1. Slide 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
1

2. Talk 1
I Y F W I th k f i t t iI am Yu-Feng Wang. I thank everyone for interest in
my topic. It is my great pleasure to address the
neurochemical mechanisms underlying breastfeedingneurochemical mechanisms underlying breastfeeding
and the therapeutic potential of oxytocin in solving
breastfeeding and associated problems.g p
2

3. Slide 2
Breastfeeding is the feeding of an infant or young child with breast
milk directly from female human breasts via lactation or nursing.
Breastfeeding has many benefits for both mother and baby, such as
d i th i id f di b t d b itreducing the incidence of diabetes and obesity.
To have the full benefit of breastfeeding, the World Health
Organization (WHO) recommends exclusive breastfeeding for the firstOrganization (WHO) recommends exclusive breastfeeding for the first
six months of life and then supplemented breastfeeding for at least
one year.
Breastfeeding is a natural human activity, while nursing difficulties are
not uncommon. According to the Centers for Disease Control (CDC),
among children born in 2007 in the USA , the rate of breastfeeding atg , g
early postpartum was 75.0%, at 6 months of age was 43.0%, and at
12 months was 22.2%. Apparently, more than 50% of mothers failed
to breastfeed their babies adequately, and thus face a risk of lactation-
failure associated diseases such as postpartum depression and
premenopausal breast cancer. 3

4. Talk 2
Recently, enormous efforts have been made to study the
failure to start lactation for about 25% of mothers, such as
those with preterm babies. However, mechanisms ofthose with preterm babies. However, mechanisms of
lactation-failure for about 32% of mothers who have once
started breastfeeding are largely unknown. To improve
breastfeeding rates and prevent lactation failure andbreastfeeding rates and prevent lactation failure and
associated diseases, understanding the mechanisms that
promote normal breastfeeding is an essential step. Our
approach of the last 17 years tries to achieve better
understanding of neurochemical mechanisms responsible
for the milk-letdown reflex, or milk-ejection reflex. Infor the milk letdown reflex, or milk ejection reflex. In
addition, we have also started to consider the translation
of our results into treatment and prevention of lactation-
failure associated diseasesfailure associated diseases.
4

5. Slide 3
In this lecture, I will first discuss neurochemical mechanisms
underlying the milk letdown reflex, particularly suckling-elicited burst
fi i f OXT di t d b OXTfiring of OXT neurons mediated by OXT.
Then, I will use data from a lactation-failure rat model to show what
happens to OXT neurons during lactation failure and to confirm thehappens to OXT neurons during lactation failure and to confirm the
potential to use oxytocin to treat lactation failure.
Following this section I will discuss how oxytocin may be useful inFollowing this section, I will discuss how oxytocin may be useful in
treating or preventing lactation-failure associated diseases.
Finally, I will briefly mention some of my considerations of how to usey, y y
OXT appropriately.
5

6. Talk 3
S f l b tf di i l ilk d tiSuccessful breastfeeding involves milk production,
secretion and ejection controlled by a series of
humoral and neural processes. Among them,p g
neurochemical events leading to milk letdown or milk
ejection are the most fragile processes. Thus,
studying the milk-letdown reflex is critical to solvingstudying the milk-letdown reflex is critical to solving
breastfeeding problems.
6

7. Slide 4
The reflex involves five basic links: first, sensing the baby’s sucking at
the receptors at nipples; second conducting neural impulses along thethe receptors at nipples; second, conducting neural impulses along the
afferent pathway mediated by mammary nerves; third, relay stations in
the spinal cord and integration in the brain; fourth, conduction along
efferent pathways via neural stalk, neurohypophysis and bloodp y , yp p y
circulation and; fifth, myoepithelial effectors in the mammary gland. In
humans, this reflex can be conditioned by auditory, olfactory and visual
stimuli, which can indirectly activate the efferent pathway of the letdown
reflex at the hypothalamus.
In studying this reflex, the most mysterious processes are the afferent
l l di t th ti ti f ll l t ineural processes leading to the activation of magnocellular oxytocin
(OXT) neurons in the supraoptic and paraventricular nuclei in the
hypothalamus. These two nuclei are also called as, SON and PVN.
7

