Oxytocin: the key to treating lactation failure and associatedlactation-failure and associated

diseasesInvited video lecture by Translational Biomedicine

Yu-Feng Wang, MD, PhD

Department of Cellular Biology and AnatomyLouisiana State University Health Sciences Center-

Shreveport LA USAShreveport, LA, USA

Breastfeeding/lactationBreastfeeding/lactation1. Breastfeeding is the feeding of an infant or young child with breast

milk directly from female human breasts via lactation or nursing.

2. Breastfeeding has many benefits for both mother and baby.

3. The World Health Organization (WHO) recommends exclusive breastfeeding for the first six months of life and then supplemented breastfeeding for at least one yearsupplemented breastfeeding for at least one year.

4. Breastfeeding is a natural human activity, while nursing difficulties are not uncommon.

5. Lactation failure is associated with postpartum depression (Ip et al, 2009), and premenopausal breast cancer (Stuebe, et al, 2009).

Outline of this lecture

1. Neurochemical mechanisms underlying the milk letdown reflex: Suckling-elicited burst firing in oxytocin (OXT) neurons

2 Therapeutic roles of OXT in lactation-failure2. Therapeutic roles of OXT in lactation failure

3. OXT and lactation failure-associated diseases

4. Issues about publicly using OXT

The milk letdown reflexThe milk-letdown reflex

Oxytocin (OXT)-

Brain

Tactile,

Oxytocin (OXT)-Secreting System

NS

SON/PVN

auditory,olfactory, or visual, stimuli

OXT

NH

PRLstimuli

Spinal cordMyoepithelia

Milk

Mammary nerve

CirculationBreast

Milk

S kli i b fi i f OXTSuckling triggers burst firing of OXT neurons in the supraoptic nucleus (SON)

Paired extracellular recordings

kes/

sLeft SON

40 sp

ik

Right SON

Intramammary pressure OX

T

g

2 min

1 m

U

Wang and Hatton, 2004

Burst firing of OXT neurons evoked inBurst firing of OXT neurons evoked in brain slicesBurst

Wang and Hatton, 2004

Wang and Hatton, 2007g ,

I Neurochemical mechanismsI. Neurochemical mechanisms underlying the milk letdown reflex: S ckling elicited b rst firing in OXTSuckling-elicited burst firing in OXT

neurons

Levels of neurochemical modulation of oxytocin neuronal activityy y

1. Afferent suckling pathway

2. Synaptic innervation

3. Glial-neuronal interaction

4 Neurochemical environment4. Neurochemical environment

5. Receptor-mediated intracellular signaling processes

6 Electrogenic organelle activity6. Electrogenic organelle activity

I I Afferent suckling pathway in the CNSGating of bursts in

OXT neurons in

I-I. Afferent suckling pathway in the CNSSynchronization of

bursts in OXT neurons OXT neurons in the SON & PVN

Lincoln and Wakerley,1975

bursts in OXT neuronsBelin and Moos, 1986

Mesencephalic

DMH and PHDubois-Dauphin et al., 1985b

1975

Lateral cervical nucleus

MesencephalicLateral tegmentum

Juss and Wakerley, 1981

Lateral funiculus of the spinal cord

nucleus

Dubois-Dauphin et al., 1985a Fukuoka et al, 1984,

Integrative processes of the afferentIntegrative processes of the afferent pathway in the hypothalamus

1 Afferent inputs from the lateral tegmentum cross to the contralateral1. Afferent inputs from the lateral tegmentum cross to the contralateral hypothalamus (Wang et al, 1995);

2. This pathway is responsible for the summation of suckling signals, the basis for burst generation (Wang et al 1996);basis for burst generation (Wang et al, 1996);

3. Burst synchrony of OXT neurons in the SON and PVN of bilateral sides depends on signals from the ventral posterior hypothalamus (Wang et al, 1997; Yang et al, 1999); ( g , ; g , );

4. OXT neurons have mutual structural and functional connections with nuclei of the mammillary body and a special group of interneurons in the SON and perinuclear zone, which mediates periodic synaptic input to OXT neurons (Wang et al, unpublished data);

5. Mammillary body neurons innervate bilateral OXT neurons and function as a “Synchronization center” (Wang et al, 8th WCNH, 2009).

