Prolactin Is Secreted from Lactotrophs in the Anterior Pituitary Gland In

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2009 Control of Prolactin Secretion by Central Oxytocin in Cervically Stimulated Ovariectomized Rats De'Nise T. McKee

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COLLEGE OF ARTS AND SCIENCES

CONTROL OF PROLACTIN SECRETION BY CENTRAL OXYTOCIN IN CERVICALLY

STIMULATED OVARIECTOMIZED RATS

DE’NISE T MCKEE

A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Degree Awarded: Summer Semester, 2009

The members of the committee approve the dissertation of De'Nise T McKee defended on July 9, 2009.

______Marc E Freeman Professor Directing Dissertation

______Timothy M Logan Outside Committee Member

______Richard Bertram Committee Member

______Michael Meredith Committee Member

______J Michael Overton Committee Member

Approved:

______P. Bryant Chase, Chair, Department of Biological Science

The Graduate School has verified and approved the above-named committee members.

ACKNOWLEDGEMENTS

I would like to thank my professor, Marc E Freeman, for his generous support and supervision. He has provided an incredible environment for me to learn and grow scientifically and personally. I am very grateful to all of my current lab members: Richard Bertram, Ruth Cristancho, Cleyde Helena, Arturo Iglesias, Jessica Kennett, Joel Tabak, and Maurizio Tomaiuolo. I am appreciative for all their help and encouragement. I am also grateful to past lab members Marcel Egli, Mike Sellix Natalia Toporikova and Cheryl Fitch Pye for helping me transition into the lab and to Raphael Szawka for his generous assistance in the lab. A special thanks to Maristela Poletini for her unwavering dedication to my training. She has inspired me inside and outside of the lab and I am ultimately grateful for all of her guidance. Thank you to my committee members: Richard Bertram, Tim Logan, Michael Meredith and Michael Overton. They all have been incredibly supportive throughout this process. I am very thankful for the wonderful Neuroscience graduate students, an amazingly supportive group. I would like to acknowledge the technical support of Maurice Manning, Dano Fiore, and Wei Wu. I would like to acknowledge the NIH for financial support through a Minority Training grant, Minority Supplement and lab grants DK43200 and DA19356. I would also like to thank the Biology Department’s financial support through Teaching Assistantships and the support of the faculty and staff of the Neuroscience Program, Biology Department, and LAR.

Finally, I would like to express my gratitude to family and friends for their vital love and encouragement. I thank my mom and dad (Brenda and Dennis), sisters (Chanda and Dia), brothers (Chaji and LaVonne), nieces, nephews, extended family, Chevonne, Lyndsey and Melita. I am very, very grateful for all of your support.

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TABLE OF CONTENTS

LIST OF FIGURES ...... vi

LIST OF TABLES ...... viii

LIST OF ABBREVIATIONS ...... ix

ABSTRACT ...... xii

CHAPTER 1 INTRODUCTION TO PROLACTIN SECRETION ...... 1

Suckling Stimulus ...... 1

Estradiol Stimulus ...... 1

Mating Stimulus ...... 2

Pseudopregnancy...... 3

Termination of mating-induced prolactin surges...... 5

Evidence for a mating-induced mnemonic...... 6

Control of Prolactin Secretion ...... 7

Prolactin inhibiting factors...... 7

Prolactin releasing factors...... 10

CHAPTER 2 OXTYOCIN ACTION AT THE LACTOTROPH IS REQUIRED FOR PROLACTIN SURGES IN CERVICALLY STIMULATED OVARIECTOMIZED RATS ... 15

Materials and Methods ...... 17

Experimental Design ...... 23

Results ...... 24

Discussion ...... 32

CHAPTER 3 NORADRENERGIC PROJECTIONS TO THE HYPOTHALAMIC PARAVENTRICULAR NUCLEUS ARE ACTIVATED BY CERVICAL STIMULATION IN OVARIECTOMIZED RATS ...... 37

Materials and Methods ...... 39

Experimental design ...... 44

Results ...... 44

Discussion ...... 48

CHAPTER 4 ACUTE ACTIVATION OF PARAVENTRICULAR OXYTOCIN NEURONS IN CERVICALLY STIMULATED OVARIECTOMIZED RATS ...... 53

Materials and Methods ...... 54

Results ...... 59

Discussion ...... 61

CHAPTER 5 DISCUSSION ...... 64

Future Directions ...... 70

Appendix A: Copyright Permission Letters ...... 72

Appendix B: IACUC Approval Letter ...... 75

REFERENCES ...... 77

BIOGRAPHICAL SKETCH ...... 97

LIST OF FIGURES

Figure 1. Prolactin secretion in response to reproductive stimuli: ...... 4

Figure 2. Illustration of neuroendocrine dopaminergic neurons...... 8

Figure 3. Activation of signal transduction pathway by prolactin...... 9

Figure 4. Diagram of oxytocin neurons in the paraventricular nucleus ...... 13

Figure 5. Connections within the network model...... 25

Figure 6. Model simulation...... 26

Figure 7. Prolactin secretion in rats with oxytocin antagonist infusion before cervical stimulation...... 29

Figure 8. Prolactin secretion in rats with oxytocin antagonist infusion after cervical stimulation...... 30

Figure 9. Prolactin secretion in cervically stimulated rats two days after cessation of OT antagonism...... 31

Figure 10. Dopamine activity in cervically stimulated rats with and without an oxytocin antagonist...... 33

Figure 11. Representation of fluoro-gold injections...... 45

Figure 12. Immunohistochemistry of brainstem noradrenergic neurons...... 46

Figure 13. The percentage of brain stem noradrenergic neurons in cervically stimulated and non-cervically stimulated rats...... 47

Figure 14. Norepinephrine content in the paraventricular nucleus of rats with and without cervical stimulation...... 48

Figure 15. Acute activation of oxytocin neurons in the paraventricular nucleus in response to cervical stimulation...... 60

Figure 16. Prolactin secretion in response to an oxytocin injection into the lateral cerebral ventricle...... 61

Figure 17. Prominent brain regions involved prolactin secretion in cervically stimulated rats...... 67

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LIST OF TABLES

Table 1 Parameter values used in the model…………………………………………….. 20

Table 2. Antibodies and reagents used in triple-labeled immunohistochemistry……... 42

Table 3. Characteristics of filter detection systems for fluoroscent dyes……………… 43

Table 4. Antibodies and reagants used in double-labeled immunohistochemistry…… 57

Table 5. Characteristics of filter detection systems for fluoroscent dyes……………… 58

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LIST OF ABBREVIATIONS

ABC actin-biotin complex

AL anterior lobe of the pituitary gland

BCA bichinchoninic acid

BNST bed nucleus of the stria terminalis

C celsius

CS cervical stimulation

DAB 3,3'-Diaminobenzidine

DHBA dihydroxybenzylamine

DMH dorsomedial hypothalamus

DOPAC dihydroxyphenylacetic acid

E2 estradiol

EDTA ethylenediaminetetraacetic acid

EGTA ethyleneglycol-bis-(β-aminoethyl ether) N,N,N’,N’-tetraacetic acid

FG fluoro-gold

h hour(s)

H2O2 hydrogen peroxide

HPLC-ED high performance liquid chromatography coupled to electrochemical detection

ICV intracerebroventricular

IHC immunohistochemistry

IL intermediate lobe of the pituitary gland

IR immunoreactivity

LC locus coeruleus

M molarity

ME median eminence

MEA medial amygdala

MEApd posterodorsal division of the medial amygdala

MPOA medial preoptic area

mRNA messenger ribonucleic acid

NE norepinephrine

NGS normal goat serum

NiSO4 nickle sulfate

NL neural lobe of the pituitary gland

OT oxytocin

OVX ovariectomized

P4 progesterone

PBS phosphate-buffered saline

PeVN periventricular nucleus

PHDA periventricular hypophyseal dopaminergic

PRL prolactin

PVN paraventricular nucleus

SCN suprachiasmatic nucleus

SON supraoptic nucleus

TH tyrosine hydroxylase

THDA tuberohypophyseal dopaminergic

TIDA tuberoinfundibular dopaminergic

TRH thyrotropin-releasing hormone

VIP vasoactive intestinal polypeptide

VMH ventromedial hypothalamus

τ tau

ABSTRACT

Prolactin is a protein hormone predominately synthesized in and secreted from cells in the anterior pituitary gland called lactotrophs. Although prolactin is elevated during other physiological states, mating induces a unique pattern of prolactin secretion. Prolactin is elevated twice a day for several days in response to a mating stimulus, suggesting a mnemonic. The neural pathway in which the mating stimulus initiates this unique prolactin secretion is unknown. However, hypothalamic inhibitory neuroendocrine dopamine neurons and stimulatory oxytocin neurons are demonstrated to be involved. Our laboratory has shown that a peripheral injection of oxytocin initiates prolactin surges like those induced by mating. This suggests that oxytocin may initiate the mating-induced prolactin surges. Whether the bolus injection of oxytocin initiated these surges by acting peripherally or centrally was investigated in this dissertation. Results showed that oxytocin does not initiate these surges by acting peripherally since a peripheral oxytocin antagonist did not block the initiation of the surges. However, peripheral oxytocin is important for maintaining the surges. Therefore, central oxytocin must initiate twice daily prolactin surges, likely from the hypothalamic paraventricular nucleus. Since the mating stimulus must convey its signal from the uterine cervix to the hypothalamus a pathway was investigated. A relay of the mating stimulus via brainstem noradrenergic input to the paraventricular nucleus was shown. Only brainstem noradrenergic neurons that projected to paraventricular nucleus were activated by the mating stimulus, suggesting a specific pathway for the initiation of mating-induced prolactin surges. In addition, norepinephrine was released in the paraventricular nucleus in response to a mating stimulus. Lastly, oxytocin neurons in the hypothalamic paraventricular nucleus were directly investigated. Surprisingly, hypothalamic oxytocin neurons were not acutely activated by a mating stimulus, suggesting that the paraventricular nucleus is not a source of oxytocin important in initiating the mating- induced prolactin surges.

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CHAPTER 1 INTRODUCTION TO PROLACTIN SECRETION

Prolactin (PRL) is a protein hormone predominately synthesized in and secreted from cells in the anterior pituitary gland called lactotrophs. PRL receives its name from its original discovery in its role in lactation, but has been implicated in a variety of other physiological functions including fertility, maternal behavior, stress, immune function, growth and development, metabolism, and water and electrolyte balance (Freeman et al. 2000; Grattan & Kokay 2008). Thus, is it also synthesized in numerous tissues throughout the body (for references see Freeman et al. 2000). Much research has been conducted in the rat to determine the control of PRL secretion in various physiological functions, but largely its role in reproduction. This chapter will discuss the control of PRL secretion in response to reproductive stimuli in the female rat.

Suckling Stimulus

The stimulation of suckling pups induces an immediate surge of PRL that is tightly coupled with intensity and duration of suckling (Grosvenor & Mena 1971). PRL increases within 5 minutes of the suckling stimulus and reaches peak levels by 30 minutes. Upon the withdrawal of this stimulus PRL secretion subsides within 10 minutes. Increasing the number of suckling pups further increases the magnitude of PRL secretion (Grosvenor & Whitworth 1974). This is a classical neuroendocrine reflex, that is, an acute stimulus tightly accompanied by a transient response (shown in Figure 1A).

Estradiol Stimulus

The female rat undergoes a 4-5 day estrous cycle characterized by reproductive behaviors, vaginal cell types, and hormone secretions. The days of the estrous cycle are commonly referred to as proestrus, estrus, diestrus 1 and diestrus 2. The days of

the estrous cycle can be distinguished by the presence of specific cell types in vaginal smears. Each day of the estrous cycle is differentiated by the presence of the following vaginal cell types; proestrus by round nucleated epithelial cells, estrus by cornified squamous epithelial cells, and diestrus 1 and 2 by small leukocytes (Freeman 2006). These cells types indicate the condition of the corpus luteum, which immediately forms

after ovulation, on proestrous day, and begins secretion of progesterone (P4). It reaches its maximal size during diestrus 1 and in the absence of fertilization abruptly regresses by the end of diestrus 2 (Freeman 1981; Freeman 2006). PRL levels are basal throughout the estrous cycle except on the afternoon of proestrus when PRL levels increase around 1100 hr and reach peak levels by 1700 hr. PRL levels return to basal by the morning of estrus, around 0700 h (Butcher et al. 1972; Smith et al. 1975).

This surge of PRL is induced by rising levels of estradiol (E2) on diestrus1 and diestrus

2 (Neill et al. 1971; Freeman et al. 1972) and can be mimicked with exogenous E2 (Neill

1972). Once E2 levels subside, so does PRL secretion (Figure 1B). The E2 stimulus is prolonged compared to the suckling stimulus, however both responses are transient.

Mating Stimulus

Mating is dependent upon soliciting and sexual behaviors of the receptive female

when E2 levels are high (Erskine et al. 1985; Witcher & Freeman 1985). The receptive female encourages the male to participate in mating through solicitation behavior, including hopping, darting, ear wiggling, and posing (Erskine 1989). The most prominent sexual behavior of the receptive female that denotes sexual receptivity is the lordosis posture consisting of spinal dorsiflexion and tail deviation which aids in male intromission (Erskine 1989). Between intromissions, the female withdraws from the male and approaches again in certain time intervals, pacing the time between intromissions and maximizing the chances of pregnancy (Adler 1969; Edmonds et al. 1972).

In the event of successful mating, ovarian cyclicity is disrupted and vaginal smears are transformed with a prolonged appearance of small leukocytes or diestrous vaginal cell types, indicative of the extended function of the corpus luteum. The cycle of PRL secretion changes from surging every 4-5 days to twice daily for 10 days (Smith &

Neill 1976b), thus, approximately the first half of pregnancy of the 22-day rat gestation period. PRL provides luteotrophic support for the maintenance of the corpus luteum

supplying subsequent prolonged P4 secretion that is necessary for uterine implantation of the blastocyst and endometrial development (Smith et al. 1976). When rats are housed under conditions of 12 hours of light (0600-1800 h) and12 hours of dark (1800- 0600 h), mating-induced PRL surges peak consistently at 0300-0500h, referred to as the nocturnal surge, and 1700-2000 h, referred to as the diurnal surge. The nocturnal surge is consistently higher in magnitude compared to the diurnal surge (Freeman & Neill 1972; Freeman et al. 1974); shown in Figure 1C. Compared to the PRL surge induced by E2, these surges are relatively acute, returning to basal level within a couple hours after peak times. Unlike the transient response to suckling and E2, these PRL surges persist for several days after a mating episode without reinforcement of the stimulus. This phenomenon is a unique neuroendocrine reflex suggesting that a mnemonic must be present that permits the repeated expression of these PRL surges.

Pseudopregnancy. Despite the significance of the mating stimulus in inducing twice daily PRL surges for 9-10 days, they are also induced by uterine cervical stimulation (CS) from an infertile male, glass/teflon rod, or electrical current, leading to a condition called pseudopregnancy (Greep & Hisaw 1938). During this period, PRL surges maintain a prolonged corpus luteum (Smith et al. 1976) for 11 days and on the twelfth day estrous cyclicity resumes (Smith & Neill 1976b). Because these surges can be reproduced with any CS, the surges are commonly referred to as CS-induced PRL surges. Most surprisingly, CS induces these surges in the absence of ovarian steroids (Freeman et al. 1974). Compared to intact rats, these CS-induced PRL surges occur at the same time and similar duration in ovariectomized (OVX) rats however there is slight variation. PRL surges in OVX rats are reduced in magnitude (Freeman & Sterman 1978) and slowly wane as opposed to abruptly ending (Freeman et al. 1974; Smith et al.

1975; Gorospe & Freeman 1981a). Replacement of P4 increases the magnitude of the

nocturnal surge and E2 replacement increases the magnitude of the diurnal surge. E2 alone induces a diurnal surge in OVX rats (Freeman & Sterman 1978) and ovarian

steroids prolong the reoccurrence of CS-induced surges in acutely OVX rats (Gorospe & Freeman 1982).

Figure 1. Prolactin secretion in response to reproductive stimuli: A) suckling, B) estradiol, and C) mating stimuli. Grattan and Kokay 2008; J of Neuroendo

Termination of mating-induced prolactin surges. During midpregnancy, the termination of PRL surges after 10 days coincides with an increase in placental lactogens, which are hormones secreted from the placenta with similar structure and luteotrophic function as PRL (Kelly et al. 1976; Soares et al. 1991). Placental lactogens have been implicated in the termination of CS-induced PRL surges (Voogt & de Greef 1989). However administration of exogenous placental lactogens does not disrupt these surges (Smith & Neill 1976b) and in the absence of placental lactogens in pseudopregnant rats PRL surges still subside.

There is much evidence for both uterine factors and ovarian steroids in terminating the CS-induced PRL surges. Removal of uterine factors by hysterectomy prolongs the nocturnal surge and administration of a uterine extract inhibits these surges (Gorospe et al. 1981; Gorospe & Freeman 1982; Gorospe & Freeman 1984). More specifically, an inhibitory factor secreted from uterine epithelial cells terminates these surges as well (Gorospe & Freeman 1985). Thus, uterine factors have an inhibitory role on CS-induced PRL surges. Generally, ovarian steroids have a stimulatory role on these PRL surges. Elevated E2 in aging rats (Damassa et al. 1980) and injection of P4 on the day of estrus induce PRL surges similar to the CS-induced surges (Murakami et al. 1980). P4 prolongs the CS-induced nocturnal surge to 16 days in OVX rats and elevates this surge three-fold (Freeman 1979; Gorospe et al. 1981).