8. Talk 4
In studying the regulation of OXT neuronal
activity a lactating rat model is often used Toactivity, a lactating rat model is often used. To
help us understand the specifics of what
happens in OXT neurons during breastfeedinghappens in OXT neurons during breastfeeding,
let’s see a short movie revealing the feature of
the milk letdown freflex in lactating rats.
8

9. Slide 5
At the beginning of suckling, a litter of 10 pups is attached to the
nipples, and both mother rat and pups appear asleep. After a longnipples, and both mother rat and pups appear asleep. After a long
latency, all the pups suddenly suck the nipples actively, appearing as a
simultaneous stretch reaction, which indicates the occurrence of milk
letdown reflex.
9

10. Talk 5
In the whole suckling process, milk-letdown occurs
intermittently, echoing patterned firing activity in OXT
neurons and its ensuing bolus release of OXT Nowneurons and its ensuing bolus release of OXT. Now
let’s see what happens to hypothalamic OXT
neurons.
10

11. Slide 6
Here is an example of simultaneous recordings of the firing activity of
OXT neurons and intramammary pressure. In the first 10 min of
suckling, the firing rate of OXT neurons remains stable. Then, shortly
before the occurrence of milk ejection as indicated by the increase of
intramammary pressure, the firing rate of OXT neurons of the right
and left SON suddenly increases to 20-40 times higher than that
b f th ilk j ti Thi tt d fi i ti it i ll d ilkbefore the milk ejection. This patterned firing activity is called a milk-
ejection burst, or a burst. The burst and ensuing milk ejection recur
intermittently with an interval of several minutes while the basal firing
rate increases periodically between two burstsrate increases periodically between two bursts.
11

12. Slide 7
Interestingly, the burst firing can also be evoked in brain slices by
i l ti th h i l i t d t isimulating the neurochemical environment around oxytocin
neurons. The figure in the top panel shows the burst-firing of OXT
neurons evoked by phenylephrine, an alpha 1 adrenergic agonist,
in a low calcium artificial CSF The lower panel shows exemplaryin a low calcium artificial CSF. The lower panel shows exemplary
burst firing evoked by OXT.
This finding provides a novel approach to study neurochemicalThis finding provides a novel approach to study neurochemical
modulatory process of the reflex at hypothalamic level.
12

13. Slide 8
Th b t h i iti l f t f th ilk l tdThe burst phenomenon is a critical feature of the milk letdown
reflex. Synchronous burst of a large pool of OXT neurons triggers
a bolus release of OXT and causes milk letdown.
Correspondingly studying neurochemical mechanismsCorrespondingly, studying neurochemical mechanisms
responsible for the burst and the transient milk-letdown, become
the most important step in understanding the letdown reflex.
13

14. Slide 9
From suckling stimulation to the occurrence of burst firing in OXT
neurons, many modulatory levels are involved, including the afferent
neural pathway of suckling signals synaptic input and glial neuronalneural pathway of suckling signals, synaptic input and glial-neuronal
interaction, neurochemical environment, receptors and intracellular
signaling processes, and electrogenic organelle activity, etc.
14

15. Slide 10
First let’s see the afferent pathway in the schematic drawing SucklingFirst, let s see the afferent pathway in the schematic drawing. Suckling
signals carried by mammary nerves enter the spinal cord, relay in the
lateral cervical nucleus, and then cross to the contralateral side of the
brainstem at the medulla. Relayed in the lateral tegmentum of they g
midbrain, the afferent signals enter the hypothalamus diffusely, primarily
terminating in the dorsal medial and posterior hypothalamus, but
apparently not directly in the SON and/or PVN. Thus, suckling
information is likely integrated before being sent to OXT neurons via
local neural circuits.
15

