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Electronic Theses, Treatises and Dissertations The Graduate School

2005

Circadian Rhythms in the NeuroendocrineDopaminergic Neurons Regulating ProlactinSecretionMichael Timothy Sellix

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THE FLORIDA STATE UNIVERSITY

COLLEGE OF ARTS AND SCIENCES

CIRCADIAN RHYTHMS IN THE NEUROENDOCRINE DOPAMINERGIC NEURONS

REGULATING PROLACTIN SECRETION

By

MICHAEL TIMOTHY SELLIX

A Dissertation submitted to the

Program in Neuroscience

in partial fulfillment of the

requirements for the degree of

Doctor of Philosophy

Degree Awarded:

Spring Semester, 2005

The members of the Committee approve the dissertation of Michael Timothy Sellix defended on

February 7th

, 2005.

___________________________

Marc E. Freeman

Professor Directing Dissertation

___________________________

Friedrich K. Stephan

Outside Committee Member

___________________________

Debra A. Fadool

Committee Member

___________________________

Thomas C.S. Keller III

Committee Member

___________________________

Paul Q. Trombley

Committee Member

Approved:

___________________________

Timothy Moerland, Chair, Department of Biological Science

The Office of Graduate Studies has verified and approved the above named committee members.

ii

To my parents, Eleanor and Thomas, for nurturing me and giving me confidence, my wife

Michelle Lori, for her love and support, my grandfather, Eric Sellix, who gave me the chance to

succeed and to whom I will always be grateful and my grandmother Eleanor McNulty, whose

love and guidance continue to enrich and inspire.

iii

ACKNOWLEDGEMENTS

I would like to express appreciation to my mentor Marc Freeman for standing behind me

in every endeavor. I came to Dr. Freemans’s laboratory looking for guidance and mentorship; I

received that and much more. I would like to thank the members of the laboratory during my

tenure, without which much of the work included here would never have been completed. They

include Dr. Bela Kanyicska, who taught me the meaning of humility and respect, Dr. Marcel

Egli, Cheryl Fitch-Pye, Janice Dodge, De’Nise McKee, Maristela Poletini and Deann

Scarborough. Without the support and guidance of these individuals, I would certainly be a poor

scientist. I would like to acknowledge the support of the Program in Neuroscience Fellowship,

on which I depended for funding for three of my four years in the Freeman Laboratory. Further,

the outstanding mentorship I received from Drs. Frank Johnson, Richard Hyson, Thomas Houpt,

Debra Fadool, James Fadool, Paul Trombley, Tom Keller, Lloyd Epstein, Mike Rashotte, Cathy

Levenson, and the entire Neuroscience Program at Florida State University. They showed me the

path to becoming a dedicated and successful scientist, and will inspire me to continue on the road

to excellence.

Throughout my time as a graduate student I have had the privilege of working alongside

great colleagues who both challenged and inspired me. I am remisce to provide a list here for

fear of omitting a single name; however, I would like to recognize the friendship of: Dr. Nestor

Davila, Dr. Laura Blakemore, Dr. Brandon Aragona, Jessica Brann, Dr. Jacob Vanlandingham,

Glen Jesse Golden, Denesa Lockwood, Bum Sup Kwon, and Dr. Jennifer Westberry. My only

hope is that those that come after me are as fortunate in their time to be able to work with

dedicated, patient and kind individuals, without which I would never have reached this stage in

my career. Thank you all and God bless you.

iv

TABLE OF CONTENTS

List of Figures ................................................................................................................. vi

List of Abbreviations ...................................................................................................... ix

Abstract ......................................................................................................................... xi

INTRODUCTION .......................................................................................................... 1

1. CIRCADIAN RHYTHMS OF NEUROENDOCRINE DOPAMINERGIC

NEURONAL ACTIVITY IN THE FEMALE OVARIECTOMIZED FEMALE RAT 20

2. OVARIAN STEROID HORMONES MODULATE CIRCADIAN RHYTHMS OF

NEUROENDOCRINE DOPAMINERGIC NEURONAL ACTIVITY IN THE OVX

RAT ................................................................................................................................ 38

3. CLOCK GENE EXPRESSION PATTERNS IN NEUROENDOCRINE

DOPAMINERGIC NEURONS OF THE OVX RAT: CORRELATION WITH THE

CIRCADIAN AND SEMI-CIRCADIAN RHYTHMS OF DOPAMINE TURNOVER

IN NEUROENDOCRINE DOPAMINERGIC NEURONS........................................... 68

4. EFFECTS OF ACUTE PERIOD1, PERIOD2 AND CLOCK GENE KNOCKDOWN

IN THE SUPRACHIASMATIC NUCLEUS ON THE CIRCADIAN RHYTHMS OF

NEUROENDOCRINE DOPAMINERGIC NEURONAL ACTIVITY ....................... 100

DISCUSSION............................................................................................................... 125

APPENDIX…………………………………………………………………………... 135

REFERENCES ............................................................................................................ 138

BIOGRAPHICAL SKETCH ........................................................................................ 171

v

LIST OF FIGURES

1.NEUROENDOCRINE DOPAMINERGIC NEURONS .............................................. 2

2.PHYSIOLOGICAL RHYTHMS OF PROLACTIN SECRETION ............................... 6

3. THE MOLECULAR CLOCK ..................................................................................... 17

4. DRINKING ACTIVITY FROM OVARIECTOMIZED RATS BEFORE AND

AFTER TRANSITION FROM A STANDARD 12:12 L:D CYCLE TO CONSTANT

DARKNESS OR A DELAYED L:D CYCLE................................................................. 26

5. SERUM CONCENTRATIONS OF PROLACTIN AND CORT IN ADULT OVX

RATS UNDER A STANDARD 12:12 L:D CYCLE OR CONSTANT DARKNESS .. 28

6. DA TURNOVER IN THE MEDIAN EMINENCE, NEURAL LOBE AND

INTERMEDIATE LOBE OF ADULT OVX RATS UNDER A STANDARD 12:12

LIGHT:DARK CYCLE OR CONSTANT DARKNESS............................................... 31

7. DA TURNOVER IN THE MEDIAN EMINENCE, NEURAL LOBE AND

INTERMEDIATE LOBE OF ADULT OVX RATS UNDER A STANDARD 12:12

LIGHT:DARK CYCLE OR A PHASE-DELAYED L:D CYCLE ................................ 33

8.OVARIAN STEROID HORMONES DO NOT AFFECT THE CIRCADIAN

RHYTHMS OF DRINKING ........................................................................................... 44

9. ESTRADIOL MODULATES THE MAGNITUDE, BUT NOT THE TIMING, OF

THE CIRCADIAN RHYTHMS OF PROLACTIN AND CORT SECRETION ............ 46

10. ESTRADIOL AND PROGESTERONE MODULATE THE MAGNITUDE, BUT

NOT THE TIMING, OF THE CIRCADIAN RHYTHMS OF PROLACTIN AND

CORT SECRETION........................................................................................................ 48

11. ESTRADIOL AND PROGESTERONE AFFECT THE TIMING AND

MAGNITUDE OF THE CIRCADIAN RHYTHMS OF DA TURNOVER IN THE

MEDIAN EMINENCE.................................................................................................... 50

12. ESTRADIOL AND PROGESTERONE AFFECT THE MAGNITUDE, BUT NOT

THE TIMING OF THE CIRCADIAN RHYTHMS OF DA TURNOVER IN THE

NEURAL LOBE.............................................................................................................. 52

vi


THE TIMING OF THE CIRCADIAN RHYTHMS OF DA TURNOVER IN THE

INTERMEDIATE LOBE ................................................................................................ 54


THE TIMING OF THE CIRCADIAN RHYTHMS OF PROLACTIN AND

CORT SECRETION FOLLOWING ENTRAINMENT TO A PHASE-DELAYED

L:D CYCLE.................................................................................................................... 56

15. ESTRADIOL AFFECTS THE MAGNITUDE, BUT NOT THE TIMING OF THE

CIRCADIAN RHYTHMS OF DA TURNOVER IN THE MEDIAN EMINENCE,

NEURAL LOBE AND INTERMEDIATE LOBE FOLLOWING ENTRAINMENT

TO A PHASE-DELAYED L:D CYCLE......................................................................... 59

16. ESTRADIOL AND PROGESTERONE AFFECT THE MAGNITUDE, BUT NOT THE

TIMING OF THE CIRCADIAN RHYTHMS OF DA TURNOVER IN THE MEDIAN

EMINENCE, NEURAL LOBE AND INTERMEDIATE LOBE FOLLOWING

ENTRAINMENT TO A PHASE-DELAYED L:D CYCLE........................................... 61

17. RT-PCR AMPLIFICATION OF PER1, PER2, CLOCK AND BMAL1 MRNA

FROM SCN, ARN AND PITUITARY GLAND FROM ADULT OVX RATS ............ 77

18. CHARACTERIZATION OF PER1, PER2 AND CLOCK PROTEINS IN THE

BRAIN AND PITUITARY GLAND OF OVX RATS. ....................................................78

19. DRINKING ACTIVITY OF OVX RATS UNDER A STANDARD L:D CYCLE

AND CONSTANT DARKNESS .................................................................................... 80

20. CHARACTERIZATION OF PER1, PER2 AND CLOCK IMMUNO-

REACTIVITY.................................................................................................................. 81

21. CLOCK PROTEIN IMMUNOREACTIVITY WITHIN THE NUCLEUS OF

NEUROENDOCRINE DOPAMINERGIC NEURONS AND SCN NEURONS........... 82

22. PER1 EXPRESSION IN NDNS UNDER A STANDARD 12:12 L:D CYCLE


23. PER2 EXPRESSION IN NDNS UNDER A STANDARD 12:12 L:D CYCLE


24. CLOCK EXPRESSION IN NDNS UNDER A STANDARD 12:12 L:D CYCLE


25. PER1, PER2 AND CLOCK EXPRESSION IN THE ZONA INCERTA UNDER A

STANDARD 12:12 L:D CYCLE AND CONSTANT DARKNESS.............................. 91

vii

26. PER1, PER2 AND CLOCK EXPRESSION IN THE SCN UNDER A STANDARD

12:12 L:D CYCLE AND CONSTANT DARKNESS .................................................... 93

27. INJECTION OF CLOCK GENE AS-ODN COCKTAIL DISRUPTS

CIRCADIAN RHYTHMS OF DRINKING ACTIVITY OF THE OVX RAT ..............108

28. INJECTION OF CLOCK GENE AS-ODN COCKTAIL REDUCES PER1, PER2

AND CLOCK PROTEIN EXPRESSION WITHIN THE SCN..................................... 109

29. CLOCK GENE KNOCKDOWN DOES NOT DISRUPT L LIGHT-ENTRAINED

OR FREE-RUNNING RHYTHMS OF PRL SECRETION OF THE OVX RAT........ 111

30. CLOCK GENE KNOCKDOWN DISRUPT LIGHT-ENTRAINED, BUT NOT

FREE-RUNNING RHYTHMS OF CORT SECRETION OF THE OVX RAT ............ 113

31. CLOCK GENE KNOCKDOWN DISRUPTS LIGHT-ENTRAINED, BUT

NOT FREE-RUNNING RHYTHMS OF DA TURNOVER IN THE

MEDIAN EMINENCE................................................................................................... 115

32. CLOCK GENE KNOCKDOWN FAILED TO DISRUPT LIGHT-ENTRAINED

OR FREE-RUNNING RHYTHMS OF DA TURNOVER IN THE

NEURAL LOBE ........................................................................................................... 117

33. CLOCK GENE KNOCKDOWN DISRUPTS LIGHT-ENTRAINED, BUT NOT

FREE-RUNNING RHYTHMS OF DA TURNOVER IN THE

INTERMEDIATE LOBE ............................................................................................... 118

34. CLOCK GENE KNOCKDOWN DISRUPTS LIGHT-ENTRAINED, BUT NOT

FREE-RUNNING RHYTHMS OF DA CONCENTRATION IN THE

ANTERIOR LOBE......................................................................................................... 120

35. THE PHOTO-NEUROENDOCRINE SYSTEM REGULATING PRL

SECRETION IN THE ADULT FEMALE RAT ......................................................... 126

36. SYNERGY BETWEEN CLOCK GENE EXPRESSION WITHIN VIPERGIC

NEURONS OF THE SCN AND NDNS IN THE REGULATION OF DA

TURNOVER RHYTHMS .............................................................................................. 131

37. HYPOTHETICAL REGULATION OF DA SYNTHESIS ENZYME GENE

EXPRESSION BY BOTH RHYTHMIC VIP ACTIVATED CREB ACTIVATION

OF PERIOD GENE EXPRESSION AND ENDOGENOUS RHYTHMS OF

CLOCK:BMAL1 DRIVEN TRANSCRIPTION IN NDNS.......................................... 133

viii

LIST OF ABBREVIATIONS

AL anterior lobe of the pituitary gland

ARN arcuate nucleus

AS-ODN antisense deoxyoligonucleotides

AVP arginine vasopressin

BHLH basic helix-loop-helix

C Celsius

CRF corticotrophin-releasing factor

CRY cryptochrome gene

cAMP cyclic adenosine monophosphate cDNA complementary deoxynucleic acid

D2R dopamine receptor family 2

DA dopamine

DOPAC dihydroxyphenylacetate

DHBA dihydroxybenzylamine

DAR dopamine receptor

DMARN dorsomedial arcuate nucleus

DTT dithiothreitol

EDTA ethylenediaminetetraacetic acid

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

FRAS fos-related antigens

GABA γ–amino-butyric acid

G-protein GTP-binding protein

h hour(s)

HEPES N-2-Hydroxyethylpiperazine-N´-2-ethane sulfonic acid

ICC immunocytochemistry

IEG immediate early gene

IgG immunoglobulin G

IL intermediate lobe of the pituitary gland

IR immunoreactivity

LPV long portal vessels

min(s) minute(s)

M molar

m moles

ME median eminence

mRNA messenger ribonucleic acid

NaVO4 sodium orthovanadate

NDS normal donkey serum

NDN(s) neuroendocrine dopaminergic neuron(s)

NL neural lobe of the pituitary gland

OSA octane-sulfonic acid

OT oxytocin

PACAP pituitary adenylate cyclase-activating polypeptide

PAS per-arnt-sim domain

ix

PBS phosphate-buffered saline

PCR polymerase chain reaction

PER period gene

PeVN periventricular nucleus

PHDA periventricular hypophyseal dopaminergic

PRL prolactin

PRF prolactin-releasing factor

PSP pseudo-pregnancy

RARN rostral arcuate nucleus

r.t. room temperature

RT-PCR reverse transcription – polymerase chain reaction

RS-ODN random sequence deoxyoligonucleotides

SCN suprachiasmatic

SPV short portal vessels

τ tau, period

TBS tris-buffered saline

TEA triethylamine

TIDA tuberoinfundibular dopaminergic

THDA tuberohypophyseal dopaminergic

TH tyrosine hydroxylase

VIP vasoactive intestinal polypeptide

VPAC-2 vip type-2 receptor

x

ABSTRACT

The pituitary gland hormone prolactin (PRL) regulates diverse physiological functions in the

female mammal. PRL is secreted into peripheral circulation by lactotrophs in the anterior lobe of

the pituitary gland. The primary physiological regulator of PRL secretion is Dopamine (DA).

Three populations of neuroendocrine DAergic neurons (NDNs) with cell bodies in the

periventricular (PEVN) and arcuate (ARN) nuclei of the hypothalamus release DA. During the 4-

5 day estrous cycle of the rat, PRL secretion peaks on the afternoon of proestrus, due to a gradual

rise in circulating ovarian steroids. Experiments show that the proestrous afternoon rise in PRL is

timed by inputs from the biological clock located in the suprachiasmatic nucleus (SCN). Studies

verify that disruption of the connection between SCN and its targets within the hypothalamus

disrupt the timing of PRL secretion. Recently, it has been shown that the oscillatory function of

the SCN occurs due to autoregulatory negative feedback loops of transcription factor expression

within SCN neurons. These transcription factors are referred to as “clock genes”. Clock genes

drive cell autonomous oscillations of gene expression and activity in the SCN and additional

areas of the CNS, coordinating rhythms of physiological activity. Given that the timing of PRL

secretion appears to be regulated by the SCN and that NDNs receive direct input from the SCN, I

hypothesized that circadian rhythms of activity in NDNs time PRL secretion in the

ovariectomized (OVX) rat. I have shown that NDNs exhibit circadian and semi-circadian

rhythms of activity that are modulated by ovarian steroid hormones. Further, I have determined

the light-entrained and free-running rhythms of clock gene expression in NDNs. In addition, I

have found that antisense knockdown of several clock genes in the SCN modulates, but fails to

abolish, circadian rhythms of NDN activity. Results from these experiments, in agreement with

previous work, reveal that NDNs display circadian and semi-circadian rhythms of DA release,

driven by direct influence from the SCN. My results suggest a functional link between the

expression of clock genes within SCN neurons and NDNs in the control of circadian rhythms of

DA release and PRL secretion in the female mammal.

xi

INTRODUCTION

Prolactin: a peptide hormone of the anterior pituitary gland

The peptide hormone prolactin (PRL) was first identified as an extract of the pituitary

gland exceedingly efficient at stimulating crop-milk secretion in pigeons (2,3). While the

moniker “prolactin” describes PRLs effects at the mammary gland, it fails to describe the

multitude of physiological functions ascribed to this diverse hormone. PRL plays a varied role in

a wide array of systems ranging from reproductive physiology to the immune response (for

review see (1)). Translation of the PRL gene produces a prohormone of 227 amino acids that is

cleaved to produce a mature PRL molecule of 199 amino acids and a small 28 residue signaling

peptide. The PRL gene product undergoes a variable number of post-translational modifications,

producing several truncated and/or polymerized forms (1). PRL is secreted by lactotrophs or

mammatrophs that are located within the anterior lobe of the pituitary gland. PRL secretion is

synergistically regulated by hypophysiotropic hormones released into the blood supply bathing

the lactotroph (1,4-7). Lactotrophs constitutively secrete a significant amount of PRL in the

absence of inhibitory influence from hypothalamic inputs (8-10). Hypothalamic PRL -releasing

and -inhibiting factors reach the anterior lobe via two main routes, (1) through the primary long

portal vessels draining the capillary beds of the median eminence, or (2) through short portal

vessels connecting the neurointermediate lobe with the anterior lobe (11-13).

Prolactin acts on target regions in the CNS and periphery through binding to two

isoforms of the PRL receptor gene product, a short form and a long form. The PRL receptor is a

member of the class I cytokine receptor family and shares amino acid sequence homology with

other members of this family, such as growth hormone receptors (14,15). PRL receptors are

receptor tyrosine kinases with several tyrosine residues within the intracellular domain that are

phosphorylated by receptor-associated Janus kinase 2 after ligand binding (16-18). The ligand-

activated phosphorylation of the intracellular domain of the PRL receptor leads to binding and

phosphorylation of STAT (signal transducer and activator of transcription). Activated STAT

molecular homodimerize, enter the nucleus and initiate a subsequent increase in transcription

within the genome of the target cell (for review see (1,19-21)). Although a direct interaction

between STAT signaling and clock gene expression has not been determined, MAP kinase

1

Figure 1. Neuroendocrine Dopaminergic Neurons (NDNs) The

three populations of NDNs include the periventricular-hypophysial

(PHDA; A14), tuberohypophysial (THDA) and tuberoinfundibular

(TIDA) DAergic neurons (A12). Both the PHDA and THDA send

long axons down the pituitary stalk (PS) and terminate on short

portal vessels (SP) in the neural (NL) and intermediate (IL) lobes.

The TIDA neurons terminate on fenestrated capillaries in the

external zone (EZ) of the median eminence (ME) that drain into long

portal vessels (LP). The portal vasculature (SP, LP) bath the anterior

lobe (AL) where the PRL secreting lactotroph resides.

Abbreviations; third ventricle (III.v), optic chiasm (OC), internal

zone (IZ), mammillary bodies (MB). (Reprinted from (1)).

2

pathways may bridge the connection between STAT signaling and clock gene expression (for

review see (1)).

Dopaminergic Regulation of PRL Secretion

Dopamine of hypothalamic origin exerts a tonic inhibitory control over PRL secretion

(for review see (1,4,5)). Three populations of neuroendocrine dopaminergic neurons (NDNs)

with cell bodies in the mediobasal hypothalamus release dopamine (DA; Fig. 1). The (1)

tuberoinfundibular dopaminergic (TIDA) and (2) tuberohypophseal dopaminergic (A12)

(THDA) neurons have cell bodies located throughout the arcuate region of the mediobasal

hypothalamus and (3) periventricular hypophysial dopaminergic (A14; PHDA) neurons have cell

bodies located in the periventricular region of the rostral hypothalamus (Fig. 1). PHDA and

THDA axons project down the infundibular stalk terminating on fenestrated short portal vessels

within the neural (NL) and intermediate (IL) lobes of the pituitary gland (22,23). Alternatively,

TIDA axons terminate on a fenestrated capillary bed within the external zone of the median

eminence that drains into long portal vessels (LPV) carrying dopamine to the AL of the pituitary

gland (for review see (12)). The role of TIDA as the primary PRL inhibitory neurons is well

established in the literature (24). However, a growing importance has been assigned to THDA

and PHDA neurons in the regulation of PRL secretion (25). In fact, evidence from posterior

lobectomy experiments strongly support a role for the THDA and PHDA in the regulation of

PRL secretion (25-27). There is an endogenous circadian rhythm of PRL secretion in the

ovariectomized (OVX), OVX-estradiol replaced, and cervically stimulated rat wherein PRL

levels remain low in the early portion of the light phase and gradually rise to peak in the late

afternoon (24,28-30). Thus, the rhythm of PRL release is inversely correlated with DAergic tone

in a complex output putatively entrained by photic cues originating in the central biological

clock, or suprachiasmatic nucleus (SCN; (24)). NDNs express PRL receptors and show marked

responses to circulating PRL levels, suggesting a high degree of sensitivity to the

pharmacological and physiological status of the animal (31,32). A plethora of afferent inputs

from regions throughout the CNS modulate DA release, thereby affecting PRL secretion. These

factors include serotonin derived from the dorsal raphe, norepinephrine-epinephrine from the

locus coeruleus and vasoactive intestinal polypeptide (VIP)/arginine vasopressin (AVP) from the

SCN and paraventricular nucleus (for review see (1)). Though I have focused on the

3

mechanisms driving circadian rhythms of NDN activity, I acknowledge the importance of

rhythmic PRL-releasing factors in the control of PRL secretion.

Physiological Rhythms of Prolactin Secretion

PRL secretory patterns depend on the physiological status of the animal. Three

physiological states characterized by distinct patterns of PRL secretion include the estrous cycle,

lactation and pregnancy/pseudopregnancy (see Fig. 2). During the 4-5 day estrous cycle, PRL is

released on the 3rd

-4th

day or proestrus, in response to rising titers of ovarian steroids (Fig. 2; for

review see (1,33)). Experiments with OVX rats given exogenous steroid treatment suggest a

critical period for the timing of the proestrous PRL surge (34). A single bolus injection of

estradiol-17β, given to an OVX animal induces daily surges of PRL secretion around 1600h,

suggesting a circadian regulatory system modulated by the rising titer of ovarian steroids. Rising

levels of ovarian estradiol act at the pituitary gland to enhance PRL synthesis/secretion and

reduce the expression of DA receptors (35-37). Further, ovarian steroids affect the

hypothalamus to modulate action potential frequency and gene expression within NDNs (38-40).

Like estradiol, ovarian progesterone plays a clear role in the timing and amplitude of the

proestrous PRL surge (41). Further, precise timing of the proestrous PRL surge enhances female

sexual behavior (reviewed in (33)). Animals treated on proestrus with the DA agonist

bromocriptine, which blocks the proestrous PRL surge, displayed a reduced lordosis quotient that

was restored with addition of ovine PRL (42). While it is clear that ovarian steroids initiate the

proestrous surge, very little is known with respect to the termination of the surge in the absence

of mating during the estrous cycle.

While it is clear that DA release from the hypothalamus plays a role in the timing of the

proestrous surge, little evidence supports a role for a singular PRL-releasing factor (PRF). DA

levels in the median eminence and pituitary gland, as well as the activity of DA synthetic

enzymes in the arcuate nucleus, decline during the proestrous surge (43-45). The candidates for

PRL-releasing factor include, but have not been limited to, thyrotrophin releasing hormone

(TRH), vasoactive intestinal polypeptide (VIP) and oxytocin (OT) and angiotensin II (for review

see (1)). Evidence for TRH as a PRL-releasing factor comes from studies using passive

immunoneutralization of endogenous TRH (46). However, failure to observe a rise in TSH that

should accompany a PRL surge-inducing rise in TRH decreases enthusiasm for TRH as a

significant PRF. VIP has been shown to stimulate PRL secretion both in vivo (47) and in vitro

4

(48) and VIP receptors (type-1, VPAC-1) have been identified on lactotrophs and on neurons of

the medial basal hypothalamus (for review see (1,49)). Thus, VIP and TRH are both good

candidates for PRF during the estrous cycle. OT may also play a role in regulating the

proestrous PRL surge, as it has been shown to induce PRL secretion in vivo (50), and direct

antagonism of OT receptors blocks the proestrous PRL surge (51). Although several additional

factors of hypothalamic origin contribute to the family of PRFs, it remains to be seen whether a

single factor plays a prominent role in the initiation of the proestrous PRL surge. Moreover,

limited evidence exists to support a functional link between circadian oscillators in the SCN and

PRF neurons within the hypothalamus (52,53). However, as the afternoon surge induced by

estradiol in the OVX rats faithfully occurs between 1500-1700h, it is apparent that a circadian

component, most likely of hypothalamic origin, drives this daily rhythm through both PRF

activation and a decline in DAergic tone. Evidence from experiments in my laboratory support

this assertion, suggesting the function of an underlying “endogenous stimulatory rhythm”

mediated by a heretofore-unverified PRF (53-55).

