HPA responses to stress

Hypothalamus

Pituitary

Adrenals

High LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)

Hypothalamus

Pituitary

Adrenals

Low LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)



to stress

Trai

t Anx

iety

Maternal Care (Parental Bonding Index)4030201010

20

30

40

50

60

70

80

(r = -0.54, p<.01)

Parental Care & Anxiety

Pruessner et al. J Neurosci 2004

Anxiety & HPA Responses To Stress

Pruessner et al. J Neuroscience 2004

High Low0

10

20

30

40

50

60

Sta

te A

nxi

ety

*

High Low-20

-10

0

10

20

30

40

Co

rtis

ol (

∆ µ

g/d

l/min

)

*

Ventral Striatum DA Stress Response

Right

Low High

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Rac

lop

rid

e (

∆ S

tres

s -

Bas

al)

Left

Low High

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Rac

lop

rid

e B

d (

∆ S

tres

s -

Bas

al)

Pruessner et al. J Neurosci 2004

Inte

g. C

ort

iso

l (∆

Str

ess

-B

asal

)

PBI (Cr/M)

50403020-30

-10

10

30

50

(r= -0.63; p<.07)

Cortisol Response

PBI (Cr/M)

Dopamine Release

Rac

lop

rid

e (∆

Str

ess

-B

asal

)4030201010

-0.05

0.05

0.15

(r= -0.66; p<.05)

High LG Low LG

Hypothalamus

Pituitary

Adrenals

High LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)

Hypothalamus

Pituitary

Adrenals

Low LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)

Cross-fostering reveals evidence for direct, postnatal

effects of maternal care

High/High High/Low Low/High Low/Low.

.

.

.

.

GR

ir (

RO

D)

**

Cross-fostering reveals evidence for direct, postnatal

effects of maternal care on hippocampal GR expression

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

% LG

hip

po

ca

mp

al G

R m

RN

A

Within-litter variation in pup LG frequency predicts

hippocampal glucocorticoid receptor gene expression

r= 0.375, p= 0.008

Hypothalamus

Pituitary

Adrenals

Hippocampus

Glucocorticoids

(-)

CRF

ACTH

(-)

?

How might maternal licking/grooming

regulate hippocampal glucocorticoid

receptor gene activity and HPA function?

And how do

these effects persist over the lifespan

of the offspring?

Signal

Receptor

Extracellular signalMaternal

Care

DNA

Transcription

Factors

Intracellular

signal

gene

Relevant gene - environment interaction

cAMP

NGFI-A

5-HT7 rec

5-HTTactile stimulation

(maternal LG)

GR gene

PKA

(T3)

(H-P-T)

Transcription

Factor

Summary of in vivo and in vitro studies

NGFI-A overexpression in cultured hippocampal neurons

0.0

2.0

4.0

6.0

8.0

10.0

No

rmali

zed

GR

0.0

2.0

4.0

6.0

8.0

10.0

No

rmali

zed

GR

17

0.0

1.0

2.0

3.0

No

rma

lize

d N

GF

I-A

0 1 2 3 4

0

1

2

3

No

rma

lize

d G

R

Normalized NGFI-A

Ctl EV NGFI-A Ctl EV NGFI-A Ctl EV NGFI-A

NT EV OE0

1

2

3

4

5

No

rmali

zed

GA

D1

GAD1 expression in NGFI-A

overexpression cells

Hellstrom et al., in press

What are the relevant

genomic targets?

Clone the 5‟ untranslated

region of the rat

hippocampal glucocorticoid

receptor gene

cAMP

NGFI-A

5-HT7 rec

5-HT

GR gene

PKA

Gene organization

Non-coding, regulatory

region (contains enhancers,

repressors, etc.).

Coding region

responsible for protein

synthesis.

(+ or -)

Clone the 5‟ untranslated region of the rat

hippocampal glucocorticoid receptor gene

Glucocorticoid receptor gene

(~110 kb)

1 2 3 4 5 6 7 8 9 10 11 12 2 3 4 5 6 7 8 9

Variable exon 1 region Constant region

5’ 3’

Transfection studies with promoter-reporter constructs reveal exon

17 sequence has considerable transactivational capacity.