16. Our studies focus on the integrative processes of afferent neural
Slide 11
pathways in the brain for synchronizing bursting among OXT neurons.
We have found that,
1. Afferent inputs from the lateral tegmentum partially cross to the
t l t l id f th h th l (W t l 1995)contralateral side of the hypothalamus (Wang et al, 1995);
2. This crossing pathway is responsible for the summation of suckling
signals, which is a basis of burst generation (Wang et al, 1996);
3 Burst synchrony of OXT neurons in the SON and PVN of bilateral3. 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);
4 OXT neurons have mutual structural and functional connections with4. OXT neurons have mutual structural and functional connections with
the nuclei of the mammillary body and a special group of interneurons
in the SON and perinuclear zone, which provides a periodic synaptic
input to OXT neurons with an inhibitory period before the burst and anp y p
active period following the burst (Wang et al, unpublished data);
5. Mammillary body and associated structures innervate bilateral OXT
neurons simultaneously while receiving feedback modulation from
OXT neurons, and function as a “Synchronization center” (Wang et al,
8th WCNH, 2009). 16

17. Slide 12
Now, Let’s see some structural and functional features of synaptic
f O
1. Direct synaptic innervation of OXT neurons is limited to a few
brain areas including the nucleus of the solitary tract (NTS)
innervation of OXT neurons during lactation and suckling.
brain areas including the nucleus of the solitary tract (NTS),
posterior hypothalamus, dorsal medial hypothalamus, perinuclear
zone, bed nucleus of the stria terminalis (BNST), and SON and
PVN on the contralateral side (Wakerley etal, 1994).PVN on the contralateral side (Wakerley etal, 1994).
2. Lactation increases the number of direct synapses on OXT
neurons (Hatton et al, 2004; Theodosis et al,2008);
3. OXT reduces tonic EPSCs (Kombian et al, 1997, Pittman et al,
2000) and IPSCs (Brussaard, 1995), but increases intermittent
clustered EPSCs (Israel et al, 2003; Wang and Hatton, 2004,
2007, 2009) and likely clustered IPSCs as well (Moos 1995);
4 OXT li i i di h i i i f h BNST4. OXT can elicit periodic changes in synaptic inputs from the BNST
(Lambert et al, 1994), histaminergic tuberomammillary nuclear
neurons and intra-SON interneurons (Wang et al, 8th WCNH,
2009) as well as a fraction of perinuclear zone neurons (Dyball &2009), as well as a fraction of perinuclear zone neurons (Dyball &
Leng, 1986).
17

18. Slide 13
During suckling, afferent neural signals directly or indirectly activate
these neural structures having direct synaptic connections with OXT
neurons in the SON and PVN. OXT neurons of bilateral sides
simultaneously receive noradrenergic, glutamatergic, oxytocinergic,
GABAergic, and histaminergic synaptic innervation. These synaptic
i fl d ti f t ti i t hil i i thinfluences reduce continuous fast synaptic input while increasing the
incidence of intermittent clustered synaptic input on OXT neurons. In
addition, synaptic input also directly modulates OXT neuronal activity
by periodically releasing neurotransmitters time locked with theby periodically releasing neurotransmitters, time-locked with the
burst firing.
These activities form a facilitative environment around OXT neuronsThese activities form a facilitative environment around OXT neurons
while providing a trigger for burst generation.
18

19. Talk 6
While receiving presynaptic innervation, magnocellular
OXT l d t i d l ti bOXT neurons are also under tonic modulation by
surrounding astrocytes. As reported by Hatton’s lab and
Theodosis' lab, during transition from pregnancy to, g p g y
lactation, astrocytes in the SON show dramatic
morphological plasticity, which facilitates burst firing and
synchronysynchrony.
19

20. Slide 14
Most astrocytes are located in the ventral region of the SON theMost astrocytes are located in the ventral region of the SON, the
ventral glial lamina. As shown by immunostaining for GFAP, the
astrocyte cytoskeletal element, these ventrally-located astrocytes
send processes dorsally and form a natural barrier betweensend processes dorsally and form a natural barrier between
neighboring OXT neurons as indicated by combined immunostaining
of OXT-neurophysin.
The question is what happens to astrocytes during suckling/OXT
actions.
20