The best-understood and most widely studied physiological rhythm of PRL secretion is

the neuroendocrine reflex driving PRL secretion during lactation (Fig. 2; for review see (1)).

Serum PRL levels increase within minutes of the suckling stimulus, remain elevated during

nursing and are proportional the intensity of the nursing stimulus (i.e. the number of hungry

young pups; Fig. 2; (1)). Interestingly, the magnitude of PRL secretion in response to suckling

young increases in the afternoon, suggesting a synergism between the neuroendocrine reflex

controlling suckling induced PRL secretion and the under-lying circadian rhythm of PRL

secretion (55,56). While some evidence supports a role for several of the putative PRFs in the

neuroendocrine reflex, a clear candidate has remained elusive (1). Although I did not examine

the role of clock gene expression in suckling-induced PRL secretion, I can assume that the

underlying circadian rhythm is driven by the same basic mechanism we are examining in OVX

rats.

In animals housed under a standard L:D cycle (lights on 0600-1800h), stimulation of the

uterine cervix (cervical stimulation or CS; which leads to pregnancy or pseudopregnancy (PSP))

induces a PRL secretory pattern characterized by a nocturnal surge (N) between 0100h – 0500h

and a diurnal surge (D) occurring between 1600h – 1800h (Fig. 2 and (57)). The two daily

5

Figure 2. Physiological rhythms of PRL secretion. During

pseudopregancy/pregnancy (top middle) there are two daily surges of PRL, a

nocturnal surge around 0300h and a diurnal surge around 1700h. Following

application of the mechanical suckling stimulus (bottom left), serum PRL

levels increase in the dam until pups are removed. The magnitude of the

suckling-induced increase depends on the number of pups. During the 4-5

day estrous cycle (bottom right) PRL levels rise on the afternoon of proestrus,

along with the gonadotrophins LH and FSH, in response to rising titers of

ovarian steroids and immediately precedes ovulation on the morning of estrus

(graphic courtesy of M. Egli).

6

surges of PRL initiated after application of a mating stimulus to the uterine cervix provide the

primary luteotrophic support necessary to maintain the progesterone secretory activity of the

corpus luteum during early pregnancy (33,57). The twice daily surges of PRL during

pregnancy/PSP persist for 10-12 days and are driven by synergistic input from both

hypothalamic release and inhibiting hormones (57,58). Previous experiments in my laboratory

suggest that these surges are driven by OT of hypothalamic origin (59,60). Further, data indicate

that stimulation of the uterine cervix results in a significant decrease in the activity of the NDNs

at the approximate time of each surge (61). Therefore, my laboratory proposed the existence of

an endogenous stimulatory rhythm for PRL secretion, unmasked by the decline in DAergic tone

and perpetuated by the PRF activity of OT following stimulation of the uterine cervix (53,55,60).

Evidence from these experiments suggested that the nocturnal rise in OT was mediated by

vasoactive intestinal polypeptide (VIP) of SCN origin, while the diurnal surge was driven by

serotonergic input from the dorsal raphe nucleus (52,54,59,60). Recent evidence from my

laboratory suggests a more complex integration of the OT system by inputs from the central

oscillator in the SCN (58). Evidence suggests that the timing of the two daily PRL surges during

PSP is mediated by direct input from the SCN (29,62). The current model suggests that VIP of

SCN origin entrains the two surges through a direct OT-stimulating effect in the early morning.

Further, OT acts indirectly on interneurons within the hypothalamus in order to decrease

DAergic tone throughout the 10-12 days of PSP (58). Thus, the SCN plays an integral part in

controlling the timing of the two daily surges with respect to the 24-hour day and represents a

strong link between the central biological clock and the neuroendocrine circuit regulating PRL

secretion.

Given the ubiquity with respect to control of the physiologically relevant secretion of

PRL, I attempted to better understand the underlying mechanism by which the SCN controls the

timing of PRL secretion. I have utilized the proestrous PRL surge as a model due to its

simplicity and elegance. I isolated the effects of cyclic ovarian hormone secretion using the OVX

rat as a model and determined the influence of ovarian steroids using an acute steroid

replacement strategy. I chose not to investigate the link between the circadian oscillator and

lactation or pregnancy/PSP due to the increased complexity associated with the neuroendocrine

reflexes and hypothalamic networks regulating each of these distinct rhythms. However, as

stated above, the SCN appears to play a significant role in the timing of these physiological

7

rhythms. Thus, the mechanism by which the circadian clock regulates the neuroendocrine system

underlying the proestrous afternoon PRL surge may represent the fundamental timing unit of a

broad range of physiological states.

Circadian Rhythms: Form and Function

The circadian timing system consists of three primary components, the circadian

oscillator, the outputs of that oscillator and afferent inputs from the retina connecting the

biological clock to the environment. In mammals, the central circadian clock is located within

the suprachiasmatic nucleus of the anterior hypothalamus (63,64). The SCN consists of paired

symmetrical nuclei divided along the midline by the third ventricle and containing approximately

10,000 individual neurons and glial cells per nucleus (65,66). The input to the SCN arises from

distinct retinal ganglion cells and terminates predominately within the ventrolateral or core

region of the nucleus (67-73). The SCN makes an array of diverse projections throughout the

brain and spinal cord, influencing a diversity of functions; from rhythmic hormone secretion to

locomotor activity, feeding and sleep/wake cycles. Fundamental experiments have shown that

each individual SCN neuron contains the fundamental properties of an endogenous circadian

oscillator (74,75). The seminal work of Pittendrigh, Daan and Aschoff described circadian

rhythms according to three basic tenants or principles (76-82). The first, and most obvious,

principle is the well-defined period of the rhythm. The term “circadian” (latin circa-dias; around

one day) describes any rhythm with a period or tau (τ) of approximately 24 hours. This

identifies and distinguishes the rhythm from other rhythms that have either considerably shorter

τ (i.e. ultradian rhythms having a period less than 24 hours and generally on the order of minutes,

also referred to as circhoral) or periods considerably longer than 24h (t > 24h, referred to as

“infradian”) including such weekly (estrous cycle), monthly (menstrual cycle) or yearly (circa-

annual) cycles; for review see (65).

A second primary characteristic of a circadian oscillation, and most important for my

purpose, is that the oscillation continues in the absence of any external influence (78).

Therefore, in the absence of any exogenous input from the environment, a physiological rhythm

is considered to be circadian if it is maintained with a period close to 24h (80). The concept of an

endogenous, free-running (i.e., continuous oscillation in constant conditions), rhythm found it’s

origin as early as the 18th

century, when the astronomer, Jean Jacques d’Ortous de Mairan,

published his brief communication on the rhythmic leaf movements of the heliotrope plant,

8

Mimosa pudica (66). De Mairan observed a rhythm of pedicel and leaf opening in a light:dark

cycle, characterized by leaf opening during the light phase, that persisted in constant conditions

(66). However, it is generally believed that the concept of an endogenous circadian rhythm was

not truly accepted until the early 1900’s, when von Frisch and Forel published their work on the

endogenous rhythm of food seeking in honeybees (66). It is now generally accepted that free-

running circadian rhythms of hormone secretion, locomotor activity, feeding, sexual

reproduction and metabolism are ubiquitous from single cell protists to mammals (83-85). The

concept of “Circadian Time” (CT) refers to a subjective measure dependent on the activity of the

animal under constant conditions, such that CT12 identifies the time of day when activity onset

occurs and CT24 indicates the end of the activity period. In contrast, “Zeitgeber Time” (ZT) is

defined by the duration of the light and dark phases of the photoperiod, such that ZT0 refers to

the time of lights on and ZT12 refers to the time of lights off. Of course, when animals are

entrained to a defined light:dark (L:D) cycle, ZT12 and CT12 occur at approximately the same

time of day (clock time). The earliest experiments identifying “free-running” rhythms were

conducted in humans by Aschoff and colleagues, wherein the authors developed the theory of

“internal desynchronization” that describes the free-running rhythms of hormone secretion, body

temperature and locomotor activity in a constant environment and the gradual dissociation of the

free-running sleep/wake cycle and other rhythms including body temperature (for review see

(86,87)). Moreover, the abolition of free-running circadian rhythms of locomotor activity and

adrenal glucocorticoid secretion following SCN lesions have provided fundamental evidence

linking the SCN to core biological clock function (63,64,88-90).

A third and final characteristic of a circadian oscillation is the ability of that rhythm to

entrain to environmental cues (81). To be characterized as an endogenous, circadian oscillation,

a rhythm must entrain to external cues. Entrainment, within limits, may be induced by any

number of external stimuli ranging from light stimulation at the photoreceptor cell (91) to the

availability of food (92,93). Entrainment allows the organism to anticipate events in its

environment and make decisions regarding resource availability and energy expenditure that

confer an evolutionary advantage for that organism (84,94). The entrainment limits of an

oscillator and the phase response to light exposure describe the oscillator and represent species

and individual differences (79). Most, if not all, mammals can easily entrain to daylengths

between 20 and 26 hours, but find it difficult to entrain to photoperiods outside this range

9

(79,95). The nature of light entrainment is best exemplified by the phase response curve. The

phase response curve indicates the response of a parameter, such as locomotor activity, in terms

of phase advances or delays across the subjective day to a zeitgeber such as light (79,96,97). The

phase response curve is an accurate measure of the entraining power of light on the oscillator, as

each oscillator (i.e. individual animal) responds with variable phase shifts (in terms of

magnitude), following a light-pulse (82). The power of the shift (advance or delay) also relates

to the pattern of activity in the organism. Diurnal rodents and humans tend to have periods

slightly longer than 24 hours and generally respond more strongly to phase delaying light pulses

delivered during the early subjective night (circadian time (CT) 14-18) (76,77,79,81,98), while

nocturnal mammals tend to have periods slightly shorter than 24 hours and respond more

strongly to phase advancing pulses during the late subjective night (CT18-22) (79,99). The

aforementioned guidelines are referred to as “Aschoff’s rule” and are, minus a few exceptions,

generally true for both nocturnal and diurnal species (77). Therefore, phase response curves

reflect the power of the entrainment to the 24 L:D cycle of the oscillator by light and the

apparent dependence of that phase-shift on the endogenous free-running period of a given

organism (79). Although many rhythms display all three of the aforementioned characteristics,

many rhythms display only one or two of these characteristics. I have adopted the term “semi-

circadian” to categorize any rhythm that displays part, but not all, of the characteristics of a

circadian rhythm. The term “semi-circadian” should not be confused with semicircadian

rhythms, such as the biphasic pattern of PRL secretion in the pseudopregnant rat. I have

utililized the three primary features of a circadian rhythm outlined above in determining the

nature of the circadian rhythms of DA release and gene expression in NDNs of OVX-steroid-

primed rats.

Circadian Rhythms in Endocrinology and Neuroendocrinology

The relationship between the circadian timing system and the endocrine/neuroendocrine

system has been established by nearly 30 years of research. Circadian rhythms of hormone and

neurohormone secretion have been thoroughly characterized in mammals (100-102). The

hierarchical relationship between the circadian oscillator in the SCN and targets in the

hypothalamus responsible for regulating hormone secretion has been established for nearly all of

the known neuroendocrine factors (87,100,101,103). In rodents, circadian rhythms of pituitary

gland hormone secretion and hypothalamic neuroendocrine neuron activities have provided the

10

foundation for our most fundamental understanding of circadian physiology (64). The role of the

SCN in the control of rhythmic hormone secretion in females has been thoroughly studied and

well established. Classic experiments by Everett and Sawyer established the potential role of the

circadian oscillator. The authors found that blocking the proestrous surge of LH with a timed

injection of pentobarbital in the early afternoon, but not early evening, delayed the surge for

exactly 24 hours (104). Thus, the drug failed to abolish the releasing mechanism but simply

delayed the trigger for LH secretion and subsequent follicular rupture until the same time of day

on the following day (104). Similar and equally elegant studies have been conducted with regard

to the steroid-induced LH and PRL surges in OVX rats (105-108). This experiment provided a

key piece of data supporting the relationship between the circadian timing system and the

endocrine system. In mammals, circadian rhythms have been observed for almost all of the

primary hypophysiotropic hormones including growth hormone (GH), thyroid-stimulating

hormone (TSH), PRL and adrenocorticotrophic hormone (ACTH (101)). As mentioned,

circadian rhythms of hypothalamic-release and inhibiting factors have been observed for each of

the aforementioned pituitary gland hormones (100). The SCN plays the role of orchestrator,

synchronizing the rhythms of hormone secretion to maintain adequate function of the organism.

Several hormones also display ultradian (LH and ACTH) and circa-annual rhythms (PRL in

seasonal breeders) of secretion (for review see (100)). The interaction between ovarian steroid

hormone secretion and the circadian timing system is implied by the identification of steroid

receptors within SCN neurons and the observation of various steroid effects on gene expression

within the SCN (109-113). My laboratory has shown dramatic effects of ovarian steroids on

circadian rhythms of DA release from NDNs corresponding to significant changes in the timing

and amplitude of PRL secretion (see Chapter 2 and (45,114)). Thus, like general endocrine

physiology, maintenance of endocrine and neuroendocrine rhythms requires long and short -loop

feedback between the circadian timing system and the neuroendocrine-endocrine system.

The Photoneuroendocrine System

In many spontaneously ovulating species, the circadian timing system facilitates timed

events including: ovulation, mating and sexual receptivity (for review see(1,100,115)). In some

seasonal breeders, the circadian timing system is dedicated to measuring daylength, necessary for

successful fertility and reproduction (116-119). Initiation and maintenance of seasonal breeding

cycles depend on the rhythmic synthesis and release of the pineal hormone melatonin (120-122).

11

Melatonin synthesis and secretion at the level of the pineal gland is indirectly controlled by the

SCN through a multisynaptic pathway including neurons within the paraventricular nucleus and

superior cervical ganglion (123,124). Noradrenergic neurons within the superior cervical

ganglion project back to the pineal gland, where they release norepinephrine at the melatonin-

secreting pinealocytes (116,125,126). Moreover, AVP neurons may also project directly to the

pineal gland to regulate melatonin synthesis and release (127-129). Evidence from several

experiments suggest that the SCN tonically inhibits melatonin secretion during the day

(130,131). In turn, secreted melatonin affects the activity of SCN neurons in an elegant long-

term feedback loop (132-135). Melatonin receptors are also expressed within the pars tuberalis,

an embryonically derived extension of the anterior pituitary gland that encapsulates the

infundibular stalk (136-138). Hormone secreting cells of the pars tuberalis predominately secrete

gonadotrophins, although some cells also secrete PRL and novel peptide hormones referred to as

tuberalins (139-144). The primary function of melatonin in seasonal breeding species, such as

the sheep and hamster, is to initiate seasonal changes in fertility, physical appearance and

reproductive behavior (145-151). Recently, experiments indicate that rhythmic expression of

clock genes within cells of the pineal and pars tuberalis contribute to the appearance of seasonal

reproductive rhythms such as increased frequency of PRL and LH secretion (152-155). Evidence

suggest that the duration of the light period dictates the duration of melatonin secretion, which in

turn signals the melatonin sensitive cells in the pars tuberalis and pre-mammillary hypothalamus

to facilitate seasonal changes in PRL and gonadotrophin secretion (147). The role of the pineal

in regulating seasonal rhythms is supported by studies with pinealectomized animals, which

continue to display circadian, but not seasonal rhythms (116-119). The duration of the

photoperiod is processed by the SCN and transduced in the pineal where the information is

converted into the expression and activation of melatonin synthesis enzymes (116). The duration

of melatonin secretion increases with shortening of daylength in long (Syrian and Siberian

hamsters) and short (sheep) –day breeders. The increased or decreased duration of melatonin

secretion in seasonal breeders inhibit or stimulate reproductive factors, such as changing pelage,

testis weight and overall body fat content (116). The ability of photoperiod duration to dictate

seasonal reproductive function via the circadian timing system was first suggested by Bunning,

and is currently referred to as “the external coincidence model” (116). Clock genes, such as PER

and CLOCK, may act to facilitate seasonal rhythms through localized regulation of rhythmic

12

gene expression, periodically modulated by periodic pineal melatonin. In fact, evidence suggests

cycling expression of the clock gene PER1 in rodent pituitary gland cells depends on

sensitization of adenosine receptors, which occurs through nocturnal activation of MEL1a

melatonin receptors (155). I hypothesized that a similar system drives the rhythmic release of

DA from NDNs. In lieu of melatonin, the SCN drives the NDNs through direct neural input,

while clock gene expression within the DA neurons modulates rhythms of DA synthesis and

release. I cannot rule out a role for melatonin in our model system, although little evidence

supports photoperiodic regulation of PRL secretion in the rat (156).

Circadian Regulation of Neuroendocrine Dopaminergic Neuronal Activity

Although the rhythm of activity in DA release and PRL secretion is modulated by ovarian

and adrenal steroids, the endogenous rhythms persist in the OVX rat with dampened magnitude

and modest phase advance (114). Experiments reveal that estrogen replacement (28,105,106)

and cervical stimulation (157,158) result in significant daily PRL increases in the afternoon and

early morning , indicating an oscillatory mechanism influenced by ovarian steroids. Analyses in

my laboratory and others have identified an endogenous rhythm of PRL releasing factors in the

female rat (53,159) and an endogenous daily rhythm of DA turnover from NDN (114). The use

of the immediate early genes (IEG) c-fos and the fos-related antigens (FRAs) as viable markers

of neuronal excitability in neuroendocrine cells of the hypothalamus is well established

(160,161). My laboratory has shown that immediate early gene expression within the NDN of

OVX, OVX-steroid replaced, and cervically stimulated rats has a daily rhythm corresponding

with DA turnover in terminal regions of NDN inversely correlated with PRL secretion (61,162).

The rhythm of DA release from TIDA neurons and the role of circulating ovarian steroids in the

timing of these rhythms has been established in rat (24,30,114,163-165). Preliminary data

suggest that estradiol plays a role in the timing and amplitude of the circadian rhythm of NDN

activity and the afternoon PRL increase (114).

A role for the SCN in driving the timing of NDN activity has been suggested, though a

precise neural mechanism by which the SCN regulates NDN activity is still largely unknown.

Evidence from hamsters suggests that neural afferents from the SCN to hypothalamic targets are

necessary for maintenance of endocrine and neuroendocrine rhythms (166-168). However,

evidence from tract-tracing experiments in hamsters suggest that SCN efferents project primarily

to the sub-paraventricular region and the dorsomedial hypothalamus, with very few projections

13

terminating within the PeVN and ARN (169,170). Experimental evidence supports the presence

of VIP-IR fibers on and around NDN cell bodies in the OVX rat (171,172). Vasoactive intestinal

peptide is produced by cells in the ventrolateral SCN and displays a light-entrained diurnal

rhythm of mRNA (173,174) and protein expression (52,175-178). The rhythm of VIP mRNA

expression in the SCN of female rats degrades over the life of the animal, in parallel with the

amplitude and frequency of many endocrine/neuroendocrine rhythms (179,180). Further, recent

evidence suggests developmental reorganization of VIP afferents on gonadotrophin-releasing

hormone neurons in the pre-optic area of the female rat (181). These data suggest that VIP, of

SCN origin, plays a significant role in the generation and maintenance of the pre-ovulatory PRL

and LH surges. VIP receptors have been localized to neurons in the arcuate nucleus and AL of

the pituitary gland (182), and lesions of the SCN abolish the diurnal rhythm of PRL secretion

(24). Moreover, pituitary adenylate cyclase activating polypeptide (PACAP) and VIPergic

efferents, arising from the ventrolateral portion of the SCN, may play a role in relaying photic

cues directly from the retina to NDN (183-185).

Given the established role of circulating ovarian steroids in regulating the rhythm of

NDN activity and PRL release in the OVX rat (186), my laboratory determined the effects of

ovarian steroids on the expression of VIPergic fibers and VIP type-2 (VPAC2) receptors on

NDN. Data revealed that exogenous estradiol and progesterone treatment induced a significant

increase in VPAC2 receptors on NDN (172), supporting a modulatory role for steroid hormones

in VIPergic transmission of photic cues from the SCN to NDN. Additional evidence suggests

that ovarian steroids modulate clock gene and gap-junction forming protein expression within the

SCN (111-113). Therefore, varying titers of ovarian steroids, perhaps during the estrous cycle,

may act centrally within the SCN to adjust the timing of neuroendocrine DAergic neurons.

Finally, experiments show that disruption of VIP protein expression in the ventrolateral SCN

with antisense deoxyoligonucleotides (AS-ODN) against VIP mRNA affects the endogenous

rhythm of LH secretion and the circadian rhythm of activity in NDN (187,188). Data from these

experiments suggest that VIP inhibits DA release in the afternoon in steroid-primed OVX rats,

allowing for the afternoon PRL surge. The inhibitory action of VIP on the VPAC2 receptor is

novel, as VPAC2 receptors are classically associated with stimulatory G-proteins (189,190). A

similar interaction appears to regulate AVP-mediated inhibition of corticotrophin-releasing

factor (CRF)-releasing neuron activity during the first half of the light-phase in nocturnal rodents

14

(191). Additional experiments suggest that the inhibitory effects of AVP on CRH neurons is

mediated by GABAergic interneurons within the DMH and sPVN (192). Although I have

determined the presence of VIPergic afferents on DAergic neurons, a similar mechanism may

account for VIP-mediated inhibition of NDN activity. Given the apparent anatomical

relationship between the SCN and the NDN, I have determined the pattern of clock gene

expression in the SCN and both clock gene expression and DA turnover within NDNs and their

target regions in an attempt to reveal a functional correlation between the central oscillator

(SCN) and the potential “slave” oscillator (NDNs).

Molecular Mechanism of Circadian Timing

The circadian timing of neuroendocrine rhythms is vital to the response of the organism

to its environment and conserved throughout ontogeny in eukaryotic cells (94). The timing of

neuroendocrine events is vital for proper homeostatic and reproductive function in mammals,

and the ability of an organism to respond to shifts in the light:dark cycle confers a strong

evolutionary advantage. The endogenous nature of SCN generated rhythms has inspired

molecular and physiological research into specific cellular events that provide the framework of

these rhythms (193). As early as the 1970’s, experiments suggested that rhythmic gene

expression within the CNS played a fundamental role in the appearance of circadian rhythms

(194). The pivotal work begun in the laboratories of Seymour Benzer and colleagues, as well as

the experiments conducted less than 20 years later by Michael Roshbash and colleagues, opened

the door to our understanding of the molecular mechanisms driving circadian clocks (195-197).

Mammalian homologs of the cyclic Drosophila genes period and cycle have only recently

been cloned and expressed within the SCN of the anterior hypothalamus in mouse, rat and

human (198-200). However, providence opened the gateway for our understanding of the

genetic basis for molecular clocks nearly 10 years before the positional cloning of the clock

(Circadian Locomotor Output Cycle Kaput) locus in mice (198). In 1988, Ralph and Menaker

identified a novel behavioral phenotype in golden hamster characterized by a shortening of the

free-running period from ~24h to ~20h (201). While the authors failed to fully characterize the

molecular basis for the mutation, their work undoubtedly inspired interest in the molecular basis

for circadian rhythms. Following the successful cloning of CLOCK, a series of gene products

containing basic helix-loop-helix (bHLH)/PAS domains (per-arnt-sim protein interaction

domains) and displaying distinct circadian rhythm of expression in SCN cells have been cloned

15

from mammalian tissues, including period 1-3, cryptochrome 1-2, brain-muscle arnt-like protein

1 (BMAL1), Rev-erbα, retinoic acid-like orphan receptor alpha (RORα), Differentiated 1,2

(Dec1,2) and the kinases Casein kinase Iε and Casein kinase Iδ (202-207).

The mammalian clock genes comprise a complex transcriptional/translational feedback

loop that regulates the rhythmic output of the oscillator (see Fig. 2 (202,204,206)). The core

mechanisms of the circadian clock are putative “clock” gene products regulated through

transcriptional control feedback by their own protein products (204). The feedback loop

involves the regulation of the three dper homologs (per 1-3) and the two cryptochrome genes

(cry1 and cry2; (204,206)). Per and cry expression is driven by the basic helix-loop-helix/PAS

domain containing protein transcription factors BMAL1 and CLOCK (204). PER and CRY

proteins act as negative regulators in the feedback loop, translocating back to the nucleus and

disrupting the expression of bmal1:clock through interactions with CACGTG-sequence E-box

enhancer elements (208,209). Evidence from per1/2 single and double-knockout mice suggest

some functional redundancy between per1 and per2, but that at least one paralog is required for

the expression of circadian rhythms of locomotor activity (210). In CRY deficient mice mper1

and mper2 gene expression is arrhythmic and both genes are expressed at moderate levels,

indicating an essential role for cryptochrome genes in negative regulation of the rhythmic

feedback loop (208). A series of interlocking negative and positive feedback loops including the

repressor Rev-erbα and retinoic acid-like orphan receptor α (RORα) complete the regulatory

system. While CLOCK:BMAL1 heterodimers drive transcription of the PER and CRY

repressors, they also drive expression of the rev-erbα gene. REV-ERBα has been shown to

repress BMAL1 transcription through interactions with the product of the rorα gene (211,212).

