PREGNANCY STIMULATED

NEUROGENESIS IN THE

ADULT FEMALE FOREBRAIN

MEDIATED BY PROLACTIN SHINGO, T., GREGG, C., ENWERE, E., FUJIKAWA, H.,

HASSAM, R., GEARY, C., CROSS, J.C., AND S. WEISS

PRESENTERS: KIM BLOM, MATT

REITSMA CHARMAINE IP

THURSDAY MARCH 12, 2009

Presentation Outline

Background and Introduction Basic neuroanatomy lesson

Significance

Focal Paper Experiments 1 – 6

Summary of Experiments Important Questions and

Answers

Critical Assessment

Questions and Answer Period

Background: What’s That Smell

Olfaction is an ancient

sense

Mate choice, mating ,

parent-offspring bonding,

parenting

Evolutionarily conserved

Physiological

mechanisms not well

understood

Neuroanatomy

Techniques

BrdU: synthetic thymine

analog incorporated into cell

during dividing in S phases;

evidence of cell divisions

Conjugated with fluorescent

markers

Fluorescence Micrograph:

Light microscope with filter

allowing illumination of

different light waves

Possible Candidates

Potential Candidates

Estrogen

Progesterone

Prolactin

Important Periods

Postcoitium (PC)

Gestational Day (GD)

Postpartum Day (PD)

Consider These...

Why is BrdU a good candidate for identifying

cell proliferation?

What are the significant developmental dates?

Experiment 1: Cellular

Proliferation Compare virgin mice to timed pregnant

mice Inject with BrdU to label dividing cells in

forebrain SVZ

GD7: 65% increase in BrdU labelled cells

GD14: returned to baseline levels

PD7: second increase

PD14 and PD21: return to baseline

Confirmation by two additional experiments

1. Ki76

2. BrdU labelled cells in hippocampaldentate gyrus

Cell Fate?

Three fates of these cells

1. neuronal SVZ cells migrate to olfactory

2. die

3. turn into glial cells and migrate to corpus

callosum

Glial cells are non-neuronal

cells that add nutrition

supporting nerve cells by

supplying oxygen and remove

dead neurons

Experiment 2: Identification of de

novo Neuronal cells

Examined fates 4 weeks

later using a different set

of mice

Neuronal Phenotype had to

be distinguishable

BrdU/NeuN (dual-labelled)

Viewed with fluorescent

micrographs Arrows

Experiment 2: Identification of de

novo cells continued

GD7 and PD7: significantly more neurons

Pregnancy and post-partum increases SVZ

proliferation and doubles interneurons in the

olfactory bulb

Experiment 3a: Pregnancy

modulated neurogenesis?

Pregnancy: involves release of circulating

hormones and physiological signals

Need to differentiate what causes

neurogenesis

Specifically: Circulating maternal hormones vs

physiological signals arising from embryo

implantation

Experiment 3a cont’d

Mated 6-8wk old virgin

females x vasectomized

males = pseudopregnancy

Pseudopregnant females had 42 + 13% more

BrdU labelled cells in forebrain SVZ compared

to non-mated virgin females at PC 7

Returned to baseline at PC14

Pseudopregnancy: transient

alteration of maternal pituitary

and ovarian steroid hormones

that mimics the changes during

first half of normal gestation

Narrowing down our options...

Expt 3a cont’d...

Tested estrogen and progesterone alone and

together

Infused directly into brain or peripherally into:

a) Pregnant and pseudo pregnant females

b) Normal and ovariectomized females

Both hormones failed to increase the number

of BrdU cells in the SVZ

Experiment 3b: PRL, a likely

suspect

What makes PRL a good candidate?

PRL present in virtually all invertebrates and

vertebrates

PRL concentrations change over course of

pregnancy and birth

PRL receptors expressed at high levels in

areas related to neuron growth and migration

Experiment 3b: PRL... guilty?Finding out why/how

Infused PRL into 6-8wk old ovarietomized

females for 6 days

a) Subcutaneously (sc)

b) Intracerebroventricularly (icv)

Found that 6 day infusion of PRL increased

BrdU-labelled cells in forebrain SVZ

Finding similar to that observed in pregnant

mice at GD7

Experiment 3b: Results

PRL mimics

pregnancy-induced

neurogenesis

Both sc and icv routes

increased the number of

BrdU labelled cells in

forebrain SVZ (infusion for

6 days)

Experiment 3c: Functional

consequences of PRL infusion

Looked at PRL injected

mice 4 weeks later, found:

PRL doubled numbers of

new olfactory

interneurons in the

forebrain SVZ

Does PRL infusion have same functional

consequences seen in pregnant mice?