21. Slide 15
By sampling the SON at different stages of suckling and simulating the
neurochemical environment around oxytocin neurons in brain slices, we
have observed a GFAP-mediated acute astrocyte plasticity in response
t kli OXT ti l ti B f kli /OXT ti l tito suckling or OXT stimulation. Before suckling/OXT stimulation,
astrocyte processes with scaffolding of GFAP lie between membranes
of neighboring OXT neurons. Suckling reduced the abundance of GFAP
filaments which was partially reversed after the occurrence of milkfilaments, which was partially reversed after the occurrence of milk
ejection. Simulating the increased release of OXT during suckling by
incubation of the slices with OXT, we also observed a GFAP reduction;
and a reversal of OXT-elicited GFAP reduction was simulated byand a reversal of OXT elicited GFAP reduction was simulated by
transient 12 mM K+ exposure, a phenomenon observed in OXT-
secreting system accompanying the milk ejection. Thus, astrocyte
plasticity mirrors OXT neuronal activity and ensuing neurochemicalp y y g
environment changes.
21

22. Slide 16
F th t di i di t th tFurther studies indicate that,
1. 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).
3. Suckling and OXT cause acute retraction of astrocyte processes
d OXT (W d H tt 2007) b d l i iaround OXT neurons (Wang and Hatton, 2007) by depolymerizing
GFAP filaments (Wang and Hatton, 2009), which reflects the
dynamic activity of OXT neurons via neurogenic neurochemical
changeschanges.
4. GFAP plasticity modulates OXT neuronal activity by changing water
transportation, morphology, and glutamate metabolism in astrocytes.
5 In addition astrocyte plasticity is also related to increased5. In addition, 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 burstg p g
generation.
22

23. Talk 7
Contributions of synaptic input and astrocyte modulationContributions of synaptic input and astrocyte modulation
of oxytocin neuronal activity are mainly achieved via
release of neurochemicals such as OXT, norepinephrine,
l t t t l di d ATP Cl ifi ti f thglutamate, prostaglandins, and ATP. Clarification of the
regulation of local neurochemical environments will
provide further insight into the burst generation process.
Now, let’s see the relationship between local
neurochemical environment and the burst in OXTneurochemical environment and the burst in OXT
neurons.
23

24. Slide 17
1. Suckling increases intra-SON and PVN release of OXT (Neumann et
al, 1993, Bealer and Crowley, 1998).
2. OXT release during suckling depends on actions of glutamate (Parkerg g p g (
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 I d l ti f OXT l i ti i t ti b t3. 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 essential.
4 Prostaglandins (Wang and Hatton 2006) ATP and adenosine (Ponzio4. Prostaglandins (Wang and Hatton, 2006), ATP and adenosine (Ponzio
et al, 2006) from astrocytes contribute to the changes in the burst-
related extracellular milieu.
5. In addition, 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).
24

25. Slide 18
The firing activity of oxytocin neurons is closely related to their chemical
environment. As shown in the figure, during suckling, tonic synaptic input
and retraction of astrocyte processes around OXT neurons increasey p
extracellular levels of OXT, glutamate, PGs, K+, and others while GABA
and Ca2+ levels are reduced, which creates a facilitatory chemical
environment for synchronous burst firing. When OXT neurons are ready to
discharge bursts intrinsically, bolus release of glutamate will trigger burst
activity. Synchronized burst of a large pool of OXT neurons dramatically
changes the local neurochemical environment again, particularly a large
i i t ll l K+ l l hi h lt i i f t tincrease in extracellular K+ level, which results in re-expansion of astrocyte
processes. Meanwhile, ATP is converted to adenosine, and GABA and
taurine are likely released from astrocytes, contributing to postburst
inhibition of OXT neuronal activity Following these transient changes theinhibition of OXT neuronal activity. Following these transient changes, the
extracellular neurochemical environment will largely return to that at the
beginning of suckling stimulation, and a new cycle toward bursting begins.
25

26. Talk 8
Burst discharges are directly determined by membraneBurst discharges are directly determined by membrane
electrogenic activities of OXT neurons in certain spatial
and temporal orders. However, none of the suckling-
associated neurochemicals elicit bursts directly; thus, a
modification of cellullar/molecular signaling processes in
OXT neurons is needed to prepare OXT neurons toOXT neurons is needed to prepare OXT neurons to
discharge a burst.
26