RORα has been shown to act as a positive feedback regulator, driving expression of BMAL1

following nuclear translocation (212). Evidence from both experiments and mathematical

models suggest a modulatory role for the REV-ERB/ROR feedback loops (and their Drosophila

counter-part, the CLOCK driven VRILLE/PDP-1 feedback loop (207,212,213)). Results from

Rev-erbα mutant mice support a functional, albeit semi-redundant, role for this additional

feedback loop (214). Rev-erbα mutants display altered free-running periods of locomotor

activity but remain able to sustain oscillations in constant conditions and entrain to a

photoperiod. An additional loop involves CLOCK:BMAL1 driven transcription of the dec1 and

16

Figure 3. The Molecular Clock. The core molecular clock is driven by a series of

interlocked transcription/translational feedback loops. The transcription factors CLOCK

and BMAL1 interact as heterodimers and enhance transcription of the PERIOD and

CRYPTOCHROME gene families. The PER and CRY gene products heterodimerize

after phosphorylation by Casein Kinase I epsilon and act as a repressor through allosteric

interactions with CLOCK:BMAL1 heterodimers. REV-ERBα transcription is similarly

regulated by CLOCK:BMAL1 and acts as a repressor of BMAL1 transcription. Not

shown is the newly discovered role of the RORα gene product or the accessory loop

driven by DEC1/2. Modified from (204).

17

dec2 gene products (215). Like PER and CRY, DEC1/2 repress their own transcription by

blocking CLOCK:BMAL1 induced transcription and, in addition, they repress PER and CRY

transcription (215). Although significant, the RORα/Rev-erb-α and DEC1/2 feedback loops act

as peripheral loops and their role in the core oscillation appear to be only modulatory (207).

Thus, I only consider the CLOCK:BMAL1 driven PER and CRY expression feedback loop as

the central molecular oscillator. Therefore, the current model of the molecular clock includes the

fundamental negative feedback loops driven by CLOCK:BMAL, facilitated by delayed

repression of CLOCK:BMAL1 mediated transcription by PER and CRY and tuned by auxiliary

loops of ROR/REV-ERB and DEC1/2 mediated transcription (204,207).

Recently, the role of casein kinases in the molecular clock has provided additional

strength to the link between clock gene function and behavioral phenotypes. Casein Kinase Iε, a

mammalian homolog of the Drosophila gene product double-time, has been cloned in rat and

mouse and has been shown to play a central role in the molecular clockwork (216-219).

Experiments have shown that casein kinases phosphorylate PER/CRY protein complexes at

specific residues on the PER protein in order to facilitate nuclear translocation of the PER/CRY

complex (220,221). Further, disruption of the ability of the casein kinases to phosphorylate

PER/CRY through the addition of phosphotases results in reducing incorporation of nuclear

PER/CRY (221). Recently, investigators have found that a mutation of the phosphorylation site

on PER1 in rodents results in hypo-phosphorylation of PER and a build up of PER1 in the

cytoplasm, resulting in a strong advance of the free-running period, akin to the phenotype

observed in tau mutant hamsters (218,222). In fact, evidence now suggests that mutation of the

casein kinase Iε gene produced the behavioral phenotype observed by Ralph and Menaker nearly

20 years ago (222-225). Mutation of the PER2 phosphorylation site in humans is linked to a

sleep disorder commonly referred to as familial advanced sleep-phase syndrome, characterized

by an advance of wake time (as compared to WT) of approximately 4 hours (226-228). These

and other experiments have only begun to unravel the complex and exciting genetic basis for

sleep disorders. In parallel with evolutionary research into the phylogenetic prevalence of clock

gene function, these studies will provide new and exciting insights into the basis for circadian

rhythms.

In the following experiments, I have determined the endogenous nature of the circadian

rhythms of NDN activity in the female rat. In doing so, I have attempted to better understand the

18

nature of these rhythms with respect to their free-running periods and their ability to entrain to a

photoperiod. I have attempted to answer the following: 1) Do the NDNs that regulate the

precisely timed diurnal rhythm of PRL secretion from the lactotroph display free-running and

light-entrained circadian rhythms of DA release in the steroid-depleted rat?, 2) How do the

ovarian steroids estradiol and progesterone modulate the timing and amplitude of DA release

from NDNs?, 3) Are the putative clock genes, which have been implicated as the primary

components of the cellular oscillator driving autonomous rhythms, expressed in NDNs? and 4)

Are clock genes functionally linked to rhythms of DA turnover within NDNs? I have

speculated, based on several key findings within the literature, that NDNs acts as a functional

“slave”-oscillator, dependent on SCN neurons for entraining cues and long-term maintenance of

a strong free-running rhythm. However, even in the absence of SCN-derived afferent inputs, the

NDNs may retain the ability to oscillate with a period near 24h. Thus, the NDNs drive the

precisely timed PRL secretory event through both indirect timing cues transduced by the SCN

and through local timing of DA synthesis and release, precisely generated by the cells own

transcriptional machinery.

19

CHAPTER 1

CIRCADIAN RHYTHMS OF NEUROENDOCRINE DOPAMINERGIC NEURONAL

ACTIVITY IN OVARIECTOMIZED RATS

Introduction

In ovariectomized (OVX) estrogen-treated (24,28,30) or cervically stimulated (29,158)

rats, there is an endogenously controlled rhythm of PRL secretion. These rhythms of PRL

secretion are inversely correlated with the release of DA into portal blood in a complex output

entrained by photoperiod cues transduced by the suprachiasmatic nucleus (SCN) of the anterior

hypothalamus (24,62). Among the three populations of neuroendocrine dopaminergic neurons,

only the TIDA population has been characterized as under circadian control (24). Results from

SCN lesion experiments suggest that the diurnal rhythm of DA release in TIDA nerve terminals

is driven by photic cues transduced by the SCN (229). Since TIDA, THDA and PHDA all

participate in the control of rhythmic PRL secretion (25), I hypothesize that all NDNs display

endogenous circadian rhythms of activity, most likely driven by SCN efferents and entrained by

photic cues transduced by the SCN, that maintain the proper timing of PRL secretion.

In order to be considered an endogenously driven circadian rhythm, a cyclic phenomenon

such as DA turnover or PRL secretion must possess three main attributes: (1) the rhythm must

have a period (τ) of approximately 24 hours, (2) it must continue to cycle with a free-running

period of approximately 24 hours under constant conditions such as constant darkness (DD) or

constant light (LL) and (3) it should be entrained to a Zeitgeber, such as light, arousal, or some

other exogenous cue (66,76,94,230). Given current knowledge, I have characterized the

endogenous rhythms of DAergic neural activity in TIDA, THDA, and PHDA neurons by

measuring DA turnover in the ME, NL, and IL with high performance liquid chromatography

coupled to electrochemical detection (HPLC-EC), as well as serum PRL and corticosterone

(CORT) concentrations by radioimmunoassay (RIA). The circadian rhythm of serum CORT

20

levels has been thoroughly studied and was included as a positive control for proper function of

the SCN under variable lighting conditions. OVX rats were subjected to classic approaches for

defining a circadian system including: (a) a standard 12:12 L:D cycle (illumination from 0600 to

1800 h), (b) a 6 h phase-delay of the L:D cycle (illumination from 1200 to 2400 h), or (c)

constant darkness (DD). These studies were performed in OVX rats in order to isolate these

rhythms from possible influences of ovarian steroids.

Methods

Animals

Adult female Sprague-Dawley rats (> 60 days of age) weighing 250-300g (Charles River

Labs inc. Charles River N.C.) were used in all experiments. Animals were housed under varying

lighting conditions with constant temperature (25oC) and humidity, with standard rat chow and

water available ad libitum. Under 12:12 L:D cycles, the room was illuminated with four 40 W

fluorescent bulbs. Animals housed under DD conditions and those housed under a 12:12 L:D

cycle were sacrificed during the dark phase under dim red light (< 1 lux). For DD animals, all

maintenance was performed in dim red light (<1 lux) with the aid of infrared goggles (Unitec

Series, GSCI Inc., Canada) at variable times between 0900h and 1400h to avoid potential

entrainment to non-photic stimuli by disrupting the animals during the inactive period (231,232).

All experimental procedures were performed with strict adherence to the guidelines for animal

care and use established by the Florida State University Animal Care and Use Committee.

Bilateral Ovariectomy and analysis of drinking rhythm

Animals were anesthetized with halothane and OVX bilaterally. The abdominal cavity

was exposed with a 10-15 mm incision immediately to the right of the midline and bilateral

ovarian tissues were removed. Hypothalamic tissue and pituitary glands were obtained from

OVX rats a minimum of 10 days post-ovariectomy when the concentration of estradiol in serum

reached a nadir of < 8 pg/ml, as previously measured by RIA (172,233). In the rat, feeding and

drinking patterns are well established circadian rhythms which can be used for monitoring

circadian time (63,64,234). In constant conditions the rhythm of drinking activity free-runs with

a period slightly greater than 24 hours, similar to the observed free-running period of locomotor

activity in the rat (63,235-239). Drinking activities of up to 8 animals were simultaneously

monitored continuously for 24 hours over several consecutive days with an automated device

21

(“Lickometer”; Dilog Instruments, Tallahassee, FL.; (240,241)) coupled to a task-dedicated

microcomputer. The device consists of a circuit measuring individual licks in thirty second bins

over 24 hours and automated data recording software for offline analysis. Drinking rhythms were

analyzed offline with ESP500 software (Ross Henderson, Dept. of Psychology, FSU) using a

moving average function of data binned at 10 minutes over a 12-h period before and after the

onset of drinking. Such analysis allowed us to determine the onset of drinking activity,

designated circadian time 12 (CT12), under varying lighting conditions. The circadian time scale

is a non-light cycle subjective time scale based on the timing of activity unique to each animal

(78). Circadian time was utilized in order to make direct comparisons across multiple animals

with respect to the free-running rhythms of DAergic neuronal activity in a population normalized

with respect to time of day. CT12 was used as a reference for tissue collection under both

alternating L:D (12h light; onset 0600h) and DD (constant dark) conditions. Analyses of drinking

patterns recorded with the device were used to generate double plotted actograms of drinking

activity for animals under both DD (Experiment I) and a phase-delayed L:D cycle (Experiment

II) (Fig. 4; Circadia software, ver. 2.1.16; Behavioral Cybernetics, Inc.).

Tissue Preparation

Tissue samples were collected at transitional points marking the beginning and end of the

activity period (subjective night; CT12 and CT0 respectively) and every four hours from CT2-

22. Animals were briefly exposed to CO2 (50% CO2: O2) and rapidly decapitated. Trunk blood

was collected. Serum samples were frozen at –20º C until assayed for PRL and CORT

concentrations by RIA. The brain and pituitary gland were quickly removed, placed on ice, and

the ME of the hypothalamus, as well as the NL and IL of the pituitary gland were carefully

dissected and placed in homogenization buffer (0.2 N perchlorate with 50 µM EGTA) and

rapidly (<10 sec.) frozen in an arctic-ice tube transport block (USA Scientific Inc.). Tissue

samples were stored at -80oC until assayed for DA and DOPAC. On the day of analyses for

catecholamines, tissue samples were thawed, briefly sonicated at 4oC, and 20 µl of the

homogenate was removed for determination of protein content. The remaining sample was

centrifuged at 13,000 rpm for 20 minutes. Supernatant was filtered on a Costar Spin-X 0.22 µm

nylon filter (Corning, Inc, N.Y.).

22

High Performance Liquid Chromatography with Electrochemical Detection (HPLC-EC)

The HPLC-EC technique is well established in my laboratory (31,45,114). The

concentration of DA and DOPAC (Dihydroxyphenylacetate), a primary metabolite of DA, was

measured in tissue extracts from the pituitary gland and ME. Briefly, 25 µl of the filtrate was

injected into the HPLC system by an autosampler (WISP 710B, Waters, Milford MA). Mobile

phase consisted of 75 mM sodium dihydrogen phosphate monohydrate (EM Science, Gibbstown,

NJ), 1.7 mM 1-octane sulfonic acid (Acros Chemicals), 100 µl/L triethylamine (Aldrich,

Milwaukee, WI), 25 µM EDTA (Fisher Scientific), 4.5% acetonitrile (EM Science), titrated to

pH 3.0 with phosphoric acid (Fisher Scientific), and delivered by a dual-piston pump (Kratos

Analytical Instruments, Ramsey, NJ) at 0.7 ml/min. Water was purified on a U.S. Filters ultra-

pure water system with ultraviolet cartridge to 18 MΩ resistance and polished with a Sep-Pak

mini-column (Millipore). Catecholamines were separated on a reverse phase C-18 column (MD-

150, Dimensions 150 x 3 mm, particle size 3 µm, 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: -85 mV, E2: -225 mV, ESA 5011 High Sensitivity Analytical Cell,

ESA). The change in current on the second electrode was measured by a coulometric detector

(Coulochem II, ESA) and recorded using Baseline 810 software (Waters). DA and DOPAC were

identified based on their peak retention times (RT = 9 min. and 5.5 min respectively).

The amount of catecholamine in each sample was estimated by direct comparison to the

area under each peak for known amounts of catecholamine. The amount of 3,4-

dihydroxybenzylamine (DHBA, RT = 6.5 min.) recovered was compared to the amount of

DHBA added as internal standard and corrected for loss of sample (usually < 5%). The

sensitivity of the assay is 30 picograms (pg) of DA and 15 pg of DOPAC. DA turnover,

characterized by the ratio of DOPAC:DA content in tissue, has been shown to be a reliable index

of neuronal activity in neuroendocrine dopaminergic neurons (242). DA turnover is defined as

the exocytotic release of DA from neuroendocrine DAergic nerve terminals, DA re-uptake into

presynaptic terminals, and the degradation of DA to DOPAC by monoamine oxidase (MAO;

(242). Thus, DA turnover is an indirect biochemical measure of DA release and metabolism,

reflecting acute changes in DAergic neuronal activity. DOPAC and DA levels were adjusted

with tissue protein levels (pg catecholamine/mg protein) and used to calculate the DOPAC:DA

ratio.

23

Protein Assay

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 IL.). Tissue

homogenate (10 µl) was aliquoted in duplicate into 96-well plates (Corning, Corning NY) with

200 µl of BCA solution and incubated at 60o C for 30 minutes. The absorbance of each well was

measured at 562 nm by a micro-plate spectrophotometer (Molecular Devices, Palo Alto, CA).

Unknowns were compared against standards of bovine serum albumin. Assay sensitivity was 100

µg/ml and the intra-assay coefficient of variation was 5-10%.

Radioimmunoassay

The concentration of PRL in serum was determined by RIA using NIDDK materials

supplied through the National Pituitary Hormone Distribution Program (A.F. Parlow) and

Protein-A as described previously (31,61,162). Serum concentrations of PRL are expressed as

ng/ml in terms of the rat PRL RP-3 standard. Assay sensitivity was 1 ng/ml and the inter-assay

and intra-assay coefficients of variation 10% and 5%, respectively. CORT was measured using

the commercially available Coat-a-Count®

rat corticosterone kit (Diagnostic Products Corp., CA)

according to the manufacturer’s specifications.

Experiment I. Neuroendocrine DAergic neuronal activity, serum PRL, and serum CORT

in OVX rats under a standard 12:12 L:D cycle (lights on 0600h) or constant darkness (DD)

DA turnover in the ME, NL, IL, serum PRL and CORT concentrations were measured in

samples obtained every 4 hours from CT 2-22 and at the light:dark transition points CT0 and

CT12 in animals in a 12:12 L:D cycle (lights on 0600) or under constant darkness (DD). Eight

adult female OVX rats of the Sprague-Dawley strain were placed in the Lickometer device in a

12:12 L:D cycle with lights on at 0600h for 5 days. On day 6, animals either remained in a 12:12

L:D cycle, or were placed in constant darkness (DD) for five days. Four animals at each time

point were sacrificed on the fifth day (10 days after OVX), tissue was collected for determination

of DA and DOPAC content by HPLC-EC and serum was collected to determine PRL and CORT

by RIA.

Experiment II. Neuroendocrine DAergic neuronal activity, serum PRL, and serum CORT

in OVX rats under a standard 12:12 L:D cycle (lights on 0600h) or a 6-hour phase-delayed

12:12 L:D cycle (lights on 1200h)

24

DA turnover in the ME, NL, IL, serum PRL and CORT concentrations were measured in

samples obtained every 4 hours between CT2 and CT22 and at the transition points between light

and dark (CT0 and CT12) and in rats following a 6-hour phase delay in the 12:12 L:D cycle

(lights on 1200h, lights off 2400h). Again, 8 animals were placed under a 12:12 L:D cycle with

lights on at 0600h for three days. On day 4, the illumination in the animal room changed to a

light cycle from 1200-2400 h. Four animals at each time point were sacrificed after one week

under the new phase-delayed 12:12 L:D cycle (10 days after OVX) and DA turnover, serum PRL

and serum CORT levels were compared to animals exposed to 12 h of illumination from 0600h.

Data Analysis

All data points are expressed as mean + SEM of 4 animals and identical data from 8

circadian times representing one 24-hour period were double plotted. Data were double plotted to

emphasize rhythmicity and allow for extrapolation of the proposed rhythm for an additional 24h

cycle. Serum PRL, CORT and DA turnover are plotted as a function of circadian time and

aligned at CT12 for comparison. Although they exhibit a distinct rhythm, all of my data do not

conform to a sine/cosine wave function that precluded a non-linear regression analysis to

elaborate the data as a function of time and lighting condition. Moreover, as single samples were

obtained from each animal at time points over a 24 h period, it is difficult to extrapolate accurate

phase and period measures. However, data were analyzed with two-way ANOVA for time of day

effects, lighting condition effects, and lighting x time interactions, followed by Bonferroni paired

post-hoc statistical tests and one-way ANOVA for within light-treatment time effects, followed

by multiple comparisons with the Student-Neuman-Keuls post-hoc test. P<0.05 was accepted as

the limit of significance. These analyses provide us with amplitude and duration values that were

used to estimate phase and period in the absence of a repeated-measures design. ANOVA were

performed and graphs were created with Graph-pad software (San Diego, CA.)

Results

Analysis of Drinking Behavior

The onset of drinking activity was determined on the two days prior to tissue collection

for each animal and averaged to determine activity onset on the following day. This method

allowed us to predict the onset of activity under entrained and free-running conditions with an

assumed error of 10-15 minutes, given the general level of variance of drinking activity onset in

25

Figure 4. Drinking activity from OVX rats under before and after transition

from a standard 12:12 L:D cycle to constant darkness or a delayed L:D

cycle (PD). Double plotted (see below) actograms of drinking activity from (A)

two representative animals (designated DDR1 and DDR2) on the last day before

and four days after transition from a standard12:12 L:D cycle (lights on 0600h)

to constant darkness (DD; sacrifice on day 5), and (B) two representative animals

(designated PDR1 and PDR2) during the six days under a phase delayed L:D

cycle (lights on 1200-2400h) prior to sacrifice (day 7).The data were double

plotted to emphasize rhythmicity and allow for extrapolation of the proposed

rhythm for an additional 24h cycle. In A and B, gray arrowheads indicate the

approximate onset of drinking activity under a standard 12:12 L:D cycle (lights

on 0600h) for each animal. Black arrowheads above the data in Fig. 1A indicate

the first day under DD conditions. The thickness of the horizontal bars represents

the mean amplitude of drinking activities. The break in activity found on the final

day of measurements represents the termination of data collection before

sacrifice and tissue collection. For all animals the onset of drinking behavior,

designated as CT12, was calculated as the average of drinking activity onset on

the last two days prior to the day of sacrifice (day 3 and 4 L:D/DD; day 5 and 6

phase delay L:D).

26

my rats (generally 10-15 minutes from cycle to cycle), which I consider acceptable with a

sampling frequency of 2-4 hours. Figure 4A illustrates double plotted actograms from two

representative animals (designated DDR1 and DDR2) before and after transition from a normal

12:12 L:D cycle (lights on 0600h) to constant darkness or (Fig. 4B) from two representative

animals (designated PDR1 and PDR2) after transition from a standard 12:12 L:D cycle (lights on

0600h) to a phase-delayed L:D cycle (lights on 1200h). In Figure 4A and 4B gray arrowheads

indicate the approximate onset of drinking activity under a standard12:12 L:D cycle for each

animal on the day prior to transition to experimental conditions. The black arrowheads above the

data in Figure 4A indicate the first day under DD conditions. For all animals, CT12 was

calculated as the average time of drinking activity onset on the last two days prior to the day of

sacrifice (day 3 and 4 L:D/DD; day 5 and 6 phase delay L:D). Under a standard L:D cycle,

animals displayed an average onset of drinking activity near zeitgeber time (ZT) 11.5 (+ 0.5h;

clock time1730h). Five days after the transition to DD conditions (indicated by a black

arrowhead in Fig. 4A), the average onset of drinking activity was delayed approximately 2.0

hours (+ 0.5h), with a free-running period (τ) of approximately 24.4 (+ 0.1h) hours, resulting in

an onset of drinking activity at approximately ZT 14 (+ 0.5h;clock time 2000h). By one week

after a 6 h phase-delayed L:D cycle (Fig. 4B), the average onset of drinking activity phase-

delayed, resulting in a new onset of drinking activity at ZT 17.5 (+ 0.5h; clock time 2330h). For

comparison, all data were aligned to CT12 regardless of lighting condition, allowing us to

determine phase relationships between entrained and free-running rhythms. Taken together, these

data reveal a significant response of the circadian rhythm of drinking activity to a 6 h phase-

delayed L:D cycle and DD conditions.

Serum PRL and CORT in OVX rats under a 12:12 L:D cycle (lights on 0600h), constant

darkness (DD), or a 6-hour phase-delayed 12:12 L:D cycle (lights on 1200h)

In animals under a standard 12:12 L:D cycle (on 0600h-off 1800h) analysis of PRL

secretion as a function of time and lighting condition revealed an overall effect of time (F=2.99,

p<0.05) but not lighting condition (F=1.45, p>0.05) with no significant interaction of time x

lighting condition (F=1.11, p>0.05). Comparisons did not reveal a significant rhythm of PRL

secretion in OVX rats under L:D conditions, in agreement with prior results from my laboratory

(162). However, following 5 days under DD conditions, PRL secretion in OVX rats did display a

significant increase above basal secretion at CT10 (Fig. 5A; p<0.05). In addition, CORT levels

27

Figure 5. Serum concentrations of prolactin (PRL; A,C), and CORT (B,D) in adult OVX

rats under a standard 12:12 L:D cycle (lights on 0600h; dashed line) or constant

darkness (solid line). The response to a 6 h phase-delay of the 12:12 L:D cycle is shown in

panels C (PRL) and D (CORT). For comparison, data are aligned to CT12, the onset of the

drinking rhythm, as described in methods. Each point represents the mean + S.E.M. of four

animals collected every 4 hours from CT2-CT22, and at the light:dark transition points CT0

and CT12. The original single day of data collection are double plotted to emphasize the

daily rhythm under each lighting condition.Dissimilar letters (a,b,c) indicate significant

effects of time within a lighting condition (p<0.05) and * indicate significant effects of

lighting condition within a specific time of day (p<0.05).

28

in animals under L:D and DD conditions were affected by time of day (Fig. 5B; F=14.16,

p<0.0001), but did not show a significant overall effect in response to lighting condition (F=0.41,

p>0.05) and there was no interaction of time x lighting (F=0.76, p>0.05). Post-hoc comparisons

revealed a significant increase in serum CORT levels from CT6 to CT10 in animals under a

standard L:D cycle (p<0.0001) or DD (p<0.01), with a sustained high amplitude during

subjective night (between CT12 and CT22; p<0.05 under both L:D and DD) followed by a return

to basal levels by the onset of subjective day (CT24; Fig. 5B; p<0.05 when compared with peak

values at CT10). My data reveal that serum CORT displays a circadian rhythm tightly coupled to

the free-running activity rhythm with a period close to 24.5 hours, and confirm several earlier

reports (64,243) suggesting that CORT is secreted with a circadian rhythm. Moreover, they

verify that, although the activities of DAergic neurons are indeed circadian (see below), such

rhythms are not reflected in the secretion of PRL in the absence of ovarian steroids. Serum PRL

and CORT from phase-delayed animals displayed rhythms similar to those observed in animals

prior to a phase-shift of the L:D cycle. As shown in Fig. 5C, serum PRL in animals under a

phase-delayed L:D cycle display a significant daily rhythm. While two-way ANOVA revealed a

significant effect of time (p<0.05) but not lighting condition (p>0.05) or the interaction of time x

lighting condition (p>0.05), one-factor ANOVA revealed substantial increases in serum PRL at

CT10 (p<0.01) and CT14 (p<0.01) when compared with basal levels between CT0-6, CT12 and

CT18-22 (Fig. 5C). Since I did not observe a significant increase in PRL secretion in animals

under a standard L:D cycle, I hypothesize that the rhythm of PRL secretion observed in phase-

delayed animals is a result of amplitude differences in DA turnover between pre-shift and phase-

shifted animals, as evidenced by the entrained rhythms of all three populations of DAergic

neurons (see below).ANOVA revealed a significant effect of time of day in serum CORT levels

(F=14.47, p<0.0001) but not lighting condition (F=0.26, p>0.05) and did not reveal a substantial

interaction between time of day x lighting (F=2.17, p>0.05). Serum CORT levels also entrained

to the new L:D cycle within 7 days (Fig. 5D). Within lighting condition comparisons revealed a

significant increase in serum CORT from CT6 to CT10 (p<0.001) with significantly greater

levels between (CT10-12; p<0.01) and a gradual decline to basal levels by the onset of subjective

day (CT24). However, comparisons between pre-shift and phase-delayed animals revealed that

serum CORT levels were substantially lower at CT22 (p<0.05) in phase-delayed animals, most

likely an effect of delayed entrainment of the serum CORT rhythm during the shift.

29

Experiment I. Neuroendocrine DAergic neuronal activity in OVX rats under a 12:12 L:D

cycle (lights on 0600h) or constant darkness (DD)

In animals under a standard 12:12 L:D cycle, two-way ANOVA of DA turnover in the

median eminence revealed a significant effect of lighting condition (F=14.77, p<0.001), time

(F=10.46, p<0.001), and an interaction of lighting x time (F=25.76, p<0.001). As seen in Fig.

