Slide 1

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

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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

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Slide 2Breastfeeding 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 at g , gearly 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

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.

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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 use y, y yOXT appropriately.

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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 gneurochemical 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.

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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 blood p y , yp p ycirculation 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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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 the y gmidbrain, 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.

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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 an p y pactive 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

Slide 12Now, 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, perinuclearzone, 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).

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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.

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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 ylactation, astrocytes in the SON show dramatic morphological plasticity, which facilitates burst firing and synchronysynchrony.

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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.

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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 neurochemical p y y genvironment changes.

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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 burst g p ggeneration.

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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.

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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 (Parker g 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).

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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 increase y pextracellular 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.

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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.

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Slide 19In 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 tburst 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).

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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+ conductance p , g p j , ), gand 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.

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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 dramatically y yp p ycompared 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.

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As a whole, neurochemical mechanisms underlying suckling-evoked

Slide 22s 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 GqG 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.

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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.

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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 , gthe failure of breastfeeding following maternal separation.

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Slide 25

Next, let’s see the effect of lactation-interruption on interactions , pbetween 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.

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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.

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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 later p p y g ysuckling stimulation.

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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 GqG 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.

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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.

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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.

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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.

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Slide 30

In our study, it was found that maternal separation causes signs in the dams y, p gsimilar 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.

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Next, let’s see how OXT may be used in prevention of breast cancer.

Slide 31e 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.

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Talk11

Most breast cancer risk factors work through changes in hormone levels that influence the development of breast pepithelium. 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 tonic yp , papplication of OXT on hydrogen peroxide-induced Cox-2 expression in mammary glands in weaning rats.

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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.

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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 gappropriately 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.

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Slide 34

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

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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 gCorrespondingly, 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

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required to clarify the mechanisms and approaches for OXT actions before OXT is really publicly applicable as a “Cuddle chemical.”

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 benefited pp y g yfrom 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

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