(McCormick et al. Mol Endo 2000)

Critical for hippocampal GR

1 2 3 4 5 6 7 8 9 10 11 12 2 3 4 5 6 7 8 9


5’ 3’

DG CA1 CA2 CA30.00

0.05

0.10

0.15 High LG

Low LG

Rela

tive

OD

U

*

* **

1681 ccc

1741 ctctgctagt gtgacacact t1cg2cgcaact c3cgcagttgg 4cggg5cg6cgga ccacccctg7c

1801 ggctctgc8cg gctggctgtc accct9cgggg gctctggctg c10cgaccca11cg ggg12cgggct

1861 c13cgag14cggtt ccaagcct15cg gagtggg16cg gggg17cgggag ggagcctggg agaa

DNA sites that regulate glucocorticoid receptor gene

NGFI-A

GR Promoter 17 Sequence

1 2 3 4 5 6 7 8 9 10 11 12 2 3 4 5 6 7 8 9


5’ 3’

*

1.5

1.0

0.5

0.0

NGFI-A mRNA levels Day 6

cAMP

NGFI-A

5-HT7 rec

5-HT

GR gene

PKA

Day 6 pups

NGFI-A binding to the GR(17) promoter

in neonates

**

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

An

tib

od

y/i

np

ut

(RO

D) 1.0

NGFI-A

cAMP

NGFI-A

5-HT7 rec

5-HT

GR gene

PKA

NGFI-A binding to Exon 17

promoter region - P4 On/Off

hi on hi off lo on lo off0

1

2

No

rmali

zed

GR

17

NGFI-A binding to GAD1 promoterin P4 on/off model

hi on hi off lo on lo off0. 00

0. 05

0. 10

0.15

No

rma

lize

d G

AD

1

NGFI-A binding to exon 17 promoter is dynamically

regulated by mother – pup interactions

…..

Exon 1 (Noncoding region) Exons 2- 9 Coding region

5’ 3’

(+)

Glucocorticoid receptor mRNA

Glucocorticoid receptor protein

Offspring of High LG mothers

17

NGFI-A

But…..

*

1.5

1.0

0.5

0.0

1.5

1.0

0.5

0.0

Pup Adult

NGFI-A levels

So, while increased levels of NGFI-A can explain the increased

activity of the glucocorticoid receptor gene in the pup, it does not

explain why the difference is still observed in adult animals?

…..


5’ 3’

(+)




17

NGFI-A

Stable modification

of the DNA????

GR expression & HPA

function


CH3

CH3

CH3

1681 ccc

1741 ctctgctagt gtgacacact t1cg2cgcaact c3cgcagttgg 4cggg5cg6cgga ccacccctg7c

1801 ggctctgc8cg gctggctgtc accct9cgggg gctctggctg c10cgaccca11cg ggg12cgggct

1861 c13cgag14cggtt ccaagcct15cg gagtggg16cg gggg17cgggag ggagcctggg agaa

DNA sites that regulate glucocorticoid receptor gene

NGFI-A

GR Promoter 17 Sequence

1 2 3 4 5 6 7 8 9 10 11 12 2 3 4 5 6 7 8 9


5’ 3’

5’ 3’0.0

0.2

0.4

0.6

0.8

1.0

Low LG

High LG

c-M

eth

yla

tio

n

*

NGFI-A site

5‟…tgcgggggcgggg…3‟

NGFI-A

CH3CH

3

High LG Low LG

Low/Low High/High Low/High High/Low

0.0

0.2

0.4

0.6

0.8

c-m

eth

yla

tio

n

5' CpG region of NGFI-A/RE


NGFI-A

CH3 CH

3

5‟…T G C G G G G G C G G G G …… 3‟

5‟…T G C G G G G G C G G G G …….3‟

CH3

CH3

5‟…T G C G G G G G C G G G G …….3‟

CH3

CH3

5‟…T G C G G G G G C G G G G …… 3‟

(no methylation)

(methylated only at 3‟; High LG)

(methylated only at 5‟)

(methylated at both 5‟ and 3‟; Low LG)

In vitro purified NGFI-A binding to ….