6A, DA turnover in the ME displayed higher levels during the subjective day with significant

peaks at CT6 and CT 12 (p<0.05) compared with a substantial nadir at CT10 (p<0.05). DA

turnover in the ME remained low throughout the remainder of the subjective night (CT14-22) as

compared with peak levels at CT6 and CT12 (Fig. 6A; p<0.05). Following 5 days under DD

conditions, peaks of DA turnover in the ME were delayed, occurring during the subjective day at

CT10 (p<0.01) and again during the subjective night at CT18 and CT24 (Fig. 6A; p<0.001)

when compared with significantly lower levels during the subjective day at CT2 (p<0.0001),

CT6 (p<0.0001), CT12 (p<0.0001) and during the subjective night at CT22 (p<0.01). These data

suggest that the rhythm of DA turnover in the ME is a circadian rhythm, characterized by two

peaks of activity, with a free-running period (τ) greater than the free-running period of locomotor

activity, which is approximately 24.5h. Moreover, there appears to be an increase of the inter-

peak interval between CT10 and CT18 (8h) under DD when compared with peaks at CT6 and

CT12 (6h) under a standard L:D cycle and an overall increase in the amplitude of DA turnover

during the second peak under DD (from CT12 L:D to CT18 DD; Fig. 6A).

In the NL, there was a significant effect of time of day (F=2.23, p<0.05) but not of

lighting condition (F=0.63, p>0.05), and there was no interaction of lighting condition x time

(F=1.95, p>0.05). DA turnover in the NL from animals under L:D conditions displayed a rhythm

similar to DA turnover in the ME, with peaks of turnover at CT6 and CT12 (p<0.05; Fig. 6B)

compared with a significant nadir at CT2, CT10 and CT14-24 (p<.05; Fig. 6B). Following 5 days

under DD conditions, paired comparisons revealed there was no longer a significant rhythm of

DA turnover in the NL (p>0.05 for all time points), suggesting that DA neural activity in THDA

neurons is driven by a dampened oscillator activated by light (Fig. 6B).

In the IL, two-way ANOVA of DA turnover revealed a significant effect of time of day

(F=4.62, p<0.01), but not of lighting condition (F=0.001, p>0.05) and did not support a

significant interaction between lighting x time of day (F=1.44, p>0.05). Under L:D conditions,

DA turnover in the IL was high during the subjective day between CT2 and CT10, displayed a

30

Figure 6. DA turnover in the (A) ME, (B) NL, and (C) IL of adult OVX rats under a

standard 12:12 L:D cycle (lights on 0600h; dashed line) or constant darkness (solid line).

For comparison, data are aligned to CT12 as described in methods. Each point represents the

mean + S.E.M. of four animals collected every 4 hours from CT2-CT22, and at the light:dark

transition points CT0 and CT12. The original single day of data collection are double plotted

to emphasize the daily rhythm under each lighting condition. Dissimilar letters (a,b,c) indicate

significant effects of time within a lighting condition (p<0.05) and * indicate significant

effects of lighting condition within a specific time of day (p<0.05).

31

significant decrease at the onset of subjective night at CT12 (Fig. 6C; p<0.05), and then further

decreased during the subjective night between CT14 and CT18 (p<0.05) as compared with levels

during subjective day. DA turnover subsequently increased from basal levels between CT18 and

CT22 (Fig. 6C; p<0.05). Although the rhythm of PHDA neuronal activity exhibits a free-running

period of approximately 24.5 hours, the amplitude and frequency of DA turnover in the IL was

unaffected following 5 days under DD conditions. These results indicate that the rhythm of

PHDA neuronal activity is also endogenously regulated, with a free-running period of

approximately 24.5 hours.

Experiment II. Neuroendocrine DAergic neuronal activity in OVX rats under a standard

12:12 L:D cycle (lights on 0600h) or a 6-hour phase-delayed 12:12 L:D cycle (lights on

1200h)

Comparing DA turnover in the ME of animals under a standard 12:12 L:D cycle with

animals under a 6-h phase-delayed L:D cycle (Fig. 7A) revealed a significant effect of time

(F=21.27, p<0.0001) and a significant interaction of time x lighting condition (F=3.05, p<0.01)

but not of lighting condition alone (F=0.53, p>0.05). Individual comparisons show that the

circadian rhythm of DA turnover in the ME responded to a 6-hour phase-delayed L:D cycle with

complete entrainment to the new L:D cycle within 7 days (Fig. 7A). In phase-delayed animals,

DA turnover was greatest during the subjective day, with significant peaks at CT6 (p<.001) and

CT12 (p<0.001), as compared with basal levels at CT2, CT10, and the entire duration of the

subjective night (p<.001; see Fig. 7A). These data, taken together, suggest that the activity of

TIDA neurons is entrained to the 12:12 L:D cycle.

Two-factor analysis of DA turnover in the NL revealed a significant effect of time of day

(F=15.25, p<0.0001), lighting condition (F=47.48, p<0.0001), and a significant interaction of

time x lighting condition (F=5.58, p<0.0001) in animals under a phase-delayed L:D cycle (Fig.

7B). After 7 days, DA turnover in THDA neurons terminating in the NL completely entrained to

the new L:D cycle, with peak values at CT2 (p<0.01) and CT6 (p<0.01), compared to a reduced

magnitude during the subjective night between CT12-24 (p<0.01; Fig. 7B). Although the

rhythm of DA turnover in the NL entrained to the new L:D cycle within 7 days, there was a

significant reduction in the magnitude of DA turnover between CT12-CT22 (p<0.05), compared

to animals under a standard 12:12 L:D cycle. In addition, the rhythm of DA turnover in the NL

displayed an increased amplitude (peak-to-trough) in shifted animals compared with animals

32

Figure 7. DA turnover in the (A) ME, (B) NL and (C) IL of adult OVX rats

under a standard 12:12 L:D cycle (lights on 0600h; dashed line), or a phase-

delayed L:D cycle (on 1200h-off 2400h; solid line). For comparison, data are

aligned to CT12, the onset of the drinking rhythm, as described in methods. Each

point represents the mean +S.E.M. of four animals collected every 4 hours from

CT2-CT22, and at the light:dark transition points CT0 and CT12. The original

single day of data collection are double plotted to emphasize the daily rhythm

under each lighting condition. Dissimilar letters (a,b,c) indicate significant effects

of time within a lighting condition (p<0.05) and * indicate significant effects of

lighting condition within a specific time of day (p<0.05).

33

under a standard 12:12 L:D cycle. Therefore, I conclude that, like TIDA neuronal activity,

THDA neuronal activity is also entrained to the L:D cycle. Two-way ANOVA of DA turnover in

the IL of rats under a standard or phase-delayed L:D cycle revealed a significant effect of time of

day (F=5.64, p<0.001) and lighting condition (F=20.57, p<0.001) but not a significant interaction

between time x lighting (F=1.65, p>0.05). As I observed in the ME and NL, paired comparisons

reveal that DA turnover in the IL was greatest during the subjective day between CT2-CT12

(p<0.01) in animals under a phase-delayed L:D cycle, with a significant decline at CT18

(p<0.05), and a return to maximal levels by CT22 (p<0.05; Fig. 7C). Comparisons between

animals kept under a standard or phase-delayed L:D cycle revealed a significantly greater DA

turnover at CT22 (p<0.05) in control animals, which represents a greater rebound from the nadir

at CT18. Thus, as with TIDA and THDA neurons, PHDA neuronal activity is also entrained to

the 12:12 L:D cycle.

Summary and Conclusions

The purpose of these experiments was to determine if the three populations of

neuroendocrine dopaminergic neurons known to control PRL secretion are indeed under the

direct or indirect control of a circadian clock. To be considered an endogenous circadian rhythm,

a cyclic phenomenon such as DA turnover must possess three attributes: (1) the rhythm must

have a period of approximately 24 hours, (2) it should continue to cycle with a free-running


constant light (LL) and (3) it should be entrained to the environmental light:dark cycle

(66,76,94,230). The purpose of performing these experiments in OVX rats was to isolate the

rhythm from potential influences of estrogen (24) or PRL (31,32,244).

For control purposes, I monitored two well established circadian rhythms: that of water

consumption and plasma CORT concentration (66,122). In my laboratory, the 24-hour drinking

rhythm free-ran in DD, and entrained to a 6-hour phase delay of the 12:12 L:D cycle. Moreover,

the rhythm of CORT secretion was phase-locked to the activity rhythm and free-ran with a

period of approximately 24.5 hours. Under a standard L:D cycle (illumination from 0600 to

1800 h), the rhythm of DA turnover in the terminals of TIDA neurons in the ME display a

diurnal rhythm with an increased magnitude during subjective day, peaks at CT6 and CT12 and a

34

nadir during subjective night. Following 5 days of DD, the diurnal rhythm of DA turnover in

terminals of TIDA neurons in the ME free-runs with a period greater than 24.5 hours; a greater

sustained magnitude during subjective night and significant peaks of activity at CT10, CT18 and

CT24, but not CT22. In addition, these neurons entrained to a 6-hour phase delay of daily

illumination. A free-running rhythm of DA turnover in TIDA neurons under constant conditions

coupled with entrainment to a 6 hour shift in illumination are strongly suggestive of a circadian

rhythm with a free-running period of approximately 24.5 hours. In contrast, DA turnover in

THDA neurons terminating in the NL showed a period of approximately 24.5 hours and phase-

shifted after 5 days exposure to a 6 hour delay in lighting onset but did not show a rhythm of

significant amplitude under DD. These observations suggest that the rhythm of THDA neuronal

activity is under the control of single or multiple dampened oscillator(s) activated by light.

Alternatively, individual THDA neurons may adjust their activity phase with kinetics (245)

different than both TIDA and PHDA neurons. In either case, THDA neurons are clearly under

the influence of light-entrained circadian oscillator(s). Finally, much like TIDA neurons, the

rhythmic turnover of DA in PHDA neurons terminating in the IL presented with a free-running

period of approximately 24 hours and entrained to a 6-hour delay in light onset, but unlike TIDA

neurons, did not respond with a significant adjustment in amplitude under DD. Overall, these

data indicate that both TIDA and THDA neuronal activity is directly regulated by an endogenous

light-responsive circadian oscillator, with distinct free-running periods under constant

conditions. However, THDA neuronal activity rhythms are driven and entrained by light.

This and previous studies (24) leave little doubt that TIDA neurons respond to a light-

entrained circadian oscillator such as the SCN. Indeed, in OVX estrogen-treated rats, lesions of

the SCN block the proestrous-like release of PRL and the diminution of DOPAC concentrations

in the ME (24). Moreover, in animals receiving a copulomimetic stimulus, the resulting twice

daily pulses of PRL secretion are absent after lesion of the SCN (62). If the SCN, in fact,

transduces the lighting periodicity, it responds to varying forms of constant environments in

dissimilar manners. In LL, copulomimetic stimuli will not initiate twice daily surges of PRL in

OVX rats (29). However, in DD OVX rats, copulomimetic stimuli will induce surges of PRL

which are of equivalent magnitude to that of L:D rats but, though coupled in time with respect to

each other, occur at random times with respect to the 24 hour clock (29). This latter finding

suggests that these rhythms are regulated by a single oscillator or two coupled oscillators (57). In

35

addition, this finding further suggests that TIDA neurons terminating in the ME that display

endogenous, free-running (Fig. 3A) and entrainable (Fig. 4A) activity rhythms are the primary

regulators of the precisely timed PRL secretory response to mating.

I have found that THDA and PHDA neurons also play a major role in control of PRL

secretion (25). The present study reveals that, though the activities of these two populations of

DA neurons are diurnal, only the PHDA neurons describe all characteristics of a circadian

rhythm. In the case of THDA neurons, activity rhythms are not endogenous or may merely be

dampened in DD. Although both populations entrain to a new lighting regimen, my data suggest

that only PHDA neuronal activity is endogenous (Fig. 6C) whereas THDA neuronal activity may

be regulated by single or multiple dampened oscillator(s) passively driven by light signals from

the retina (Fig. 6B). I have recently shown that VIP-immunoreactive (VIP-IR) efferent fibers

from the SCN terminate upon TIDA, THDA and PHDA neurons (172). Experiments in my

laboratory employing VIP anti-sense oligonucleotides suggest that VIPergic SCN efferents are

directly involved in control of these rhythms (188).

OVX rats do not release PRL with a significant diurnal rhythm despite large changes in

the activity of TIDA, THDA and PHDA neurons throughout the day. It is well established that

estrogen is required to elaborate a proestrous-like surge of PRL which presumably sensitizes

pituitary lactotrophs to an uncharacterized PRL-releasing factor of hypothalamic origin (53)

and/or stimulates the release of such factors (1). In the rat, pseudopregnancy (PSP) induced by

mechanical stimulation of the uterine cervix involves both a reduction in inhibitory DAergic tone

and an increase in the releasing activity of putative PRL-releasing factors (57). Studies

conducted in my laboratory suggest that VIP, oxytocin, and serotonin may play substantial roles

in timing the two distinct PRL surges characteristic of PSP(53,55,159). My current experiments

do not preclude the potential activity of other PRL-releasing factors, but indicate that

endogenous, circadian rhythms of DAergic neural activity, driven by the SCN, regulate the

timing of PRL secretion in the absence of ovarian steroids(246-248).

Although are data indicate a clear lack of steroid-dependent rhythmicity, estrogen may

function to alter the timing of the activity of TIDA, THDA and PHDA neurons (114). It remains

to be seen if circulating ovarian steroids in the cycling rat exert their primary effects on the

period (τ), the amplitude, or both the period and amplitude of proposed rhythms of

neuroendocrine DAergic neural activity. Further, as ovarian steroids facilitate a significant

36

increase in circulating PRL levels on the third day (proestrus) of the 4-day estrous cycle of the

rat, it remains to be seen whether PRL-feedback on DAergic neuronal activity contributes to the

entrainment of the proposed rhythms of neuroendocrine DAergic neural activity (244,249).

Previously, my laboratory has shown a significant increase in the level of immediate early gene

expression and DA turnover in TIDA, THDA, and PHDA neurons approximately 2-3 hours after

an ovarian steroid hormone-induced PRL surge (32). Further, I have shown that

immunoneutralization of endogenous PRL significantly reduced the amplitude of DAergic

neuronal activity and immediate early gene expression in ovarian steroid-treated animals (249).

These data support a role for PRL-feedback in the regulation of neuroendocrine DAergic

neuronal activity and timed PRL secretion, but further experiments are needed to better clarify

the role of PRL feedback on the circadian rhythms of DAergic neuronal activity.

These studies, taken together, show that TIDA and PHDA neurons share the primary

attributes of an endogenous circadian rhythm while THDA neurons entrain to a new photoperiod

but do not exhibit a free-running period under constant conditions. While strong evidence from

my laboratory and others support a primary role for the SCN in the regulation of TIDA and

PHDA activity rhythms, it remains to be determined whether the activity of THDA neurons is

regulated by a dampened light-entrained oscillator and where that oscillator may be located.

37

CHAPTER 2

OVARIAN STEROID HORMONES MODULATE CIRCADIAN RHYTHMS OF

NEUROENDOCRINE DOPAMINERGIC NEURONAL ACTIVITY

Introduction

In ovariectomized (OVX), steroid hormone-treated (28,30), or cervically stimulated

(29,158) rats, there are endogenously controlled daily rhythms of PRL secretion. These rhythms

of PRL secretion are inversely correlated with the release of DA from NDN nerve terminals

(114). Evidence suggests that these rhythms are entrained by photoperiodic cues transduced by

the suprachiasmatic nucleus (SCN) (62,250,251). Experiments from my laboratory and others

suggest a direct effect of steroid hormones on tyrosine hydroxylase gene expression and neuronal

activity in NDN (40,114,252). Within the SCN there is a significant effect of ovarian steroids on

both gene expression and neuronal activity (111-113). Such data strongly support a modulatory

role for ovarian steroids at the level of the pituitary gland, NDN and SCN to strengthen

functional coupling between DAergic neuronal activity rhythms and PRL secretion.

In order to be considered a true circadian rhythm, a cyclic phenomenon such as DA

turnover must have three primary attributes: (1) the rhythm must have a period of approximately

24 hours, (2) it should maintain a free-running period of approximately 24 hours under constant

conditions such as constant dark (DD) or constant light (LL) and (3) it should display

entrainment to the environmental light:dark cycle (66,81,82). TIDA, THDA and PHDA neurons

all participate in the control of rhythmic PRL secretion (25) and ovarian steroids modulate serum

PRL levels, DA turnover and gene expression within all 3 populations of neuroendocrine

DAergic neurons (24,28,40,114,252-254). I have shown in Chapter 1 that both TIDA and

PHDA, but not THDA neurons, exhibit free-running and light-entrained circadian rhythms in the

OVX rat. Although THDA neuronal activity rhythms entrained to the 12:12 L:D cycle, they did

not maintain a free-running rhythm in constant conditions (DD) and therefore displayed some

but not all properties of a circadian oscillator (see Chapter 1, Fig. 6). Given the effects of

ovarian steroids on DA neurons and PRL secretion, I propose that ovarian steroids modulate the

38

timing of these rhythms, as well as the magnitude (overall amount of DA turnover) of each

throughout the 24-hour period, to coordinate a properly timed afternoon PRL surge. Through the

following experiments, I have determined: (1) the response of endogenous rhythms of DAergic

neural activity in TIDA, THDA, and PHDA neurons to ovarian steroid treatment by measuring

DA turnover in the ME, NL, and IL by high performance liquid chromatography with

electrochemical detection (HPLC-EC) and (2) the response of serum PRL and corticosterone

(CORT) concentrations by radioimmunoassay (RIA). DA turnover is an indirect biochemical

measure of DA release and metabolism that is a reliable index of acute changes in NDN activity

(23,242,255). Serum CORT was measured to verify a functional output of the circadian

oscillator in my animals under varying lighting conditions. OVX rats were subjected to classic

approaches for defining a circadian system, including; (a) a standard 12:12 L:D cycle (lights on

0600h; L:D), (b) constant darkness (DD), or a phase-delayed 12:12 L:D cycle (lights on 1200h;

pdL:D) and treated with or without estradiol-17β (E) or estrogen and progesterone (E+P).

Methods

Animals

As outlined in Chapter 1, all experiments used adult female Sprague-Dawley rats (> 60

days of age) weighing 250-300g (Charles River Labs inc., Wilmington, MA) that were housed

under varying lighting conditions in constant temperature (25C) and humidity with standard rat

chow and water available ad libitum. The room was illuminated with four 40 W fluorescent

bulbs, producing a minimum illumination of 100 lux at cage level. For animals housed under

DD all maintenance was performed in dim red light (< 1 lux) or with the aid of infrared goggles

(Unitec Series, GSCI Inc., Canada). Under both L:D and DD conditions maintenance was

performed every third day between 0900h and 1400h (the first half of the 12-hour light phase) to

avoid potential entrainment to non-photic stimuli by disrupting the animals during the inactive

period (232). Animals housed under DD conditions were sacrificed in dim red light (<1 lux). All

experimental protocols were approved by the Florida State University Animal Care and Use

Committee (ACUC).

39

Ovariectomy and Steroid Hormone Treatment

Animals were anesthetized with Halothane and OVX bilaterally. Animals were injected

with ovarian steroids a minimum of 10 days post-ovariectomy when the concentration of

estradiol in serum reached a level below 8 pg/ml, as previously determined by RIA (172,233).

All animals were placed under a standard L:D cycle (lights on 0600h-1800h) for 5 days for

habituation to the home cage. On day 6, animals were divided into treatment groups based on

steroid injection and lighting condition. Animals under a standard L:D cycle (lights on 0600h-

1800h) or constant darkness for 5 days were injected with estradiol-17β (E; 20 µg/rat i.p. in corn

oil vehicle; Sigma) at 1000h on the fourth day under L:D or DD, followed by progesterone (P;

1mg/rat i.p. in corn oil vehicle; Sigma) or corn oil vehicle at 1300h on the fifth day (L:D-E or

DD-E, L:D-E+P or DD -E+P). Animals under a delayed L:D cycle (pdL:D) for seven days were

injected with E on the sixth day and corn oil vehicle or P on the seventh day (pdL:D-E, pdL:D-

E+P). Therefore, regardless of lighting condition, all animals received E injections on the day

before sacrifice (simulated diestrus-2) and P on the day of sacrifice (simulated proestrus). Given

that E (1000h) and P (1300h) injections were given regardless of circadian time, P-treatments

were given after CT6 in L:D, while they were given immediately before tissue collection at CT6

under DD. The steroid-replacement paradigm used in these studies simulated circulating ovarian

steroid hormone levels on proestrus (256) and did not assume a free-running circadian rhythm of

ovarian steroid hormone synthesis and secretion.

Analysis of Drinking Rhythm

As in Chapter 1, drinking was measured over the 24-hour day with an automated device

(Dilog Instruments, Tallahassee, FL.) counting individual licks in 30-second bins over 24 hrs and

Circadian Time 12 (CT12; onset of subjective activity period). CT12 was used as a reference for

tissue collection regardless of lighting condition or steroid treatment. In all experiments,

samples were collected at the beginning and end of the subjective night (CT12 and CT0

respectively) and every four hours from CT2-22 (a total of 24h). Double plotted actograms of

drinking activity (12-hour moving average of drinking activity around a central peak of activity)

were produced with Circadia software (ver. 2.1.16; Behavioral Cybernetics, Inc., Tallahassee,

FL.)

40

Tissue Preparation and Serum Collection

Animals were briefly sedated by inducing hypercapnia (50% CO2: O2) and then rapidly

decapitated. Trunk blood was collected. Serum samples were frozen at –20C until assayed for

PRL and corticosterone (CORT) concentrations by RIA. The brain and pituitary gland were

quickly removed, placed on ice, and the median eminence, as well as neural and intermediate

lobes of the pituitary gland were carefully dissected, placed in homogenization buffer (0.2 N

perchlorate with 50 µM EGTA) and rapidly (~30 sec.) frozen in an ArticIce tube transport block

(USA Scientific Inc., Ocala FL.). Tissue samples were stored at -80C until assayed for DA and

DOPAC. On the day of analysis for catecholamines, tissue samples were thawed and processed

for HPLC-EC analysis as previously described (Chapter 1).

Measurement of Dopamine (DA) and Dihydroxyphenylacetate (DOPAC) by High

Performance Liquid Chromatography with Electrochemical Detection (HPLC-EC)

The HPLC-EC technique has been well established in my laboratory (114) and was

thoroughly described in Chapter 1. The concentrations of DA and DOPAC, a primary metabolite

of DA, were measured in tissue extracts from the pituitary gland and mediobasal hypothalamus

as previously described (Chapter 1). The amount of catecholamine in each sample was estimated

by direct comparison to the area under each peak for known amounts of catecholamine. The

amount of 3,4-dihydroxybenzylamine (DHBA, RT = 6.5 min) recovered was compared to the

amount of DHBA added as internal standard and corrected for sample loss (usually < 5%). The

assay detects 30 pg of DA and 15 pg of DOPAC. DA turnover is defined as the exocytotic

release of DA from neuroendocrine DAergic nerve terminals, DA re-uptake, and the degradation

of DA to DOPAC by monoamine oxidase (MAO) in the presynaptic terminal (242).

Protein Assay

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, IL) as previously

described (Chapter 1). Assay sensitivity was 100 µg protein and the intra-assay coefficient of

variation was 5-10%.

Radioimmunoassay

The concentration of PRL in serum was determined by radioimmunoassay (RIA) using

NIDDK materials supplied through the National Pituitary Hormone Distribution Program (A.F.

Parlow) and Protein-A as previously described (31). Serum CORT concentration was determined

41

using the commercially available Coat-a-Count®

rat corticosterone RIA kit (Diagnostic Products

Corp., Los Angeles, CA) according to the manufacturer’s specifications.

Experimental Design. Effects of ovarian steroids on the timing and magnitude of the

circadian rhythms of serum PRL, serum CORT, and DA turnover in the ME, NL and IL

DA turnover in the ME, NL, IL, serum PRL and serum CORT concentrations were

measured in samples obtained at 4 hour intervals from CT2-22 and at the light-dark transition

(CT0 and CT12) in animals under L:D , constant dark (DD) or phase-delayed L:D (pdL:D)

conditions. Four adult female Sprague-Dawley rats were OVX and housed individually in cages

attached to the automated drinking device under L:D conditions for 5 days. On day 6, animals

remained under L:D conditions, or were placed in either (1) DD for five days or (2) a pdL:D

cycle for 7 days and injected with ovarian steroids as described in methods. For comparison,

data from non-injected control OVX animals are presented. Animals were sacrificed on the fifth

day (pdL:D) under their respective lighting condition (10-12 days after OVX). In each

experiment tissue was collected for HPLC-EC determination of DA and DOPAC content and

serum was collected to determine serum PRL and CORT by RIA. Although data from non-

injected OVX rats were previously shown (see Chapter 1, Fig. 6) but were collected at the same

time as steroid-treated animals.