27. Slide 19
In studying potential signaling processes determining the inherent
1. OXT receptor (OTR) is expressed in both neurons and astrocytes
study g pote t a s g a g p ocesses dete g t e e e t
burst capacity, it has been found that,
1. OXT receptor (OTR) is expressed in both neurons and astrocytes
in the SON (Wang and Hatton, 2006).
2. The major signaling pathway of the 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. In OTR signaling, dynamically changing the phosphorylation of
ERK1/2 in a unique spatiotemporal order can trigger burst (Wang
and Hatton 2007b)and Hatton, 2007b).
5. Moreover, OXT induces Cox-2 and promotes prostaglandin (PG)
synthesis in OXT neurons and astrocytes, promotes actin
polymerization (Wang and Hatton 2006) and facilitates burstspolymerization (Wang and Hatton, 2006), and facilitates bursts
(Wang and Hatton, 2007b).
27

28. Slide 20
As shown in the schematic drawing, suckling-elicited synaptic input and
astrocyte plasticity lead to increases in somatodendritic release of OXT in
the hypothalamus. By mobilizing the Gβγ-dominant signaling cascade,
OXT i h h l ti f ERK 1/2 Ph h ERK 1/2 t thOXT increases phosphorylation of ERK 1/2. PhosphoERK 1/2 together
Gαq subunit signaling, induces Cox-2 and synthesis of prostaglandins.
PhosphoERK 1/2 coordinated with prostaglandin-elicited protein kinase A
(PKA) signaling causes reorganization of actin filaments and ensuing(PKA) signaling causes reorganization of actin filaments, and ensuing
changes in the activity of electrogenic organelles. The functions of
posphoERK 1/2 and PKA are antagonistic in general in OTR signaling.
However by eliciting their activities in a burst-favorable spatiotemporalHowever, by eliciting their activities in a burst favorable spatiotemporal
order, such contradictory signals are highly coordinated to change the
state of membrane electrogenic organelles (e.g., ion channels, pumps,
transporters, gap junctions, etc.), leading to decreases in K+ conductancep , g p j , ), g
and increases in Na+, Ca2+ and non-selective cation currents. When
changes in the local neurochemical environment are phase-locked with
these intracellular signaling processes, a burst will occur.
28

29. Relative to other levels, electrical features supporting burst
Slide 21
firing are not well understood yet.
To explain the transition between low-frequency basal firing and high-
frequency synchronized burst, a gating mechanism and a synchronization
mechanism were proposed in early studies.
Later, Hatton and colleagues identified an increased incidence of junctional
coupling among OXT neurons in lactating rats, which likely facilitates thecoupling among OXT neurons in lactating rats, which likely facilitates the
burst gating and synchrony. Then, Stern and Armstrong found, there is a
rebound depolarization, following transient hyperpolarization of membrane
potential in OXT neurons in lactating rats, which supports a short burst firing.
I i it b t fi i d l h l f d th t di b tIn an in vitro burst firing model, we have also found that preceding a burst,
the rising slope of the afterhyperpolarization is decreased while the rising
slope of spikes is increased. Further analysis reveals, in burst firing neurons,
the decay time course of the afterhyperpolarization is reduced dramaticallyy yp p y
compared to the non-burst firing neurons (Wang and Hatton, 2004). All these
features give OXT neurons a specific capacity to fire spikes in bursts, likely
via a dramatic decrease in K+ currents, an increase in the Na+
currents and a larger hyperpolarization activated inward current on the basiscurrents and a larger hyperpolarization-activated inward current on the basis
of a general excitation of OXT neuronal activity.
29