On the other hand, E2 implants prolong the diurnal surge by 16 days. E2 and P4 implants extend the nocturnal and diurnal surge to 16 days, but the nocturnal surge is significantly less than the nocturnal surge prolonged by P4 alone (Gorospe & Freeman

1981a). During midpregnancy there is a slight increase in E2 and a decrease in P4 (Welschen et al. 1975). These changes in ovarian steroids are attributed to the termination of CS-induced PRL surges Gorospe & Freeman 1981a).

Further evidence demonstrates the combination of uterine factors and ovarian steroids for the termination of CS-induced PRL surges. CS-induced PRL surges are extended to 16 days by hysterectomy. However, hysterectomy and ovariectomy extends only the nocturnal surge. Hysterectomy and P4 further increase the nocturnal surge and prolong the diurnal surge to16 days in OVX rats (Freeman 1979). More so,

hysterectomy prolongs the nocturnal surge in OVX rats treated with E2 and P4 (Gorospe et al. 1981) and OVX prevents the prolonging of nocturnal surge induced by

hysterectomy. However, replacing P4 overrides the effects of OVX on the nocturnal surge (Voogt 1980). Taken together, these data show that CS-induced PRL surges are terminated by the combination of inhibitory uterine factors and changes in ovarian steroids.

Evidence for a mating-induced mnemonic. Pseudopregnancy can be initiated immediately when CS is applied during ovulation. Immediately following ovulation, constant diestrous smears are present along with twice daily PRL surges beginning within 14 hours after the uterine cervical stimulus (Beach et al. 1975). The fact that these CS-induced PRL surges continue for several days after CS is the first evidence for a “memory” or mnemonic. Such that, the signal from CS is stored and is expressed as twice daily PRL surges for 10-12 days without additional CS (Everett and Quinn 1966). In addition, evidence of a mnemonic is present in delayed pseudopregnancy. This condition occurs when CS is applied two days after ovulation and rats display constant diestrous smears (Greep & Hisaw 1938) and twice daily PRL surges (Beach et al. 1975) following the next ovulation. This suggests a mnemonic of CS that is stored but not expressed until after ovulation. Delayed pseudopregnancy can be provoked by delaying ovulation with administration of sodium pentobarbital, a short-acting barbiturate, (Everett 1967) or disruption of the corpus luteum (de Greef & Zeilmaker 1976). Moreover, PRL surges are not required during this delay (Beach et al. 1975). CS and insemination can be separated by 3 days and still induce pregnancy (Terkel et al. 1990) and immature 23 day old rats delay expression of the surges for 2-3 days after the stimulus (Smith & Ramaley 1978). A mnemonic is also suggested for processing the amount and pattern of CS (mating or artificial). During the mating episode, 10-15 intromissions are necessary for the induction of pseudopregnancy (Diamond 1970). When intromissions are spaced apart with certain time intervals, fewer intromissions are required (Gilman 1979; Erskine 1989). During the episode of artificial CS, multiple stimuli appropriately spaced apart are also necessary for the induction of CS-induced PRL surges (Gunnet and Freeman 1983). These data suggest that time intervals

between cervical stimuli permits the neural processing of the cervical stimulus for the induction of the CS-induced mnemonic. The hypothalamus is proposed for storage since stimulation of the hypothalamus induces pseudopregnancy (Everett & Quinn 1966) and surgical separation of the hypothalamus from the pituitary abolishes the CS- induced PRL surges (Freeman et al. 1974). Antagonizing hypothalamic neurohormones unmasks these PRL surges in OVX rats (Arey et al. 1989; Arey & Freeman 1991). Taken together, this demonstrates that CS elicits a mnemonic, likely stored in the hypothalamus and retained for several days until it is expressed through twice daily PRL surges.

Control of Prolactin Secretion

Prolactin inhibiting factors. Dopamine (DA), a catecholamine neurotransmitter, controls PRL secretion by its tonic inhibition. Its source is three populations of neuroendocrine DA neurons located in the hypothalamus, designated tuberoinfundibular dopaminergic (TIDA) and tuberohypophyseal dopaminergic (THDA) neurons, located throughout the arcuate nucleus, and the periventricular hypophyseal dopaminergic (PHDA) neurons located in the periventricular nucleus. The TIDA axons terminate on a fenestrated capillary bed in the external zone of the median eminence, THDA axons terminate on short portal vessels in the neural lobe and intermediate lobe (Kawano & Daikoku 1987), and PHDA axons terminate solely on short portal vessels in the intermediate lobe (Goudreau et al. 1995) (Figure 2). Long and short portal vessels carry DA to the anterior pituitary gland where DA acts on its D2 receptors to tonically inhibit PRL secretion (for references see Ben Jonathan & Hnasko 2001).

Figure 2. Illustration of neuroendocrine dopaminergic neurons. AL, anterior lobe; EZ, exterior zone; IL, interior lobe; IZ, interior zone; LP, long portal vessels; MB, midbrain; ME, median eminence; NL, neural lobe; OC, optic chiasm; PHDA, periventricular hypophyseal dopaminergic neurons; PS, pituitary stalk; SP, short portal vessels; THDA, tuberohypophyseal dopaminergic neurons; TIDA, tuberoinfundibular dopaminergic neurons

Release of DA’s inhibitory tone is required for PRL secretion. Therefore when PRL is

elevated in response to suckling (de Greef & Visser 1981), E2 (Ben Jonathan et al. 1977; Lerant & Freeman 1997), and mating stimuli (Lerant et al. 1996) DA levels are low. As PRL is secreted it binds to the long form of its receptor on neuroendocrine DA neurons (Lerant & Freeman 1998; Kokay & Grattan 2005). This binding induces cross phosphorylation of the janus kinase 2 (JAK) and subsequent recruitment and phosphorylation of a Signal Transducer and Activator of Transcription (STAT5), both intracellular transcription factors involved in PRL secretion (Lerant et al. 2001; Grattan et al. 2001; Ma et al. 2005). Phosphorylated STAT5 undergoes dimerization and translocation into the nucleus upregulating tyrosine hydroxylase, a catecholamine rate- limiting enzyme. This results in an increase in DA synthesis and release, depicted in Figure 3 (for references see Freeman et al. 2000; Grattan et al. 2008), and creates a negative feedback loop since PRL inhibits its own secretion. In response to CS, this feedback loop is expressed as an anti-phase DA release and PRL secretion. That is, at

the time of the PRL surges DA levels are low, but when PRL returns to basal levels DA levels are high. PRL’s feedback on DA neurons implies that PRL must be reaching the brain. This may occur via an incomplete blood brain barrier (Cheunsuang et al. 2006) or the transport of PRL across the choroid plexus epithelial cell where long and short forms of PRL receptors are present (Walsh et al. 1987; Walsh et al. 1990).

Figure 3. Activation of signal transduction pathway by prolactin. Prolactin (PRL) inhibits its own secretion by stimulation of dopamine synthesis and release creating a negative feed-back loop. It does so by activation of its long form receptor (PRL-RL) on neuroendocrine dopaminergic neurons inducing phosphorylation of the janus kinase (JAK) and signal transducers and activator of transcription (STAT5), intracellular signaling proteins involved in PRL secretion. Translocation of STAT5 into the nucleus upregulates tyrosine hydroxylase expression, a catecholamine rate-limiting enzyme, for subsequent dopamine synthesis and release. Grattan et al 2008; J of Neuroendo

Prolactin releasing factors. Despite low DA tone enabling PRL secretion, this alone does not produce the magnitude of physiological PRL levels in response to suckling, thus PRL releasing factors are proposed (de Greef et al. 1981). Numerous factors stimulate PRL secretion including hypothalamic peptides, neurotransmitters, and opiods (for references see Freeman et al. 2000). Neuronal markers aid in determining the involvement of these PRL releasing factors. c-fos is an immediate early gene that is commonly used as a generic marker of neuronal activation in neuroendocrine systems (Morgan & Curran 1989). It is rapidly and transiently expressed without de novo protein synthesis in response to synaptic activation, which results in changes of synthesis and secretion of neuroendocrine neurons (Luckman et al. 1994). c-fos and its protein c-Fos are used to determine the involvement of these neurons in response to various stimuli including light/dark, hormones, odor, mating, suckling, and stress (Kovacs 2008). Therefore c-Fos is used as an aid to determine activation of neurons in response to PRL releasing stimuli. The prominent releasing factors are briefly discussed in the following section.

Thyrotropin-releasing hormone (TRH) is a tripeptide synthesized in the periventricular, dorsomedial, arcuate and paraventricular nuclei (Brownstein et al. 1974). TRH reaches its receptors on the lactotroph (Hinkle & Tashjian, Jr. 1973) by way of long portal vessels (Lechan & Jackson 1982).It stimulates PRL secretion in vitro

(Aizawa & Hinkle 1985) and in vivo in response to E2 (de Greef et al. 1985). However, TRH does not stimulate PRL secretion in response to suckling (Riskind et al. 1984; Sheward et al. 1985) or CS.

Vasoactive intestinal peptide (VIP) is synthesized in the suprachiasmatic and paraventricular nuclei and traces of VIP are present in portal blood (Lam 1991). Consistent evidence of VIP’s stimulatory role on PRL secretion is shown in vitro (Samson et al. 1980) and in vivo in OVX rats (Vijayan et al. 1979). Disruption of VIP decreases the surge of PRL in response to E2 (Harney et al. 1996; Van Der Beek et al. 1999). VIP controls PRL surges induced by CS and VIP in the PVN is elevated at times of the CS-induced PRL surges (Arey & Freeman 1992b). Disruption of VIP expression abolishes the diurnal surge (Egli et al. 2004) and antagonism of VIP abolishes both CS-

induced PRL surges (Arey & Freeman 1990). The suprachiasmatic nucleus is the master clock that controls the circadian rhythms of the central nervous system and peripheral tissues (Reppert & Weaver 2001). As mentioned previously, CS-induced PRL surges peak at approximately 0300 h (nocturnal surge) and 1700 h (diurnal surge) during the first half of pregnancy, but also in the absence of pregnancy and ovaries (for ref see Introduction). Despite the various conditions and stimuli in which CS-induced surges are produced, the timing of these surges remains the same (Gorospe & Freeman 1981b) even when the stimulus is applied at different time intervals (Smith & Neill 1976a). But the timing of the surges will shift according to lighting conditions (Bethea & Neill 1979; Yogev & Terkel 1980; Naito et al. 1981) and lesions of the suprachiasmatic nucleus prevents the initiation of these surges (Yogev & Terkel 1980; Kawakami & Arita 1981), suggesting an entrainment of these surges by this nucleus. It seems this entrainment occurs through VIP neurons in the suprachiasmatic nucleus that project to the paraventricular nucleus (Watts & Swanson 1987; Watts et al. 1987; Teclemariam-Mesbah et al. 1997b), presumably at VIP receptors on oxytocin neurons (Sheward et al. 1995; Egli et al. 2004; Kennett et al. 2008b).

Vasopressin is a peptide synthesized primarily in the paraventricular and supraoptic nuclei and its axons terminate directly in the posterior pituitary and on long portal vessels in median eminence (Sofroniew 1983). Vasopressin receptors are present in the pituitary gland (Antoni 1984) and vasopressin induces PRL secretion (Mai & Pan 1990). Antagonizing vasopressin reduces the suckling-induced PRL surge (Nagy et al. 1991). However, vasopressin does not seem to play a role in CS-induced PRL surges (Benoussaidh et al. 2005).

Oxytocin (OT) is a peptide synthesized primarily in the hypothalamic paraventricular and supraoptic nuclei. Similar to vasopressin, its axons terminate in the posterior pituitary and on long portal vessels in the median eminence (Samson & Schell 1995); depicted in Figure 4. OT travels to the anterior pituitary via portal vessels or peripherally and binds to its receptors on the lactotroph (Chadio & Antoni 1989; Breton et al. 1995). Inhibition of OT abolishes the PRL secretion in response to suckling

(Samson et al. 1986), E2 (Johnston & Negro-Vilar 1988; Sarkar 1988) and uterine

cervical stimuli (Arey & Freeman 1990). Removal of the posterior lobe prevents the suckling-induced PRL surge (Murai & Ben Jonathan 1987b). OT secretion coincides with PRL secretion induced by suckling (Grosvenor et al. 1986). Initial evidence of OT release in response to CS is evident during parturition. Stimulation of the uterine cervix, via contraction, results in OT secretion that induces additional uterine contractions and OT secretion, creating a positive feedback of OT release (Ferguson 1941). Furthermore, stimulus from the uterine cervix is transmitted via from the pelvic and hypogastric nerves reaching the lumbar sacral region of the spinal cord and transcends the spinals cord via ascending noradrenergic pathways (Lodder & Zeilmaker 1976; Steinman et al. 1992). c-Fos expression is increased in the lumbar sacral region in response to CS (Chinapen et al. 1992). There are direct projections from the spinal cord to OT neurons in the hypothalamic paraventricular nucleus (Burstein et al. 1990; Teclemariam-Mesbah et al. 1997a). In response to CS, OT neurons are activated (Flanagan et al. 1993) and OT is released (Sansone et al. 2002) increasing uterine contraction for the aid of sperm transport (Toner & Adler 1986). Furthermore, there is much evidence of OT’s stimulatory role on CS-induced PRL surges, as OT neurons in the paraventricular nucleus are activated during times of the surges (Arey & Freeman 1992a) and a bolus injection of OT initiates the CS-induced PRL surges (Egli et al. 2006). In addition, blocking OT receptors blocks these surges as well (Arey & Freeman 1990; Mckee et al. 2007). Unlike other PRL releasing factors, OT has a clear role in CS-induced PRL surges thus this dissertation will focus on OT’s involvement in the CS- induced PRL surges.

Figure 4. Diagram of oxytocin neurons in the paraventricular nucleus Axons of oxytocin neurons in the paraventricular nucleus of the hypothalamus terminate in the median eminence and the neural lobe. Oxytocin reaches lactotrophs in the anterior lobe of the pituitary via long and short portal vessels, respectively (depicted in red). AL, anterior lobe; IL, interior lobe; NL, neural lobe; OT, oxytocin; PRL, prolactin; PVN, paraventricular nucleus of the hypothalamus; III V, third ventricle

In addition to the hypothalamic peptides, there are many neuromodulators that stimulate PRL secretion including serotonin, an amine neurotransmitter synthesized in

the raphe nuclei (Ungerstedt 1971), in response to suckling (Barofsky et al. 1983), E2 (Pan & Gala 1987) and mating (Arey & Freeman 1990). Salsolinol, a DA metabolite, is synthesized in the posterior pituitary gland (Toth et al. 2001) and increases PRL

secretion and its antagonism abolishes PRL secretion in response to suckling and E2 (Radnai et al. 2004). Nitric oxide is a free-radical gas synthesized throughout the brain (Bredt & Snyder 1994). Nitric oxide neurons increase in activation in response suckling to (Ceccatelli et al. 1993) and mating (Yang & Voogt 2002). Norepinephrine, a catecholamine neurotransmitter, is synthesized in six major cell groups in the brainstem, designated as A1, A2 and A4-A7, (Ungerstedt 1971) and stimulates PRL secretion in ovariectomized rats (Vijayan & McCann 1978; Negro-Vilar et al. 1979) and in response to E2 (Carr et al. 1977; Vijayan & McCann 1978). The A1 and A2 neurons are activated in response to CS (Luckman 1995; Yang & Voogt 2001; Cameron et al. 2004b) and lesions of the A6 disrupt the proestrus (Anselmo-Franci et al. 1997) and E2-induced PRL surge (Poletini et al. 2004).

CHAPTER 2 OXTYOCIN ACTION AT THE LACTOTROPH IS REQUIRED FOR PROLACTIN SURGES IN CERVICALLY STIMULATED OVARIECTOMIZED RATS

In response to CS, PRL is secreted from lactotrophs in the anterior pituitary gland in two daily surges during the first half of pregnancy; a nocturnal surge peaking at 0300 h and a diurnal surge at 1700 h (Butcher et al. 1972; Smith & Neill 1976b). These PRL surges are partially responsible for inhibition of cyclic ovarian activity and the promotion of luteal function and development (Soares et al. 1991). CS induces these PRL surges in the absence of ovaries and they persist for 10-12 days following brief electrical CS (Freeman et al. 1974; Smith & Neill 1976b). Due to the persistence of the PRL surges, it has been suggested that there is a mnemonic present that allows the surges to occur for several days without additional stimuli and this mnemonic has been suggested to reside in the hypothalamus (Freeman et al. 1974; Freeman & Banks 1980).

