This invited video lecture in Translational Biomedicine presents the current understanding of neurochemical mechanisms underlying milk-letdown reflex, and experimental evidence supporting a therapeutic role of oxytocin in some lactation failure-associated diseases.

1. Oxytocin: the key to treating
lactation failure and associatedlactation-failure and associated
diseases
Invited video lecture by Translational Biomedicine
Yu-Feng Wang, MD, PhD
Department of Cellular Biology and Anatomy
Louisiana State University Health Sciences Center-
Shreveport LA USAShreveport, LA, USA

2. Breastfeeding/lactationBreastfeeding/lactation
1. 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).

3. 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

4. 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
PRL
stimuli
Spinal cordMyoepithelia
Milk
Mammary
nerve
Circulation
Breast
Milk

6. S kli i b fi i f OXTSuckling triggers burst firing of OXT
neurons in the supraoptic nucleus (SON)
Paired extracellular recordings
kes/s
Left SON
40spik
Right SON
Intramammary pressure
OXT
g
2 min
1mU
Wang and Hatton, 2004

7. Burst firing of OXT neurons evoked inBurst firing of OXT neurons evoked in
brain slices
Burst
Wang and Hatton, 2004
Wang and Hatton, 2007g ,

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

9. 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

10. I I Afferent suckling pathway in the CNS
Gating of bursts in
OXT neurons in
I-I. Afferent suckling pathway in the CNS
Synchronization of
bursts in OXT neurons OXT neurons in
the SON & PVN
Lincoln and Wakerley,
1975
bursts in OXT neurons
Belin and Moos, 1986
Mesencephalic
DMH and PH
Dubois-Dauphin
et al., 1985b
1975
Lateral cervical
nucleus
Mesencephalic
Lateral tegmentum
Juss and Wakerley, 1981
Lateral funiculus
of the spinal cord
nucleus
Dubois-Dauphin
et al., 1985a Fukuoka et al, 1984,

11. 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).

12. I II Synaptic innervationI-II. Synaptic innervation
1. 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, )

13. B t i t d ti tBurst-associated synaptic events
BNST, PNZ, TMTakano et al.,
Boudaba and Tasker, 2006
Israel et al 2003
Pittman et al, 2000
SON
PVN
Takano et al.,
1990
B d
Israel et al, 2003
Dorsal medial
hypothalamus
PVN
Wakerley
and Lincoln 1973
Brussaard,
1995
Ventral posterior
hypothalamus
hypothalamus and Lincoln, 1973
Takano et al., 1992
Honda & Higuchi, 2007
NTS
hypothalamus
Weiss et al., 1989
Tribollet et al., 1985
Moos et al., 2004 Cumbers et al, 2007,

14. 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
AVP
neurons
lamina
Astrocytes (GFAP staining)
Hatton and Wang , 2008
neurons

15. 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
Milk
l td
OXT
12 mM K+
i OXT
Modified from Wang and Hatton, 2009
letdown in OXT
20 μM

16. 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.

17. I IV N h i l i t d b tI-IV. Neurochemical environment and bursts
1. 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).

18. Burst-associated neurochemicalBurst associated neurochemical
environment around OXT neurons
Suckling/OXT Suckling/OXT
Before burst After burst
Ca2+
OXT
Glu
Oxytocin
neuron
Glu
K+K+
K+ HAGl
Oxytocin
neuron
Oxytocin
neuron
PGsATP
Ca2+
Glu Glu
GABAGABA
NE
K
Glu Glu
GABAAdenosine
K
K+K+Ca2+Ca2+
K+ HAGlu
Astrocyte
Hatton and Wang , 2008; Wang and Hamilton, 2009
AstrocyteAstrocyte

19. 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).

20. OXT receptor signaling and bursts

21. 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).

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

23. II. Roles of oxytocin in lactation-y
failure

24. 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

25. 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

26. 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

27. 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 Gq
G 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.

28. III. Oxytocin and lactation-failure
associated diseasesassociated diseases

29. III I P t t d iIII-I. Postpartum depression
1. 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.

30. Depressive signs in lactation interruptedDepressive signs in lactation-interrupted
rat dams
Wang and Hatton, Frontiers in Neuroscience, 2009

31. III II OXT and breast cancerIII-II. OXT and breast cancer
1. 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.

32. Effects of OXT on H O evokedEffects of OXT on H2O2-evoked
expression of Cox-2 in mammary glands
from weaning ratsfrom weaning rats

33. Th ti t ti l f OXTTherapeutic potential of OXT
1. 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.

34. IV. Issues about publicly using OXT

35. 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