I II Synaptic innervationI-II. Synaptic innervation1. Direct synaptic innervation of OXT neurons is limited to a few brain

areas including the nucleus of the solitary tract (NTS), posterior hypothalamus, dorsal medial hypothalamus, perinuclear zone (PNZ), bed nucleus of the stria terminalis (BNST), and SON and PVN on the contralateral side (Wakerley et al, 1994).

2 L t ti i th b f di t ti i ti f OXT2. Lactation increases the number of direct synaptic innervation of OXT neurons (Hatton et al, 2004; Theodosis et al,2008);

3. Reduced tonic EPSCs (Kombian et al, 1997, Pittman et al, 2000) and IPSCs (Brussaard 1995) in response to OXT stimulation;and IPSCs (Brussaard, 1995) in response to OXT stimulation;

4. Increases in intermittent clustered EPSCs (Israel et al, 2003; Wang and Hatton, 2004, 2007, 2009) and likely IPSCs (Moos 1995);

5 OXT-elicited periodic changes in synaptic inputs from the BNST5. OXT elicited periodic changes in synaptic inputs from the BNST (Lambert et al, 1994), histaminergic tuberomammillary neurons and intra-SON interneurons (Wang et al, 8th WCNH, 2009), and a fraction of PNZ neurons (Dyball & Leng, 1986).( y g, )

B t i t d ti tBurst-associated synaptic events

BNST, PNZ, TMTakano et al.,Boudaba and Tasker, 2006

Israel et al 2003Pittman et al, 2000

SON

PVN

Takano et al., 1990

B d

Israel et al, 2003

Dorsal medialhypothalamus

PVN

Wakerleyand Lincoln 1973

Brussaard,1995

Ventral posteriorhypothalamus

hypothalamus and Lincoln, 1973

Takano et al., 1992Honda & Higuchi, 2007

NTShypothalamus

Weiss et al., 1989Tribollet et al., 1985Moos et al., 2004 Cumbers et al, 2007,

I III I t ti b t t t dI-III. Interactions between astrocytes and OXT neurons in the SON

OXT neurons(OXT-neurophysin staining)Histology of the SON

OXT neurons

Ventral glial l i

AVPneurons lamina

Astrocytes (GFAP staining)Hatton and Wang , 2008

neurons

GFAP plasticity during suckling in lactating rats or OXT stimulation in brain slices

Nuclei GFAP NPs Merge Nuclei GFAP NPs Merge

In Vivo In Vitro

Non-Suckling

Suckling

Control

OXTSuckling

Milkl td

OXT

12 mM K+

i OXT

Modified from Wang and Hatton, 2009

letdown in OXT

20 μM

Contribution of astrocyte plasticity to theContribution of astrocyte plasticity to the activation of OXT neurons

1 Acute astrocyte plasticity is essential for suckling evoked burst firing1. Acute astrocyte plasticity is essential for suckling-evoked burst firing in OXT neurons and ensuing milk letdown (Wang and Hatton, 2009).

2. Astrocytes promote glutamate release, and partially mediate effects of OXT on tonic and clustered EPSCs (Wang and Hatton 2009)of OXT on tonic and clustered EPSCs (Wang and Hatton, 2009).

3. Suckling and OXT cause acute retraction of astrocyte processes around OXT neurons (Wang and Hatton, 2007, 2009), which reflects OXT neuronal activities via neurogenic neurochemical changes.g g

4. GFAP plasticity modulates OXT neuronal activity by changing water transportation, morphology, and glutamate metabolism in astrocytes.

5. Astrocyte plasticity is also related to increased prostaglandin synthesis (Wang and Hatton, 2006) and ATP metabolism (Ponzio et al, 2006), which together with bolus glutamate release provide an external driving force for burst generation.

I IV N h i l i t d b tI-IV. Neurochemical environment and bursts1. Suckling increases intra-SON and PVN release of OXT (Neumann et

al 1993 Bealer and Crowley 1998)al, 1993, Bealer and Crowley, 1998).2. OXT release during suckling depends on actions of glutamate (Parker

and Crowley, 1993, 1995), norepinephrine (NE, Bealer and Crowley, 1998), and histamine (HA, Bealer and Crowley, 1999, 2001), releases of which are increased during suckling.

3. In modulation of OXT release, synergistic interactions between glutamate and NE (Parker and Crowley, 1993) and between HA and NE (Bealer and Crowley 1999) are essentialNE (Bealer and Crowley, 1999) are essential.