DA binds to its receptors on the lactotrophs to inhibit PRL secretion (for references see Ben Jonathan 1985). Release of this inhibitory tone is required for PRL secretion, and PRL, in turn, up regulates the activity of DA neurons, by enhancing tyrosine hydroxylase activity (Moore et al. 1980; Hentschel et al. 2000). DA is released from three subpopulations of hypothalamic DA neurons, designated as tuberoinfundibular dopaminergic (TIDA) and tuberohypophyseal dopaminergic (THDA) neurons located throughout the arcuate nucleus, and the periventricular hypophyseal dopaminergic (PHDA) neurons located in the periventricular nucleus. The TIDA axons terminate on a fenestrated capillary bed in the external zone of the median eminence, THDA axons terminate on short portal vessels in the neural lobe and intermediate lobe, and PHDA axons terminate solely on short portal vessels in the intermediate lobe. DA

supply reaches lactotrophs in the anterior lobe of the pituitary gland from each of these regions via these long or short portal vessels (for references see Ben Jonathan & Hnasko 2001).

Oxytocin (OT) is a neurohormone classically known for its role in milk let down (for references see Gimpl & Fahrenholz 2001) and parturition (Ferguson 1941). Both OT and PRL are released in response to the suckling (Grosvenor et al. 1986) and mating stimuli (Freeman et al. 1974; Sansone et al. 2002). There is evidence that OT plays a physiological role by acting at the lactotroph. There are OT receptors on lactotrophs in the anterior pituitary gland (Morel et al. 1988; Chadio & Antoni 1989; Breton et al. 1995) and OT reaches the lactotroph via long and short portal vessels (Samson & Schell 1995). Immunoneutralization of OT attenuates the surge of PRL on proestrous day (Sarkar 1988),and inhibition of OT abolishes this surge (Johnston & Negro-Vilar 1988) as well as suckling-induced PRL increase (Samson et al. 1986). It is known that CS produces an immediate surge of OT in rats (Moos & Richard 1975), sheep (Kendrick et al. 1991), pigs (Gilbert et al. 1997), and humans (for references see Lippert et al. 2003) and is followed by rhythmic PRL secretion in rats (Freeman & Sterman 1978). Our laboratory has found that OT stimulates the secretory activity of the lactotrophs (Egli et al. 2004) and that a single injection of OT initiates rhythmic PRL surges in OVX rats similar to those seen in OVX-cervically stimulated rats (Egli et al. 2006). These results together give a foundation for OT’s physiological control of PRL secretion.

The known interactions between DA and PRL, and the suggested role of OT, were previously illustrated by our laboratory with a mathematical model (Bertram et al. 2006). According to this model, CS induces a surge of OT and results in a long-lasting inhibition of DA neuronal activity. The reduction in DA tone, along with the direct stimulatory influence of OT on lactotrophs, facilitates rhythmic PRL secretion. The continued interaction between DA neurons and lactotrophs leads to a rhythmic secretory PRL pattern for several days. Thus, it is suggested that this mechanism is the basis of the mnemonic for the PRL rhythm that resides in the hypothalamus, while the secretion of PRL surges is due to actions of DA and OT on the lactotroph.

The aim of this study is to test two hypotheses. The first is that the direct stimulatory action of OT on lactotrophs is necessary for rhythmic PRL secretion in OVX- cervically stimulated rats. The second is that the cervically stimulated-induced mnemonic of the PRL rhythm resides in the hypothalamus, and is therefore not affected by actions of OT on lactotrophs. An OT antagonist was used that does not cross the blood-brain barrier to remove the direct stimulatory effects of OT on lactotrophs. According to our hypotheses, the cervically stimulated-induced mnemonic should still be triggered, even though PRL surges do not occur due to the inhibitory effects of the OT antagonist on the lactotrophs. However, when the OT antagonist has cleared from the circulation, it is expected that the rhythmic PRL secretion would return, since the mnemonic was activated by CS and should (according to our second hypothesis) be unaffected by the OT antagonist that acts in the pituitary. If hypothalamic DA neurons are involved in PRL surges, then it is predicted that DA neuronal activity will be high when PRL secretion is low. That is, DA activity will be elevated in anti-phase with the PRL surges. Because it is predicted that PRL surges will be abolished in the presence of the OT antagonist, it is also expected that DA neuronal activity will be low due to the lack of stimulation from PRL. That is, both PRL secretion levels and DA neuronal activity will be low in the presence of OT antagonist. The model predictions were tested using measurements of serum PRL levels and the activity of DA neurons that project to the median eminence, intermediate lobe and neural lobe.

Materials and Methods

Mathematical Model. A mathematical model was used to help with the experimental design. This was described in detail in a previous publication (Bertram et al. 2006), but will be briefly outlined here. The results of experiments described herein have helped to clarify the role of OT. In addition, an inhibitory line from lactotrophs to hypothalamic OT neurons was added. Thus, some aspects of the earlier model have been modified.

The essential components of the model are illustrated in Figure 5. This is a mean field model, where the activity level of each cell population is described by a single variable. Four cell populations are included: hypothalamic dopaminergic neurons (DA), OT neurons of the paraventricular nucleus (OT), vasoactive intestinal polypeptide

neurons of the suprachiasmatic nucleus (VIP), and pituitary lactotrophs (PRL). Differential equations for the dynamics of each of these variables are briefly developed below.

The DA neurons are stimulated by PRL, but with a time delay (DeMaria et al.

2 1999). The stimulatory term is Td + k p PRLτ , where Td represents constant stimulatory drive, PRLτ represents PRL secretory activity delayed by τ = 3 h, and kp is the strength of the PRL feedback. The inhibitory terms include a term for first order decay of DA neuron activity, − q DA , and inhibition due to VIP synaptic input from the

suprachiasmatic nucleus, − rνVIP ⋅ DA . Here q is a decay or clearance rate and rv is the strength of the VIP inhibition. The differential equation for DA neuron activity (DA) is:

d DA = T + k PRL2 − qDA − r VIP ⋅ DA . (1) dt d p τ v

The differential equation for PRL secretory activity (PRL) is:

d PRL Tp +ν oOT = 2 − q PRL . (2) dt 1+ kd DA

The lactotrophs are subject to inhibition by DA and stimulation by OT. The first term in (2) includes both of these effects, with OT in the numerator and DA in the denominator of the expression. The strength of these feedback effects is determined by the parameters ν o and kd. Also in the numerator is a constant stimulatory drive factorTp . Finally, there is a first-order decay of lactotroph activity, − q PRL . An OT antagonist is

simulated by setting ν o = 0.

It is postulated that CS activates OT neurons of the paraventricular nucleus (PVN) and that these or another population of OT neurons (such as those in the supraoptic nucleus) trigger the PRL rhythm by indirectly inhibiting hypothalamic DA neurons. The motivation for this mechanism is the focus of an earlier publication [see

(Bertram et al. 2006)]. The DA neuron inhibition is achieved in the model by setting

Td = 0 .

The two sources of OT are designated OTcs (the first pulse of OT induced by CS) and OTPVN (the subsequent pulses of OT on the following days) The total OT level is

OT = OTcs + OTPVN . The first component of OT released as a direct result of CS and is transient in nature:

d OT cs = p CS − q OT , (3) dt cs cs where CS = 1for 2 h immediately following CS, otherwise CS = 0 . The other OT source is due to the activity of OT neurons of the paraventricular nucleus. There is evidence that PRL has a rapid inhibitory influence on the electrical activity of these neurons

(Kokay et al. 2006). Therefore, this inhibitory action in the differential equation for OTPVN was included. The OTPVN differential equation is:

d OTPVN To = 2 − qOTPVN , (4) dt 1+ ko PRL

where To = 0 prior to CS. The rapid inhibitory action of PRL is achieved by placing PRL in the denominator of the first term. Note that this is a rapid PRL effect, not a delayed PRL effect as was used in Eq. 1.

Under normal lighting conditions the activity of VIP neurons of the suprachiasmatic nucleus is high during the morning and low the rest of the day (Okamoto et al. 1991; Shinohara et al. 1993; Gerhold et al. 2002). Therefore their activity was modeled as a square pulse that is elevated for three hours during the morning (VIP = 2 ) and is 0 for the rest of the day. This VIP is input to the DA equation (1).

Parameter values for this mean field model were set to produce the PRL rhythm and are given in Table 1 below. The equations were solved numerically using the 4th- order Runge-Kutta method implemented in the XPPAUT software package (Ermentrout

G.B. 2002). The variables are in arbitrary units (except time, in hours), so curves are presented in normalized form.

Table 1 Parameter values used in the model

Tp=1 kd=1 q=0.5 Td=10

kp=0.3 τ = 3 h rv=2 ν o = 1

Pcs=1000 ν = .0 0002 rn=0.2 T = 3 cs o

ko = 1

Animals. Adult female Sprague-Dawley rats (200–250 g; Charles River, Raleigh, NC) were kept in standard rat cages under a 12:12-h light-dark cycle (lights on at 0600), with water and rat chow available ad libitum. All rats were bilaterally ovariectomized under Halothane anesthesia. Animal procedures were approved by the Florida State University Animal Care and Use Committee.

Cervical Stimulation. The uterine cervix was stimulated with an electrode constructed from a Teflon rod (diameter, 5 mm), with two platinum wires protruding from the tip. Each rat was stimulated twice, the first time at 1700 h and the second time on the following morning at 0900 h; times which mimic normal mating on proestrus evening and the morning of estrus. Stimulations were applied as two consecutive trains of electric current of 10-s duration (rectangular pulses, 1 ms of 25 V at 200 Hz). This procedure has been shown to yield the highest success rate in initiating two daily PRL surges that are characteristic of mated rats (Gorospe & Freeman 1981b).

Jugular vein catheter implantation and OT antagonist infusion. A polyurethane catheter tube (Micro-Renathane; Braintree Scientific, Braintree, MA) was inserted into the jugular vein as the rats were anesthetized with Halothane. Blood was collected in 200 μL

volumes every 2 hours over a period of 24 h. Blood loss during sampling was compensated by sterile saline replacement. Serum samples were stored at –40 C until analysis for PRL concentration. A tube was extended from the jugular vein, filled with heparinized saline (50 U/mL), fitted subcutaneously and exteriorized at the back of the animal’s neck. To prevent stress from effecting PRL levels, the external tube was connected at least 4 hours prior to the first blood collection.

2 4 The selective OT antagonist desGly-NH2-d(CH2)5[D-Tyr ,Thr ]OVT was generously provided by Dr. Maurice Manning, Toledo, Ohio (Manning et al. 1995). The OT antagonist was dissolved in sterile saline and infused at 0.5µg/Kg⋅min via osmotic pumps (Alzet Osmotic pumps, model AP-2001D, rate=8 μL/h, duration=24 h; Braintree Scientific, Braintree, MA). The osmotic pumps were filled with the OT antagonist or sterile saline and infused intravenously using the jugular vein catheter implantation described above. The osmotic mini pump was implanted subcutaneously and connected to the end of the tubing. All infusions persisted for 24 h.

Preparation of the median eminence, intermediate lobe, and neural lobe. Rats were sacrificed at 0900 h, 1200 h, and 1700 h. The median eminence, intermediate lobe, and neural lobe was separated and stored in vials with 200μL of homogenization buffer (0.15 N perchloric acid, 50μM EGTA, 13.6 nM dihydroxybenzylamine) at -80 C until the day of assay.

Radioimmunoassay. Serum concentrations of PRL were estimated in duplicate by the rat PRL radioimmunoassay kit as previously described (Freeman & Sterman 1978). Rat PRL RP-3 standard was supplied by Dr. Albert Parlow through the National Hormone and Pituitary Program (Torrance, CA). To prevent interassay variation, all samples were assayed in the same radioimmunoassay. The lower limit of detection for PRL was 0.10 ηg/mL. The intra-assay coefficient of variation was 5%.

DA and dihydroxyphenylacetic acid (DOPAC) measurement by high performance liquid chromatography coupled to electrochemical detection (HPLC-ED). DOPAC:DA ratio was measured as an index of DA neuronal activity. DA is synthesized and metabolized to DOPAC by monoamine oxidase on the outer membrane of the mitochondria before or

after release and reuptake of DA. The presence of DOPAC in axon terminals of the median eminence, neural lobe, and intermediate lobe is indicative of the activity of DA neurons of the TIDA, THDA, and PHDA, respectively (Annunziato & Weiner 1980; Lookingland et al. 1987). HPLC-ED is a well established procedure in our laboratory (DeMaria et al. 1998; Sellix et al. 2004). Median eminence, neural lobe and intermediate lobe samples were thawed, homogenized, and sonicated in 1.5 N perchloric acid and 50 μM EGTA. The samples were centrifuged (20 min at 8000 ×g). The supernatant was filtered through a 0.2mm nylon microfiltration unit (Osmonics, Livermore, CA.), and then placed into autosampler vials. The concentration of DA and DOPAC was measured using HPLC-ED. Twenty microliters of each sample was injected by an autosampler (Model 542 Autosampler, ESA, Inc., Chelmesford, MA). Mobile phase consisted of 75 mM sodium dihydrogen phosphate monohydrate (EM Science, Gibbstown, NJ), 1.7 mM 1-octane sulfonic acid (Fisher Scientific), 100 mL/L triethylamine (Aldrich, Milwaukee, WI), 25 μM EDTA (Fisher Scientific), 6% Acetonitrile (EM Science), titrated to pH 3.0 with phosphoric acid (Fisher Scientific), was delivered by a dual piston pump (LC-20AD Shimadzu Co. Analytical & Measuring Instruments Division, Kyoto, Japan) at 600μL/min. Water was purified on a Milli-Q system (Millipore, Bedford, MA) to 18 MΩ resistance and further polished with a Sep-Pak mini-column (Millipore). Catecholamines were separated on a reverse phase C18 column (MD-150, Dimensions 150×3 mm, particle size 3μm, ESA, Chelmsford, MA), oxidized on a conditioning cell (E: +300 mV, ESA 5010 Conditioning Cell, ESA) and then reduced on a dual channel analytical cell (E1: -65 mV, E2 : -225 mV, ESA 5011 High Sensitivity Analytical Cell, ESA). The change in current on the second analytical electrode was measured by a coulometric detector (ESA Coulochem II, ESA) and recorded using EZStart 7.3 SP1 (Shimadzu, Kyoto, Japan). DA and DOPAC were identified on the basis of their peak retention times. The amount of catecholamine or internal standard, dihydroxybenzylamine, in all sample peaks was estimated by comparison to the area under each peak for known amounts of each. Recovery of dihydroxybenzylamine as the internal standard corrected for any loss of sample. The sensitivity of the assay was 6 ρg of DA.

Protein Assay. In order to insure the accuracy of each dissection, the amount of protein in each sample was measured using a micro-modified form of the Pierce Bichinchoninic Acid (BCA) Protein Assay Kit (Pierce, Rockford, Ill., USA). Tissue homogenate (10 μL) was aliquoted in duplicate into 96-well plates (Corning, Corning, N.Y., USA) with 200 μL of BCA solution and incubated at room temperature for 20 min. The absorbance of each well was measured at 562 nm by a micro-plate spectrophotometer (Molecular Devices, Palo Alto, Calif., USA). Unknowns were compared against standards of bovine serum albumin. Assay sensitivity was 100 μg/mL. There was no significant difference in amount of protein within each dissected group of the median eminence, intermediate lobe, and neural lobe (data not shown).

Data analysis. All values are expressed as means ± SE. Two-way analysis of variance (treatment × time) was used for the comparison of differences between treatment groups, followed by Bonferroni comparison. One-way analysis of variance was used for comparison of differences within treatment groups, followed by Bonferroni comparison. Statistical analyses were performed and graphs were created using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA). Differences were considered significant at the level of P ≤ 0.05.

Experimental Design

Experiment 1. Effects of OT antagonist on initiation of CS-induced rhythmic PRL secretion. Ten days after OVX, rats were infused with OT antagonist or saline through the jugular vein via osmotic pumps beginning at 1300 h, 4 h prior to the first CS (day 0), and continuing for 24 h (1300 h, Day 1). Blood sampling was begun 6 hours later (1900 h, Day 1) and continued every two hours for 24 h through 1900 h on Day 2. In experiments 1-3, the OT antagonist was infused through one jugular vein and blood samples were taken from the other. The two catheters were not detrimental to the animals based upon pilot studies.

Experiment 2. Effects of OT antagonist on maintenance of CS-induced rhythmic PRL secretion. Ten days after OVX, rats were cervically stimulated and infused with OT antagonist for 24 h through the jugular vein via osmotic pumps beginning 4 h (1300 h)

after the second CS on Day 1 and continuing through 1300 h on Day 2. Blood sampling was begun at 1900 h on Day 1 and continued every two hours for 24 h through 1900 h on Day 2.

Experiment 3. Return of rhythmic PRL secretion after clearance of OT antagonist. Ten days after OVX, rats were infused with OT antagonist or saline for 24 hours through the jugular vein via osmotic pumps beginning at 1300 h on Day 0 (4 hours before CS) and continuing through 1300 h on Day 1. Blood was collected beginning 36 hours later (1900 h, Day 2) and continued every two hours for 24 h through 1900 h on Day 3.

Experiment 4. Activity of DA neurons during OT antagonist treatment. Ten days following OVX, rats were treated as described below and sacrificed at 0900 h, 1200 h, or 1700 h and the median eminence, intermediate lobe, and neural lobe were rapidly dissected. The rats were divided into three groups: 1. OVX: rats were ovariectomized and 10 days later sacrificed. 2. OVX-cervically stimulated: OVX rats were cervically stimulated (as previously described) and sacrificed. 3. OVX-cervically stimulated/OT antagonist: OVX rats were cervically stimulated then infused with OT antagonist (as previously described in experiment 1) and sacrificed on Day 2.