30. As a whole, neurochemical mechanisms underlying suckling-evoked
Slide 22
s a o e, eu oc e ca ec a s s u de y g suc g e o ed
burst firing in OXT neurons through OXT can be summarized as
follows:
During suckling, a tonic afferent suckling message from the nipples
increases OXT level in the SON and PVN. By activating OTR and Gq
G proteins, OXT increases phosphoERK 1/2 in the somata and PKA in
the processes in a burst-associated temporal order. In astrocytes,
OXT t ti f t t f th di fOXT causes retraction of astrocyte processes from the surrounding of
OXT neurons, creating a facilitatory neurochemical environment. In
OXT neurons, reorganization of actin filaments occurs, preparing OXT
neurons to discharge bursts intrinsically Meanwhile presynaptic tonicneurons to discharge bursts intrinsically. Meanwhile, presynaptic tonic
inhibition of glutamatergic transmission facilitates bolus release of
glutamate, forming a trigger for burst firing. Together, recurring
retraction and expansion of astrocyte processes changing the localretraction and expansion of astrocyte processes, changing the local
neurochemical environment, activating intrinsic signaling processes in
OXT neurons, adding a bolus release of glutamate from presynaptic
terminals result in rhythmic, synchronous bursting of OXT neurons,y , y g ,
bolus release of OXT, and therefore, intermittent milk ejections.
30

31. Slide 23
Theoretically, disrupting the modulatory process at any level will result inTheoretically, disrupting the modulatory process at any level will result in
a failure of activation of OXT neurons and the letdown reflex. Lacking
suckling stimulation is the most common reason for lactation failure,
particularly at the start of breastfeeding; however, neural mechanisms
underlying lactation failure in those with successful breastfeeding
experience remain unknown. Moreover, experimental evidence
regarding therapeutic roles of OXT in lactation-failure mothers is still
controversial. To restore breastfeeding efficiently, it is necessary to
examine what happens to OXT neurons during lactation failure and how
oxytocin can be used to restore breastfeeding.
In the next section, let’s see the role of oxytocin in
lactation failure.
31

32. Slide 24
Here, I will use the data from a lactation-failure rat model, to show
what happens to OXT neurons and to confirm the potential of using
O fOXT to treat lactation failure.
In this study, we separated a rat dam from her pups for 20 h per
day for four days and pups were nursed by another mother duringday for four days, and pups were nursed by another mother during
the separation. We then observed electrical responses of OXT
neurons in the SON in the anesthetized mother rats after the four-
day separation As we can see in the figure suckling caused aday separation. As we can see in the figure, suckling caused a
burst discharge in the normal lactating rat, but failed to trigger burst
firing in the lactation-interrupted rat. This result indicates
malfunction of OXT neurons, accounting for the lack of OXT and, g
the failure of breastfeeding following maternal separation.
32

33. Slide 25
Next, let’s see the effect of lactation-interruption on interactions, p
between OTR and its downstream signals. We first immuno-
precipitated OTR, and then detected Gaq/11 subunits and total ERK
(tERK) 2 in Western blot. As shown in the figure, lactation interruption
significantly reduced molecular association between OTR and tERK 2
in the upper panel, and OTR and Gaq/11 subunits in the lower panel,
compared to those in normal lactating rats.
Thi lt i di t f ti l li f OTR ith G /11 GThis result indicates functional uncoupling of OTRs with Gaq/11 G
proteins and ERK proteins, resulting in the failure of burst in OXT
neurons.
33

34. Talk 9
From the results presented above, we can see, lactation
failure is due to malfunctions of OXT neurons. Since
activation of OXT neurons by suckling is mediated byactivation of OXT neurons by suckling is mediated by
local release of OXT, nasal delivery of OXT can access
brain without the blockade of blood brain barrier, thus,
nasal OXT delivery should maintain OXT neuronal
activity during lactation interruption.
To test this hypothesis, we further examined effects of
nasal OXT on intramammary pressure changes during
suckling stimulation in lactation interrupted ratssuckling stimulation in lactation-interrupted rats.
34