Results virtually identical to that seen in

GD7 pregnant mice

Experiment 3 cont’d

•Supports specific and selective action of PRL

•Can safely suggest that PRL is the most likely

hormone mediating this response

•Cannot rule out possible action of PRL on

neuroblast migration

Experiment 4: cross gender

PRL and its

receptors also

present in males

Infused 6-8wk old

mice with PRL or

PRL-releasing

peptide for 6 days

• Increased proliferation of BrdU labelled

cells in forebrain SVZ observed in both

non-ovariectomized females and males

Experiment 5: PRLR Locations

PRLR (receptor) mRNA

located in:

1. Choroid plexus

secretes growth factors (TGF

α)

PRL indirect effect

2. Dorsolateral corner of SVZ

neuronal progenitors depart

for migration

rostral migratory stream

PRL role in initiating migration

Experiment 5a: Continued

Western Blot with

1. Cultured in vitro

NSCs

2. Spleen PRLR

(known)

• PRLR on cultured NSCs

• In vitro NSCs

equivalents of the

precursors to olfactory

interneuron

Experiment 5b: Functional Link –

PRL + Pregnancy Induced ....

Proliferation

1. NSC + PRL = no proliferation

2. NSC + PRL + EGF = proliferation (35%) neurospheres

PRL dose dependent

Self-Renewal 1. Neurospheres + 30nM PRL = secondary neurospheres (25%)

• but PRL in

pregnancy induces

100% neurogenesis

• only observed 40-

60% increase

•Other reasons?

Experiment 5b: Continued

Is there differentiation?

1. Neurospheres + EGF =

insignificant differentiation

2. Neurospheres + EGF +

30nM PRL = significant

differentation

Addition of PRL doubles

number of neurons

1. PRL can directly activate NSC’s

2. Augment proliferation and differentation

Experiment 6: PRL Signal

Requirement

Lifr genes: maintaining mammalian forebrain

neural stem cells

Heterozygous mutants have significant reduction of

olfactory neurons

PRLR-/- cannot be pregnant or pseudopregnant

Unable to test

Homozygous mutant and heterozygous

nulliparous females have reduced maternal

behaviour towards foster pups

Dose dependent (allele copies)

Experiment 6: PRL Signal

Requirement

Hypothesized:

Heterozygous mutant Prlr gene has 50%

reduction in amount of BrdU labelled cells

A decrease in amount of receptors will reduce the

amount forebrain neurogenesis

They then compared the number of BrdU-

labelled cells in timed pregnant Wild type and

Heterozygous Prlr females

Experiment 6: PRL Signal

Requirement

prior to mating, heterozygous and wildtype

have same number of BrdU labelled cells

At GD7 number of BrdU labelled cells:

Wildtype: increase 62%

Heterozygous mutant increase 33%

Summary

Experiment 1: Identification that Cellular

Proliferation occurs in the forebrain SVZ

Used BrdU and compare to Ki67/dentate gyrus

Found to increase at the proliferation at GD7 and

PD7

Experiment 2: Identified that the newly

proliferated cells end up the olfactory bulb and

are an increase in neuronal cells

Summary Continued

Experiment 3a: how pregnancy modulates

neurogenesis (pseudopregnancies)

Experiment 3b: prolactin as a likely suspect –

effects when artificially infused

Experiment 3c: we know prolactin increases

no. of BrdU, does it also stimulate

neurogenesis?

Experiment 4: gender differences – does

prolactin work the same in each?

Summary Continued

Experiment 5a: Determine location of PRLR

Choroid plexus, dorsolateral ventricle

Experiment 5b: Function of PRL on neurons

PRL directed NSC proliferation, differentiation

Experiment 6: PRLR Control

Gene disruption decreases Prl, decreases

proliferation

Conclusion: The Bigger Picture

Given the results...

PRL is the most likely hormone to mediate

pregnancy-stimulated neurogenesis

Lends support to the theory of brain plasticity

Critique: Strengths and

Weaknesses

Strengths

Thorough

Prl inducing neurognesis

from many angles

Genetic modulation

Gene knock out

In vitro and In vivo

Ruled out other candidates

Weakness

Lack of headings

Not identifying VEH or PRP

Make more connections to

developmental stages and

evolution

Future Directions

Longer experiment for further Prl effects

Experiment 6: did not definitively check if decreased number of neurons (NeuN)

Investigate what makes GD7 and PD7 important times for neurogenesis

GD7: mother avoid eating bad foods because gastrulation, neurolation and organogenesis occuring

“It is not birth, marriage or death, but gastrulationwhich is truly the most important time in your life” –Wolpert

PD7: ???

Up To Speed!!

PRL required for corpus luteum survival

(Frasor and Gibori 2003)

neovascularization and increase in size

PRLR-/- apoptosis and degeneration

PRLR-/- males are fertile (modest role)

Hyperlactation, autoimmune diseases,

compromise immune function

This study was first to demonstrate

hormone can stimulate

Genesis, migration, differentiation of

neurons

Pictoral References

http://www.wildriverreview.com/4/images/art-sd_fate.jpg

http://www.cartage.org.lb/en/themes/Sciences/Lifescience/GeneralBiology/Physiolog

y/NervousSystem/Neuron/08705a.jpg

http://www.cancerhelp.org.uk/cancer_images/brain-ventricles.gif

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Literature Cited

Bachelot, A., and N. Binart. 2007.

Reproductive role of prolactin. Society for

Reproduction and Fertility 1741-7899.

Shingo, T., Gregg, C., Enwere, E., Fujikawa,

H., Hassam, R., Geary, C., Cross, J.C., and S.

Weiss. 2003. Pregnancy-stimulated

neurogenesis in the adult female forebrain

mediated by prolactin. Science 299:117-120.

Questions