Results

Modeling Predicts that an OT Antagonist will Suppress the PRL Rhythm but not the Mnemonic of CS. In (Bertram et al. 2006), our laboratory developed a mathematical model for the initiation, maintenance, and termination of the PRL rhythm. As described in Methods, the model has been modified to include the rapid inhibitory influence of PRL on the activity of OT neurons that is described in (Kokay et al. 2006). The role that the direct stimulatory action of OT has on lactotrophs was increased, so that now this action is necessary (but not sufficient) for a PRL surge. In this model (illustrated in Figure 5) CS increases OT release from the hypothalamus into the pituitary gland. OT within the hypothalamus acts on a population of interneurons that inhibit hypothalamic DA neurons, triggering the PRL rhythm.

Figure 5. Connections within the network model. A filled arrow represents a stimulatory influence, while an open arrow represents an inhibitory influence. The connection from lactotrophs to DA neurons is delayed by τ = 3 h. The dashed arrow represents an indirect inhibitory connection. VIP input to DA neurons is applied periodically in the morning of each day for 3 h. A version of this model was described in an earlier publication (Bertram et al. 2006). DA, dopamine; OT, oxytocin; PRL, prolactin; VIP, vasoactive intestinal peptide

The rhythm, once triggered, results from the interaction between PRL (which stimulates DA neurons) and DA (which inhibits PRL). This is shown in Figure 6 (A,B). The OT surge (panel C) induced by CS inhibits DA neurons causing a surge in PRL (panel A). Since PRL stimulates DA neurons with a time delay (τ), an increase of DA neuronal activity occurs following the PRL surges (panel B). This inhibits lactotrophs, so PRL levels decline. The decline in PRL levels causes a delayed decline in DA, which allows PRL secretion to increase. In this way, a circadian PRL-DA rhythm is established, with DA surges occurring between surges of PRL. The DA surges occur at noon, and are marked with asterisks in Figure 6B. The phase of the oscillation is set by VIP input from

the suprachiasmatic nucleus. In the model, there is a VIP surge each morning for 3 h, prior to and after CS (Figure 6B, E, dashed curve). Each VIP surge lowers DA, as is evident in Figure 6 .

Figure 6. Model simulation. (A) Cervical stimulation on day 0 induces a circadian PRL rhythm. (B) DA neuronal activity peaks at noon (asterisks) and is out of phase with the PRL surges. VIP surges (dashed) occur every morning for 3 h and have an inhibitory action on DA neurons. The DA neuronal activity time course has been translated upward by one unit for clarity. (C) Cervical stimulation results in a surge in OT that triggers PRL surges. Cervical stimulation also activates OT neurons in the paraventricular nucleus, which provide stimulatory drive to the lactotrophs. The lactotrophs feedback onto and inhibit the OT neurons, producing an OT rhythm. (D-F) Same variables, but

with an OT antagonist present through day 3 (simulated by setting ν o = 0 ). Once the OT antagonist is removed, DA neurons interact with lactotrophs to produce a circadian rhythm in PRL and DA, with DA neuronal activity peaking at noon (asterisks); CS, cervical stimulation; DA, dopamine; OT, oxytocin; OTA, oxytocin antagonist; PRL, prolactin; VIP, vasoactive intestinal peptide

The trigger for the secretory PRL rhythm in Figure 6(A-C) is the CS-induced OT surge. One result of CS is that hypothalamic OT neurons are activated, and remain activated after the surge has subsided (panel C). This provides stimulatory drive to lactotrophs, which acts in conjunction with reduced inhibitory DA input to allow the circadian PRL-DA rhythm to emerge. The OT level itself exhibits oscillations, due to the rapid negative feedback from PRL.

The model suggests that the mnemonic induced by CS is in the hypothalamus, but the secretory PRL rhythm itself involves the interaction of hypothalamic DA neurons with lactotrophs. Thus, it should be possible to suppress PRL surges induced by CS by inhibiting the lactotrophs, while not interfering with the mnemonic of CS. To demonstrate this with the model, the application of an OT antagonist that does not cross the blood-brain barrier was simulated. This will eliminate the direct stimulatory influence of OT on the lactotrophs, without affecting the influence of OT in the hypothalamus. The results of this computer simulation are shown in Figure 6 (D-F). The OT antagonist

is simulated by setting ν o = 0 in Eq. (2), so that the direct influence of OT on lactotrophs is removed. The simulated OT antagonist is applied until day 3 (dashed curve, panel D). The CS is given on day 0. As before, CS induces a surge of OT followed by activation of hypothalamic OT neurons, so that OT peaks and then falls to a lower oscillatory level (panel F). Also as before, the OT surge inhibits the DA neurons (panel E). Although the OT neuronal activity is elevated, the OT receptors on lactotrophs are antagonized by the OT antagonist. Without the direct stimulatory influence of OT, a reduction of the DA tone is insufficient to allow significant lactotroph activation and PRL release. Thus, the PRL level remains low following CS, showing only small fluctuations. The DA level also lacks a rhythm, and in particular there are no surges in DA neuronal activity at noon (asterisks) as there were in the case of no OT antagonist Figure 6 (D-F). This prediction is somewhat counter-intuitive, since one typically associates low PRL secretion with an elevated level of DA tone. However, in the model simulation, OT antagonist suppresses the PRL rhythm and, indirectly, the noontime DA neuronal activity elevations.

Although the PRL rhythm does not occur when OT antagonist is present in the model, the mnemonic mechanism that responds to CS has been activated. This is seen in Figure 6 (D-F) as a reduction in DA tone and an elevation in OT neuronal activity. Therefore, when the OT antagonist is removed at the end of day 3, disinhibiting the OT receptors on the lactotrophs, the secretory PRL rhythm begins almost immediately (panel D). The rhythm in DA neuronal activity begins at the same time, peaking at noon (asterisks). When the PRL surges begin, they occur at the right time of the day (early morning and late afternoon). This is because the timing of the pulses is influenced by the daily VIP pulses, which are not affected by the OT antagonist.

In summary, the model makes several predictions:

(1) The CS-induced secretory PRL rhythm should be suppressed while the OT antagonist is present, but should resume soon after the OT antagonist leaves the system,

(2) When the PRL rhythm begins, the PRL surges should occur at the right time of day (early morning and late afternoon),

(3) In rats subject to CS but without the OT antagonist, the DA activity peaks at around noon. If, however, the CS-induced secretory PRL rhythm is suppressed by an OT antagonist, the DA activity will be low at noon. When the OT antagonist leaves the system and the secretory PRL rhythm begins, one should observe DA activity peaks at around noon.

These predictions were tested in the following studies.

Experiment 1. Prevention of the induction of PRL surges by CS during infusion of OT antagonist.

Figure 7 shows plasma PRL levels obtained every 2 h on Day 2 from rats exposed to an OT antagonist for 24 h prior to and during CS on Days 0 and 1. In saline-infused OVX- cervically stimulated rats, PRL levels were elevated at 0300 h (P < 0.01) and 1700 h (P < 0.05). These are time points at which the nocturnal and diurnal surges occur. The OT

antagonist infusion prevented the induction of PRL surges by CS. These data support our model prediction Figure 6 that the OT antagonist abolishes PRL surges induced by CS, leaving only small, non-significant variations of PRL levels throughout the day.

Figure 7. Prolactin secretion in rats with oxytocin antagonist infusion before cervical stimulation. The induction of prolactin surges by cervical stimulation was prevented during infusion of an oxytocin antagonist. Rhythmic secretion of PRL on Day 2 after 24h infusion of the OT antagonist (dotted line) or saline (solid line) in OVX rats before/during cervical stimulation. Infusion of the OT antagonist 24h before cervical stimulation prevented initiation of PRL surges. Values are expressed as mean ηg/mL of PRL ± SE (n=3-10 serial samples/point). *Significantly lower PRL levels than corresponding times in saline infused rats (P < 0.05). There were no significant elevation in PRL secretion at any time in the OT antagonist-treated group. aStatistical difference from all other time points within the saline infused rats.

Experiment 2. Decrease of the nocturnal surge and abolition of the diurnal surge of PRL by an OT antagonist infusion after CS.

Figure 8 shows the pattern of plasma PRL secretion during a 24 h period, beginning at 1900 h on day 2 after CS. In the saline-infused OVX-cervically stimulated rats, PRL

levels were elevated at 0300 h (P < 0.01) and 1700 h (P < 0.05). These are time points at which the nocturnal and diurnal surges occur. In the OT antagonist- infused rats, PRL levels were elevated at only 0100 h and 0300 h (P < 0.05), representing only the time of the nocturnal surge. PRL levels did not increase at the times corresponding to the diurnal surge. However, the concentration of PRL in the OT antagonist-treated rats was significantly lower than that of saline infused rats at 0300 h and 1700 h (P < 0.05), the times of both the nocturnal and diurnal PRL surges. Together, these data show that the OT antagonist infused after CS decreases the nocturnal and abolishes the diurnal PRL surges. Thus, the PRL rhythm, induced by CS, was diminished and later eliminated by the OT antagonist.

Figure 8. Prolactin secretion in rats with oxytocin antagonist infusion after cervical stimulation. Rhythmic secretion of prolactin with oxytocin antagonist after cervical stimulation when the OT antagonist infusion (dotted line) or saline infusion (solid line) began after cervical stimulation in OVX rats. The OT antagonist decreases the nocturnal and abolishes the diurnal surges of PRL when infused after cervical stimulation. Values are expressed as mean ηg/mL of PRL ± SE (n=3-10 serial samples). *Significantly lower PRL levels than corresponding times in saline infused rats (P < 0.05). aStatistical difference from all other time points within the OT antagonist or saline infused rats.

Experiment 3. Return of rhythmic PRL secretion after treatment with an OT antagonist. Figure 9 shows plasma PRL levels obtained every 2 h on Day 3 from rats exposed to the OT antagonist for 24 h prior to and during CS on Days 0 and 1. In the saline- infused OVX-cervically stimulated rats, PRL levels were elevated at 0300 h (P < 0.01) and 1700 h (P < 0.05). These are time points at which the nocturnal and diurnal surges occur. In OVX-cervically stimulated rats infused with an OT antagonist, PRL levels were also significantly elevated at 0300 h and 1700 h (P < 0.05) on day 3. The PRL surges observed in the OT antagonist and saline treated rats were equivalent in timing and magnitude, suggesting clearance of the OT antagonist.

Figure 9. Prolactin secretion in cervically stimulated rats two days after cessation of OT antagonism. Rhythmic secretion of PRL returns two days after cessation of OT antagonism. OVX-cervically stimulated rats were infused with the OT antagonist (dotted line) or saline (solid line) for 24 h. Blood samples were obtained for 24 h beginning 30 h after cessation of infusion. Values are expressed as mean ηg/mL of PRL ± SE (n=3-10 serial samples). There were no differences in serum concentrations of PRL within any time point between rats infused with saline or OT antagonist. aStatistical difference from all other time points within the saline or OT antagonist infused rats.

These findings support our model prediction Figure 6 that the OT antagonist will not interfere with the triggering of the mnemonic of CS, so that once the OT antagonist is cleared from the system the rhythmic PRL surges will appear. This suggests that the CS-induced mnemonic resides at a location other than the pituitary, most likely in the hypothalamus.

Experiment 4. Activity of DA neurons during the OT antagonist treatment. The data in Figure 10 represent the DOPAC:DA ratio, an index of DA neuronal activity (Annunziato & Weiner 1980; Lookingland et al. 1987). Rats were either OVX, OVX-cervically stimulated or OVX-cervically stimulated and treated with an OT antagonist. The latter group was exposed to an OT antagonist for 24 h prior to and during CS on Days 0 and 1. In the OVX-cervically stimulated group, the DOPAC:DA ratio in the median eminence and neural lobe was significantly elevated at 1200h on Day 2 (P < 0.05 and P < 0.001, respectively). This elevation was not observed in the intermediate lobe (Figure 10). These data indicate that DA neuronal activity was increased at 1200 h in anti-phase with PRL secretion in TIDA and THDA neurons but not in the PHDA neurons. In the OT antagonist/cervically stimulated group, the DOPAC:DA ratio in the median eminence was elevated at 0900h and decreased at 1700h (P < 0.05), and in the neural lobe was elevated at 0900 h and decreased at 1700 h (P < 0.05). The presence of the OT antagonist disrupted the anti-phasic DA neuronal activity. In addition, the OT antagonist eliminated the increase in the DOPAC:DA ratio at 1200 h in the cervically stimulated group in the neural lobe (P < 0.01). Together, these data support our model prediction (Figure 6) that CS induces an increase in DOPAC:DA ratio at 1200 h in anti-phase with PRL secretion in TIDA and THDA neurons and that this increase is abolished by OT antagonism.

Discussion

In the present study, parallel but differing approaches were taken to describe the role of OT and DA in control of mating-induced PRL secretion. First, mathematical models were developed to predict their respective roles and then tested the predictions with the use of an OT antagonist, which does not cross the blood brain barrier. It was shown

Figure 10. Dopamine activity in cervically stimulated rats with and without an oxytocin antagonist. DOPAC:DA ratio in the median eminence, intermediate lobe, and neural lobe indicating DA neuronal activity of the TIDA, PHDA, THDA, respectively of OVX, OVX-cervically stimulated, and OVX-cervically stimulated/OT antagonist treated rats at 0900 h, 1200 h, and 1700 h. Cervical stimulation induces an increase in DA neuronal activity in the TIDA and THDA neurons at 1200 h (P < 0.05), in anti-phase with PRL surges.

that, in the presence of the OT antagonist, rhythmic CS-induced PRL secretion is prevented or abolished and, after the probable clearance of the OT antagonist, rhythmic PRL secretion returns. Thus, the mnemonic of CS was maintained even though the rhythmic PRL surges were inhibited by the OT antagonist. This further supports that the mnemonic, which is not affected by the OT antagonist, is not in the pituitary gland. In response to CS hypothalamic DA neuronal activity is elevated in antiphase with rhythmic PRL surges, the OT antagonist disrupts this CS-induced elevation.

It is hypothesized that OT is necessary for PRL surges in OVX-cervically stimulated rats by directly acting at the lactotroph. Using mathematical modeling it was predicted that in the presence of an OT antagonist, CS-induced PRL surges would be abolished with only small variations in PRL secretion. To investigate our hypothesis, OT antagonist was infused for 24h before CS and blood collections began after the OT antagonist infusion in OVX rats. As predicted by our model, the OT antagonist blocked the initiation of CS-induced PRL surges. To further investigate our hypothesis, an OT antagonist was infused for 24h after CS and blood collection began during the OT antagonist infusion. It was that found the OT antagonist decreased the nocturnal and abolished the diurnal surges of PRL. These findings support our predictions and previous suggestions of OT’s stimulatory role on lactotrophs (Egli et al. 2004) and the effects of the OT antagonist on PRL surges in OVX-cervically stimulated rats, previously demonstrated in our laboratory Arey and Freeman 1990. The decrease and not abolition of the nocturnal surge in rats infused with an OT antagonist after CS is attributed to the shorter period of time between the beginning of the OT antagonist infusion and blood collections, thus not permitting the OT antagonist to exert its full effect.

It is hypothesized that the mnemonic of CS is independent of OT actions at the lactotroph. Therefore, even in the presence of the OT antagonist, CS should impose a mnemonic for rhythmic PRL secretion in the hypothalamus and after the OT antagonist is cleared, rhythmic PRL secretion should begin. To investigate our hypothesis, an OT antagonist was infused before CS for 24 h in OVX rats and blood collections began 30 hours after ending the infusion. As predicted (Figure 6), rhythmic PRL secretion

returned on Day 3, suggesting the clearance of the OT antagonist. To further investigate our hypothesis, an OT antagonist was infused after CS for 24 h in OVX rats and blood collections began 5 days later. Rhythmic PRL secretion also returned on Day 6, after the antagonist was presumably cleared (data not shown). These findings support our hypothesis that OT actions at the lactotroph are essential for the occurrence of PRL surges, but are not involved in the CS-induced mnemonic.

Our laboratory has previously found that VIP neurons in the SCN are involved in the control of rhythmic activity of DA neurons in the hypothalamus (Bertram et al. 2006). In the present study, it was also hypothesized that VIP neurons, controlling the time of day of the PRL surges, would not be affected by an OT antagonist; therefore after the OT antagonist clears, the PRL surges would return at the time of day of the nocturnal and diurnal PRL surges. As shown in Figure 9, the PRL surges return at the same time of day as the nocturnal and diurnal surges in control rats. This supports our hypothesis that the timing of the PRL surges is controlled from outside of the pituitary, most likely by VIP neurons of the SCN.

In our model, rhythmic PRL secretion is produced by interaction with hypothalamic DA neurons, such that DA neuronal activity peaks at noon, in anti-phase with the PRL surges. Model simulations suggested if the OT antagonist inhibits the PRL surges, then it will also inhibit the noontime peak in DA neuronal activity, since this peak is due to the stimulatory effects of PRL. To investigate the hypothesis, neuroendocrine DA neuronal activity (TIDA, PHDA, THDA) was determined by measuring the DOPAC:DA ratio in the median eminence, intermediate lobe, neural lobe, in OVX, OVX- cervically stimulated, and the OT antagonist/cervically stimulated groups. As our model predicted, DA neuronal activity of OVX-cervically stimulated rats was elevated at 1200 h, in anti-phase with PRL surges, in the TIDA and THDA neurons and not elevated at 1200 h in the PHDA neurons (Figure 10). Therefore, this suggests that the DA neurons in the arcuate nucleus are involved in the cervically stimulated-induced PRL surges and not DA neurons in the periventricular nucleus. The presence of the OT antagonist disrupts the anti-phasic DA neuronal activity. Because hypothalamic DA neurons display a spontaneous rhythmic pattern of activity in unstimulated OVX rats (Mai et al.