35. Slide 26
In the figure, the top row shows, suckling caused intermittent
i i i t i l l t ti t thincreases in intramammary pressure in normal lactating rat; the
middle row shows, lactation-interruption suppressed the recurrence of
milk ejections; and the bottom row shows, when OXT was supplied
nasally during lactation interruption regularity of the milk ejectionsnasally during lactation interruption, regularity of the milk ejections
was largely restored.
Noteworthy is, the magnitudes of OXT-elicited intramammary
pressure changes are the same among the three groups supportingpressure changes are the same among the three groups, supporting,
the failure of the milk-letdown reflex was due to the lack of OXT.
Thus, nasal application of OXT during lactation interruption can
maintain the responsive capacity of the OXT-secreting system to laterp p y g y
suckling stimulation.
35

36. Th l f t i i l t ti f il b i d f ll
Slide 27
The roles of oxytocin in lactation-failure can be summarized as follows:
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 t i d ERK 1/2G protein and ERK 1/2.
3. As a consequence, the malfunction of the OXT-secreting system
leads to the failure of OXT secretion into the blood during
suckling and the failure of milk letdownsuckling, and the failure of milk letdown.
4. Finally, nasal application of OXT during lactation interruption can
rescue the milk letdown reflex.
36

37. Talk 10
This result is directly meaningful for those service women
or working mothers who have to be separated from their
babies for weeks or longer while having the desire tobabies for weeks or longer while having the desire to
breastfeed their babies later. This result also sets a strong
experimental basis for using OXT to treat lactation failure-
associated diseases.
37

38. Slide 28
In the next section, I want to look at potential use of OXT to
treat or prevent lactation-failure associated diseases, such as
t t d i d l b tpostpartum depression and premenopausal breast cancer.
38

39. First let’s consider postpartum depression
Slide 29
First, let s consider postpartum depression.
Lactation failure is associated with a high incidence of anxiety and
other mood disorders, including postpartum depression (PPD) .other mood disorders, including postpartum depression (PPD) .
Studies have revealed that postpartum depression affects up to 15%
of mothers (Pearlstein et al, 2009). At one hand, women with
depressive symptoms in the early postpartum period may be at
increased risk for negative infant-feeding outcomes (Dennis and
McQueen, 2009). On the other hand, early cessation of breastfeeding
or not breastfeeding was associated with an increased risk of
maternal depression (Ip, et al, 2009).
However, almost all the data have been gathered from observational
studies and the causal relationship between postpartum depressionstudies, and the causal relationship between postpartum depression
and breastfeeding failure remains to be verified.
39

40. Slide 30
In our study, it was found that maternal separation causes signs in the damsy, p g
similar to those seen in postpartum depression in humans (Wang and Hatton,
2009). As shown in the figure, lactation interruption significantly reduced the
interest of mother rats in their offspring, as indicated by the longer latency
d d d f t li k th I dditi t l b d i htand reduced frequency to lick the pups. In addition, maternal body weight
gains are also reduced significantly compared to normal lactation rats. It is
clear, dam-pup separation caused maternal depression.
As observed in the lactation restoration, nasal OXT also increased the
interest of the dams in their pups in the rescue of lactation. Thus, curing
lactation failure should also relieve maternal depression.
40

41. Next, let’s see how OXT may be used in prevention of breast cancer.
Slide 31
e t, et s see o O ay be used p e e t o o b east ca ce
1. According to the American Cancer Society, over a woman's lifetime,
the chance of developing invasive breast cancer is about 12%.the chance of developing invasive breast cancer is about 12%.
2. Interestingly, 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 risk 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).
3. However, a systematic review of the literature of all types of studies
failed to reveal a consistent effect of insufficient milk supply onfailed to reveal a consistent effect of insufficient milk supply on
breast cancer risk (Cohen et al, 2009).
4. Thus, a causal relationship between lactation failure and general
breast cancer probability remains to be establishedbreast cancer probability remains to be established.
41

42. Talk11
Most breast cancer risk factors work through changes in
hormone levels that influence the development of breastp
epithelium. OXT induces a significant differentiation of
epithelial cells during lactation, and reduces breast
cancer susceptibility If breastfeeding really has anticancer susceptibility. If breastfeeding really has anti-
cancer roles, pulsatile OXT actions like that during
nursing should be more effective than tonic actions
under other phsiological conditions in suppressing the
proliferative activity in mammary tissues. To test this
hypothesis, we observed effects of pusatile versus tonicyp , p
application of OXT on hydrogen peroxide-induced Cox-2
expression in mammary glands in weaning rats.
42