4. Prostaglandins (Wang and Hatton, 2006), ATP and adenosine (Ponzio et al, 2006) from astrocytes contribute to the changes in the burst-related extracellular milieu.related extracellular milieu.

5. OXT neuronal activity-elicited changes in ion levels also modulate the activity of the OXT-secreting system, such as K+ level (Leng and Shibuki, 1987).

Burst-associated neurochemicalBurst associated neurochemical environment around OXT neurons

Suckling/OXT Suckling/OXT

Before burst After burst

Ca2+

OXTGlu

Oxytocin neuron

GluK+K+

K+ HAGl

Oxytocin neuron

Oxytocin neuron

PGsATP

Ca2+

Glu GluGABAGABA

NEK

Glu GluGABAAdenosine

KK+K+Ca2+Ca2+

K+ HAGlu

Astrocyte

Hatton and Wang , 2008; Wang and Hamilton, 2009

AstrocyteAstrocyte

I V Receptor mediated intracellularI-V. Receptor-mediated intracellular signaling processes

1. OXT receptor (OTR) has been identified in both SON neurons and1. OXT receptor (OTR) has been identified in both SON neurons and astrocytes (Wang and Hatton, 2006).

2. The major signaling pathway of OTR involves Gq/11-type G-proteins (Sanborn et al, 1998; Gimpl and Fahrenholz, 2001).

3. OTR-associated Gβγ- subunit is a dominant signal in OXT-evoked bursts (Wang and Hatton, 2007a), can activate ERK1/2 (extracellular signal-regulated protein kinase 1/2) and protein kinase A (PKA) (Sanborn et al, 1998; Zhong et al., 2003).

4. Phosphorylation of ERK1/2 in a unique spatiotemporal order can trigger burst (Wang and Hatton, 2007b).

5 OXT induces Cox 2 and prostaglandin (PG) synthesis in OXT5. OXT induces Cox-2 and prostaglandin (PG) synthesis in OXT neurons and astrocytes, promotes actin polymerization (Wang and Hatton, 2006), and facilitates bursts (Wang and Hatton, 2007b).

OXT receptor signaling and bursts

I VI El t i ll ti itI-VI. Electrogenic organelle activity and burst of OXT neurons

1. Burst firing in OXT neurons has strong features of gating (Lincoln and Wakerley, 1975) and synchronization (Belin and Moos, 1986).

2. Lactation increases junctional communication between OXT ti b t h (H tt d Y 1994)neurons, promoting burst synchrony (Hatton and Yang, 1994).

3. There is a rebound depolarization following a transient hyperpolarization of membrane potential, which underlies the burst gating (Stern and Armstrong 1997)gating (Stern and Armstrong, 1997).

4. During seconds preceding a burst, the rising slope of the afterhyperpolarizations (AHPs) is decreased while the rising slope of spikes is increased; in burst firing neurons the decay timeof spikes is increased; in burst firing neurons, the decay time course of the AHPs is shortened dramatically, which favors burst onset (Wang and Hatton, 2004).

Neurochemical process of OXT elicited burstsNeurochemical process of OXT-elicited bursts

II. Roles of oxytocin in lactation-yfailure

Lactation interruption leads to the failureLactation-interruption leads to the failure of burst firing during suckling

Normal lactating rats Burst

Lactation-interrupted ratsLactation interrupted rats

Wang and Hatton, Frontiers in Neuroscience, 2009

L t ti i t ti l d t liLactation-interruption leads to uncoupling of OTR with its downstream effectors

tERK 2-WB42 KDa

OTR-IP

Gαq/11 subunits WB40 KDa

66 KDa

OTR-IP

Gαq/11 subunits-WB

66 KDa

40 KDa

Nasal OXT restores milk ejection patternNasal OXT restores milk ejection pattern-intramammary pressure assay

1 mU OXT (i.v.)

Normal lactation

1 mU OXT (i.v.)

Lactation interruption

Interruption plus OXT

5 min5 min

Roles of OXT in lactation-failure

1. Lactation interruption-caused lactation failure is due to a malfunction of OXT neurons and their failure to respond to suckling stimulation.

2. The malfunction of OXT neurons is related to an uncoupling between OXT receptors and the downstream signals, such as GqG protein and ERK 1/2.