Data Analysis

Serum PRL, serum CORT and DA turnover are expressed as mean (ng/ml, ng/ml and

DOPAC:DA ratio, respectively) + SEM of 4 animals, presented as a function of circadian time

and double plotted to emphasize rhythms (see above). Although they exhibit a distinct rhythm,

all of my data do not conform to a sine/cosine wave function, which prohibits a non-linear

regression analysis to present the data as a function of time and lighting condition. Moreover, as

samples were obtained from each animal at individual time points over a 24 h period (CT0/24-

CT22), it is difficult to extrapolate accurate phase and period measures. It is clear that the

preferable approach when performing circadian studies would be serial sampling of individual

animals. However, analyses of recovered tissue preclude such an approach. Data for steroid

treated animals were compared with previously collected data from OVX animals at identical

circadian times. To facilitate direct comparisons, all data points regardless of steroid treatment or

lighting condition were aligned by circadian time. Due to this method, rhythms with free-

running periods >24.5h appear as phase-delays under DD when compared with free-running

42

rhythms having a period closer to 24 hours. Data were analyzed with two-way ANOVA for (A)

time of day effects, steroid hormone effects and the interaction between lighting and steroid

treatment or (B) light cycle effects, time of day effects and the interaction between light cycle

and circadian time, followed by Bonferroni paired post-hoc statistical tests. In addition, data

were analyzed with one-way ANOVA within light-treatment, time or steroid effects followed by

multiple comparisons with the Student Neuman-Keuls post-hoc test. P<0.05 was accepted as the

limit of significance. Thus, though limiting, these analyses provide us with amplitude and

duration values that were used to provide an approximation of phase and period in the absence of

a serial sampling and a repeated-measures design. ANOVA were performed and graphs were

created with Graph-pad Prism software (Graphpad Software Inc., San Diego, CA.)

Results

Analysis of Drinking Behavior

The beginning of the 12-hour activity period, identified as CT12, was determined on the two

days prior to tissue collection for each animal and averaged to predict the onset of activity on the

following day. CT12 was predicted under entrained and free-running conditions with an assumed

error of 10-15 minutes, given a variance in activity onset among my rats (generally 10-15

minutes from cycle to cycle), which I consider acceptable with a sampling frequency of 2-4

hours. In L:D-E and L:D–E+P rats (Fig. 8A), CT12 was approximately 1730+0.2h. Five days

after the transition to DD, CT12 was delayed approximately 2 hours to 1930+ 0.2h, resulting in

an approximate free-running period (τ) of 24.4 hours (Fig. 8B). After 7 days under pdL:D, CT12

in E and E+P treated animals was 2330+0.5h (Fig. 8C), indicating complete entrainment of the

circadian drinking activity rhythm to the new L:D cycle. Taken together, analysis of drinking

rhythms in steroid-primed rats under L:D, DD and pdL:D conditions verify a free-running, light

entrained rhythm of drinking activity with a period of approximately 24h.

Effects of Estradiol-17β on the circadian rhythms of serum PRL and serum CORT in OVX

animals under a standard 12:12 L:D cycle or constant darkness

In L:D, analysis of PRL secretion as a function of time and E-treatment revealed an effect

of steroid treatment (p<0.01; F=13.12) and time-of-day (p<0.01; F=4.96) without a significant

interaction of time x steroid treatment (p>0.05; F=1.56). Non-injected control OVX animals did

43

Figure 8. Ovarian steroid hormones do not affect the circadian rhythms of drinking

activity. Double plotted (see below) actograms of drinking activity from two representative

animals under (A) a standard12:12 L:D cycle (lights on 0600h-1800h; L:D), (B) before and after

transition from a standard 12:12 L:D cycle to constant dark (DD), or (C) before and after a 6h

phase-delay (pd) in the 12:12 L:D cycle (lights on 1200h-2400h; pdL:D) treated with E or E+P.

Treatment with E or E+P did not affect the (A,C) light-entrained or (B) free-running components

of the circadian drinking activity rhythm. I observed a (B) free-running rhythm of drinking

activity with a period of approximately 24h that (C) entrained to a novel L:D cycle within 7 days.

In A-C, (4) indicate the approximate onset of drinking activity under L:D for each animal. In

1B and C, (4) = the first day (plotted on the actogram just before the onset of activity) after

transition to (B) DD or (C) pdL:D conditions. The time of E-treatment (1000h in L:D and D:D,

1600h in pdL:D) is indicated by ( ), while ( ) = the time of P-treatment (1300h in L:D and DD,

1900h in pdL:D). The width of the horizontal bars represents the mean amplitude of drinking

during the activity period, presented as a 12 hour moving average around the center of activity.

The break in activity found on the final day of measurements represents the termination of data

collection before sacrifice and tissue collection. Horizontal bars above the data indicate the dark

phase and time labels are in hours and minutes.

44

not display a significant diurnal rhythm of PRL secretion under L:D (Chapter 1). Following E-

treatment (Fig. 9A) I observed a significant diurnal rhythm of serum PRL characterized by a

significant peak at CT10 (p<0.001) that was significantly greater in L:D-E than L:D-OVX rats

(p<0.01; Fig. 9A). Following 5 days in DD (Fig. 9B), analysis of PRL secretion from E-treated

animals revealed a significant effect of time-of-day (p<0.0001; F=10.39) and steroid treatment

(p<0.0001; F=40.05) with a significant interaction between time x steroid treatment (p=0.0001;

F=5.60). OVX E-treated rats in DD (Fig. 9B) displayed a serum PRL peak between CT10

(p<0.05) and CT12 (p<0.001) and was significantly greater than DD-OVX rats at both times

(CT10 (p<0.05), CT12 (p<0.05); Fig. 9B). Thus, I observed a significant increase in the

magnitude but not the timing of the free-running rhythm of PRL secretion in OVX-E-treated rats.

Serum CORT levels in OVX E-treated rats under L:D conditions were affected by

steroids (p<0.01; F=7.62) and time (p<0.001; F=9.83) but did not display an interaction between

steroid treatment x time (p>0.05; F=1.46). Post-hoc comparisons revealed a significant peak in

serum CORT at CT10 (p<0.05) and CT14 (p<0.05; Fig. 9C). Compared with L:D-OVX, serum

CORT from L:D-E rats declined more rapidly to basal level between CT14 and CT22 (p<0.05;

Fig. 9C). In contrast, although there was a significant effect of time-of-day (p<0.0001;

F=12.69), E did not affect the free-running rhythm of CORT secretion under DD (p>0.05;

F=1.89) and did not reveal an interaction between steroid treatment x time (p>0.05; F=1.23).

Comparisons reveal a significant increase in CORT between CT6 and CT10 (p<0.01) with a

sustained level through CT22 (p<0.01) compared with basal levels at CT24 (Fig. 9D). There was

no significant difference in serum CORT levels between OVX-untreated and E-treated animals

under DD throughout the entire subjective day (Fig. 9D). Taken together, these data indicate that

E induces minor effects on the magnitude of serum CORT under L:D conditions but has little or

no effect on its free-running rhythm.

Effects of Estradiol-17β and Progesterone on the circadian rhythms of serum PRL and

serum CORT in OVX animals under standard 12:12 L:D cycle and constant darkness

In L:D, E+P-treatment (p<0.0001; F=133.4) and time (p<0.0001; F=21.10) exerted

significant effects on the diurnal rhythm of serum PRL, with a significant interaction between

time x steroid treatment (p<0.0001; F=20.14). Comparisons within L:D-E+P animals reveal a

significant increase in serum PRL between CT6 and CT10 (p<0.001) with a further rise to peak

at CT12 (p<0.05; Fig. 10A). Peak levels of serum PRL in L:D-E+P animals were significantly

45

Figure 9. Estradiol modulates the magnitude, but not the timing, of the circadian rhythms

of serum PRL and serum CORT. Serum concentrations of prolactin (PRL; A,B), and

corticosterone (CORT; C,D) in non-injected OVX ( ------- ) and OVX estradiol 17-β-treated

( OVX+E; _______

) animals under a standard 12:12 L:D cycle (lights on 0600h); or DD. E-

treatment affected the magnitude of the light-entrained and free-running rhythms of serum PRL

(A,B) and serum CORT (C,D) at various times throughout the subjective day, but did not affect

the overall phase or period of these rhythms. In each figure, ( ) = the approximate time of E and

corn oil vehicle (CO) injections and do not apply to non-injected OVX rats. Each point

represents the mean (ng/ml) + SEM of four animals collected every 4 hours from CT2-CT22, and

at the light-dark transition points CT0 and CT12. Dissimilar letters (a,b,c) indicate significant

effects of time within lighting condition (p<0.05), (#) = significant effects of steroid treatment

within a specific time of day (p<0.05) and ( ) = a significant peak value within lighting

condition and hormone treatment in the absence of adjacent differences across circadian time

(p<0.05).

46

greater than levels in L:D-OVX rats at CT10 (p<0.001), CT12 (p<0.001) and CT14 (p<0.01).

Following 5 days under DD, post-hoc comparisons revealed a significant increase in serum PRL

from DD-E+P rats between CT2 and CT6 (p<0.05) with a further increase to peak levels

between CT10-CT12 (p<0.001 compared with CT2; Fig. 10B). After the afternoon peak of

serum PRL, I observed a sustained high level of serum PRL between CT14-CT22 (p<0.05; Fig.

10B). Further, peak levels of PRL secretion in DD-E+P rats were significantly greater than DD-

OVX animals between CT6 and CT22 (p<0.01). These data agree with numerous reports

showing that E+P stimulate a diurnal rhythm of PRL secretion in OVX rats (1). In addition, I

observed a slight advance of the free-running rhythm of PRL secretion that may result from

broadening of the PRL secretory pattern following E+P treatment (Fig. 10B).

Analysis of serum CORT in L:D-E+P rats revealed a significant effect of steroid treatment

(p<0.05; F=6.04) and time (p<0.05; F=15.19) and a significant interaction between steroid

treatment x time (p<0.0001; F=15.09). Post-hoc comparisons show a significant increase in

serum CORT between CT6 and CT10 (p<0.05) followed by a sustained level between CT12-

CT18 (p<0.05; Fig. 10C). CORT levels at CT22 are significantly lower in L:D-E+P than L:D-

OVX rats (p<0.05), indicating a more rapid return to basal levels in E+P-treated animals (Fig.

10C). Thus, my data suggest a small but significant effect of E+P-treatment on the phase of the

light-entrained CORT secretory rhythm. After 5 days in DD, serum CORT in DD-E+P rats

exhibited a significant response to time-of-day (p<0.0001; F=6.02) but not steroid treatment

(p>0.05; F=0.26), and an interaction between steroid treatment x time (p<0.001; F=4.39).

Comparisons revealed a significant increase in CORT between CT24 and CT12 in DD-E+P

animals (p<0.05; * in Fig. 10D). Comparison between DD-OVX and DD-E+P animals revealed

a significantly greater CORT level at CT6 in E+P-treated animals (p<0.05), indicating an

advance of the free-running CORT rhythm in response to steroid treatment. However, as

reported above for the free-running rhythm of CORT secretion in E-treated rats, I did not observe

a significant overall change in the timing of the CORT secretory rhythm in DD following

response to treatment with both E+P (Fig. 10D).

47

Figure 10. Estradiol and progesterone modulate the magnitude, but not the timing, of the

circadian rhythms of serum PRL and serum CORT. Serum concentrations of PRL(A,B), and

CORT (C,D) in non-injected OVX (--------) and OVX-estradiol-17β and progesterone-treated

(OVX-E+P______

) animals under a standard 12:12 L:D cycle (L:D, lights on 0600h); or DD.

Treatment with both E and P further increased the magnitude of the light-entrained (A) and free-

running (B) components of the circadian rhythm of PRL secretion, but did not induce a

significant change in either component (C,D) of the circadian rhythm of CORT secretion. In

each figure, ( ) =the approximate time of E and P injections and do not apply to non-injected

OVX rats. Each point represents the mean (ng/ml) + SEM of four animals collected every 4

hours from CT2-CT22, and at the light-dark transition points CT0 and CT12. Dissimilar letters

(a,b,c) indicate significant effects of time within lighting condition (p<0.05), (#) = significant

effects of steroid treatment within a specific time of day (p<0.05) and ( ) indicates a significant

peak value within lighting condition and hormone treatment in the absence of adjacent

differences across circadian time (p<0.05).

48

Effects of Estradiol-17β and Progesterone on the circadian rhythm of DA turnover in the

ME of OVX under a standard 12:12 L:D cycle or constant darkness

I have previously reported that TIDA neurons in OVX rats display a circadian rhythm of

DA turnover, which is entrained by light and has a free-running period of approximately 25h (see

Chapter 1 and Fig. 6). Analysis of DA turnover in TIDA nerve terminals from L:D-E rats (lights

on 0600h) reveal a significant effect of time (p<0.0001; F=16.46), but not steroid treatment

(p>0.05; F=0.002) and a significant interaction between steroid treatment x time (p<0.001;

F=5.02). One-factor ANOVA revealed peaks of DA turnover at CT6 (p<0.01) and CT18

(p<0.001) surrounding a nadir at CT14 (p<0.001; Fig. 11A). Compared with L:D-OVX rats,

L:D-E animals displayed a reduced level of DA turnover at CT12 (p<0.01; Fig. 11A). After 5

days in DD, two-factor analysis of DA turnover in DD-E rats revealed a significant effect of

steroid treatment (p<0.01; F=8.13) and time (p<0.0001; F=12.63), with a significant interaction

between time x steroid treatment (p<0.0001; F=11.11). In DD-E rats DA turnover peaked at

CT2 followed by a gradual decline to a nadir at CT12 (p<0.01; Fig. 11B). DA turnover was

subsequently depressed in TIDA nerve terminals throughout a majority of the subjective night

(CT14, CT22; p<0.05 vs. CT2; Fig. 10B). This parabolic pattern of DA turnover corresponds to

a broad increase in PRL secretion above basal levels (see above; Fig. 10B). In contrast to DD-

OVX rats, DA turnover in TIDA nerve terminals of DD-E animals was significantly greater at

CT2 (p<0.001) and lower at CT18 (p<0.001). These data suggest that E-treatment may prevent

delays under DD and/or facilitate an estimated free-running period closer to 24h in TIDA

neurons, closer to the estimated period of PRL secretion and drinking behavior rhythms. Two-

factor analysis of DA turnover in L:D-E+P rats reveals a significant effect of steroid treatment

(p<0.05; F=5.73) and time (p<0.0001; F=9.99), with a significant interaction between steroid

treatment x time (p<0.0001; F=13.01). Post-hoc tests identified a significant peak of DA

turnover at CT2 in L:D-E+P animals followed by a gradual decline to basal levels by CT12-14

(p<0.001 vs. CT2; Fig. 11C). Compared with L:D-E animals, L:D-E+P rats displayed a broader

reduction in DA turnover between CT12-CT14 (Fig. 11C). However, like E-treated rats, L:D-

E+P animals did not exhibit a significant increase in DA turnover at CT12 (p<0.001) when

compared with L:D-OVX rats (Fig. 11C). The increased duration of basal DA turnover level in

E+P-treated animals is associated with a broader increase in PRL secretion between CT6 and

CT14 when compared with both L:D-OVX and L:D-E rats. The free-running rhythm of DA

49

Figure 11. Estradiol and Progesterone affect the timing and magnitude of the circadian

rhythm of DA turnover in the ME. Dopamine (DA) turnover in the median eminence (ME) of

adult OVX-untreated (A-D ---------), OVX E-treated (OVX+E; A, B ______

) and E+P-treated

(OVX+EP; C, D _______

) animals under a standard 12:12 L:D cycle (lights on 0600h; A, C); or

DD (B, D). In OVX rats, E-treatment affected the magnitude of the (A) light-entrained

component and the period of the (B) free-running component of the circadian rhythm of DA

turnover in the ME. The addition of exogenous P further advanced the (D) free-running rhythm

and reduced the magnitude of the (C) light-entrained rhythm of DA turnover in the ME. In each

case the reduction in DA turnover in the ME as a result of E+P treatment corresponds to a

significant increase in the level of PRL secretion. In A-D ( ) = the approximate time of E and

P, or corn oil vehicle (CO), injections and do not apply to non-injected OVX rats. Each point

represents the mean (ratio) + SEM of four animals collected every 4 hours from CT2-CT22, and

at the light-dark transition points CT0 and CT12. Dissimilar letters (a,b,c) indicate significant

effects of time within lighting condition (p<0.05), (#) indicates significant effects of steroid

treatment within a specific time of day (p<0.05) and ( ) indicates a significant peak value within

lighting condition and hormone treatment in the absence of adjacent differences across circadian

time (p<0.05).

50

turnover in DD-E+P animals was affected by steroid treatment (p<0.0001; F=20.49) and time

(p<0.0001; F=21.33), with a significant interaction between time x steroid treatment (p<0.0001;

F=13.31). Individual comparisons revealed a peak of DA turnover at CT2 followed by a

significant and immediate decline to basal levels at CT6 (p<0.001; Fig. 11D). Compared with

DD-OVX rats, DD-E+P animals had significantly greater DA turnover at CT2 (p<0.001) and

significantly lower DA turnover at CT10 (p<0.001) and CT18 (p<0.001; Fig. 11D). These data

indicate an advancing effect of E+P-treatment on the estimated free-running rhythm of TIDA

neuronal activity. Thus, my data support a role for ovarian steroids in modulating the timing of

DA turnover in TIDA nerve terminals to strengthen functional coupling between the circadian

rhythms of DA release and PRL secretion in the female rat.

Effects of Estradiol-17β and Progesterone on the rhythm of DA turnover in the NL of OVX

rats under a standard 12:12 L:D cycle or constant darkness

In L:D-OVX animals I have reported a significant diurnal rhythm of DA turnover in

THDA nerve terminals with significant peaks at CT6 and CT12 (Chapter 1). Two-factor

ANOVA of DA turnover in L:D-E rats revealed a significant effect of steroid treatment

(p<0.0001; F=88.38) and time (p<0.0001; F=14.04) with a significant interaction between time x

steroid treatment (p<0.001; F=4.44). Pairwise comparisons show that THDA neuronal activity

in L:D-E animals peak between CT2-CT6 when compared with basal levels during the remainder

of the subjective day (p<0.05; Fig. 12A). As seen in Fig. 12A, DA turnover in the NL in L:D-E

rats was significantly lower at CT10 (p<0.001), CT12 (p<0.001) and CT18-24 (P<0.01). Thus,

direct comparison of DA turnover between L:D-OVX and L:D-E animals suggests that E-

treatment decreases the amount of DA release from THDA nerve terminals, without affecting the

estimated period or phase of the light-entrained rhythm.

After 5 days in constant conditions I did not observe a significant rhythm of DA turnover

in the NL, indicating that THDA neurons do not fulfill all of the requirements of a circadian

oscillator (Fig. 12B, see also Chapter 1). However, following E-treatment, two-factor analysis of

DA turnover in THDA nerve terminals revealed a significant effect of circadian time (p<0.001;

F=4.59) and E-treatment (p<0.001; F=13.65) with a significant interaction between time x

steroid treatment (p<0.0001; F=6.17). In Figure 12B, one-factor ANOVA within lighting

condition revealed a consistently elevated level of DA turnover throughout the subjective day

(between CT2-CT12), characterized by a significant peak at CT10 (p<0.05 vs. CT12).

51

Figure 12. Estradiol and Progesterone affect the magnitude, but not the timing, of the

circadian rhythm of DA turnover in the NL. DA turnover in the neural lobe (NL) of non-

injected OVX (A-D ---------), OVX E-treated (OVX+E; A, B ______

) and OVX EP-treated

(OVX+EP; C, D _____

) animals under a standard 12:12 L:D cycle (lights on 0600h; A, C); or DD

(B, D). E and E+P-treatment reduced the magnitude of DA turnover in the NL at various times

throughout the subjective day under both (A,C) light-entrained and (B,D) free-running

conditions. Further, although the response of the free-running rhythm of DA turnover in the NL

to exogenous steroids indicates a free-running rhythm of activity, this affect is most likely due to

transient changes in the magnitude of DA turnover during the late subjective night and does not

represent an emergent property of the rhythm under DD. In A-D ( ) =the approximate time of E

and P, or corn oil vehicle (CO), injections and do not apply to non-injected OVX rats. Each

point represents the mean (ratio) + SEM of four animals collected every 4 hours from CT2-

CT22, and at the light-dark transition points CT0 and CT12. Dissimilar letters (a,b,c) indicate

significant effects of time within lighting condition (p<0.05), (#) indicates significant effects of

steroid treatment within a specific time of day (p<0.05) and ( ) indicates a significant peak

value within lighting condition and hormone treatment in the absence of adjacent differences

across circadian time (p<0.05).

52

In contrast to the arrhythmia observed in DD-OVX rats, DD-E animals displayed a distinct

rhythm, potentially initiated by the increased levels of circulating steroid hormone (Fig. 12B).

However, the free-running rhythm of THDA neuronal activity in DD-E rats did not correlate

with the free-running rhythm of PRL secretion. Therefore, my data suggest that ovarian steroids

exert their primary effects on the magnitude of DA release from THDA neurons under DD.

Analysis of DA turnover in THDA nerve terminals of L:D-E+P animals with two-factor

ANOVA revealed a significant effect of time (p<0.0001; F=9.01) and E+P treatment (p<0.0001;

F=80.41) with a significant interaction between time x steroid treatment (p<0.0001; F=6.21). In

L:D-E+P rats DA turnover in the NL peaked between CT2-CT6 (p<0.001) compared to a nadir

at CT12 (Fig. 12C). When compared with L:D-OVX rats DA turnover in the NL of L:D-E+P

animals was significantly lower between CT10-CT18 (p<0.05) and CT24 (p<0.05;Fig. 12C).

Such data suggest that steroid treatment affects the magnitude but not the timing of the light-

entrained rhythm of THDA neuronal activity. Two-factor analysis of DA turnover in DD-E+P

rats revealed a significant effect of time (p<0.01; F=4.00) but did not show a significant effect of

E+P treatment (p>0.05) or an interaction between E+P treatment x time (p>0.05). While

pairwise comparisons did reveal a significant peak in DA turnover at CT22 (p<0.01) when

compared with CT14 and CT24, this rhythm does not correspond to the timing of PRL secretion

in DD-E+P animals (Fig. 12D). Surprisingly, this monophasic rhythm appears inverted with

respect to the rhythm of DA turnover in DD-E rats (Fig. 12D vs. Fig. 12B). Thus, in both L:D-

E+P and DD-E+P animals, E+P treatment appeared to affect the magnitude but not the estimated

period of the light-entrained and free-running rhythms of THDA neuronal activity.

Effects of Estradiol-17β and Progesterone on the circadian rhythm of DA turnover in the

IL of OVX rats under a standard 12:12 L:D cycle or constant darkness

Although two-factor analysis of DA turnover in PHDA nerve terminals from L:D-E rats

did not reveal a significant effect of steroid treatment (p>0.05; F=1.966), I did observe a

significant effect of time (p<0.0001; F=6.870) and an interaction between time x steroid

treatment (p<0.0001; F=9.01). As seen in figure 13A, DA turnover in the IL increased between

CT2 and CT6 (p<0.001) followed by a sustained baseline of DA turnover throughout the dark

phase (Fig. 13A). DA turnover in the IL returned to peak level by the end of the dark phase (Fig.

12A). Compared with data from L:D-OVX, L:D-E animals displayed a lower level of DA

turnover in the IL at CT10 (p<0.001) and CT22 (p<0.001; Fig. 13A). However, the overall

53

Figure 13. Estradiol and Progesterone affect the magnitude, but not the timing, of the

circadian rhythm of DA turnover in the IL. DA turnover in the intermediate lobe (IL) of adult

OVX-untreated (A-D, --------), OVX E-treated (OVX+E; A, B _______

) and OVX EP-treated

(OVX+EP; C, D _______

) animals under a standard 12:12 L:D cycle (lights on 0600h; A, C); or

DD (B, D). In parallel with THDA neurons, the magnitude of the (A,C) light-entrained and

(B,D) rhythms of DA turnover in the IL were affected by E or E+P-treatment, although this

affect did not extend to the period or phase of either component of these circadian rhythms. In

A-D ( ) d= the approximate time of E and P, or corn oil vehicle (CO), injections and do not

apply to non-injected OVX-untreated rats. Each point represents the mean (ratio) + SEM of four

animals collected every 4 hours from CT2-CT22, and at the light-dark transition points CT0 and

CT12. Dissimilar letters (a,b,c) indicate significant effects of time within lighting condition

(p<0.05), (#) indicates significant effects of steroid treatment within a specific time of day

(p<0.05) and ( ) indicates a significant peak value within lighting condition and hormone

treatment in the absence of adjacent differences across circadian time (p<0.05).

54

diurnal rhythm, characterized by greater overall activity during the light phase, was not affected

by the addition of ovarian steroids. Following 5 days under DD, two-factor analysis of DA

turnover in PHDA terminals from E-treated rats revealed a significant effect of time (p<0.0001;

F=5.815) and E-treatment (p<0.01; F=11.98) without a significant interaction between E-

treatment x time (p>0.05; F=1.71). Pairwise comparisons indicate a free-running rhythm of DA

turnover with a peak at CT6 (p<0.01; Fig. 13B). The free-running rhythm of DA turnover in

PHDA nerve terminals following E-treatment did not differ in magnitude or estimated period

from DD-OVX animals with the exception of CT22 (p<0.05; Fig. 13B). These data suggest that

acute E-treatment had a minor effect on the overall magnitude of DA release but did not affect

the free-running rhythm of PHDA neuron activity. Two-factor analysis of DA turnover in the IL

of L:D-E+P rats as a function of ovarian steroid treatment and circadian time revealed a

significant effect of time (p<0.0001; F=10.72) and E+P treatment (p<0.0001; F=18.12) with a

significant time x E+P treatment interaction (p<0.0001; F=12.01). As seen in figure 12C, DA

turnover in the IL peaked at CT2 (p<0.001) and CT6 (p<0.001; Fig. 13C). When compared with

L:D-OVX rats, DA turnover in the IL of L:D-E+P animals was significantly lower at CT10,

CT14 and CT22 (p<0.001); but was significantly greater at CT24 (p<0.001). These data indicate

a significant reduction in PHDA neuronal activity beginning 2-3 hours before the onset of the

activity period (dark phase), associated with a steroid-induced increase in PRL secretion.

Therefore, my data provide further evidence to support a primary effect of ovarian steroids on

the magnitude of DA release from PHDA nerve terminals throughout the day.