43. Slide 32
As shown in the figure, treatment of the mammary tissues with 50
μM hydrogen peroxide for 40 min significantly increased Cox-2
levels. Simultaneous application of 0.1 nM OXT with hydrogen
peroxide produced different results depending on the patterns of
OXT application. It was only the pulsatile application of OXT that
significantly reduced hydrogen peroxide-induced Cox- 2
i Thi lt l l i di t th t l til OXT tiexpression. This result clearly indicates that pulsatile OXT actions
rather than tonic OXT actions have strong anti-proliferative effects
following oxidative stress in mammary glands, and demonstrates a
causal relationship between lactational OXT and reducedcausal relationship between lactational OXT and reduced
mammary tumorigenesis.
43

44. From our preliminary studies presented above, we consider the
potential of OXT in treating or preventing lactation-failure
Slide 33
associated diseases as follows:
1. Lactation failure is also accompanied by signs of depression, which are related to
the lack of brain OXT from the SON and/or PVN.
2 OXT i t i l (Y hid t l 2009) d l k f t i i2. 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 mothers with postpartum depression.ac a o a u e o e s pos pa u dep ess o
3. Lactational pattern of OXT actions is also important for prevention of breast cancer.
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 of mammary glands to carcinogens following sufficientreduction in susceptibility of mammary glands to carcinogens following sufficient
breastfeeding.
4. Finally, lactation failure increases the incidence of premenopausal breast cancer,
and nasal application of OXT can restore the regulation of milk letdown. Thus,pp g
appropriately applying OXT has the potential to reduce the risk of breast cancer in
non-breastfeeding mothers or mothers with insufficient lactation. From our present
result, we can also predict that OXT can specifically reduce breast cancer incidence
in years following the weaning one of the surges of breast cancer occurrencein years following the weaning, one of the surges of breast cancer occurrence.
44

45. Slide 34
In the final section, I would like to address several issues regarding
public use of OXTpublic use of OXT.
45

46. OXT is likely beneficial for a large population beyond
b tf di th R tl OXT h b h t t i
Talk 12
breastfeeding mothers. Recently, OXT has been a hot topic
for its effects on social cognition, pair-bonding, enhancing sex
quality, reducing fear, anti-autism, and so on.y g
Correspondingly, OXT nasal spray has become available
commercially. The availability of oxytocin puts forward a
serious question regarding its potential risk to public health Itserious question regarding its potential risk to public health It
is urgent to define the appropriate targets for OXT use, since
unwanted side effects of OXT may occur while pursuing its
b fi i l ff t M d fi i th i t tibeneficial effects. Moreover, defining the appropriate times
and doses as well as patterns of application is also important,
since the action of OXT is strongly time-, dose- and pattern-
dependent. Inappropriately applying OXT may cause effects
opposite to those expected. Thus, further studies are
required to clarify the mechanisms and approaches for OXT
46
required to clarify the mechanisms and approaches for OXT
actions before OXT is really publicly applicable as a “Cuddle
chemical.”

47. Slide 35
At the end of this lecture, I would like to thank my previous supervisors,
who contributed to my studies at different stages of my career. Dr. Negoro,
th fi t t idi t t d th l td fl f i ithe first mentor guiding me to study the letdown reflex from in vivo
approaches, Dr. Yamashita and his associates, teaching me the in vitro
approaches to study OXT neurons, and Dr. Hatton who was my strongest
supporter of translational studies. My studies have also greatly benefitedpp y g y
from many senior scientists; and here I could only name a few of them.
Specific thanks to Dr. Hamilton, who has strongly encouraged me to study
interactions between olfaction and hypothalamic neuroendocrine process. I
would also like to thank Dr Knight for kindly revising this lecture and Drswould also like to thank Dr. Knight for kindly revising this lecture, and Drs.
Liu and yang for video editing. And I appreciate all members of this and
previous labs for ongoing discussions and advice. I also thank the
sponsors of my research.y
Any questions and comments are welcome
47