3. The malfunction of the OXT-secreting system leads to the failure of OXT secretion into the blood during suckling, and the failure of milk letdown.

4 Nasal application of OXT during lactation interruption can rescue4. Nasal application of OXT during lactation interruption can rescue the milk letdown reflex.

III. Oxytocin and lactation-failure associated diseasesassociated diseases

III I P t t d iIII-I. Postpartum depression1. Postpartum depression (PPD) affects up to 15% of mothers

(P l t i t l 2009)(Pearlstein et al, 2009).

2. Women with depressive symptoms in the early postpartum period may be at increased risk for negative infant feedingperiod may be at increased risk for negative infant-feeding outcomes (Dennis and McQueen, 2009).

3 Early cessation of breastfeeding or failure to breastfeed was3. Early cessation of breastfeeding or failure to breastfeed was associated with an increased risk of maternal postpartum depression (Ip, et al, 2009).

4. However, almost all the data were gathered from observational studies so the causality of postpartum depression and breastfeeding failure remains to be verified.

Depressive signs in lactation interruptedDepressive signs in lactation-interrupted rat dams

Wang and Hatton, Frontiers in Neuroscience, 2009

III II OXT and breast cancerIII-II. OXT and breast cancer1. According to the American Cancer Society, over a woman's lifetime,

the chance of developing invasive breast cancer is about 12%. 2. Investigations based on special populations reveal a strong cancer

preventive effect of breastfeeding. For instance, among women with a first-degree family history of breast cancer, breastfeeding cut the i k f b t b 59 t (St b t l 2009) A drisk of breast cancer by 59 percent (Stuebe et al., 2009). And

among younger African-American women, up to 68% of basal-like breast cancer could be prevented by promoting breastfeeding and reducing abdominal adiposity (Millikan et al 2008)reducing abdominal adiposity (Millikan et al, 2008).

3. Systematic review of the literature of all types of studies failed to reveal a consistent effect of insufficient milk supply on breast cancer risk (Cohen et al 2009)risk (Cohen et al, 2009).

4. Thus, a causal relationship between lactation failure and general breast cancer probability remains to be established.

Effects of OXT on H O evokedEffects of OXT on H2O2-evoked expression of Cox-2 in mammary glands

from weaning ratsfrom weaning rats

Th ti t ti l f OXTTherapeutic potential of OXT1. Lactation failure is also accompanied by depressive signs, which are related to

the lack of brain OXT from the SON and/or PVN.2. OXT can increase serotonin release (Yoshida et al, 2009), and lack of

serotonin is related to the occurrence of postpartum depression. Thus, timely application of OXT may prevent maternal depression during lactation interruption and prevent lactation failure in depressive mothersinterruption and prevent lactation failure in depressive mothers.

3. The intermittent pattern of OXT actions during suckling can effectively suppress the proliferative reaction of mammary tissue to oxidative stress, accounting for the reduction in susceptibility to carcinogens following sufficient breastfeeding.

4. Lactation failure increases the incidence of premenopausal breast cancer, whereas nasal application of OXT can restore the regulation of milk letdown. Thus appropriately applying OXT has the potential to reduce the risk ofThus, appropriately applying OXT has the potential to reduce the risk of premenopausal breast cancer in non-breastfeeding mothers or mothers with insufficient lactation. From present result, we predict that OXT can also reduce breast cancer incidence in years following the weaning.

IV. Issues about publicly using OXT

AcknowledgmentsAcknowledgments• Previous mentors of this work• Hideo Negoro, PhD

Academic advices and helps• Williams R. Crowley, PhDg ,

• Hiroshi Yamashita, PhD• Glenn I. Hatton, PhD

y• Harold Gainer, PhD• Joan Y. Summy-Long, PhD• Jeffrey G. Tasker, PhD

• Critical Reading of David S. Knight, PhD.

• Video editing of Xiaoli Liu, PhD.

y ,• Jonathan B. Wakerley, PhD• John A. Russell, PhD• Takashi Higuchi, PhDVideo editing of Xiaoli Liu, PhD.

and Hai-Peng Yang, MD.

• All members of the laboratory

g ,• Kazumasa Honda, PhD• Diansheng Yang, MD

for ongoing discussions • Kathryn A. Hamilton, PhD

Research has been supported by NIH, Japanese Monbusho, Jiamusi University, UC-Riverside, and LSUHSC-Shreveporty, , p