After transition to DD, analysis of DA turnover in the IL of E+P-treated rats with two-

factor ANOVA did not reveal a significant effect of E+P treatment (p>0.05; F=2.23) but did

reveal an effect of time (p<0.001; F=4.755) and a significant interaction between E+P treatment

x time (p<0.01; F=3.19). DA turnover in the IL of DD-E+P rats displayed a free-running rhythm

with a significant peak at CT22 (p<0.01; Fig. 13D). Although I observed a significantly greater

level of DA turnover at CT22 (p<0.05) in DD-E+P rats when compared with DD-OVX animals,

I did not see a substantial effect of E+P treatment on the free-running rhythm of DA turnover in

PHDA terminals. These data further suggest that E+P-treatment affects the magnitude of DA

release from PHDA nerve terminals at specific times during the subjective day, without affecting

55

Figure 14. Estradiol and progesterone affect the magnitude, but not the timing, of the

circadian rhythms of serum PRL and serum CORT following entrainment to a phase-

delayed L:D cycle. Serum concentrations of PRL(A,B), and CORT (C,D) in E-treated

(OVX+E; A, B) and E+P-treated (OVX+EP; C,D) animals under a standard 12:12 L:D cycle

(lights on 0600h; --------); or a 6h phase-delayed 12:12 L:D cycle (lights on 1200h;_________

).

After entrainment to a pdL:D cycle, treatment with E increased the magnitude of the afternoon

(A) PRL surge but did not affect the newly entrained rhythm of (B) CORT secretion. Likewise,

E+P-treatment affected the magnitude of (C) PRL secretion following entrainment but had little

affect on the rhythm of (D) CORT secretion. Each point represents the mean (ng/ml) + SEM of

four animals collected every 4 hours from CT2-CT22, and at the light-dark transition points CT0

and CT12. Dissimilar letters (a,b,c) indicate significant effects of time within lighting condition

(p<0.05), (#)=significant effects of steroid treatment within a specific time of day (p<0.05) and

( )=a significant peak value within lighting condition and hormone treatment in the absence of

adjacent differences across circadian time (p<0.05).

56

the estimated period or phase of the free-running rhythm of PHDA neuronal activity. Effects of

Estradiol-17β and Progesterone on the entrainment of serum PRL and serum CORT

rhythms in OVX rats

`Two factor analysis of serum PRL from E-treated rats under a standard L:D cycle (lights

on 0600h; L:D) and a phase-delayed L:D cycle (lights on 1200h; pdL:D) revealed a significant

effect of light cycle (p<0.0001; F=17.48), time-of-day (p<0.0001; F=48.38) and an interaction

between time x lighting (p<0.0001; F=18.54). Individual comparisons within pdL:D-E animals

show a significant peak in serum PRL at CT10 (p<0.001) compared to basal PRL levels

throughout the remainder of the day (Fig. 14A). The peak in serum PRL at CT10 in pdL:D-E

animals was significantly greater than the peak in observed in L:D-E animals (p<0.001; Fig.

14A). Therefore E-replacement increases the level of serum PRL secretion without exerting

observable effects on entrainment. After treatment with both E+P, two factor analysis of serum

PRL revealed a significant effect of time-of-day (p<0.0001; F=24.92) and an interaction between

time x lighting (p<0.0001; F=10.04), but did not show a significant effect of light cycle (p>0.05;

F=3.27). Pairwise comparisons within pdL:D-E+P animals verify a significant increase in serum

PRL between CT10 and CT12 (p<0.001), followed by a further rise-to-peak between CT12 and

CT14 (p<0.05; Fig. 14C). When compared to L:D-E+P animals, pdL:D-E+P rats displayed a

delayed increase in serum PRL (from between CT6-CT10 under standard L:D to CT10-12 under

pdL:D) resulting in significantly lower serum PRL levels in pdL:D rats at CT10 (p<0.001) and

greater serum PRL levels at CT14 (p<0.001; Fig. 14C). Thus, although E+P treatment exerts

minor effects on the timing of PRL secretion they did not disrupt entrainment to a delayed L:D

cycle. As a means to verify functional regulation of the neuroendocrine system by the central

circadian oscillator, I measured serum CORT in E-treated animals before and after transition to a

pdL:D cycle. Previous data suggest that E+P affects the magnitude, but not the estimated phase

and period of the light-entrained rhythm of CORT secretion (257). Two-factor ANOVA of

serum CORT levels within E-treated rats under L:D and pdL:D conditions revealed a significant

effect of time (p<0.0001; F=7.36) but did not show a significant effect of light cycle (p>0.05;

F=3.49) or a significant interaction between time x light cycle (p>0.05; F=1.65). Comparisons

within light cycle of serum CORT from pdL:D-E rats reveal a significant rhythm of CORT

secretion with peak values at CT12 compared to basal levels at CT24 (p<0.001; Fig. 14B).

57

Comparisons within E-treated rats as a function of light cycle (L:D vs. pdL:D) did not

reveal an overall effect of shifting the L:D cycle, supporting the observation that E-treatment in

OVX rats does not effect the ability of light to entrain the rhythm of CORT secretion (Fig. 14B).

Comparisons of serum CORT secretion within E+P-treated OVX rats delineated a significant

effect of circadian time (p<0.0001; F=14.09) and light cycle (p=0.006; F=8.30) but failed to

show a significant interaction between time x light cycle (p=0.05; F=2.18). Pairwise

comparisons within pdL:D-E+P rats as a function of time reveal a circadian rhythm of serum

CORT with a significant increase to peak values at CT10 (p<0.001) opposing a nadir at CT24

(Fig. 14D). Comparison within steroid treatment as a function of light cycle divulged greater

serum CORT levels at CT10 following the transition to pdL:D conditions (Fig. 14D). These

results support a slight phase-advance of the light-entrained serum CORT rhythm after 7 days

under the pdL:D cycle in E+P treated rats.

Effects of Estradiol-17β alone on the entrainment of DA turnover rhythms in the ME, NL

and IL of OVX rats

I have shown that TIDA neurons from L:D-E rats display a significant diurnal rhythm

with peak levels during the early subjective day between CT2 and CT6 (Fig. 15A). Moreover, I

have shown that this diurnal rhythm is rapidly and strongly entrained by the daily photoperiod

(Chapter 1). I have determined the effects of ovarian steroid treatment on the ability of these

rhythms to entrain to a pdL:D cycle. Two-factor ANOVA of DA turnover from the ME of L:D-

E and pdL:D-E rats revealed a significant effect of light cycle (p<0.0001; F=55.07), time

(p<0.0001; F=16.60) and a significant interaction between light cycle x time (p=0.003). As seen

in figure 15A, pairwise comparisons within pdL:D-E animals avow a diurnal rhythm of DA

turnover in TIDA nerve terminals with a significant increase between CT0 and CT2 (p<0.05).

DA turnover in TIDA nerve terminals from pdL:D-E rats was significantly lower at CT6, 10, 12

(p<0.05) and CT18 (p<0.001) when compared with L:D-E animals (Fig. 15A).

Analysis of DA turnover in the NL from LD-E and pdL:D-E animals with two-factor

ANOVA revealed a significant effect of light-cycle (p=0.006; F=8.12) and time (p<0.0001;

F=16.85) but failed to show an interaction between light cycle x time (p>0.05; F=1.61l; Fig.

15B). In parallel with the ME, DA turnover in the NL of pdL:D-E animals display a significant

58

Figure 15. Estradiol affects the magnitude,

but not the timing, of the circadian rhythms

of DA turnover in the ME, NL and IL

following entrainment to a phase-delayed

L:D cycle

DA turnover in the (A) ME, (B) NL and (C) IL

of OVX-E–treated rats under a standard 12:12

L:D cycle (L:D; on 0600h,----------) or a 6-hour

phase-delayed 12:12 L:D cycle (pdL:D; on

1200h, ________

). Following entrainment to a

pdL:D cycle the magnitude of DA turnover in

the (A) ME and (B) NL of E-treated rats were

modestly reduced during both the subjective

day (ME and NL) and subjective night (ME).

In addition, I observed a delaying transient in

the rhythm of DA turnover in the (C) IL.

However, these affects do not support a

significant change in the overall phase of these

rhythms following E-treatment. Each point

represents the mean (ratio) + SEM of four

animals collected every 4 hours from CT2-

CT22, and at the light-dark transition points

CT0 and CT12. Dissimilar letters (a,b,c)

indicate significant effects of time within a

particular light cycle (p<0.05), (#) indicates

significant differences across light cycle within

a specific time of day (p<0.05) and ( )

indicates a significant peak value within a

particular light:dark cycle in the absence of

adjacent differences across circadian time

(p<0.05).

59

diurnal rhythm of DA turnover characterized by a rise to peak values in the early subjective day

between CT0 and CT2 (p<0.01) with a peak at CT6 (p<0.001 vs. CT24; Fig. 15B). Photoperiod

comparisons reveal that pdL:D-E animals have a small but significantly higher level of DA

turnover at CT6 (p<0.01) but are otherwise not significantly different from L:D-E rats (Fig.

15B).

Two-factor ANOVA for DA turnover in the IL of L:D-E and pdL:D-E animals reveals a

significant effect of time (p<0.0001; F=16.61) and an interaction between time x light cycle

(p<0.0001; F=16.13) but failed to reveal a significant effect of light cycle (p>0.05; F<0.01).

Comparisons across circadian time within pdL:D-E rats expose a significant diurnal rhythm

marked by a substantial increase from basal levels at CT0 to peaks at CT6 and CT12 (p<0.001).

This biphasic rhythm is followed by basal DA turnover throughout the remainder of the

subjective night (Fig. 15C). Thus, data from pdL:D-E rats support my previous result and verify

that all 3 populations of neuroendocrine dopaminergic neurons display circadian rhythms, which

are strongly entrained to the daily light:dark cycle and show significant magnitude, but not

estimated phase or period responses to E-treatment.

Effects of Estradiol-17β and Progesterone on the entrainment of DA turnover rhythms in

the ME, NL and IL of OVX rats

Analysis of DA turnover in TIDA nerve terminals from L:D-E+P and pdL:D-E+P rats

with two-factor ANOVA revealed a significant effect of light cycle (p<0.001; F=14.78), time

(p<0.0001; F=15.95) and a significant interaction between light cycle x time (p<0.01; F=3.77).

Within pdL:D-E+P rats I observed a significant diurnal rhythm with a rise to peak by CT2

(p<0.05) and a sustained elevation through CT6 (p<0.05), which opposes a nadir at CT10 (Fig.

16A). When compared with L:D-E+P animals, pdL:D animals display a reduced level of DA

turnover at CT10 (p<0.05) and again at CT22 (p<0.05). Two-factor analysis of DA turnover in

the NL of L:D-E+P and pdL:D-E+P rats revealed a significant effect of light cycle (p<0.05;

F=6.96) and time (p<0.0001; F=18.63) with no interaction between light cycle x time (p>0.05;

F=0.97). Post-hoc tests within photoperiod show that DA turnover in the NL of pdL:D-E+P

animals entrain to the pdL:D cycle with a significant diurnal rhythm (Fig. 16B). The newly

entrained rhythm is characterized by an increase in the early subjective day between CT0 and

CT2 (p<0.01), followed by a plateau through CT6 and a precipitous decline to basal levels by

CT12 (p<0.001 vs. peak at CT6; Fig. 16B).

60

Figure 16. Estradiol and Progesterone

affect the magnitude, but not the timing, of

the circadian rhythms of DA turnover in

the ME, NL and IL following entrainment

to a phase-delayed L:D cycle. DA turnover

in the (A) ME, (B) NL and (C) IL of OVX-

E+P–treated rats under a standard 12:12 L:D

cycle (L:D; on 0600h, ----------) or a 6h phase-

delayed 12:12 L:D cycle (pdL:D; on 1200h, ________

). After the addition of both E+P and

entrainment to a pdL:D cycle the magnitude of

DA turnover in the (A) ME was reduced

during both the subjective day and night and I

observed a small delaying transient in the

rhythm of DA turnover in the (C) IL. I did not

observe any change in the magnitude or phase

of the light-entrained rhythm of DA turnover

in the (B) NL. Albeit significant, these effects

do not comprise a substantial change in the

period or phase of these rhythms. Each point

represents the mean (ratio) + SEM of four

animals collected every 4 hours from CT2-

CT22, and at the light-dark transition points

CT0 and CT12. Dissimilar letters (a,b,c)

indicate significant effects of time within a

particular light:dark cycle (p<0.05), (#)

indicates significant differences across

light:dark cycle within a specific time of day

(p<0.05) and ( ) indicates a significant peak

value within a particular light:dark cycle in the

absence of adjacent differences across

circadian time (p<0.05).

61

Two-factor ANOVA of DA turnover in the IL of L:D-E+P and pdL:D-E+P rats revealed

a significant effect of time (p<0.0001; F=38.61) and an interaction between light cycle x time

(p<0.0001; F=23.62), but failed to support a significant effect of light cycle (p>0.05; F=0.22).

Analysis of DA turnover in the IL of pdL:D-E+P animals as a function of time reveal that DA

turnover in PHDA neurons entrain to the delayed L:D cycle (Fig. 16C). The entrained rhythm is

characterized by an increase in the early subjective day between CT2 and CT6 (p<0.001),

followed immediately by a precipitous decline to basal levels by CT10 (p<0.001 vs. peak at CT6;

Fig. 16C). Two-factor ANOVA did not indicate a significant difference across photoperiod at

any time when comparing L:D-E+P and pdL:D-E+P rats, indicating that DA release from PHDA

neurons entrained to the pdL:D cycle with no effect on the estimated phase or magnitude of the

rhythm.


The purpose of these experiments was to determine the role that ovarian steroids play in

adjusting the timing and amplitude of the circadian rhythms of neuroendocrine DAergic neuronal

(NDN) activity. Previously, I reported significant circadian rhythms of serum PRL and NDN

neuronal activity in the OVX rat (Chapter 1). As in those experiments, I have monitored two

established rhythms: fluid intake and plasma CORT concentration, to verify the adequate and

consistent physiological function of the central circadian oscillator (Chapter 1). In nocturnal

rodents, both display distinct free-running and light entrained circadian rhythms (66,81,82). In

my laboratory, the circadian rhythm of drinking activity in ovarian steroid-treated rats entrained

to the L:D cycle and free-ran with a period of approximately 24.4 hours. As I sacrificed animals

on the simulated proestrus (day of P injection) and did not continue behavioral measurements

beyond that day, it is difficult to verify a delayed response on the phase or period of the 24h

drinking rhythm to exogenous steroids in my rats. However, I did not observe a significant acute

response of the free-running and light-entrained rhythms of drinking behavior to ovarian steroid

hormones.

The circadian rhythms of serum PRL and serum CORT in E or E+P-treated animals were

entrained by light and free-ran with an estimated period of approximately 24.4 hours. Although I

observed a decline in the level of serum CORT during the subjective night in steroid treated rats

62

in L:D, my results imply that ovarian steroid hormones did not significantly affect the free-

running rhythm of CORT secretion. While the rhythm of serum PRL in E+P-treated rats

appeared to show a slight delay following entrainment to the new L:D cycle, this does not

constitute a significant change in the estimated period of the rhythm and may be due to a

delaying transient during the shift to the new L:D cycle. In agreement with many previous

reports, I conclude that the primary effects of ovarian steroid hormones are on the synthesis and

release of PRL (1). However, it remains to be seen whether the lactotroph maintains a

pacemaker function, allowing for precisely timed secretion, with gain supplied by ovarian steroid

hormones.

I have previously reported a light-entrained diurnal rhythm of DA turnover in TIDA

nerve terminals within the ME of L:D-OVX rats with a biphasic pattern during the subjective

day, followed by a sustained trough during the remainder of the subjective night (Chapter 1).

Steroid treatment reduced the rise in DA turnover, which follows the afternoon PRL increase but

otherwise had no effect on the rhythm under L:D conditions. These data suggest a possible

reduction in the effects of PRL-feedback on the activity of TIDA neurons after acute steroid

treatment (254). In DD-OVX rats, the rhythm of DA turnover in TIDA neurons free-runs with a

period of approximately 25 hours and exhibits a greater activity during subjective night. After

treatment with ovarian steroids, the free-running rhythm of DA turnover in the ME displayed an

estimated period of approximately 24.4 hours. In L:D-E+P rats I observed a more rapid decline

to basal levels, followed by a gradual rise to peak levels throughout the remainder of the

subjective night. Further, DD-E+P animals displayed a more gradual rise to peak values during

the subjective night, providing further evidence for a decreased response of TIDA neurons to

PRL feedback (31,254). Therefore, as a result of steroid treatment, it appears that DA turnover in

TIDA nerve terminals free-runs with an estimated period of 24.4h. In agreement with my

previous studies, DA turnover in the ME of E and E+P-treated rats effectively entrained to a

pdL:D cycle within 7 days. However, I did observe a significant decline in the amount of DA

turnover in the ME after transition to the new L:D cycle, suggesting a transient rebound after the

delay. Such data suggest that ovarian steroids modulate the rhythm of TIDA neuron activity by

advancing the free-running rhythm of DA turnover in TIDA neurons or by strengthening the

coupling between free-running TIDA neurons and the SCN. Regardless of the mechanism, it

would appear that the resulting rhythm of DA turnover in the ME facilitates the timing of the

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diurnal rhythm of PRL secretion. This effect may be mediated by direct actions of ovarian

steroids on SCN pacemaker cells (41,111-113,258,259) and/or TIDA neurons (39,40,114,260).

Previous reports from my lab suggest that VIPergic afferents of SCN origin synapse on all 3

populations of NDN (171,172) and ovarian steroids increase the expression of VPAC2 receptors

(172). Therefore, ovarian steroids may affect the timing of DA release by strengthening synaptic

communication between the SCN and TIDA neurons through upregulation of VPAC2 receptor

expression in the arcuate nucleus (171,172,187,261,262). DA turnover in the NL of L:D-OVX

rats displays a significant diurnal rhythm with biphasic peaks during the subjective day. In L:D-

E animals I observed a similar diurnal rhythm with a single peak between CT2 and CT6. Acute

E-treatment initiated a significant decline in DA turnover just prior to the onset of the dark phase

and again during the second half of the night. These data suggest a significant effect of ovarian

steroid treatment on the magnitude of DA release at the time of the diurnal PRL surge and

immediately following that surge.

After treatment with E+P, DA turnover in THDA nerve terminals from L:D rats also

displayed a diurnal rhythm with characteristics similar to L:D-OVX and L:D-E animals.

However, in comparison with L:D-OVX rats, DA turnover in L:D-E+P animals declined to a

much lower level during the PRL increase. A decrease in DA turnover in the NL at the onset of

subjective night indicates a diminished effect of PRL feedback on the activity of THDA neurons

(31,254). After 5 days in DD, DA turnover in the NL of L:D-OVX rats does not display a

significant free-running rhythm (see Chapter 1, Fig. 6). I have hypothesized that THDA

neuronal activity may be passively driven by photic cues transduced by the SCN and/or driven

by oscillatory activity with kinetics significantly different from the core circadian oscillator (i.e.

local autonomous cellular oscillations with a free-running period significantly greater than 24.5

hours; (245,263,264). In contrast with DD-OVX rats, I observed a significant decrease in DA

turnover in the NL of DD-E rats during the subjective night. The free-running rhythm of DA

release from THDA neurons of DD-E+P rats appeared inverted when compared with DD-E

animals. These data suggest that E alone may induce a significant rhythm of DA turnover in the

NL that is modified by the addition of exogenous P. Although I did observe a significant

increase in the magnitude of THDA neuronal activity at the middle of the subjective day in

pdL:D-E rats, both E and E+P rats showed complete entrainment to the pdL:D cycle within 7

days. My results agree with previous reports which have indicated antagonistic effects of E+P

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on daily rhythms of locomotor activity, DA turnover, tyrosine hydroxylase and PRL-receptor

expression (31,40,254,265,266). Given that THDA nerve terminals do not display a free-running

rhythm of DA turnover in OVX rats (see Chapter 1, Fig, 6), it remains to be seen whether the

effects of ovarian steroids under DD are due to steroid induced changes in the density of afferent

inputs from the SCN or locally regulated effects on activity at the THDA neuron.

Previous studies indicate that PHDA neurons display a circadian rhythm of activity that is

entrained by light and free-runs with an estimated period near 24 hours (Chapter 1). In OVX-

untreated rats under L:D conditions I observed a significant rhythm of DA turnover in the IL

characterized by sustained levels throughout the light period and a gradual decline during the

early dark phase. Following E-treatment, DA turnover in the IL of L:D animals displayed a

significant diurnal rhythm with peaks in the early subjective day, followed by a rapid decline to

sustained basal levels. Compared with L:D-OVX rats, the light-entrained rhythm of PHDA

neuronal activity in L:D-E animals declined earlier in the day and remained at a basal level.

After treatment with E+P, the light-entrained rhythm of DA turnover in the IL displayed a

distinct pattern with peaks during the early morning and late night. Thus, the light-entrained

rhythms of DA turnover in the IL from both E and E+P-treated animals correspond to the

increasing amplitude of serum PRL in each steroid environment. DA turnover in the IL of DD-

OVX rats displayed a free-running rhythm with an estimated period of approximately 24.5 hours,

which did not differ significantly in form from the rhythm of DA turnover in L:D animals.

Treatment with E slightly advanced the free-running rhythm of DA turnover in the IL and

abolished the rebound that occurred during late subjective night in DD-OVX rats. After

treatment with both E+P, the effect of E was reversed, with a significant peak induced at CT22

surrounded by a maintained level of DA turnover throughout the remainder of the subjective day.

Thus, in PHDA neurons, as in THDA neurons, ovarian steroids modify the overall magnitude but

not the phase or period of the respective light-entrained and free-running rhythms of activity. My

data agree with previous work and support the hypothesis that ovarian steroids adjust the timing

of the diurnal PRL surge through changes in the magnitude of DA release from all 3 populations

of NDNs.

I have shown that TIDA and PHDA neurons display circadian rhythms of activity,

characterized by entrainment to varying L:D cycles and an estimated free-running period of

approximately 24.5 hours (Chapter 1). Further, although THDA neuronal activity is entrained by

65

light, it does not free-run in DD (Chapter 1). I have speculated that ovarian steroids play a

significant role in establishing both the phase and general magnitude of NDN neuronal activity

through dramatic effects on the amount of DA release and metabolism during the 24-hour day.

Diverse effects of ovarian steroids on the activity of NDN and PRL secretion have been well

documented (1,186). I have found that ovarian steroids prevent the dramatic phase-delay in

TIDA neuronal activity by reducing the amount of DA turnover during the afternoon PRL

increase. Although treatment with ovarian steroids modulates the amount of DA turnover within

THDA and PHDA neurons, they do not have a robust effect on the frequency of their DA

turnover rhythms. Ample experimental evidence suggests that photic, behavioral and chemically

induced phase-shifts display “time of day” dependent effects, characterized by a various “dead-

zones” throughout the subjective day (232,267-272). These”dead-zones” are times during the

subjective day wherein exogenous cues such as light fail to induce significant phase shifts.

Although I utilized an acutely delivered steroid hormone injection schedule designed to simulate

hormone titers seen during the rat estrous cycle and did not assume a free-running rhythm of

steroid hormone secretion, I cannot rule out a similar effect of steroid hormones on the SCN. I

hypothesize that ovarian steroids modulate both the magnitude and timing of DA turnover in

NDN terminals to facilitate timed PRL secretion. Moreover, light can also affect the expression

of physiological or behavioral events that are otherwise controlled by the clock, but do so

without an effect on the phase or period (273-275). For example, activity is suppressed by

exposure to light even in rats bearing SCN lesions (276). Thus, while I have concluded that

ovarian steroids modulate the phase and or period of these rhythms, I cannot rule out a potential

masking effect of light. Alternatively, I cannot rule out the potential role of an endogenous

stimulatory rhythm mediated by an unknown PRL-releasing factor (for review see (1)). In fact,

my data neither support nor deny the role of a PRF in the timing of the circadian rhythm of PRL

secretion and experiments are underway to determine the role of a putative PRF in the regulation

of precisely timed PRL secretion.

TIDA neurons display a free-running rhythm of activity with an estimated period greater

than 24.5h (Chapter 1). These data imply that TIDA neurons act as semi-autonomous or slave

oscillators entrained by photic cues transduced by the SCN. The product of the period gene (a

mammalian homolog of the Drosophila clock gene) has been localized to neurons of the arcuate

nucleus in rats (245,263) and mice (277). Further, Kriegsfeld and colleagues have co-localized

66

mPER and tyrosine-hydroxylase within neurons of the arcuate nucleus of mice (277). However,

it remains to be seen whether clock gene expression confers autonomous oscillatory function on

NDN in rats, and more importantly, how specific clock-controlled genes play a fundamental role

in regulating their physiological function. Analysis of clock gene expression patterns in NDN

and models of DAergic function under semi-autonomous control of the SCN are underway and

will add new insight to our current understanding. I conclude that ovarian steroids affect the

magnitude and timing of DA turnover in TIDA, THDA and PHDA nerve terminals to strengthen

functional coupling between DA release and PRL secretion.