1994; DeMaria et al. 2000; Sellix & Freeman 2003), the low levels of DA neuronal activity observed at 1700 h might be the expression of an endogenous circadian rhythm of these neurons in OVX-cervically stimulated and OVX-cervically stimulated rats treated with OT antagonist. Interestingly, the OT antagonist did not affect the expression of this rhythm. Yet, even with low DA activity, a PRL surge is not seen in either the OVX rats (data not shown) or OVX cervically stimulated rat under OT antagonist treatment, confirming our hypothesis that the PRL surge is due to the combination of a decrease in DA neuronal activity and the actions of a PRL releasing factor. Our results suggest that OT is the PRL releasing factor required for the CS- induced PRL surges.

In our mathematical model it is predicted that CS triggers the PRL rhythm by indirectly inhibiting hypothalamic DA neurons. The evidence for this is indirect and based on our previous finding that an OT injection initiates a long-lasting circadian PRL rhythm (Egli et al. 2006) and a parallel modeling study (Bertram et al. 2006) showing that partial inhibition of DA neurons is the only way to initiate and maintain the PRL rhythm. In the modeling study, it is postulated a population of bistable OT-sensitive interneurons that are switched from the "off" to the "on" state by the OT bolus, and which innervate and inhibit DA neurons (which themselves don't have OT receptors). This is but one potential mechanism for transducing the OT bolus into a sustained PRL rhythm. However there is no direct evidence for these OT-sensitive interneurons. Additional mechanisms are being investigated in which CS triggers rhythmic PRL surges by exploring other parts of the brain that project to the hypothalamus and are activated by CS. In turn, these brain regions may also be involved in the sustained PRL rhythm. Taken together, this study confirms the stimulatory role of OT on lactotrophs in the production of PRL surges in response to CS.

CHAPTER 3 NORADRENERGIC PROJECTIONS TO THE HYPOTHALAMIC PARAVENTRICULAR NUCLEUS ARE ACTIVATED BY CERVICAL STIMULATION IN OVARIECTOMIZED RATS

CS induces a pattern of PRL secretion from lactotrophs of the anterior pituitary gland characterized by twice daily surges (Freeman et al. 2000). The neural pathway by which the stimulus produces this PRL secretion is under investigation. The signal of CS is transmitted from the pelvic nerve to the lumbarsacral region of the spinal cord. The signal transcends the anterolateral columns of the spinal cord to the brain stem where it is conveyed to brain regions via noradrenergic bundles (Terkel 1988; Erskine 1995). Beyond this, it is not clear how the signal is processed and specific neural connections that stimulate the CS-induced PRL surges.

Hypothalamic DA’s tonic inhibition is a well-known mechanism controlling PRL secretion (Ben Jonathan & Hnasko 2001). However the decrease in DA’s inhibition cannot fully account for the magnitude of physiological PRL secretion (de Greef et al. 1981). Several factors have been shown to stimulate PRL secretion, of which, many are present in the hypothalamic paraventricular nucleus (PVN). OT neurons in the PVN are activated by CS (Arey & Freeman 1992a; Flanagan et al. 1993; Polston & Erskine

1995), and OT stimulates PRL secretion in vitro (Egli et al. 2004) and in response to E2 (Kennett et al. 2008a) and CS (Arey & Freeman 1990). In addition to OT, norepinephrine (NE) is known to stimulate PRL secretion. Intracerebroventricular NE injections increase basal (Vijayan & McCann 1978; Negro-Vilar et al. 1979) and E2- induced PRL secretion (Vijayan & McCann 1978), and central inhibition of NE prevents

E2-induced PRL secretion (Carr et al. 1977). More importantly, it is predicted that NE stimulates PRL secretion by activating PRL releasing neurons in the PVN. Bilateral

injections of NE in the medial basal hypothalamus (MBH), which encompasses the PVN, increase basal PRL levels (Day et al. 1982). Increased NE release in the MBH

(Jarry et al. 1986) coincides with the E2-induced PRL surge as well. NE stimulates PVN neurons in vitro and this response is blocked by noradrenergic receptor antagonists (Daftary et al. 2000). In addition, injections of NE agonist and antagonist in the PVN stimulates and inhibits PRL secretion, respectively (Dodge & Badura 2004). Together, these studies strongly suggest that NE activates neurons of the PVN.

Central NE originating from the A1 (caudal ventrolateral medulla) and A2 (nucleus of the solitary tract) comprise the ventral noradrenergic bundle (Ungerstedt, 1971; Moore and Bloom, 1979). In response to CS, A1 and A2 neuronal activity increases (Luckman 1995; Yang & Voogt 2001; Cameron et al. 2004b). Moreover, lesion of ventral noradrenergic bundle prevent CS-induced PRL secretion (Hansen et al. 1980). NE originating from A6 (Locus Coeruleus; LC), where the dorsal noradrenergic bundle arises, (Ungerstedt 1971; Moore & Bloom 1979) also stimulates PRL secretion, since lesions of the LC disrupt the proestrus (Anselmo-Franci et al. 1997) and E2- induced PRL surges (Poletini et al. 2004). The A1, A2 and LC noradrenergic nuclei are therefore involved with stimulation of PRL secretion.

The purpose of this study is to determine if CS activates A1, A2, and LC neurons that project to the PVN, suggesting a pathway by which CS induces twice daily PRL surges. A retrograde tracer, Fluoro-Gold (FG), was used to delineate the connectivity of neural circuits involved in CS-induced PRL surges in the following study. FG was injected into the PVN. Sections of the A1, A2, and LC were triple-labeled using immunohistochemistry for c-Fos (a neuronal marker for activation), FG (a marker for projection of neurons to the PVN), and tyrosine hydroxylase (a rate-limiting enzyme for catecholamine synthesis and marker for identification for noradrenergic neurons). To determine if NE is released in the PVN after CS, the amount of NE in the PVN was measured after CS using micropunch technique and high performance liquid chromatography coupled to electrochemical detection.

Materials and Methods

Animals. Adult female Sprague Dawley rats (200–250 g; Charles River, Raleigh, NC) were housed in standard rat cages under a 12-h light, 12-h dark cycle (lights on at 0600 h), with water and rat chow available ad libitum. All rats were bilaterally ovariectomized under an isoflurane/oxygen gas mixture; isoflurane gas was set at 2.5-3% and the oxygen gas to 2.0 liter/min during surgery. Animal procedures were approved by the Florida State University Animal Care and Use Committee.

Fluoro-Gold (FG) injection. Rats were anesthetized with Ketamine (0.7 mL; 49 mg/mL) and Xylazine (0.3 mL; 1.8 mg/mL) and placed in a stereotaxic apparatus (David Kopf, Tujunga, CA). A midline incision exposed the skull and a small opening in the skull was made with a dental drill. FG (Flurochrome, LLC; Denver, CO) was injected unilaterally into the right paraventricular nucleus using the following coordinates: anterior-posterior (AP): -1.7 mm posterior to bregma, medial-lateral (ML): 0.3 mm right from midline and dorsoventral (DV): 7.6 mm below dura (Paxinos & Watson 1998). Sixty ηL of 4% FG, diluted in 0.9% NaCl, was injected for the duration of 5 minutes with a 10-µL syringe coupled to an infusion pump. The cannulae were left in place for an additional 10 minutes for complete diffusion. Fourteen days after injection rats were subjected to CS and perfusion (described below).

Cervical stimulation. The uterine cervix was stimulated with an electrode constructed from a Teflon rod (diameter, 5 mm), with two platinum wires protruding from the tip. Each rat was stimulated twice, the first time at 1700 h and the second on the following morning at 0900 h. Stimulations were applied as three consecutive trains of electric current of 10-sec duration (rectangular pulses, 1 msec of 25 V at 200 Hz). This procedure has been shown to yield the highest success rate in initiating two daily PRL surges that are characteristic of mated rats (Gorospe & Freeman 1981b).

Tissue preparation. Rats were deeply anesthetized with 83 mg/kg body wt of pentobarbital sodium solution (Nembutal, Abbott Laboratories, Chicago, IL) and were transcardially perfused with 40 mL of sterile saline containing heparin (5 IU/mL), followed by 200 mL of 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) in 0.1 M

PBS (pH = 7.2). After perfusion, brains were removed, postfixed in 4% paraformaldehyde for 2 h, and cryoprotected in 20% sucrose in 0.1 M PBS at 4°C for at least 24 h. Coronal sections of 30 µm were cut on a sliding microtome (Richard-Allan Scientific, Kalamazoo, MI) for the following brain regions PVN and LC (Paxinos & Watson 1998) and A1/A2 (Paxinos 1999).

Immunohistochemistry. For analysis of triple-labeling of c-Fos, FG and TH, sections from the A1, A2 and LC were rinsed 5 times for 10 min each in 0.01M PBS (pH=7.35) containing 0.1% Triton-X 100 (Sigma-Aldrich) to remove cryoprotectant. To reduce binding of residual aldehydes and ketones, tissues were incubated in 1% NaBH4

(Sigma-Aldrich) for ten minutes and 3% H2O2 (from a 30% H2O2 solution VWR International) for an additional ten minutes, separated by washes. Nonspecific binding was blocked with 10% normal goat serum (NGS). All primary and secondary antibodies were diluted in 0.01 M PBS (pH = 7.35) containing 0.4% Triton-X100 and 2% NGS. Primary antibodies were incubated for 40 h at 4ºC and secondary antibodies for 2 h at room temperature. The primary antibodies used and dilutions: rabbit anti-cFos 1:10000 (EMD; Gibbstown, New Jersey), rabbit anti-FG 1:15000 (AB 153; Chemicon, Temecula, CA, EUA), and mouse anti-TH 1:15000 (MAB 308; Chemicon) respectively. Secondary antibodies used and dilutions: biotinylated goat anti-rabbit IgG 1:600 (Vector, Burlingame, CA), goat anti-rabbit Alexa Fluor 488 1:2000 (Invitrogen), and goat anti- mouse Alexa Fluor 555 1:2000, respectively. c-Fos immunoreactivity was enhanced by biotin amplification procedure modified from previous protocols (Adams 1992; Berghorn et al. 1994). After biotinylation of c-Fos, tissue sections were washed, incubated for 30 min in ABC solution (Vector), washed with 0.175 M acetate buffer (pH= 7.5), and incubated with a DAB (0.2 mg/mL), NiSO4 (25 mg/mL), 3% H2O2 solution. After rinsing, sections were mounted on SuperFrost slides (Fisher Scientific) and underwent a dehydration process of 5 minutes in each of the following solutions in sequence: distilled water, 50%, 75%, 90%, and 100% alcohols (× 2), and 100% xylene (× 2). Slides were cover-slipped using KrystalonTM (EMD; Gibbstown, New Jersey). The reagents and antibodies used are displayed in Table 2 below. Images were taken with a Leica DMLB microscope coupled to a digital camera (SPOT-Real Time monochrome, Diagnostic instruments, Inc MI USA). Image acquisition and analysis were made with

Metamorph software (Universal Imaging, Downingtown, PA.). Each section was acquired 3 times using filter GFP (excitation 450-490 nm and emission 500-550 nm) for green Alexa Fluor 488, filter Y3 (excitation 530-560 nm and emission 573-647 nm) for red Alexa Fluor 555, and white light (displayed in Table 3 below). The images were overlaid and the numbers of double-and triple-labeled neurons were counted. For cannula replacement, sections from the PVN underwent similar immunohistochemical procedures. However, the rabbit anti-FG primary, used above, was with a 1:25000 dilution and visualization of FG staining was performed using the biotinylation and DAB/nickel procedures as described above for c-Fos visualization.

PVN microdissections. Rats were rapidly sacrificed and brains were removed and placed on dry ice. The frozen brains were fixed on a sliding microtome (Microm HM 430, Thermo Scientific, Walldorf, Germany) at -15 C. Two consecutive coronal slices of 500 μm were obtained from the PVN and bilateral punches were taken with a 1.00-mm diameter micropunch (Electron Microscopy Sciences, Hatfield, PA) under a stereomicroscope (Leica Microsystems, Bannockburn, IL) and stored at -80 C until the day of assay.

Norepinephrine (NE) measurement using high performance liquid chromatography coupled to electrochemical detection (HPLC-ECD). HPLC-ECD was used to measure NE was as an indirect index of noradrenergic neuronal activity. PVN punches were sonicated in 300 µL of mobile phase (described below) then centrifuged for 20 min at 8000 × g. The supernatant was filtered through a 0.2-mm nylon microfiltration unit (Osmonics, Livermore, CA.) and placed into autosampler vials. Twenty microliters of each sample was injected by an autosampler (model 542 autosampler; ESA, Inc., Chelmsford, MA). The mobile phase consisted of 75 mM sodium dihydrogen phosphate monohydrate (EM Science, Gibbstown, NJ), 2.0 mM octane sulfonic acid (Fisher Scientific, Pittsburgh, PA), 25 µM EDTA (Fisher Scientific), and 10% acetonitrile (EM Science), titrated to pH 3.0 with phosphoric acid (Fisher Scientific) and delivered by a dual piston pump (LC-20AD; Shimadzu Co. Analytical and Measuring Instruments Division, Kyoto, Japan) at 400 µL/min. Water was purified on a Milli-Q system

Table 2. Antibodies and reagents used in triple-labeled immunohistochemistry

Incubation Step Antibody Host Vendor Working Dilution Time/Temperature 1 anti-cFos rabbit EMD; Gibbstown, New Jersey 1:10000 40 h at 4ºC 2 biotinylated anti-rabbit IgG goat Vector; Burlingame, CA 1:600 2 h at room temperature 30 min at room 3 ABC solution Vector; Burlingame, CA 1:1 temperature

4 DAB (0.2 mg/mL), NiSO4 (25 mg/mL), 15 min at room H2O2 (1 µL/mL) solution in 0.175 M, pH=7.5 acetate buffer temperature 5 anti-FG rabbit Chemicon; Temecula, CA 1:15000 40 h at 4ºC anti-TH mouse Chemicon; Temecula, CA 1:15000 40 h at 4ºC 6 anti-rabbit Alexa Fluor 488 goat Invitrogen 1:2000 2 h at room temperature anti-mouse Alexa Fluor 555 goat Invitrogen 1:2000 2 h at room temperature

Table 3. Characteristics of filter detection systems for fluoroscent dyes

Emission Dye Filter Excitation spectrum spectrum Alexa Fluor 488 GFP 450-490 nm 500-550 nm Alexa Fluor 555 Y3 530-560 nm 573-647 nm

(Millipore, Bedford, MA) to 18 mΩ resistance. NE was separated on a reverse-phase C18 column (MD-150, Dimensions 150 × 3 mm, particle size 3µm; ESA), further polished on a conditioning cell (E, + 300 mV; ESA 5010 conditioning cell) and then oxidized and reduced on a dual channel analytical cell (E1, 100 mV; E2, -225 mV; ESA 5011 high-sensitivity analytical cell). The change in current on the second analytical electrode was measured by a coulometric detector (ESA Coulochem II) and recorded using EZStart 7.3 SP1 (Shimadzu). NE was identified by its peak retention time and its amount was determined by area under the peak compared to external standards. Assay sensitivity was 7.8 ρg.

Protein assay. The amount of protein in each sample was measured using the Pierce bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Tissue homogenate of 10 µl was assayed in duplicate into 96-well plates (Corning, Corning, NY) with 200 µL BCA solution and incubated at 60 C for 20 min. The absorbance of each well was measured at 600 nm by a microplate spectrophotometer (Molecular Devices, Palo Alto, CA). Unknowns were compared against standards of BSA. Assay sensitivity was 0.0625 mg/mL.

Data analysis. Statistical analyses were performed using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA). Data are represented as mean ± SE. Statistical differences were determined by two-way ANOVA followed by Bonferroni test, one-way ANOVA followed by Newman–Keuls test or Student’s unpaired t-test. Differences were considered significant at P ≤ 0.05.

Experimental design

Experiment 1: Activation of brainstem noradrenergic neurons that project to the PVN. Three days following ovariectomy, rats were unilaterally injected with FG into the PVN. Ten days later, rats were submitted to artificial CS or manipulated without CS at 1000 h and perfused with a fixative solution 90 minutes later. Manipulations consisted of handling the rats in the same position and for the same duration as cervically stimulated rats, but without CS. Brains were removed and sectioned from the PVN, for injection placement. The A1, A2, and LC were sectioned and triple-labeled for c-Fos, FG, and tyrosine hydroxylase using immunohistochemistry.

Experiment 2: NE content in the PVN after CS. Ten to thirteen days following ovariectomy, rats were submitted to artificial CS or manipulated without CS at 1000 h and rapidly sacrificed 0, 30, 90, or 180 minutes later. Brains were quickly removed and immediately frozen. Brains were mounted on a sliding microtome at -15 C and two, 500 μm sections were taken and immediately placed on dry ice. The PVN was removed using a 1.00-mm micropunch under a stereomicroscope. NE was measured using HPLC-ECD.