5‟…T G C G G G G G C G G G G …… 3‟

5‟…T G C G G G G G C G G G G …….3‟

CH3

CH3

5‟…T G C G G G G G C G G G G …….3‟

CH3

CH3

5‟…T G C G G G G G C G G G G …… 3‟

(no methylation)

(methylated only at 3‟; High LG)

(methylated only at 5‟)

(methylated at both 5‟ and 3‟; Low LG)

NGFI-A binding to ….

(High LG)

(Low LG)

*

1.5

1.0

0.5

0.0

1.5

1.0

0.5

0.0

Pup Adult

NGFI-A levels

So while levels of NGFI-A are similar in animals reared

by High or Low licking/grooming mothers, the NGFI-A

site in the adult offspring of Low LG mothers is methylated

and therefore cannot interact with NGFI-A.

But….

…..


5’ 3’

(+ or -)



Offspring of Low LG mothers

17

NGFI-A

CH3

1.5

1.0

0.5

0.0

Adult NGFI-A

…..


5’ 3’

(+ or -)




17

NGFI-A

1.5

1.0

0.5

0.0

Adult NGFI-A

0.1

0.3

0.5

0.7

0.9

LowHigh

Exo

n 1

7le

vels

In vivo binding of NGFI-A to the glucocorticoid

receptor regulatory site in adult animals as a function

of maternal care.

Maternal Care

Glucocorticoid receptor

gene expression

CRF expression

Individual differences in stress reactivity

GC feedback

sensitivity

Differential methylation of GR promoter

… differences in NGFI-A

binding.

CmG

MeCP2

mSIN

HDAC

HDAC

TF

TF

Ac

Ac

C G

Ac

TFTF

Ac

C G

Ac

TFTF

Ac

CmG

MeCP2

mSIN

HDAC

HDAC

TF

TF

Ac

Ac

X

HDAC inhibitor - trichostatin A

X

X

DNA methylation attracts HDACs and block

the opening of chromatin

High/Ctl High/TSA Low/Ctl Low/TSA0.0

0.2

0.4

0.6

0.8

1.0

Rela

tive

RO

D

*

Weaver et al. Nature Neuroscience 2004

C G

Ac

TFTF

Ac

Histone Acetylation

C G

Ac

TFTF

Ac

High/Veh High/TSA Low/Veh Low/TSA

0.0

0.2

0.4

0.6

0.8

1.0

An

tib

od

y/I

np

ut

*

Transcription Factor (NGFI-A) Binding

CmG

MeCP

2

mSINHDAC

HDAC

TF

TF

Ac

Ac

X

C G

Ac

TFTF

Ac

0

0.1

0.2

0.3

0.4

Vehicle 100 ng/ml TSA

GR

IR

(R

OD

-IO

D)

*

Low LG

High LG

Glucocorticoid Receptor

(Hippocampus)

Low/Veh Low/TSA High/Veh High/TSA

Basal Stress Stress + 40 Stress + 60 Stress + 900

10

20

30

40

50

Co

rtic

os

tero

ne

(

µg

/dl)

Time (min)

Low/Veh

Low/TSA

High/Veh

High/TSA

Glucocorticoid stress response

Weaver et al. Nature Neuroscience 2004

GGAGCCTGGG AGAA CCAAGCCTCG GAGTGGGCG GGGGCGGGAGCH

3CH

3

GGAGCCTGGG AGAA CCAAGCCTCG GAGTGGGCG GGGGCGGGAGCH

3

Offspring of Low LG mothers


Lower levels of GR in hippocampus -

Increased stress response

High levels of GR in hippocampus -

Modest stress response

How does maternal care alter the DNA

methylation?

cAMP

NGFI-A

5-HT7 rec


(maternal LG)

GR gene

PKA

(T3)

(H-P-T)

Do these „maternal‟ signals alter the methylation

of the exon 17 GR promoter?

E .

.

.

.

.

.

HighLow

C -

meth

yla

tion


A period of methylation of the 5’ CpG site (E20 to PND 1)

followed by a demethylation (PND 1 to PND 6).