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CHAPTER 3

CLOCK GENE EXPRESSION PATTERNS IN NEUROENDOCRINE DOPAMINERGIC

NEURONS OF THE OVX RAT: CORRELATION WITH THE CIRCADIAN AND SEMI-

CIRCADIAN PATTERNS OF DA TURNOVER IN NEUROENDOCRINE

DOPAMINERGIC NEURONS

Introduction

I have established, through multiple experiments, endogenous circadian and semi-

circadian rhythms of DA turnover in the nerve terminals of TIDA, THDA and PHDA neurons in

the OVX rat (see Chapters 1, 2). Recent data suggest that neural targets of the SCN, the primary

circadian oscillator, express the putative clock genes with a circadian rhythm

(245,263,264,278,279). These tissues appear to function as “slave” oscillators that are both

entrained by photic cues transduced by the SCN and actively stimulated to maintain rhythmicity

via SCN efferents (263). Therefore, in the absence of SCN input, these tissues cannot sustain a

circadian rhythm for more than 3-5 days. These areas include the ARN, paraventricluar nucleus

(PVN), pineal gland and pituitary gland (263). However, a notable exception to this pattern is

the olfactory bulb, which appears to maintain free-running oscillations of PER1 expression in

SCN lesioned rats (279). Moreover, recent experiments suggest that cultured NIH3T3 fibroblasts

express the gene Rev-erbα in a self-sustained cell-autonomous rhythm that continues in daughter

cells after mitosis (280, 378). Recent reports suggest that neuroendocrine DAergic neurons

within the RARN express PER1 with a diurnal rhythm (277). However, these studies failed to

characterize the free-running rhythm of PER1 expression in NDN neurons. As several studies

indicate that clock genes play an active role in cellular physiology through regulation of protein-

protein interactions and gene transcription, it seems plausible that clock genes may act within the

neuroendocrine DAergic neuron to drive expression of enzymes responsible for synthesis of DA

within the pre-synaptic cell and/or release from the pre-synaptic terminal (281-283). In fact, a

rudimentary examination of the hypothalamic specific expression promoter region of the tyrosine

hydroxylase gene reveals a significant (>10) number of E-box (CACGTG) –like sequence motifs

((284) and Sellix and Freeman unpublished observation). Evidence suggests that

CLOCK:BMAL1 heterodimers drive gene transcription through DNA recognition at the E-box

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motif, E-box sequences within the TH promoter may suggest a level of clock-controlled gene

expression (CCGE) in NDNs.

Given the novel and diverse rhythms of DA turnover I have observed from the TIDA,

THDA and PHDA neurons under light-entrained and free-running conditions, I hypothesized that

rhythms of clock gene expression in NDNs neurons corresponds to the rhythmic synthesis and

release of DA from nerve terminals. Therefore, using RT-PCR, Western blot and

immunocytochemistry, I have attempted to verify the expression of several of the clock gene

products within tissues of the hypothalamo-pituitary-gonadal axis and to further characterize the

rhythmic expression of these gene products within each of the three populations of DA neurons

in the OVX rat. I have utilized antibodies against the DA synthesis rate limiting enzyme tyrosine

hydroxylase (TH) to identify DAergic neurons within the periventricular nucleus (PeVN; PHDA

neurons), rostral ARN (RARN; THDA neurons) and dorsomedial ARN (DMARN; TIDA

neurons) and specific primary antibodies against PER1, PER2 and CLOCK to identify those

DAergic neurons expressing clock genes in their nuclear compartment under both L:D and DD

conditions. Further, I have determined the expression pattern of PER1, PER2 and CLOCK in

TH-IR neurons of the Zona Incerta as a control for DAergic neurons that are not involved in the

timing of PRL secretion. In addition, I have verified the light-entrained and free running

rhythms of PER1, PER2 and CLOCK expression within the SCN core and shell (285-287). I

believe that these data will add to our general understanding of the functional role for clock

genes in “slave oscillators” that may or may not depend on the SCN for their synchronization.

Materials and Methods

Animals

Adult female Sprague-Dawley rats (> 60 days of age) weighing 250-300g (Charles River

Labs inc., Wilmington, MA) were housed under a standard 12:12 L:D cycle with lights on at

0600 or constant darkness (DD) with constant temperature (25C) and humidity. As always,

standard rat chow and water were available ad libitum. The room was illuminated with four 40

W fluorescent bulbs, producing a minimum illumination of 100 lux at cage level. Under DD

conditions all maintenance was performed in dim red light (< 1 lux) every third day between

0900h and 1400h (approximately the first half of the 12-hour light phase) to avoid potential

entrainment to non-photic stimuli by disrupting the animals during the inactive period (232). All

69

animals were sacrificed in dim red light (<1 lux). All experimental protocols were approved by

the Florida State University Animal Care and Use Committee (ACUC).

Bilateral Ovariectomy and Analysis of Drinking Rhythms

Animals were anesthetized with Halothane and OVX bilaterally. All animals were

placed under a standard L:D cycle (lights on 0600h-1800h) for 2-5 days for habituation to the

home cage. Animals were subsequently placed into DD by extension of the 12-hour dark phase

or were maintained under 12:12 L:D conditions for an additional 5 days. In constant conditions

(DD or LL) the rhythm of drinking activity free-runs with a period of approximately 24.5 hours

(63,235,236). Feeding and drinking patterns are established methods for determining circadian

time, a subjective measure based on the activity of the animal, independent of the L:D cycle

(63,64,234). Drinking was measured over the 24-hour day with an automated device (Dilog

Instruments, Tallahassee, FL.) counting individual licks in 30-second bins over 24 hrs and

Circadian Time 12 (CT12; onset of subjective activity period) was calculated. CT12 was used as

a reference for tissue collection. In all experiments, samples were collected at the beginning

(CT0) and end (CT12) of the subjective inactivity period, as well as the midpoints of both the

inactivity (CT6) and activity (CT18) periods. Double plotted actograms of drinking activity (12-

hour moving average of drinking activity around a central peak of activity) were produced with

Circadia software (ver. 2.1.16; Behavioral Cybernetics, Inc., Tallahassee, FL.)

Tissue preparation

Animals were deeply anesthetized with halothane and transcardially perfused through the

ascending aorta with 60 mls of pre-wash (0.1M PBS containing 0.5% sodium nitrite and 10,000

U/L of heparin), followed by 200 mls of ice-cold 4% paraformaldehyde (Sigma) in 0.1M PBS.

Following perfusion, brains were removed and placed in 4% paraformaldehyde to post-fix at 4C

overnight. The following morning brains were blocked immediately anterior to the optic chiasm

and just posterior of the mammillary bodies and then cryoprotected in a 20% sucrose solution for

36-48h. Brains were sectioned on a sliding microtome (Richard-Allan Scientific, Kalamazoo,

MI) at 40 µm thickness and collected in 4 adjacent series. Sections were collected in 12 well

plates containing cryoprotectant solution (288) and stored at -20C.

Immunocytochemistry

Sections were processed for PER1, PER2, CLOCK and tyrosine hydroxylase

immunoreactivity. Each series of sections was rinsed three times for 15 min in 0.1M PBS

70

containing 0.1% triton-X 100 and 0.1% sodium azide (PBX) to remove cryoprotectant (triton-X

100 and sodium azide, Sigma-Aldrich, St. Louis, MO). Non-specific binding was blocked in

10% normal goat serum (Chemicon, Temecula, CA) in PBX for 1h. Polyclonal primary

antibodies against mouse PER1 (rabbit host, polyclonal used at 1:10,000; kind gift of Dr. David

Weaver), mouse PER2 (rabbit host, polyclonal used at 1:1000, Alpha Diagnostics Inc, San

Antonio, TX) or mouse CLOCK (goat host, polyclonal used at 1:10,000; Santa Cruz

Biotechnology, Santa Cruz, CA) were incubated with monoclonal anti-mouse tyrosine

hydroxylase (1:10,000; Chemicon, Temecula, CA) for 48h at 4C on a rotating bench top shaker.

All primary and secondary antibodies were diluted in PBX. Sections were washed three times

for 10 min with PBX between each step. Anti-rabbit or anti-goat donkey CY3 (Excitation=550

nm; Emission=570 nm) conjugated and anti-mouse CY2 (Excitation=492 nm; Emission=510

nm) conjugated secondary antibodies were added (1:600 in PBX; Jackson Immunochemicals,

West Grove, PA) and sections were again incubated at 4C for 12-18h. Sections were then rinsed

with PBX three times for 15 min, mounted and coverslipped with diluted aquapolymount

(Polysciences, Warrington, PA) After several hours the edges of the coverslips were sealed with

nail polish. Controls included sections wherein primary antibody was excluded or PER1, PER2

and CLOCK primary antibodies were pre-absorbed with a substantial amount (10-100 fold

higher concentration) of the peptide fragment they were raised against (See Fig. 20). PER1,

PER2 and CLOCK immunoreactivity within the SCN was verified in wild-type and PER1/PER2

double-knockout mouse brain (Fig. 20; kind gift of Dr. David Weaver) and support previous

findings (210,289).

Microscopy and Data Analysis

Neurons in the rostral ARN (THDA), dorsomedial ARN (TIDA), PeVN (PHDA) and ZI

were identified as DAergic based on the presence of TH-immunoreactivity as previously

described (31,162,249,254). PER1, PER2 or CLOCK and TH double-labeled neurons were

identified and counted within the ARN (DMARN and RARN), PeVN and ZI. Although clock

proteins are translocated back to the nucleus, they do spend some time in the cytoplasm(221).

However, their transcriptional activation/repression function occurs within the nucleus.

Therefore, I counted PER1, PER2 and CLOCK-IR nuclei within TH-IR neurons. Given the

absence of nuclear staining for TH, I was able to clearly and efficiently identify clock protein

immunoreactive (IR) nuclei in TH-IR neurons (see Figs.21-25). Clock gene nuclei/TH staining

71

in the ZI was included as a positive control for DA staining outside of the dorsomedial

hypothalamus. Sections containing the ZI were also included as a negative control, as they

synthesize DA but do not participate in the regulation of PRL secretion. Single-labeled PER1,

PER2 or CLOCK immunoreactive nuclei were counted within the SCN core and shell (see Fig.

25). SCN core and shell have been well defined by the distribution of specific neurotransmitters

within each compartment (175,290). The SCN core contains primarily VIP-IR neurons, while an

excess of AVP-IR neurons defines the SCN shell. However, for the purpose of counting single-

labeled PER1, PER2 and CLOCK –IR nuclei, I have simply divided the SCN along its

dorsocaudal axis and counted the number of nuclei within each anatomically defined sub-region.

While I refer to these as “core” and “shell”, I acknowledge my method lacks specificity with

respect to transmitter phenotype. However, given the distance between the counted regions and

the high probability of AVP or VIP expression within my “core” and “shell”, I feel very

confident in my methodology. Images were taken with a Leica DMLB compound

stereomicroscope fitted with short-pass dichroic filters (CY2, 488 nm; CY3 596 nm) and a

SPOT-RT cooled CCD camera attached to a microcomputer. Image acquisition and analysis was

conducted using Metamorph software (Universal Imaging, Downingtown, PA.). Grayscale

images of clock gene and TH immunoreactivity were overlaid and pseudocolored in Metamorph.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis of Clock Gene

expression in OVX Female Rats

RT-PCR was used to determine the tissue distribution of several clock gene mRNAs in

OVX female rat brain and pituitary gland. Animals kept under a standard L:D cycle were briefly

anesthetized under hypercapnic (50% CO2) conditions and sacrificed by decapitation at CT6 (the

approximate time of peak per1 and per2 mRNA expression in the SCN (see (204) for review).

Brains were rapidly removed and placed in a coronal brain matrix on ice (ASI instruments,

Warren, MI). Individual 2 mm thick sections containing the suprachiasmatic nucleus (SCN) or

the medial basal hypothalamus (including the arcuate nucleus (ARN) and median eminence) are

placed on a DEPC treated glass slide. Individual 4 mm2 cubes including the entire SCN or ARN

were placed in 1 ml of TRIZOL reagent (Invitrogen, Carlsbad, CA.) and homogenized on ice.

After homogenization, 100 µl of chloroform is added to each tube. Tubes were briefly vortexed

and centrifuged at 10,000 rpm (12,000 x g). The aqueous phase was removed from each tube

and pooled according to sample. An equal volume of isopropanol was added to each tube and all

72

tubes were placed in a –80C freezer. After 6 hours at –80C, precipitated RNA is pelleted by

centrifugation at 13,000 rpm for 20 minutes at 4C. The remaining isopropanol is removed and

the pellets are washed twice with 70% ethanol, allowed to air-dry and then re-suspended in

DEPC-treated water. Total RNA concentration was analyzed by UV/vis spectrophotometry and

diluted to normalize the sample to 500 ng/µl, of which 5 µg was used for reverse transcription.

Messenger RNA is reverse transcribed to cDNA using the SuperScript tm

First-Strand Synthesis

System for RT-PCR (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol.

Following RT, clock genes are amplified using 2 µl of RT reaction with the Platinum tm

PCR

SuperMix (Invitrogen, Carlsbad, CA.) and gene specific primers for clock genes including per1,

per2, clock and bmal1, as well as primers for the rat ribosomal protein L32 as a positive control

(10 pmoles/reaction, 1 µl; final vol. of 50 µl). PCR is performed in an MJ research (Watertown,

MA) PTC-200 thermocycler at 94C for 1 min. followed by 35 cycles of amplification (94C for 1

min, 58C for 1 min, 72C for 4 min) and a final extension at 74C for 15 min. Sample are stored at

–20C until use. Reaction products are separated with 2% agarose (analytical grade; Invitrogen,

Carlsbad, CA) gels in Tris:Acetate:EDTA (TAE; Fisher Scientific, Suwanee, GA) buffer (see

Fig. 17). Gels are stained by immersion in ethidium bromide solution (0.1 µg/ml in TAE) for 10

min followed by a 20-30 min. TAE rinse. Gels are photographed and analyzed using a KODAK

Gel Logic 100 Imaging and Analysis System (Eastman Kodak Company, Rochester, NY).

Remaining PCR products were purified with a Qiagen PCR purification Kit (Qiagen, Valencia,

CA) and sequenced at the Florida State University core molecular analysis facility. NCBI blast

sequence analysis confirmed amplification of rat clock gene products with mouse primers, and

supported high homology (>90% in all cases) between mouse and rat clock genes.

Western blotting and immunodetection of clock gene products in neuroendocrine tissues

I. Extraction of clock proteins.

Proteins were extracted from neuroendocrine tissues according to the method of Lee and

colleagues (221). Briefly, animals maintained under a standard 12:12 L:D cycle were sacrificed

at CT12 under hypercapnic conditions and decapitated (CT12 represents the approximate peak of

PER1 and PER2 –ir in the SCN; (204,210,289)). The brain and pituitary gland were rapidly

removed and the brain was placed in a chilled brain-sectioning matrix (ASI instruments Inc.,

Warren MI) on ice while the pituitary gland was rapidly frozen in liquid nitrogen. Separate 2

mm thick frontal sections of the brain including the suprachiasmatic nucleus (SCN) and the

73

arcuate nucleus (ARN) were made with a sterile razor blade and placed on a chilled glass slide.

Using a scalpel, a 3mm x 3mm cube including the SCN and a 3mm x 4mm angled dissection of

the ARN were removed, as well as the cerebellum and a majority of the piriform cortex from

both hemispheres. Following dissection, tissues were rapidly frozen in liquid nitrogen and stored

at –80C until protein extraction. Tissues were thawed on ice in chilled homogenization buffer

containing: 20 mM HEPES, 100 mM NaCl, 0.05% Triton-X 100, 1mM DTT, 5 mM sodium-

β−glycerophosphate, 1 mM Na orthovanadate, 1 mM EDTA, 0.5 mM PMSF, and a cocktail of

protease inhibitors including aprotonin (10µg/ml), leupeptin (5µg/ml) and pepstatin A (2µg/ml;

(221)). Tissue samples were sonicated on ice with a mini-homogenizer and disposable pestle

(Kimble-Kontes, Vineland, NJ) using 10-15 strokes (3-4 sec/stroke). After centrifugation at

12,000 g for 15 min, supernatant was transferred to a fresh tube and re-spun. Supernatant was

removed and extracted protein concentration was determined by the micro-modified

bichonchoninic acid protein detection system (Pierce, Rockford IL) as described in Chapter 1,2.

II. SDS-PAGE and Electroblotting.

Extracted proteins were resolved by SDS-PAGE electrophoresis according to the

established protocol of Lee and colleagues (221). As clock proteins vary significantly in MW

(i.e., CLOCK MW ~85-100 kD, PER1 and PER2 ~180 kD) I utilized separate gel concentrations

for each to maximize resolution (CLOCK 8%, PER1/2 7%). Samples were mixed with equal

volume of 2X sample buffer containing: 100 mM Tris-HCL pH 6.8, 4% SDS, 20% glycerol, 5%

β-mercaptoethanol (β-ME), 2 mM EDTA and 0.1 mg/ml bromophenol blue and heated at 95C

for 5 min (221)). Samples were briefly centrifuged at 12,000g and cooled to room temperature.

Samples and MW markers were loaded into 10 well pre-set polyacrylamide gels using the mini-

protean II system (Bio-Rad, Hercules CA) and electrophoresis was carried out using 150 V of

constant voltage for 45 min-1h in Tris-glycine-SDS buffer (8mM Tris-base, 40 mM glycine,

0.1% SDS). Electroblotting was carried out with standard buffers. Briefly, gels were placed in

transfer buffer (Tris-glycine-SDS-methanol; 8mM Tris-base, 40mM glycine, 0.1% SDS and 20%

methanol) for 1h to overnight before electroblotting with constant 100 V for 90 min. Transfer

was verified by visual inspection of Pagemarker Pre-stained molecular weight marker (VWR

Inc.) and following brief staining with fast-green protein stain.

III. Immunodetection and Quantification.

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Following verification of successful transfer, blots were placed in 5% non-fat milk for 30

min at RT, followed by primary antibody (anti-CLOCK guinea pig 1:1000, anti-PER1/2 guinea

pig 1:1000 (kind gift of Dr. Choogon Lee, The Florida State University College of Medicine,

Tallahassee, FL.) for 12-16h at 4C. Primary antibody was washed off with three 10 min washes

of Tris-buffered saline with 0.1% tween-20 (TTBS) and blots were placed in secondary antibody

(anti-guinea pig IgG conjugated to horseradish peroxidase at 1:5,000, Jackson Immunolabs, West

Grove, PA) for 1h at RT. Following 6-8 10 min washes in TTBS, CLOCK or PER1/2 protein

levels were visualized using ECL chemiluminescence (Amersham Inc., Piscataway NJ)

according to the manufacturer’s specifications. Verification of clock gene staining within tissue

extracts was verified by comparison with protein extracts from liver of wild-type (WT) and

PER1/2 double knock-out mice or kidney proteins from WT and CLOCK KO mice (kindly

provided by Dr. Choogon Lee). Blots were stripped of primary/secondary antibody complex

according to the protocol provided by Pierce using their stripping and re-probing buffer and re-

probed with anti-actin mouse monoclonal primary antibody (1:1000, Sigma-Aldrich, St. Louis,

MO) to verify equal loading of protein.

Experimental Design

In order to establish the relationship between the light-entrained and free-running

circadian and semi-circadian rhythms of DA turnover in TIDA, THDA and PHDA neurons and

the expression of clock genes I have identified the pattern of PER1, PER2 and CLOCK

immunoreactivity in DAergic neurons under both L:D and DD conditions. Moreover, I utilized

RT-PCR and Western blotting for clock gene products in order to verify their expression within

tissues of the HPG axis, including the ARN and pituitary gland. Adult OVX female rats were

placed in 12:12 L:D or DD conditions for 5 days and perfused at CT0, 6, 12 and 18. Brain

sections were stained for PER1, PER2, CLOCK and the DA synthesis rate limiting enzyme

tyrosine hydroxylase (TH). The percentage of PER1, PER2 or CLOCK/TH double-labeled cells

was determined in the PeVN, ARN and ZI. The ZI was included as a positive control for

DAergic neurons that are not involved in the timing of PRL secretion. In addition, I determined

the free- running rhythms of PER1, PER2 and CLOCK expression in the SCN core and shell in

the OVX female rat.

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Data Analysis

The percent of PER1, PER2 or CLOCK/TH double-labeled cells in the DMARN, RARN,

PeVN and ZI, and the number of clock protein single-labeled cells in the SCN core and shell

represent the mean + SEM of 5 images/region/animal in a total of 3 animals (15

images/region/timepoint), as a function of circadian time. Although they exhibit a distinct

rhythm, all of my data do not conform to a sine/cosine wave function, which prohibits a non-

linear regression analysis to present the data as a function of time and lighting condition.

Moreover, as samples were obtained from each animal at individual time points over a 24 h

period (CT0-CT18), it is difficult to extrapolate accurate phase and period measures. Data were

analyzed with two-way ANOVA for (A) time of day and (B) lighting condition effects, followed

by Bonferroni post-hoc tests. P<0.05 was accepted as the limit of significance. ANOVA were

performed and graphs were created with Graph-pad Prism software (Graphpad Software Inc.,

San Diego, CA.)

Results

Clock gene mRNA and protein expression in the SCN, ARN and pituitary gland from OVX

female rats.

As shown in figure 17, per1, per2, bmal1 and clock gene mRNA are expressed within the

SCN, ARN and pituitary gland of OVX female rats. Gene products were detected with RT-PCR

and were verified by DNA-sequencing. Further, I have verified expression of PER1, PER2 and

CLOCK protein within tissue extracts containing the SCN, ARN, pituitary gland, cerebellum and

piriform cortex (Fig. 18). PER1 and PER2 (both approximately 180 kD) are abundantly

expressed within each region, with particularly high levels of expression in the SCN, pituitary

gland and piriform cortex (Fig. 18A). In order to verify the specificity of the clock protein

antibodies I probed liver extracts from wild type and PER1/PER2 double KO mice, as well as

liver extracts from clk/clk mutant mice (Fig. 18B, kind gift of Dr. Choogon Lee (198,291)). I

verified an absence of immunostaining for PER1 and PER2 in the liver of PER1/2 double KO

76

Figure 17. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) amplification of

per1, per2, clock and bmal1 mRNA from SCN, ARN and pituitary gland from adult female

OVX rats. Animals were sacrificed at CT6 under hypercapnic conditions. Individual 2 mm

thick tissue punches containing the suprachiasmatic nucleus (SCN) or the medial basal

hypothalamus (including the arcuate nucleus (ARN) and median eminence) and the entire

anterior lobe of the pituitary gland were dissected and used for total RNA extraction. RT using

oligo-DT primers was carried out to amplify cDNA. Gene specific primers for per1, per2, clock

and bmal were used to amplify cDNA. PCR products were separated with agarose gel

electrophoresis and visualized with ethidium bromide. Ribosomal prtotein L32 was also

amplified as a positive control and displayed equal amplification to clock gene products (data not

shown).

77

clk/clk

B

Figure 18. Characterization of PER1, PER2 and CLOCK proteins in the CNS of adult

OVX rats. PER1, PER2 and CLOCK protein expression in the SCN, ARN, anterior lobe of the

pituitary gland (PIT), prifirom cortex (PIR. CTX) and cerebellum (CBM) was determined with

high specificity monoclonal antibodies (kindly provided by Dr. Choogon Lee). (A) PER1/PER2

(~180 kD) and CLOCK (~85-100 kD; arrowhead) protein was detected within the SCN, ARN,

pituitary (PIT), piriform cortex (PIR. CTX) and cerebellum (CBM). (B) Binding specificity for

the antibody was verified with clock protein liver extracts from wild-type (WT) and truncated

mutant (clk/clk) mice. In all rCLOCK samples the arrow indicates non-specific binding of the

antibody while the arrowhead indicates variable CLOCK isoforms. Specificity of PER1, PER2

and CLOCK antiserum is exemplified by the absence of labeling in PER1/PER2 double KO mice

and a shift in the size of labeled bands in the clk/clk mutant liver.

78

mice and a shift in the size of the CLOCK-IR band in liver extracts from clk/clk mutant mouse

liver (221).

Light-entrained and free-running rhythms of drinking behavior in the OVX rat

As outlined in methods, drinking was measured over the 24-hour day with an automated

device (Dilog Instruments, Tallahassee, FL.) counting individual licks in 30-second bins for 24

hrs. Circadian time 12 (CT12) was designated as the onset of drinking activity. Following

ovariectomy, animals were placed in the lickometer device for 2-3 days under standard L:D

conditions (lights on 0600h; off 1800h), followed by 5 additional days under either L:D or DD

conditions. Actograms of drinking activity from adult OVX rats are shown in figure 19. Animals

under a standard L:D cycle displayed a value for CT12 near 1730+0.2h. Following 5 days under

DD conditions, CT12 was delayed to 1900+0.2h. These values indicate a τ~24.2h and verify a

functioning free-running circadian oscillator within the adult OVX rat prior to sacrifice and

perfusion. These data agree with similar analysis from published reports from my laboratory

(see Chapter 1).

Validation of PER1, PER2 and CLOCK antibodies

Given the scarcity of published results with the antibodies used in the present study, I

validated their use and specificity in the adult female rat. I stained brain sections from adult WT

mice and PER1/2 double KO mice sacrificed at CT12 (a time of peak PER1/2 expression; both

gifts from Dr. David Weaver) with anti-mouse PER1, PER2 and CLOCK polyclonal antibodies.

As reported by Bae and colleagues, I detected robust PER1, PER2 and CLOCK –IR within the

SCN of WT mice at CT12 (Fig. 20A,C,E (210)). I failed to detect a significant nuclear signal for

PER1, PER2 within the SCN of PER1/2 double KO mice, but I were able to detect a clear, albeit

more diffuse, CLOCK-IR within the SCN of KO mice (Fig. 20B,D,F). Pre-absorption of the

primary antisera with a 100-fold excess of the peptide fragment used for immunization

effectively eliminated PER1, PER2 and CLOCK staining in the SCN of adult OVX female rats

(Fig. 20G). Further, incubation without the primary antisera also eliminated nuclear staining for

PER1, PER2 and CLOCK (Fig. 20H). These results agree with previous findings with the same

antibodies, used at the same dilution and incubation period (210,289). Clock protein

immunoreactivity within both NDNs and SCN neurons is primarily limited to the nucleus,

characterized by dense, granular staining and an absence of staining within the nucleolus (Fig.

21A,B).