Results

Experiment 1: Activation of brainstem noradrenergic neurons that project to PVN. Figure 11A conveys a representation of an accurate FG injection in the PVN and Figure 11B illustrates the collective FG injections for this study. Figure 12 displays experimental immunohistochemistry examples of triple-labeled neurons in the LC, A1, and A2. c-Fos was used to determine activation of neurons, FG to determine projections of neurons to the PVN, and TH for identification of noradrenergic neurons. Figure 13 shows the percentage of double-labeled (c-Fos+/TH+/FG-) and triple-labeled (c-Fos+/FG+/TH+) neurons in the LC, A1, and A2 in ovariectomized rats 90 minutes after CS or manipulation without CS. Within the LC, A1, and A2, the number of double- and triple-labeled neurons, in rats non-cervically stimulated (n=9) or cervically stimulated (n=5), were quantified and compared by Student’s unpaired t-test following normalization. There were no differences between the percentages of c-Fos+/TH+

neurons within the LC, A1, and A2. However, CS increased the percentage of c- Fos+/FG+/TH+ neurons in the LC and A1 (P < 0.05). It is important to note that the LC consists of three major divisions (Grzanna & Molliver 1980) that were considered during data analysis. However, no differences were found between these divisions (data not shown) , therefore these data were grouped and presented in totality in the present study.

Figure 11. Representation of fluoro-gold injections. A: Immunohistochemistry representation of Fluoro-Gold injections in the paraventricular nucleus; 3V (3rd Ventricle), scale bar = 500 µm; B: Illustration of coronal brain sections collectively representing FG injections in the paraventricular nucleus; numbers indicate the distance behind bregma (Paxinos & Watson 1998).

Experiment 2: NE content in the PVN after CS. Figure 14 displays the amount of NE in the PVN 0, 30, 90, and 180 minutes after CS or manipulation without CS. Two-way ANOVA followed by Bonferroni test was used to determine the difference between treatment groups and time points. Results show an increase in NE in the PVN 180 minutes after CS compared to rats without CS. There was a decrease in NE in both cervically stimulated and non-cervically stimulated groups at 90 minutes.

Figure 12. Immunohistochemistry of brainstem noradrenergic neurons. Immunohistochemistry representation of the LC (Locus Coeruleus), A1, and A2. Panels A,E,I show tyrosine hydroxylase (TH) positive (+) neurons represented in red; B,F,J show FG (Fluoro-Gold) + neurons represented in green; C,G,K show FG+/TH+ neurons represented in yellow; D,H,L show c-Fos+ neurons represented in black. Arrows indicate triple-labeled neurons (c- Fos+/FG+/TH+); 4V (4th Ventricle); scale bar = 100 µm.

Figure 13. The percentage of brain stem noradrenergic neurons in cervically stimulated and non-cervically stimulated rats. Column A: The percentage of double-labeled neurons (c-Fos+/TH+/FG-) in the LC, A1, and A2; Column B: The percentage of triple-labeled neurons (c-Fos+/FG+/TH+) in the LC, A1, and A2. Data was normalized and compared for each brain region using Student’s unpaired t-test. Cervical stimulation induced an increase in the percentage of triple-labeled neurons in the LC and A1. Differences were considered significant when P ≤ 0.05 and are represented with an *. 47

400 non-cervically stimulated cervically stimulated 300

g protein) 200 µ *

100 # # NE (pg/

0 0 30 90 180 Time (min)

Figure 14. Norepinephrine content in the paraventricular nucleus of rats with and without cervical stimulation. Norepinephrine (NE) content in the paraventricular nucleus 0, 30, 90, and 180 minutes in rats with or without cervical stimulation. Two-way ANOVA was used to determine differences between time points and treatment groups ≤(P 0.05; *represents significant difference) as cervical stimulation decreased NE in the paraventricular nucleus after 180 minutes, indicative of an increase in noradrenergic activity. One-way ANOVA followed by Newman–Keuls test was used to determine differences within cervically stimulated or non-cervically stimulated groups.# represents differences from all other groups (P ≤ 0.05). Both cervically stimulated and non- cervically stimulated groups had a decrease in NE after 90 minutes, indicative of an increase in noradrenergic activity due to manipulation of rats.

Discussion

This study is unique in that it considers the activation and projection of noradrengeric neurons to the PVN, suggesting a specific neural pathway in which CS induces twice daily PRL surges. This was determined by FG injections in the PVN and subsequent triple labeling using immunohistochemistry to observe activation (represented by c-Fos) and projection (represented by FG) of noradrenergic neurons (represented by TH) in the LC, A1, and A2. Results show that LC and A1 neurons that project to the PVN (FG+) are activated by CS (represented as c-Fos+/FG+/TH+) but A2

neurons are not. Also, those neurons in the A1 and LC that do not project to the PVN (FG-) are not activated in response to CS (represented as c-Fos+/TH+/FG-). The fact that there is only a difference between noradrenergic neurons that project to the PVN, strongly suggests a selective neural pathway by which CS initiates twice daily PRL surges. In addition, NE content in the PVN decreases after 90 minutes in both non- cervically stimulated and cervically stimulated rats. After 180 minutes, however, this decrease is only observed in the cervically-stimulated rats. These results suggest that CS induces NE release in the PVN after 180 minutes and a stress-related NE release occurs after 90 minutes.

The phenotype of the retrograde noradrenergic neurons in the brainstem was revealed by staining of TH, a rate-limiting enzyme in the synthesis of catecholamines. However, A1 and A2 are adjacent to C1 and C2 adrenergic neurons, respectively (Paxinos 1999), which also stain for TH. To insure that only noradrenergic neurons of the A1 and A2 were analyzed, sectioning of the brainstem began caudal to the obex, which has been described to be the division of noradrenergic neurons from adrenergic neurons (Tucker et al. 1987). Previous studies show that mating increases c-Fos activation of noradrenergic neurons in the A1 and A2, but not in the LC (Yang & Voogt 2001; Cameron et al. 2004b). These studies do not consider the projections of these neurons to the PVN. However, in the present study CS only activated those noradrenergic neurons in the LC and A1 that projected to the PVN. The discrepancy is likely due to absence of E2 and P4 since ovariectomized rats were used in our study. These ovarian hormones are known to increase c-Fos expression in the A2 (Jennes et al. 1992) and ovariectomy significantly reduces the mating-induced c-Fos activation in these neurons (Cameron et al. 2004b). Although CS did not significantly activate the A1, A2, and LC that did not project to the PVN, there is a slight increase in c-Fos expression, perhaps the presence of ovarian steroids could have further activated these neurons.

The PVN consists of two major divisions, the magnocellular and the parvocellular. The magnocellular consists of large neurosecretory neurons that project to the posterior lobe. The parvocellular division contains small neurosecretory neurons

that project to the median eminence and other brain regions (Sawchenko & Swanson 1982). It is well established that the A1, A2, and LC are the primary sources of NE to the PVN (Ungerstedt 1971; Moore & Bloom 1979; Sawyer & Clifton 1980; Sawchenko & Swanson 1982; Petrov et al. 1993) and our present studies confirm this as well. The A1 (lateral portion of the ventral medulla) and A2 (medial portion of the nucleus of the solitary tract) noradrenergic neurons project to the PVN via the ventral noradrenergic bundle. The LC (the lateral portion of the pontine central gray) projects to the PVN via the dorsal noradrenergic bundle (Sawchenko & Swanson 1982). Amongst the three, the A1 has the most dense of projections and projects to essentially all parts of the parvocellular PVN and lightly to magnocellular regions (McKellar & Loewy 1981; Sawchenko & Swanson 1982). The A2 projections are quite similar, except the A1 are generally less dense and do not include projections to magnocellular PVN regions. The LC has relatively light projections to the PVN but specifically to the medial portion of the parvocellular PVN (Sawchenko & Swanson 1982). Perhaps, in the present study, CS did not activate A2 neurons projecting to the PVN because the A2 neuronal projections lack the density and specificity that are present in the A1 and LC neuronal projections.

Several neuronal phenotypes are located in the PVN, among them many have been shown to stimulate PRL secretion including those that secrete thyrotropin releasing hormone, vasointestinal peptide, vasopressin and OT (Freeman et al. 2000). My laboratory has demonstrated that OT stimulates PRL secretion in vitro and in vivo. Administration of OT to lactotrophs initiates PRL release that is preceded by intracellular Ca+2 release, suggesting that OT stimulates PRL secretion via a calcium-dependent mechanism (Egli et al. 2004). It was also shown that blocking OT receptors prohibits

suckling- and E2-induced PRL secretion (Kennett et al. 2008a). Most notably, a bolus injection of OT initiates CS-like PRL surges (Egli et al. 2006) and blocking peripheral OT receptors blocks CS-induced PRL secretion (Mckee et al. 2007). This suggests that OT is important for PRL secretion. And lastly, OT neurons in the PVN are activated during CS-induced PRL surges (Arey & Freeman 1992a).

In addition, NE may activate OT neurons that in turn stimulate PRL secretion. This is likely, as central NE stimulates OT in different physiological conditions in rats

(described above) and other species(Ginsberg et al. 1994; Vacher et al. 2002). NE also stimulates OT secretion induced by hemorrhaging (Rodovalho et al. 2006), angiotensin II release, and conditioned fear (Zhu & Onaka 2002). When injected intracerebroventricularly, NE stimulates OT neurons of the PVN (Ji et al. 1998) and NE stimulates OT secretion during late gestation (Lipschitz et al. 2004). Suckling induces an increase in OT and NE in the PVN and blocking NE receptors prevents this increase in OT (Bealer & Crowley 1998). Importantly, A1 and A2 noradrenergic neurons synapse on somata of OT neurons in the PVN (Michaloudi et al. 1997) and excitation of these noradrenergic neurons stimulates OT neurons in the PVN (Day et al. 1984; Tanaka et al. 1985). This suggests that the projections of noradrenergic neurons to the PVN, in the present study, synapse on OT neurons. Therefore, it is predicted that the projections of noradrenergic neurons to the PVN synapse on OT neurons.

NE content in the PVN was also measured as an indirect approach of determining noradrenergic activity. Because NE was measured at nerve terminals in the PVN, an increase in noradrenergic activity is reflected by a depletion of NE due to transient release and degradation of the neurotransmitter (Glowinski & Baldessarini 1966). NE in the PVN was measured 0, 30, 90, and 180 minutes after CS. The results show an increase in noradrenergic activity 180 minutes after CS compared to non- cervically stimulated rats, since a decrease in NE reflects an increase in neuronal activity. There was also an increase in noradrenergic activity at 90 minutes in both cervically stimulated and non-cervically stimulated groups. This is attributed to the stress of handling the rats. However, this effect subsided since the noradrenergic activity decreased to its original level in non-cervically stimulated rats at 180 minutes. This observation of stress is similar to previous studies, as several stressors lower brain NE (Glowinski & Baldessarini 1966). Also, it well established that NE is released in the PVN to activate corticotrophin-releasing factor containing neurons and the hypothalamic-pituitary-adrenocortical (HPA) axis in response to stress (Pacak 2000; Dunn et al. 2004). The LC, A1, and A2 respond to stress by increasing NE production and release. Under all stress conditions, these neurons return to basal level within two hours (Lachuer et al. 1991), which is reflected in the present data.

It is concluded that CS selectively activates A1 and LC noradrenergic neurons projecting to the PVN. This demonstrates a specific neural pathway involved in the initiation of the CS-induced PRL rhythm, presumably by activation of the A1and LC that stimulate OT neurons in the PVN necessary for the PRL rhythm.

CHAPTER 4 ACUTE ACTIVATION OF PARAVENTRICULAR OXYTOCIN NEURONS IN CERVICALLY STIMULATED OVARIECTOMIZED RATS

In response to CS, PRL is secreted from lactotrophs of the anterior pituitary gland twice daily. These surges of PRL are responsible for the rescue of the corpus luteum during the first half of pregnancy (Smith et al. 1976). However, they can be reproduced in the absence of pregnancy (pseudopregnancy) and ovaries (Freeman et al. 1974). Because these surges continue for 10-12 days after its stimulus, a mnemonic is suggested (Freeman et al. 1974; Smith & Neill 1976b). There is an ongoing investigation to determine brain regions and neuromodulators that initiate and maintain the mnemonic of CS-induced PRL surges.

OT is a hormone synthesized in the PVN that is implicated in the control of the CS-induced PRL surges. Its neuronal axons terminate in the posterior pituitary and median eminence, where OT reaches the anterior pituitary via short and long portal vessels, respectively, and from peripheral circulation (Samson & Schell 1995). Removal of posterior pituitary prevents the suckling-induced PRL secretion (Murai & Ben Jonathan 1987a). OT stimulates PRL secretion in vitro (Lumpkin et al. 1983). OT precedes the suckling-induced PRL surge and antiserum of OT attenuates this PRL surge (Samson et al. 1986). The proestrous PRL surge is also proceeded by OT release (Sarkar & Gibbs 1984) and immunoneutralization of OT or antagonizing its receptors decreases this surge (Johnston & Negro-Vilar 1988; Sarkar 1988). OT in the PVN is elevated preceeding the proestrus PRL surge (Greer et al. 1986). Also, lesions of the PVN suppress suckling-induced PRL secretion (Kiss et al. 1986; Bodnar et al. 2002). Our laboratory has also demonstrated that OT stimulates PRL secretion in vitro

and in vivo. Administration of OT to lactotrophs initiates PRL release that is preceded by intracellular Ca+2 release, suggesting that OT stimulates PRL secretion via a Ca+2 - dependent mechanism (Egli et al. 2004). Our laboratory has also shown that blocking

OT receptors prohibits suckling and E2-induced PRL secretion (Kennett et al. 2008a).

In addition, a bolus injection of OT initiates CS-like PRL surges (Egli et al. 2006). CS induces OT release in the spinal cord (Sansone et al. 2002) and central OT promotes uterine contractility (Benoussaidh et al. 2005). In the PVN, the expression of the immediate early genes c-fos (Pfaus et al. 1993) and egr-1 (Polston & Erskine 1995) increase acutely after CS. OT neurons in the PVN increase in c-Fos expression after

CS in E2 and P4 treated rats (Flanagan et al. 1993) and during the times of the CS- induced PRL surges (Arey & Freeman 1992a; Polston & Erskine 1995). Taken together, these studies suggest that OT from the PVN is important for stimulating CS- induced PRL surges. The goal of the present study is to determine the role of central OT in the initiation of CS-induced PRL surges by examining the acute activation of OT neurons in the PVN 90 minutes after CS using immunohistochemistry for OT and c-Fos co-localization. In addition, radioimmunoassay was used for the presence of PRL surges after an OT agonist injection in the lateral cerebral ventricle.

Materials and Methods

Animals. Adult female Sprague Dawley rats (200–250 g; Charles River, Raleigh, NC) were housed in standard rat cages under a 12-h light, 12-h dark cycle (lights on at 0600 h), with water and rat chow available ad libitum. All rats were bilaterally ovariectomized under an isoflurane/oxygen gas mixture (Aerrane; Baxter, Deerfield, IL); isoflurane gas was set at 2.5-3% and the oxygen gas to 2.0 liter/min during surgery and allotted a 1 week recovery. Animal procedures were approved by the Florida State University Animal Care and Use Committee.

Cervical stimulation. The uterine cervix was stimulated with an electrode constructed from a Teflon rod (diameter, 5 mm), with two platinum wires protruding from the tip. In experiment 1, each rat was stimulated at 1000 h. In experiment 2, each rat was stimulated twice, the first time at 1700 h and the second time on the following morning

at 0900 h. Stimulations were applied as three consecutive trains of electric current of 10-sec duration (rectangular pulses, 1 msec of 25 V at 200 Hz).

Tissue preparation. Rats were deeply anesthetized with Ketamine (0.7mL; 49 mg/mL) and Xylazine (0.3mL; 1.8 mg/mL) and were transcardially perfused with 40 mL of sterile saline containing heparin (5 IU/mL), followed by 200 mL of 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) in 0.1 M PBS (pH = 7.2). After perfusion, brains were removed, postfixed in 4% paraformaldehyde for 24 hours, and cryoprotected in 20% sucrose in 0.1 M PBS at 4°C for at least 24 hours. Coronal sections of 30-µm were cut on a sliding microtome (Richard-Allan Scientific, Kalamazoo, MI) from the PVN (Paxinos & Watson 1998).