Hippocampal GR(17) methylation


NGFI-A

CH3

E .

.

.

.

.

.

C -

meth

yla

tio

n


Demethylation corresponds to period of differences

in maternal LG

- - - - -

HighLow

Days of Age

Lic

kin

g/g

roo

min

g

cAMP

NGFI-A

5-HT7 receptor


(maternal LG)

GR gene

PKA CBP

Summary of in vivo and in vitro mechanisms

NGFI-A (egr-1, zif-268, krox-24)

High LG high NGFI-A levels

5‟…tgcgggggcgggg…3‟CH

3CH

3

Acetyl

Lys9

Lys9

NGFI-A CBP

CBP (CREB binding protein)

is increased in High LG

offspring - CBP is a

histone acetyltransferase

High LG high NGFI-A levels

5‟…tgcgggggcgggg…3‟CH

3

CH3

NGFI-A CBP

MBD2b

demethylase


CH3

CH3

CH3

DNA methylation serves as an interface between

the dynamic environment and the fixed genome

DNA methylation serves to imprint social factors,

such as maternal behavior, upon the offspring‟s genome.

CpG Islands

H3K9ac

DNA Me

mRNA EXP

Genes

NR3C1 (GR) gene locus (Chromosome

18)

PCDH clusters

Do comparable processes occur in humans?

• Post-mortem studies of hippocampus.

• Samples from suicide victims/controls.

• QSBB (Gustavo Turecki) - forensic

phenotyping.

• Human exon 1F promoter (Turner &

Muller, J Molec Endo, 2005)

Hippocampal samples from humans

• Human brain bank (suicide victims vs controls).

• All suicide victims (and none of the controls)

experienced verified abuse in childhood.

Human glucocorticoid receptor gene

A B C D E F G H I J 2 3 4 5 6 7 8 9


5’3’

Human glucocorticoid receptor gene

A B C D E F G H I J 2 3 4 5 6 7 8 9


5’3’

Control Suicide0.0

0.5

1.0

1.5GRtotal

GR

mR

NA

Control Suicide0.0

0.3

0.6

0.9

1.2 GR1F

GR

1-F

vari

an

ts

McGowan et al. PlosOne 2008, Nature Neuroscience 2009

Suicide vs abuse - GR expression

GRtotal

GR

mR

NA

/GA

PD

H (

log

co

nc

.)

Control Suicide Suicide

- Abuse + Abuse

0.0

0.4

0.8

1.2

*

GR

-1F

mR

NA

/GA

PD

H (

log

co

nc

.)


- Abuse + Abuse

0.0

0.5

1.0

1.5

*


GR

1-F

Cp

G M

eth

yla

tio

n (

%)


- Abuse + Abuse

0

20

40

60*

0

20

40

60

30 31 32

CpG sites

Suicide vs abuse - CpG methylation


Control NGFI-A expression

Co-transfection studies (NGFI-A vector w/

human GR exon 1F-luciferase construct)

0

2000

4000

6000

8000

10000

12000

14000

Lu

cif

era

se

ac

tivit

y

mPlasmid nmPlasmid Antisense


GR1F promoter methylation levels in WBC of PTSD patients and controls (new subjects)

CpG sites

Meth

ylation leve

ls (0

/ 0)

0

10

20

30

40

50

PTSD (n=8)

Control (n=4)

s2 s3

s1

4

s1

3

s2

4

s3

4s4

s3

0

s3

1

s5

s3

5

s1

5

s2

5s6

s2

6

s1

6s7

s3

7

s1

7

s2

7

s1

8

s3

8

s8

s2

9

s2

0

s1

2

s1

0s1

1

s1

9

s2

1s2

2

s2

3

s2

8

s3

6

s3

3

s3

2


CH3

CH3

CH3

DNA methylation serves as an interface between

the dynamic environment and the fixed genome

DNA methylation serves to imprint social factors,

such as maternal behavior, upon the offspring‟s genome.

Maternal care and Gene - Environment Interactions Defining ... Meaney, Maternal... · Maternal care...

Documents

Transcript of Maternal care and Gene - Environment Interactions Defining ... Meaney, Maternal... · Maternal care...