Immunohistochemistry. For analysis of triple-labeling of c-Fos and OT, PVN sections were rinsed 5 times for 10 min each in 0.01M PBS (pH=7.35) containing 0.1% Triton-X 100 (Sigma-Aldrich) to remove cryoprotectant. To reduce binding of residual aldehydes and ketones, tissues were incubated in 1% NaBH4 (Sigma-Aldrich) for ten minutes and

3% H2O2 (from a 30% H2O2 solution, VWR International) for an additional ten minutes, separated by washes. Nonspecific binding was blocked with 10% normal goat serum (NGS). All primary and secondary antibodies were diluted in 0.01 M PBS (pH = 7.35) containing 0.4% Triton-X100 and 2% NGS. Primary antibodies were incubated for 40 h at 4ºC and secondary antibodies for 2 h at room temperature. The primary antibodies used and dilutions were: rabbit anti-cFos 1:10000 (EMD; Gibbstown, New Jersey) and mouse anti-OT 1:10000 (MAB 308; Chemicon) respectively. Secondary antibodies used and dilutions were: biotinylated goat anti-rabbit IgG 1:600 (Vector, Burlingame, CA) and goat anti-mouse Alexa Fluor 555 1:2000, respectively. c-Fos immunoreactivity was enhanced by biotin amplification procedure modified from previous protocols (Adams 1992; Berghorn et al. 1994). After biotinylation of c-Fos, tissue sections were washed, incubated for 30 min in ABC solution (Vector), washed with 0.175 M acetate buffer (pH= 7.5), and incubated with a DAB (0.2 mg/mL), NiSO4 (25 mg/mL), and 3%

H2O2 solution. After rinsing, sections were mounted on SuperFrost slides (Fisher Scientific) and underwent a dehydration process of 5 minutes in each of the following solutions in sequence: distilled water, 50%, 75%, 90%, and 100% alcohols (× 2), and

100% xylene (× 2). Slides were cover-slipped using KrystalonTM (EMD; Gibbstown, New Jersey). Images were taken with a Leica DMLB microscope coupled to a digital camera (SPOT-Real Time monochrome, Diagnostic instruments, Inc MI USA). Image acquisition and analysis were made with Metamorph software (Universal Imaging, Downingtown, PA.). Each section was acquired twice using filters Y3 (excitation 530- 560 nm and emission 573-647 nm) for red Alexa Fluor 555 and white light, respectively. The images were overlaid and the numbers of double-labeled neurons were counted.

Table 4. Antibodies and reagants used in double-labeled immunohistochemistry

Step Antibody Host Vendor Working Dilution Incubation Time/Temp 1 anti-cFos rabbit EMD; Gibbstown, New Jersey 1:10000 40 h at 4ºC 2 biotinylated anti-rabbit IgG goat Vector; Burlingame, CA 1:600 2 h at RT 3 ABC solution Vector; Burlingame, CA 1:1 30 min at RT

4 DAB (0.2 mg/mL), NiSO4 (25 mg/mL), H2O2 (1 µL/mL) solution in 0.175 M, pH=7.5 acetate buffer 15 min at RT 5 anti-OT mouse Chemicon; Temecula, CA 1:15000 40 h at 4ºC 6 anti-mouse Alexa Fluor 555 goat Invitrogen 1:2000 2 h at RT

Table 5. Characteristics of filter detection systems for fluoroscent dyes

Emission Dye Filter Excitation spectrum spectrum Alexa Fluor 555 Y3 530-560 nm 573-647 nm

Intracerebroventricular cannulation. Rats were anesthetized with 100 μl/100 g body weight of ketamine (Ketaset; Fort Dodge, IA; 49 mg/mL) and xylazine (Anased; Lloyd Laboratories, IO; 1.8 mg/mL) and positioned in a stereotaxic apparatus with the incisor bar at –3.3 mm. A 22-gauge guide cannula (C313G, Plastics One, Inc., Roanoke, VA) was implanted in the right cerebral lateral ventricle with the following coordinates: 1.0 mm posterior to bregma, 1.6 mm lateral to the midline, and 3.5 mm ventral to the outer surface of the skull and attached to the skull with stainless-steel screws and acrylic cement. After surgery, the cannula was protected with a plastic-capped mandril (C313DC, Plastics One, Inc., Roanoke, VA) and a 1 week recovery was allotted to all rats.

OT injection. Rats were anesthetized with an isoflurane/oxygen gas mixture; isoflurane gas was set at 2.5 - 3% and the oxygen gas to 1.0 liter/min during injection. An OT agonist (Bachem) was dissolved in sterile saline (60 µg/µL). The OT solution or saline was injected into the third ventricle via a stainless steel needle (0.2 mm diameter) with PE-10 polyethylene tubing connected to a 50 µl Hamilton syringe (Hamilton; Reno, NV) controlled by an injection pump (KDS100, KD Scientific, Holliston, MA). The pump was set to dispense 5 μL/min thus at total of 300ng of OT agonist was injected, a dose known to stimulate uterine contraction (Benoussaidh et al. 2005). After each injection the needle was left inside the cannula for an additional minute to avoid solution reflux.

Jugular catheter implantation. A polyurethane catheter tube (Micro-Renathane; Braintree Scientific, Braintree, MA) was inserted into the jugular vein as the rats were anesthetized with isoflurane gas mixture (described above), fitted subcutaneously and exteriorized at the back of the animal’s neck. To prevent stress from effecting PRL levels, the external tube was extended. The extension was filled with gentamicine

sulfate (Alexis, San Diego, CA) to prevent bacterial growth and to maintain catheter patency (Thrivikraman et al. 2002) immediately after surgery until the first blood sample. Blood was collected in 300 μL volumes in heparinized syringes at times referenced in Figure 16. Blood loss during sampling was replacement by a sterile heparinized solution (50 U/mL). Serum samples were stored at –40 C until analysis for PRL concentration.

Data analysis. Statistical analyses were performed and graphs were created using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA). Data are represented as mean ± SE. In experiment 1, statistical differences were determined by Student’s unpaired t-test. In experiment 2, two-way analysis of variance (treatment × time) was used for the comparison of differences between treatment groups, followed by Bonferroni comparison. One-way analysis of variance was used for comparison of differences within treatment groups, followed by Bonferroni comparison. Differences were considered significant at P ≤ 0.05.

Results

Experiment 1: Acute activation of OT neurons in the PVN in response to CS. Figure 15 shows the percentage of OT neurons in the PVN that express c-Fos 90 minutes after CS or handling in cervically stimulated and non-cervically stimulated rats, respectively. Using one-way ANOVA and two-way ANOVA followed by Bonferroni analyses, no significant differences were determined.

non-cervically stimulated cervically stimulated

0.6

0.5

0.4

0.3

0.2

0.1 % of OT immunoreactive OT of %

neurons expressing c-Fos expressing neurons 0.0

Figure 15. Acute activation of oxytocin neurons in the paraventricular nucleus in response to cervical stimulation. The percentage (%) of oxytocin (OT) neurons in the PVN expressing c-Fos were quantified to determine activation 90 minutes after CS in ovariectomized rats. Data was normalized and compared using Student’s unpaired t-test. No significant differences were found between groups.

Experiment 2: Effects of central OT on PRL secretion. Figure 16 depicts the PRL levels in rats injected with OT or sterile saline into the lateral cerebral ventricle. Using Student’s unpaired t-test analysis following normalization, no significant difference was detected.

OT Saline 175 OT 150 125 100 75

PRL(ng/mL) 50 25 0 1300 1700 1900 0300 0900 1300 1700 1900 0300 0900 1300 Time of day

Day 1 Day 2 Day 3

Figure 16. Prolactin secretion in response to an oxytocin injection into the lateral cerebral ventricle. Ovariectomized rats were implanted with a guide cannula into the lateral cerebral ventricle. After 1 week of recovery, a catheter tube was inserted into the jugular vein and the following day oxytocin (OT) (300 ηg/5µL) or sterile saline (5 µL) was injected intracerebroventricularly. Blood samples were taken at times depicted above.

Discussion

In this study, the effects of central OT on CS-induced PRL surges were directly investigated. Previous studies in our laboratory show that there is an increase in c-Fos expression of OT neurons in the PVN during the times of the CS-induced PRL surges (Arey & Freeman 1992a), suggesting OT from the PVN is important in maintaining the CS-induced PRL surges. In the present study, the purpose of the first experiment was to determine if OT from the PVN is important in initiating the CS-induced PRL surges. Activation of OT neurons in the PVN was measured by c-Fos expression 90 minutes after CS to detect optimal c-Fos expression (Kovacs 1998). CS did not increase c-Fos expression in OT neurons in the PVN after 90 minutes compared to non-cervically stimulated rats. Other studies have shown no increase c-fos expression in the PVN 60 minutes after mating in intact rats (Rowe & Erskine 1993; Polston & Erskine 1995) but

an increase is observed when measuring erg-1, another immediate early gene, in the

PVN (Polston & Erskine 1995). However, in OVX E2 and P4 treated rats, there is an increase in c-Fos expression in OT neurons after mating (Flanagan et al. 1993). OVX rats were used in the present study and the lack of E2 and P4 likely contributes to the lack of an increase in c-Fos expression. E2 stimulates OT secretion physiologically, as elevated OT secretion in portal blood corresponds with increased E2 levels during proestrus (Sarkar & Gibbs 1984) along with an increased firing rate in PVN neurons

(Negoro et al. 1973). In the presence of E2 during late gestation, there is an increase in peripheral OT secretion (Numan 1988) and OT immunoreactivity in the PVN (Caldwell et al. 1987) as well as a decrease in OT neurons’ proximity to blood vessels (Jirikowski

1992). Exogenous E2 excites OT neurons (Akaishi & Sakuma 1985) and long-term E2 treatment increases OT mRNA (Crowley et al. 1995) in the PVN. An intracerebroventricular injection of OT stimulates sexual receptive lordosis behavior in

E2 and P4 (Arletti & Bertolini 1985; Gorzalka & Lester 1987), and E2 treated OVX rats (Caldwell et al. 1986). OT release in the spinal cord in response to CS is increased in

E2 and P4 treated rats (Sansone et al. 2002). In addition, E2 and P4 increase c-fos mRNA and protein in the PVN after CS compared to untreated rats (Pfaus et al. 1993; Pfaus et al. 1996).

Alternatively, the PVN may not be the source of OT that is important for the initiation of CS-induced PRL surges. The SON is another region classically known to

synthesize OT. However this brain region is not activated is after CS in E2 and P4 treated rats (Pfaus et al. 1993; Flanagan et al. 1993). These surges may be important for maintaining the CS-induced PRL surges because OT neurons in the SON increase in c-Fos expression at the time of the nocturnal surge (Polston et al. 1998). Conversely, OT neurons in the SON project primarily to the posterior pituitary (Sofroniew 1983) and results from Chapter 2 show that that CS-induced PRL surges are not initiated by OT actions at the pituitary gland (Mckee et al. 2007). There are accessory OT neurons located throughout the brain including in the MPOA (Rhodes et al. 1981). The presence of OT immunoreactivity is also modulated by E2 in these areas (Jirikowski et al. 1988). Injections of OT in the MPOA increase lordosis behavior (Caldwell et al. 1989). Further studies are required for the role of accessory OT neurons on CS-induced PRL surges. 62

Additional studies in our laboratory show that a bolus injection of peripheral OT initiates CS-like PRL surges (Egli et al. 2006). It is not known whether OT does this by peripheral or central actions. Chapter 2 results show that CS-induced PRL surges are not initiated by peripheral OT actions, suggesting central OT actions are important for the initiation of CS-induced PRL surges. Despite controversy over OT’s ability to cross the blood brain barrier, there is evidence that this may occur (Banks & Kastin 1987). This suggests that the peripheral OT injection may have crossed the blood brain barrier to initiate twice daily PRL surges. The purpose of the second experiment in this study was to determine if central OT initiates the CS-induced PRL surges. Administration of OT intracerebroventricularly did not initiate CS-induced PRL surges in OVX rats compared to saline-treated rats. However, there is some fluctuation in basal PRL secretion during the times of the CS-induced PRL surges. The saline-treated rats seem to have elevated PRL levels as basal PRL levels are typically 0-20 ηg/mL. These results may be attributed to unknown stress to the rats. PRL secretion is typically elevated in response to stress (Smith & Gala 1977) but only when PRL levels are low, as they typically are in ovariectomized saline-treated rats. Conversely, when PRL levels are elevated stress decreases PRL secretion (Morishige & Rothchild 1974), specifically during CS-induced PRL surges (Morehead & Gala 1987; Morehead & Gala 1989). This likely occurred in the present study. Additional experiments must be carried out to determine the role of central OT on CS-induced PRL surges. However, our laboratory has shown that OT neurons in the PVN increase in c-Fos expression during the times of the CS-induced PRL surges (Arey & Freeman 1992a). Also, Chapter 2 shows that peripheral OT antagonism blocks the maintenance of the CS-induced PRL surges. Taken together, these results show that OT is important for maintaining the CS-induced PRL surges by peripheral actions at the lactotroph.

CHAPTER 5 DISCUSSION

The experiments presented in this dissertation explored the stimulatory role of OT in the initiation and maintenance of the mnemonic of CS-induced PRL surges. Previous studies in our laboratory showed that a peripheral bolus injection of OT initiates CS-like PRL surges and anti-phase DA release (Egli et al 2004). These results suggest that OT is involved in the initiation of the mnemonic of CS-induced PRL surges. However, if the peripheral OT injection acted peripherally at the pituitary or centrally to initiate these surges is unknown. Therefore, in Chapter 2 the peripheral effects of OT were investigated. These studies confirmed the known anti-phase DA release and PRL secretion, as activity in two populations of hypothalamic DA neurons was elevated in anti-phase with CS-induced PRL surges. They also confirmed OT’s involvement in the anti-phase DA release and PRL secretion, as peripheral OT antagonism abolished this relationship. Additional evidence for the hypothalamic mnemonic was shown by peripheral OT antagonism disrupting CS-induced PRL surges but the mnemonic remained since the surges returned after OT antagonism ceased. This suggests that OT does not act peripherally to initiate the mnemonic of the CS-induced PRL surges, but centrally. If central OT is important in initiating twice daily PRL surges, then the source of OT is likely the PVN (Arey & Freeman 1992a). Therefore, the source of central OT in the PVN was proposed and a neural pathway that would activate the PVN was investigated in Chapter 3. It was demonstrated that CS selectively activated two populations of noradrenergic neurons projecting to the PVN resulting in an increase in norepinephrine release in the PVN. This suggests a functional pathway by which the cervical stimulus is relayed to the PVN. Therefore, the peripheral OT injection must have initiated the CS-like PRL surges by reaching the brain or by inducing uterine contraction which in turn would stimulate central OT. The latter hypothesis was ruled out since peripheral OT injections induced CS-like PRL surges in hysterectomized rats 64

(data not shown). Despite controversy over OT’s ability to cross the blood brain barrier there is evidence that this may occur (Banks & Kastin 1987). This suggests that peripheral OT crossed the blood brain barrier to initiate these PRL surges in the previous study in our laboratory (Egli et al. 2006). Therefore, in Chapter 4 the direct effects of central OT on the initiation of CS-induced PRL surges were investigated. However, OT neurons in the PVN were not acutely activated by CS. Also, an OT injection administered in the lateral ventricle did not initiate CS-like PRL surges compared to saline treated rats. However, the latter experiment must be repeated as unknown stresses may have increased PRL levels in saline treated rats. As predicted in Chapter 2, DA neurons are inhibited by some unknown population of neurons that allows for CS-induced PRL secretion. This inhibition is essential for the expression of CS-induced PRL surges. The ventrolateral portion of the ventromedial hypothalamus (VMHvl) may be this unknown population of neurons since it receives projections from the PVN (Palkovits 1999), contains OT receptors and projects to the arcuate nucleus (Canteras et al. 1994). In addition, infusion of an OT antagonist in the VMHvl prior to mating decreases the occurrence of CS-induced PRL surges in intact rats (Northrop & Erskine 2008). Therefore, the activation and projection of these neurons to the PVN 90 minutes after CS was investigated (data not shown). FG was injected into the VMHvl and the PVN was sectioned and stained for FG (for projection), c-Fos (for neuronal activation) and OT (for detection of OT neurons). However, only 1.5% of OT neurons in the PVN projected to the VMHvl. Moreover, the neurons that did project did not increase in c-Fos expression. These results further suggest that OT neurons in the PVN are not important for the initiation of CS-induced PRL surges. The role of the

VMHvl cannot be concluded from these results. However the absence of E2 in OVX animals used in this study likely contributed to the results since binding sites in this

region are dependent upon E2 (Johnson et al. 1991). Also, E2 enhances OT actions in the VMHvl in vivo (Caldwell et al. 1989) and in vitro (Kow et al. 1991). Even so, the results from this dissertation leads to two major predictions: 1) The PVN is not the source of OT that is essential for the initiation of CS-induced PRL surges or 2) Other brain regions and neuromodulators are more important in initiating the CS-induced PRL surges. Both possibilities will be discussed in combination below.

In addition to the brain regions investigated in this dissertation, there are other brain regions implicated in the mnemonic of CS-induced PRL surges, which gives way to the complexity of the mnemonic. For the role of neural activation and long-term memory, the involvement of immediate early genes c-fos, egr-1 and arc (Guzowski et al. 2001; Davis et al. 2003) will be discussed in the following brain regions: bed nucleus of the stria terminalis (BNST), dorsal medial hypothalamus (DMH), medial amygdala (MEA), medial preoptic area (MPOA), and ventromedial hypothalamus (VMH), shown in Figure 17. It is suggested that these brain regions are also important for the mnemonic of CS-induced PRL surges such that any delivery of CS activates these neurons. In response to artificial mating stimuli an increase in c-Fos expression is seen in the

BNST, MEA, MPOA, and VMH in E2 and P4 treated rats (Pfaus et al. 1996), an increased firing rate occurs in the MPOA and VMH (Dafny & Terkel 1990), c-Fos staining cells increase in the MPOA and VMH (Auger et al. 1996), and metabolic activity in the BNST and MPOA increases (Allen et al. 1981). Elevated c-Fos mRNA is seen in the BNST, MPOA, MEA, and VMH in response to a manual-artificial or a natural mating stimulus (Pfaus et al. 1993) and increased c-Fos expression in the BNST, DMH, MPOA, the posterodorsal division of the MEA and the VMH with manual and mating stimulus

(Tetel et al. 1993). In response to the mating stimulus, the MPOA and VMH in E2 and

P4 treated rats increase in c-Fos expression (Flanagan et al. 1993), elevated c-Fos expression is observed in the MEA and MPOA (Erskine 1993), elevation of c-fos and erg-1 expression is present in the BNST, MPOA, MEA, and VMH (Polston & Erskine 1995). In addition, the increase in c-Fos expression in these brain regions is blocked with pelvic nerve transection (Lodder & Zeilmaker 1976; Rowe & Erskine 1993; Pfaus et al. 2006) and lesions of the ventral adrenergic bundles prevent the induction of CS- induced PRL surges and deplete norepinephrine in these brain regions as well (Hansen et al. 1980; Hansen et al. 1981). Stimulation of the DMH/VMH initiated CS-like surges in intact and OVX rats (Freeman & Banks 1980; Gunnet et al. 1981) and lesions of this area abolish the diurnal surge in CS rats (Gunnet et al. 1981).

Figure 17. Prominent brain regions involved prolactin secretion in cervically stimulated rats. ARC, arcuate nucleus; BNST, bed nucleus of the stria terminalis ; MEA, posterodorsal divison of the medial amygdala; MPOA, medial preoptic area; VMH, ventromedial hypothalamus

The MEA has been implicated in the mnemonic for CS-induced PRL surges by long-term potentiation, an increase in synaptic efficacy due to learning and memory, requires the activation of NMDA receptors in the MEA (Gean et al. 1993; Shindou et al. 1993; Maren 1999). Long-term chemical lesions of the MEA decrease the occurrence of CS-induced PRL surges in mated rats (Coopersmith et al. 1996). As mentioned previously, the posterodorsal division of the MEA (MEApd) is important for the induction of pseudopregnancy in its processing the summation and spacing of intromissions needed for the induction of pseudopregnancy (Erskine et al. 1989). The increase in c- Fos expression of the MEApd in response to glutamatergic stimulation also increases c- Fos expression in the BNST, MPOA, PVN, and VMH (Lehmann & Erskine 2005). Three subthreshold infusions of NMDA spaced thirty minutes apart is more effective in inducing pseudopregnancy compared to one threshold dose or three subthreshold infusions with inadequate spacing time (Lehmann et al. 2005). There is also an increased expression of egr-1, an immediate early gene known to be involved in the storage of long-term memory in other brain regions, in the MEApd in response to adequate VCS (Yang et al. 2007). This suggests that the MEA is the storage location for the mnemonic of CS. In addition, the MEA is also important for the induction of CS- induced PRL surges. Glutamate, an abundant excitatory neurotransmitter, infused in the MEApd increases c-Fos expression in the MEApd (Lehmann & Erskine 2005). Bilateral infusion of NMDA, a glutamate agonist, in the MEApd induces a nocturnal prolactin surge (Polston et al. 2001) and disrupts ovarian cyclicity (Lehmann et al. 2005). NMDA receptor antagonist infusion in the MEApd before mating prevents the induction of CS-induced PRL surges and CS-induced activation of the VMH and BNST, however it has no effect after CS (Polston et al. 2001). Also infusion of other excitatory amino acids into the MEApd induces CS-induced PRL surges, but has no effect on these PRL surges when infused after CS (Lehmann et al. 2005). Long-term potentiation requires de novo protein synthesis (Huang et al. 2000) and infusion of a protein synthesis inhibitor into the MEApd does not prevent the induction of pseudopregnancy (Polston et al. 2001), suggesting that long-term potentiation in the MEApd is not required for the induction of CS-induced PRL surges.

In addition, norepinephrine release in the MEA is also important for the induction of CS-induced PRL surges (McGaugh et al. 1996; Huang et al. 2000). This would be transmitted by ventral noradrenergic bundle projects to the MEA (Petrov et al. 1993) and β-adrenergic, α1, α2, β1, β2 -noradrenergic receptors in the MEA (Young, III & Kuhar 1980; Rainbow et al. 1984). There is an elevation of NE in the MEApd in response to CS, and the source of NE is A1 and A2 noradrenergic neurons (Cameron et al. 2004a). c-Fos expression of the noradrenergic neurons increases in response to CS compared to non-mounted and mounted only females (Yang & Voogt 2001).

Lesions of the MPOA results in repetitive prolonged diestrous vaginal smears with nocturnal surges of PRL but no diurnal surges (Freeman & Banks 1980; Arita & Kawakami 1981; Jakubowski & Terkel 1986). MPOA lesion after CS results in the continued presence of a nocturnal surge and disruption of the diurnal surge and MPOA lesions in OVX rats produces a nocturnal surge (Freeman & Banks 1980). These data suggest an inhibitory role of the MPOA on the nocturnal surge and a stimulatory role on the diurnal surge. However, stimulation of the MPOA during the time of the nocturnal and diurnal surges in CS rats abolishes both CS-induced surges and simulation of the MPOA in OVX rats has no effect on basal prolactin levels (Gunnet & Freeman 1984). This suggests a stimulatory role of the MPOA on both surges. Despite the conflicting evidence, MPOA’s role in the CS-induced PRL surges is evident. In addition, neurons that synthesize gonadotropin releasing hormone increase in c-Fos expression in response to CS in the POA (Pfaus et al. 1994).

Paced mating, which involves the female rat determining the frequency between intromissions (or stimulations), increases the incidence of CS-induced PRL surges (Edmonds et al. 1972; Erskine et al. 1989), therefore it has been used as a marker for a mnemonic for CS. Chemical lesions of the MPOA lengthen the time between female- male contacts during mating (Guarraci et al. 2004). In addition, c-Fos expression in the BNST, MEApd, MPOA, and VMH increase with the number of artificial (Pfaus et al. 1996) and natural mating stimulations (Polston & Erskine 1995), suggesting these regions store the summation of stimulations for the mnemonic of CS. In response to paced mating c-Fos and Egr-1 expression increases in the MEApd, as mentioned

above, but also in the CA1 region of the hippocampus. Modulation of synapses in these regions after CS may also contribute to the mnemonic of CS (Oberlander & Erskine 2008). Arc expression is also increased in these regions and other regions of the amygdala (basolateral, central and cortical) (Yang et al. 2007). Thus storage of the mnemonic of CS may lie in these nuclei.

In conclusion, this dissertation presents additional evidence for OT’s stimulatory role on CS-induced PRL surges. Results showed that OT acts at the pituitary to maintain CS-induced PRL surges but not to initiate these surges. A noradrenergic pathway that stimulates OT neurons in the PVN was also proposed. However, the role of central OT remains uncertain. Thus OT’s role in the mnemonic for CS-induced PRL is uncertain as well. The processing and storage of the mnemonic for CS-induced PRL surges is essential for the maintenance of pregnancy in the rat. For such a significant role, it is possible that a mnemonic lies not in one distinct brain region but across many.

Future Directions

Additional investigation of the role of OT in the mnemonic for CS-induced PRL surges is necessary. It is not known whether a peripheral OT injection initiates CS-like PRL surges by reaching the brain. Although studies in Chapter 2 suggest that OT does act centrally to initiate twice daily PRL surges there is conflicting evidence presented in Chapter 4. To directly investigate this hypothesis experiments should be carried out with a peripheral OT injection in conjunction with a central infusion of an OT antagonist. If the peripheral OT injection acts centrally to induce CS-like PRL surges then in this experiment no PRL surges should be present. If not, then CS-like PRL surges will be observed. In addition, to more accurately determine if the PVN is an important source of OT for initiating CS-induced PRL surges, other techniques such as microdialysis can be used as a measure of OT release in the PVN after CS. In addition, central sources of OT other than the PVN should be investigated. Accessory OT neurons in the MPOA increase OT release in response to CS and accessory OT neurons in the anterior hypothalamus, lateral subcommissural nucleus, and medial basal hypothalamus may be involved as well (Caldwell 1992). Measuring expression of Fos related antigens, which are expressed for longer duration than c-Fos, can also help determine chronic activation

of neurons after CS. Determining differences in expression of Fos related antigens during the CS-induced PRL surges and after the termination of these surges may decipher what brain regions are involved in the mnemonic of CS. Lastly, further investigation of the unknown population of neurons that inhibit neuroendocrine DA neurons can also determine brain regions essential for the mnemonic of CS-induced PRL surges.

APPENDIX A: COPYRIGHT PERMISSION LETTERS

The Endocrine Society 8401 Connecticut Avenue, Suite 900 Chevy Chase, MD 20815-5817 Telephone: 301-941-0200 Fax: 301-941-0259 www.endo-society.org TIN Number: 73-0531256 Form was submitted by: [email protected] Original author to publish in a Dissertation. Date: June 11, 2009 Reference Number: Name: De'Nise McKee Organization: Florida State University Department: Address: 107 Chieftan Way City, State and Postal Code: Tallahassee, Florida 32306 Country: United States of America Phone: 850-644-9807 Fax: Email: [email protected] Journal: Endocrinology Author Name: D McKee Title: Oxytocin Action at the Lactotroph Is Required for Prolactin Surges in Cervically Stimulated Ovariectomized Rats Year: 2007 Volume: 148

Page Range: 4649-4657 Figure Reproduction: Figure Number(s): Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Table 1, Table 2 Where will the figures appear: - Dissertation - Title: CONTROL OF PROLACTIN SECRETION BY CENTRAL OXYTOCIN IN CERVICALLY STIMULATED OVARIECTOMIZED - Publisher: UMI/PQIL The Endocrine Society grants permission to reproduce tables/figures Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Table 1 and Table 2 from the selected article stated above contingent upon the following conditions: 1) That you give proper credit to the author(s) and to include in your citation, the title of journal, title of article, volume, issue number, date, and page numbers. 2) That you include the statement Copyright 2007, The Endocrine Society. Please understand that permission is granted for one- time use only. Permission must be requested separately for future editions, revisions, translations, derivative works, and promotional pieces. Title: Journal Publications Coordinator Date: June 11, 2009

Dear De'Nise Mckee, Thank you for your email request. Permission is granted for you to use the material below for your thesis/dissertation subject to the usual acknowledgements and on the understanding that you will reapply for permission if you wish to distribute or publish your thesis/dissertation commercially.

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-----Original Message----- From: De'Nise McKee [mailto:[email protected]] Sent: Wednesday, May 20, 2009 12:14 AM To: [email protected] Subject: Copyright Permission Form Journal of Neuroendocrinology-Blackwell Publishing: I am writing to inquire about copyright permission for two recent review articles for updated figures for my dissertation with University Microfilms Inc. Dissertation Publishing/ProQuest Information and Learning (UMI/PQIL) at Florida State University. The articles include:

1. Grattan DR, Kokay IC; Prolactin: a pleiotropic neuroendocrine hormone, 2008 Jun;20(6):752-63. Figures 1, 2, and 5

2. Grattan DR, Steyn FJ, Kokay IC, Anderson GM, Bunn SJ; Pregnancy-induced adaptation in the neuroendocrine control of prolactin secretion, 2008 Apr;20(4):497-507. Epub 2008 Figure 3

Thank you for your time. I did not see a form on your website, but if you would like to direct me to this that would be great as well. De'Nise McKee

APPENDIX B: IACUC APPROVAL LETTER

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BIOGRAPHICAL SKETCH

EDUCATION: Florida State University, Neuroscience Program, Tallahassee, Florida Ph.D. candidate 2007; Ph.D. degree expected: 08/2009 Concentration: Reproductive Neuroendocrinology

University of Indianapolis, Indianapolis, Indiana 2004 B.S., magna cum laude Major: Biology, Minor: Chemistry

RESEARCH SKILLS: Extensive experience with the following rodent surgical procedures: jugular catheterization, stereotaxic brain surgeries, and ovariectomy ] Other technical experience includes: high performance liquid chromatography-electrochemical detection, hormone measurement using radioimmunoassay and immunohistochemistry

TEACHING EXPERIENCE: Teaching Assistant; 2005, 2006, 2009 Courses: Animal Diversity Laboratory, General Genetics, Biological Science I Laboratory AWARDS AND SUPPORT: NIH Minority Supplement, 2006-2008 International Neuroendocrine Federation Young Investigator Travel Award, 2006 NIH Minority Training Grant, 2004-2006

PROFESSIONAL SERVICE: Florida-Georgia Louis Stokes Alliance for Minority Participation (FGLSAMP) Program Mentor, 2008 Tallahassee Capital Regional Science and Engineering Fair Judge, 2008-2009 North Florida Brain Bee Judge; 2007, 2008 Raa Middle School Science Fair Judge, 2007,2008 Brain Awareness Week Committee Member, 2007 Neuroscience Graduate Student Organization; Treasurer, 2005-2006 Black Graduate Student Association Community Service Chair; 2005-2006, 2007-2008 Secretary, 2005-2006 Mentor, 2006-present

PROFESSIONAL MEMBERSHIPS AND MEETINGS: The Society for Neuroscience Member, 2008 Annual Society for Neuroscience Meetings; 2005-2008

The Endocrine Society Member, 2008-present Annual Meetings of the Endocrine Society; 2006-2007 International Congress of Neuroendocrinology, 2006

PRESENTATIONS/ABSTRACTS: De’Nise T. McKee, Maristela O. Poletini, Raphael E. Szawka, Janete A. Anselmo-Franci and Marc E. Freeman 2008. Noradrenergic pathways involved in mating induced prolactin secretion. Abstract presented at the 38th annual meeting of the Society for Neuroscience.

C. V. Helena, D. T. McKee, M. O. Poletini, R. Bertram, M. E. Freeman 2008. Induction of the cervical stimulation prolactin secretory rhythm by a central but not peripheral injection of ovine prolactin in ovariectomized rats. The 38th annual meeting of the Society for Neuroscience.

R.E. Szawka, M.O. Poletini, D.T. McKee, G.E. Hoffman, J.A. Anselmo-Franci, ME Freeman 2008. Ovarian steroids regulate the activity of locus coeruleus and A2 noradrenergic neurons projecting to the medial preoptic area. International Congress of Neuroscience.

De’Nise T. McKee, Maristela O. Poletini, Richard Bertram and Marc E. Freeman 2007. The role of the locus coeruleus in the control of prolactin surges induced by cervical stimulation. Abstract presented at the 37th annual meeting of the Society for Neuroscience.

De’Nise T. McKee, Maristela O. Poletini and Marc E. Freeman 2006. Oxytocin Controls Prolactin Secretion in Ovariectomized-Cervically Stimulated Rats. Abstract presented at the Sixth International Congress of Neuroendocrinology.

Maristela O. Poletini, De’Nise T. McKee, Jessica E. Kennett, John Sarandria and Marc E. Freeman 2007. Clock proteins in the suprachiasmatic nucleus are involved with the mechanism controlling time of prolactin surges in mated rats. The 37th Annual Meeting of the Society for Neuroscience.

Marc E. Freeman, De’Nise T. McKee, Marcel Egli and Richard Bertram 2007. Biological and Mathematical Modeling Approaches to Defining the Role of Oxytocin and Dopamine in the Control of Mating-Induced Prolactin Secretion. Parental Brain Conference.

Maristela O. Poletini, De’Nise T. McKee, Jamie Doster and Marc E. Freeman 2007. Knockdown of clock genes in the suprachiasmatic nucleus blocks the estradiol-induced prolactin surge and alters the locus coeruleus circadian rhythm of ovariectomized rat. The 89thAnnual Meeting of the Endocrine Society.

Maristela O. Poletini, De’Nise T. McKee and Marc E. Freeman 2006. The Role of Clock Genes in the Suprachiasmatic Nucleus on the Prolactin Proestrus Surge in Rats. The 88th Annual Meeting of the Endocrine Society.

De’Nise T. McKee 2005. Oxytocin Effects on Prolactin Secretion. Florida State University, Neuroscience Program Summer Seminar

PUBLICATIONS: Cleyde V. Helena, De’Nise T. McKee, Richard Bertram, Ameae M. Walker, Marc E. Freeman. The rhythmic secretion of mating-induced prolactin secretion is controlled by prolactin acting centrally. Endocrinology 2009

Maristela O. Poletini, De’Nise T. McKee, Jessica E. Kennett, Jamie Doster and Marc E. Freeman. Knockdown of clock genes in the suprachiasmatic nucleus blocks prolactin surges and alters FRA expression in the locus coeruleus of female rats. Am J Physiol Endocrinol Metab. 2007 Nov;293(5) :E1325-34.

De’Nise T. McKee, Maristela O. Poletini, Richard Bertram and Marc E. Freeman. Oxytocin action at the lactotroph is required for prolactin surges in cervically stimulated ovariectomized rats. Endocrinology. 2007 Oct;148(10):4649-57.

Michael T. Sellix, Marcel Egli, Maristela O. Poletini, De’Nise T. McKee, Matthew D. Bosworth, Cheryl A. Fitch and Marc E. Freeman. Anatomical and functional characterization of circadian clock gene expression in neuroendocrine dopaminergic neurons, American Journal of Physiology- Regulatory, Integrative and Comparative Physiology. 2006 May; 290(5):R1309-23.