79

Figure 19. Drinking activity in OVX rats under a standard 12:12 L:D cycle and

constant conditions (DD). Animals were placed in my lickometer device for 3-5

days under a standard 12:12 L:D cycle with lights on from 0600h-1800h. After 5

days, animals were divided into two groups and were maintained under either (1) a

12:12 L:D cycle (L:D) or constant darkness (DD) for an additional 5 days. Animals

displayed clear light-entrained and free-running circadian rhythms of drinking activity

characterized by a free-running t~24h. Grey arrowheads indicate activity onset or

circadian time 12 (CT12) under a standard L:D cycle for each animal. The black

arrow indicates the first day of constant conditions in the DD animal.

80

Figure 20. Characterization of PER1, PER2 and CLOCK-immunoreactivity (IR). PER1,

PER2 and CLOCK protein expression in the SCN and the NDNs was determined with high

specificity polyclonal antibodies. (A,B) PER1, (C,D) PER2, and (E,F) CLOCK -IR in the SCN

of WT (A,C,E) or PER1/2 double KO mouse sacrificed at CT12 (B,D,F; mice kindly provided

by Dr. David Weaver). (G) Fluorescent signal in the SCN of an adult OVX female rat was not

present after preincubation with a 100-fold excess of the blocking peptide (staining for PER2

shown; similar results were obtained for PER1 and CLOCK (data not shown)). (H) Signal was

also eliminated from female rat SCN by removal of the primary antiserum. (as above, staining

for PER2 is shown as a representative; similar results were obtained for PER1, CLOCK). Scale

bar in A = 500 µm.

81

Figure 21. Clock gene expression within the nucleus of NDNs and SCN neurons. Clock

gene immunoreactivity (red stain) within (A) NDNs (TH-IR neuron; green stain) and (B)

SCN neurons is primarily limited to the nucleus. In A and B, CLOCK-IR nuclei are

identified by arrowheads. An absence of granular staining within the nucleolus is a clear

indicator of nuclear staining. Scale bar in B = 20 µm.

82

Light-entrained and free-running rhythms of PER1-IR in NDNs of the DMARN, RARN

and PeVN

PER1-IR nuclei were clearly labeled within TIDA, THDA and PHDA neurons located

within the DMARN, RARN and PeVN, respectively (arrowhead; Fig.22A,C,E). Generally, more

than 50% of NDNs within the DMARN, RARN and PeVN expressed nuclear PER1-IR during

the subjective day. I observed several PER1-IR nuclei that were not double-labeled with TH

(arrow), as well as several TH-IR neurons that did not express PER1 (asterisk; Fig. 22A,C,E).

Analysis of PER1-IR within the DMARN of the hypothalamus as a function of lighting condition

(L:D vs. DD) and circadian time (CT) revealed a main effect of time (F=4.85, p<0.05), but not

lighting condition (F=1.26, p>0.05) or the interaction between lighting condition x time (F=

0.31, p>0.05; Fig. 22A,B). Pairwise comparisons as a function of circadian time within lighting

condition revealed a significant diurnal rhythm of nuclear PER1 expression in TIDA neurons

with a peak at CT18 (p<0.05) compared with a nadir at CT6 (Fig. 22B). After 5 days in DD this

rhythm was abolished, although the shape of the rhythm remained similar (Fig. 22B). Therefore,

I observed a significant light-entrained, but not free-running, circadian rhythm of PER1-IR

within TIDA neurons. These data suggest a potential relationship between a diurnal rhythm of

DA synthesis/release from TIDA nerve terminals and PER1-IR in TIDA neurons. I have

previously determined that TIDA neurons display a biphasic light-entrained circadian rhythm of

DA release with a significant acrophase between CT0 and CT6. These increases occur

approximately 6-12 hours after the peak of PER-IR in TIDA neurons (Fig. 22B).

Analysis of PER1-IR within the RARN of the hypothalamus as a function of lighting

condition (L:D vs. DD) and circadian time (CT) did not reveal a main effect of time (F=0.52,

p>0.05), lighting condition (F=4.29, p=0.06) or the interaction between lighting condition x time

(F= 0.87, p>0.05; Fig. 22C,D). Individual comparisons support a lack of main effects for time or

treatment and show that PER1 expression within THDA neurons did not display either light-

entrained or free-running circadian rhythms of nuclear expression (Fig. 22D). These data agree

with my previous results showing that THDA neurons fail to express a free-running rhythm of

DA turnover within the NL (Chapter 1 and Fig. 6). However, unlike data from TIDA and PHDA

neurons, they fail to indicate a role for clock genes in the regulation of the light-entrained diurnal

rhythm of DA release from THDA nerve terminals. Analysis of PER1-IR within the PeVN of

the hypothalamus as a function of lighting condition (L:D vs. DD) and circadian time (CT)

83

Figure 22. PER1 expression in NDNs under a standard 12:12 L:D cycle and

DD. I observed strong nuclear PER1 expression (red; arrowhead) within (A)

TIDA neurons (green), (C) THDA neurons and (E) PHDA neurons. Several

neurons within these regions expressed TH-IR but failed to show strong PER1

staining (asterisk) or exhibited strong PER1 staining without detectable TH-IR

(arrow). PER1 expression peaked in (B) TIDA neurons at CT18 under L:D but

was arrhythmic in DD. (D) I failed to detect a significant rhythm of PER1

expression within THDA neurons under either condition. (F) PER1 expression

peaked at CT12 under L:D in PHDA neurons but also failed to display a free-

running rhythm in DD. In B,D and F, differing letters indicate significance across

lighting condition within time and # indicates a significant acrophase within

lighting condition. Scale bar in A = 50 µm

84

revealed a main effect of time (F=4.91, p<0.05), but not lighting condition (F=0.004, p>0.05) or

the interaction between lighting condition x time (F= 2.83, p>0.05; Fig. 22E,F). Comparisons

within animals housed under a standard L:D cycle as a function of time reveal a significant light-

entrained rhythm of PER1-IR within PHDA neurons characterized by an acrophase at CT12,

compared with basal levels at CT0 (p<0.05), CT6 (p<0.01) and CT18 (p<0.01; Fig. 22F). PER1

expression did not display a free-running rhythm within PHDA neurons. As I observed for TIDA

neurons, these data suggest a potential relationship between a diurnal rhythm of DA

synthesis/release from PHDA nerve terminals and PER1-IR in PHDA neurons. Unlike TIDA

neurons, my previous experiments show that PHDA neurons display free-running rhythms of DA

turnover with a τ near 24h (Chapter 1 and Fig. 6). Under both L:D and DD conditions, PHDA

neurons display a peak of DA release near CT18, approximately 6 hours after the observed peak

of PER1-IR within PHDA neurons (see Chapter 1 and Fig. 22F). Thus, my data suggest that

light-entrained diurnal rhythms of DA synthesis and release from TIDA and PHDA neurons may

be regulated by both SCN efferents and local regulation by nuclear PER1.

Light-entrained and free-running rhythms of PER2-IR in TIDA, THDA and PHDA

neurons

PER2-IR nuclei were clearly labeled within TIDA, THDA and PHDA neurons located


than 50% of NDNs within the DMARN, RARN and PeVN expressed nuclear PER2-IR during

the subjective day. I observed several PER2-IR nuclei that were not double-labeled with TH

(arrow), as well as several TH-IR neurons that did not express PER2 (*; Fig. 23A,C,E). Two-

factor analysis of PER2-IR within TIDA as a function of lighting condition (L:D vs. DD) and

circadian time (CT) revealed a main effect of time (F=5.10, p<0.05), but not lighting condition

(F=3.73, p>0.05) or the interaction between lighting condition x time (F= 2.46, p>0.05; Fig.

23A,B). Pairwise comparisons within L:D animals as a function of time exposed a significant

diurnal rhythm of PER-IR within TIDA neurons characterized by peak levels of PER2

expression at CT6 (p<0.05) and CT12 (p<0.05), compared with a nadir at CT0. PER2-IR levels

returned to basal levels at CT18 (p<0.05; compared with peak levels at CT12). After 5 days of

constant darkness, PER2-IR in TIDA neurons peaked at CT12 (p<0.05) compared to a nadir at

CT18. In contrast with PER1-IR, which peaked at CT18 under a standard L:D cycle in TIDA

neurons, PER2-IR peaked between CT6 and CT12 in animals housed under a standard L:D cycle

85

Figure 23. PER2 expression in NDNs under a standard 12:12 L:D cycle and

DD. I observed strong nuclear PER2 expression (red; arrowhead) within (A)

TIDA neurons (green), (C) THDA neurons and (E) PHDA neurons. Several

neurons within these regions expressed TH-IR but failed to show strong PER2

staining (asterisk) or exhibited strong PER2 staining without detectable TH-IR

(arrow). PER2 expression peaked in (B) TIDA neurons at CT6 and CT12 under

L:D and at CT12 in DD. (D) PER2 expression peaked at CT6 within THDA

neurons under L:D conditions but failed to display a significant free-running

rhythm (F) PER2 expression peaked at CT6 under L:D in PHDA neurons but also

failed to display a free-running rhythm in DD. In B,D and F, differing letters

indicate significance across lighting condition within time and # indicates a

significant acrophase within lighting condition. Scale bar in A = 50 µm

86

(Fig. 23B). Further, while PER1-IR failed to display a free-running rhythm of expression within

TIDA neurons, PER2-IR peaked at CT12, compared with a nadir at CT18 (Fig. 23B). As I have

shown, TIDA neurons display a light-entrained rhythm of DA release with significant peaks at

both CT6 and CT12 that occur at or near the peak of PER2 expression I have observed for TIDA

neurons (see Chapter 1, Fig. 6 and Fig. 23). Further, TIDA neurons display a free-running

rhythm of DA turnover with significant peaks at CT12 and CT18, also approximately the same

time as the peak of PER2 expression in TIDA neurons shown above (Chapter 1 and Fig. 23).

These data provide strong support for nuclear PER2 function in the timing of DA synthesis and

release from NDNs.

Two-factor analysis of PER2-IR within the RARN of the hypothalamus as a function of

lighting condition (L:D vs. DD) and circadian time (CT) revealed a main effect of time (F=3.70,

p<0.05) and lighting condition (F=5.58, p<0.05), but not an interaction between lighting

condition x time (F= 0.95, p>0.05; Fig. 23C,D). Comparisons within L:D animals as a function

of time delineate a significant diurnal rhythm of PER2 expression within THDA neurons defined

by a significant increase between CT0 and CT6 (p<0.05), followed by a sustained level

throughout the remainder of the subjective day (Fig. 23D). After 5 days under DD conditions

PER2-IR did not display a significant free-running rhythm (Fig. 23D). According to previous

experiments (Chapter 1), THDA neurons display a diurnal rhythm of DA turnover with increased

levels between CT0 and CT6, which corresponds to the increase of PER2-IR nuclear expression

in THDA neurons (Chapter 1, Fig. 6 and Fig. 23D). Further, prior data suggest that THDA

neurons fail to display a free-running rhythm of DA turnover, in agreement with my inability to

observe a free-running rhythm of PER2 expression within THDA neurons (Chapter 1, Fig. 6 and

Fig. 23D). Data from these experiments suggest a role for PER2 expression in the timing and

magnitude of the light-entrained rhythm of DA turnover within THDA neurons.

Two-factor analysis of PER2-IR within the PeVN of the hypothalamus as a function of

lighting condition (L:D vs. DD) and circadian time (CT) did not expose a main effect of time

(F=0.74, p>0.05), lighting condition (F=3.25, p>0.05) or an interaction between lighting

condition x time (F= 2.23, p>0.05; Fig. 23E,F). Although I did not see a main effect of time,

individual comparisons within animals housed under a standard 12:12 L:D cycle as a function of

time revealed a significant diurnal rhythm of PER2 expression with a significant peak at CT6

(p<0.05; compared with a nadir at CT0), followed by a sustained level of PER2 expression

87

throughout the remainder of the subjective day (Fig. 23F). Following 5 days in DD, I did not

observe a significant free-running rhythm of PER2 expression within PHDA neurons (Fig. 23F).

As stated above, PHDA neurons display significant light-entrained and free-running rhythms of

DA turnover in the OVX rat (Chapter 1). Under a standard L:D cycle, DA turnover in the IL

peaks between CT0 and CT12, decreases by CT18 and returns to peak level by CT2 (Chapter 1,

Fig. 6). Current data reveal that PER2-IR within PHDA neurons peaks between CT0 and CT6

and remains elevated throughout the subjective day, but fails to display a free-running rhythm of

expression (Fig. 23F). These data suggest a potential role for PER2 expression within PHDA

neurons in the regulation of light-entrained, but not free-running, circadian rhythms of DA

turnover.

Light-entrained and free-running rhythms of CLOCK-IR in TIDA, THDA and PHDA

neurons

CLOCK-IR nuclei were clearly labeled within TIDA, THDA and PHDA neurons located


than 50% of NDNs within the DMARN, RARN and PeVN expressed nuclear CLOCK-IR during

the subjective day. I observed several CLOCK-IR nuclei that were not double-labeled with TH

(arrow), as well as several TH-IR neurons that did not express CLOCK (*; Fig. 24A,C,E).

Twin-factor analysis of CLOCK-IR within the DMARN of the hypothalamus as a function of

lighting condition (L:D vs. DD) and circadian time (CT) revealed a significant effect of lighting

condition (F= 17.46, p<0.01), but not time (F= 2.03, p>0.05) or the interaction between lighting

condition x time (F= 0.30, p>0.05; Fig. 24A,B). Post-hoc tests supported a negative main effect

of both circadian time and lighting. Therefore, I did not observe a significant light-entrained or

free-running rhythm of CLOCK expression within TIDA neurons (Fig. 24B).

Two-factor analysis of CLOCK-IR within the RARN of the hypothalamus as a function

of lighting condition (L:D vs. DD) and circadian time (CT) revealed a significant effect of

lighting condition (F= 6.941, p<0.05), but not time (F= 0.05, p>0.05) or the interaction between

lighting condition x time (F= 0.50, p>0.05; Fig. 24C,D). Although two-factor analysis avowed a

significant effect of lighting condition, pairwise comparisons failed to detect a significant rhythm

of CLOCK expression under both L:D or DD conditions (Fig. 24D). Two-factor analysis of

CLOCK-IR within the PeVN of the hypothalamus as a function of lighting condition (L:D vs.

DD) and circadian time (CT) did not show a significant main effect of lighting condition

88

Figure 24. CLOCK expression in NDNs under a standard 12:12 L:D cycle

and DD. I observed strong nuclear CLOCK expression (red; arrowhead) within

(A) TIDA neurons (green), (C) THDA neurons and (E) PHDA neurons. Several

neurons within these regions expressed TH-IR but failed to show strong CLOCK

staining (asterisk) or exhibited strong CLOCK staining without detectable TH-IR

(arrow). I failed to detect a significant light-entrained or free-running rhythm of

CLOCK expression the (B) TIDA or (D) THDA neurons. However, (F) PHDA

neurons failed to display a light-entrained rhythm, although they did exhibit a

free-running rhythm with a significant acrophase at CT0. In B, D and F,

differing letters indicate significance across lighting condition within time and #

indicates a significant acrophase within lighting condition. Scale bar in A = 50

µm

89

(F= 1.11, p>0.05) time (F=3.07, p>0.05) or an interaction between lighting condition x time

(F=1.59, p>0.05; Fig. 24E,F). Pairwise comparisons within animals housed under a standard

L:D cycle as a function of time failed to expose a significant light-entrained rhythm of CLOCK-

IR within PHDA neurons (Fig. 24F). Unlike both TIDA and THDA neurons, PHDA neurons

display a free-running rhythm of CLOCK expression with a significant peak at CT0 (p<0.05),

compared with basal levels at both CT12 and CT18. Overall, data from these experiments

suggest that CLOCK protein is constitutively expressed within all three populations of NDNs

(Fig. 24B,D,F). Several experiments have concluded that CLOCK protein is also constitutively

expressed within the SCN (202,205,206,292-294). Data from my laboratory supports a similar

pattern in NDNs (see Fig. 24). Therefore, it would appear that constitutive CLOCK expression

is a defining feature of the molecular clock found within both the central circadian oscillator in

the SCN and its primary central targets, including the NDNs of the hypothalamus.

Light-entrained and free-running rhythms of PER1, PER2 and CLOCK-IR in the DAergic

neurons of the ZI

As described above, PER1, PER2 and CLOCK -IR nuclei were clearly labeled within

TH-IR neurons located within the ZI (arrowhead; Fig. 25A,C,E). In contrast with areas

containing NDNs, I observed fewer clock protein-IR nuclei within ZI neurons (generally less

than 50%; arrowhead) and even fewer PER1, PER2 or CLOCK-IR nuclei that were not double-

labeled with TH (arrow; Fig. 25A,C,E). Two-factor analysis of PER1-IR within the ZI of the

hypothalamus as a function of lighting condition (L:D vs. DD) and circadian time (CT) did not

reveal a main effect of time (F=1.51, p>0.05), lighting condition (F=4.224, p=0.06) or the

interaction between lighting condition x time (F= 0.87, p>0.05; Fig. 25A,B). Individual

comparisons show that PER1-IR within the ZI did not display a significant rhythm under either a

standard 12:12 L:D cycle or DD (Fig. 25B). Two factor analysis of PER2-IR within the ZI of the

hypothalamus as a function of lighting condition (L:D vs. DD) and circadian time (CT) revealed

a main effect of time (F=4.20, p<0.01), lighting condition (F=19.12, p<0.01) and an interaction

between lighting condition x time (F=6.16, p<0.01; Fig. 25C,D). Pairwise comparisons within

animals housed under a standard L:D cycle as a function of time establish a diurnal rhythm of

PER2 expression within the ZI with significant peaks at CT6 (p<0.01), CT12 (p<0.01) and CT18

(p<0.01), compared with a nadir at CT0 (Fig. 25D). Animals placed under DD conditions for 5

days failed to display a significant free-running rhythm of PER1 expression (Fig. 25D). Analysis

90

Figure 25. PER1, PER2 and CLOCK expression in the Zona Incerta (ZI)

under a standard 12:12 L:D cycle and DD. I observed strong nuclear (A,B)

PER1, (C,D) PER2 and (E,F) CLOCK expression (red; arrowhead) within TH-IR

neurons of the Zona Incerta. Several neurons within this region expressed TH-IR

but failed to show strong PER1, PER2 or CLOCK staining (asterisk) or exhibited

strong clock protein staining without detectable TH-IR (arrow). Although I failed

to detect a light-entrained or free-running rhythm of (B) PER1 and (F) CLOCK

expression within ZI, (D) PER2 expression exhibited a light-entrained rhythm

with a significant acrophase at CT6, followed by a gradual decline to basal levels

at CT0. In B, D and F, differing letters indicate significance across lighting

condition within time and # indicates a significant acrophase within lighting

condition. Scale bar in A = 50 µm

91

of CLOCK-IR within the ZI of the hypothalamus as a function of lighting condition (L:D vs.

DD) and circadian time (CT) revealed a main effect of lighting condition (F=5.77, p<0.05), but

not time (F=1.16, p>0.05) or a significant interaction between lighting condition x time (F= 1.07,

p>0.05; Fig. 25E,F). Although I detected a main effect of lighting condition, individual

comparisons within animals under a standard L:D cycle or DD as a function of time did not

support significant light-entrained or free-running rhythms of CLOCK expression within the ZI

(Fig. 25F). Zona incerta neurons express TH-IR but do not play a role in the neuroendocrine

regulation of PRL secretion. Given the complex nature of the ZI’s precise role in

neurophysiology, it is difficult to define the functional significance of the diurnal rhythm of

clock gene expression within this region.

Light-entrained and free-running rhythms of PER1, PER2 and CLOCK immunoreactivity

in the SCN core and SCN shell

As described above, PER1, PER2 and CLOCK -IR nuclei were clearly labeled within

SCN shell and core neurons (Fig. 26A,C,E). In contrast with areas containing NDNs, I did not

determine the phenotype of SCN neurons containing PER1, PER2 or CLOCK protein.

Designation of SCN core and shell was made by rough anatomical boundaries, in the absence of

double labeling for VIP or AVP. On average, the number of CLOCK-IR neurons was nearly

twice as high as the number of PER1 or PER2 –IR nuclei within both the SCN core and shell

(Fig. 26A,C,E). In general, the number of PER-IR nuclei was greater within SCN shell than SCN

core, while the number of CLOCK-IR nuclei was generally more broadly dispersed across the

nucleus (Fig. 26A,C,E). Analysis of PER1-IR within the SCN core and shell as a function of

lighting condition (L:D vs. DD) and circadian time (CT) revealed a robust main effect of time

(F=108.2, p<0.001), lighting condition (F=116.5, p<0.001) and the interaction between lighting

condition x time (F=45.60, p<0.001; Fig. 26A,B). Pairwise comparisons within SCN shell under

a standard 12:12 L:D cycle as a function of time support a significant diurnal rhythm of PER1

expression with significant peaks at both CT6 (p<0.001) and CT12 (p<0.001) when compared

with basal levels at CT0 (Fig. 26B). Comparisons show that the number of PER1-IR nuclei at

CT6 is significantly greater in SCN shell than SCN core (p<0.05; Fig. 26B). Comparisons avow

an identical rhythm within SCN core, with significant peaks at CT6 (p<0.001) and CT12

(p<0.001) when compared with a nadir at CT0 (Fig. 26B). Comparisons within SCN shell and

core under DD as a function of time failed to reveal a significant free-running rhythm of PER1

92

Figure 26. PER1, PER2 and CLOCK expression in the SCN under a standard

12:12 L:D cycle and DD. I observed strong nuclear (A) PER1, (C) PER2 and (E)

CLOCK expression within the SCN shell and SCN core. (B) PER1 expression

peaked at CT6 and CT12 in both SCN-S and SCN-C in animals under a standard

L:D cycle, but not DD. (D) PER2 expression peaked at CT18 in animals under L:D

conditions, but also failed to display a free-running rhythm of expression. (F)

Although the number of CLOCK-IR nuclei was significantly greater within SCN-S

and SCN-C in DD animals at CT6, I did not detect an overall free-running or light

entrained rhythm of CLOCK expression within SCN core or SCN shell. In B,D and

F, differing letters indicate significance across lighting condition and region within

time and # indicates a significant acrophase within lighting condition and region.

Scale bar in A = 200 µm

93

expression. Paired comparisons within circadian time as a function of lighting condition show

that peaks of PER1 expression under a L:D cycle at CT6 within SCN shell (p<0.001) and core

(p<0.001) and CT12 within SCN shell (p<0.001) and core (p<0.001) are significantly greater

than similar values in DD (Fig. 26B). Although my data for PER1-IR within SCN shell/core

agree with data from several previous experiments (210,289), I were unable to confirm prior

results with respect to a free-running rhythm of PER1 expression (210). While disappointing,

these data may reflect high phase variability among my animals after 5 days under DD

conditions. A significant decrease in the absolute number of PER1-IR nuclei in DD animals

supports a dampening of this rhythm due to individual differences between animals. However, a

low level of variance within regions and time fails to support this argument.

Analysis of PER2-IR within the SCN core and shell as a function of lighting condition

(L:D vs. DD) and circadian time (CT) revealed a main effect of lighting condition (F=6.01,

p<0.01), but not time (F=2.50, p>0.05) or the interaction between lighting condition x time

(F=1.05, p>0.05; Fig. 26C,D). Pairwise comparisons exposed a significant diurnal rhythm of

PER2 expression within the SCN shell characterized by a significant peak at CT18 (p<0.05),

when compared with basal levels at CT0. Comparisons did not show a similar rhythm within the

SCN core (Fig. 26D). Further, comparisons did not reveal a significant free-running rhythm of

PER2 expression in either the SCN shell or core (Fig. 26D). Several experiments have shown a

circadian rhythm of PER2 expression within the SCN defined by a significant peak between

CT12 and CT18 (204,206,209,210). My data agree with previous reports regarding the light-

entrained rhythm of PER2 expression. However, I failed to observe a significant free-running

rhythm of PER2 expression within the SCN (Fig. 26D). As with PER1 expression, my inability

to observe a free-running rhythm of PER2 expression within the SCN may stem from variability

in phase induced by individual differences in τ across animals. However, as with PER1

expression, the level of error I observed within region and circadian time does not appear to

support the existance of a dampened rhythm resulting from between animal phase variation.

Regardless, I was able to observe significant light-entrained circadian rhythms of PER1 and

PER2 expression within the SCN that agree with published literature (210,289).

Analysis of CLOCK-IR within the SCN core and shell as a function of lighting condition

(L:D vs. DD) and circadian time (CT) revealed a robust main effect of lighting condition

(F=4.61, p<0.01) but not time (F= 1.26, p>0.05) or an interaction between lighting condition x

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time (F= 0.81, p>0.05; Fig. 26E,F). Paired comparisons failed to exhibit a significant light-

entrained or free-running circadian rhythm of CLOCK expression within SCN shell and core

(Fig. 26F). However, comparisons as a function of region and lighting condition within

circadian time revealed a significant greater level of CLOCK-IR within SCN core (p<0.05) and

shell (p<0.05) under DD, compared with levels from the same regions of animals housed under a

standard L:D cycle (Fig. 26F). Although this represents a significant increase in CLOCK-IR

nuclei in animals housed in DD, it does not represent a significant peak in CLOCK expression

across the subjective day (Fig. 26F). Therefore, I found that CLOCK expression within the SCN

was constitutively expressed, in agreement with numerous previous studies indicating acyclic

CLOCK expression within the SCN.


The purpose of these experiments was two-fold: (1) to determine the pattern of PER1,

PER2 and CLOCK expression within NDNs and (2) to ascertain the potential relationship

between light-entrained and free-running rhythms of clock gene expression within NDNs and

circadian rhythms of DA turnover within TIDA, THDA and PHDA neurons. Previously, I

reported significant circadian rhythms of DA turnover in the OVX rat (see Chapter 1). I

hypothesized that these circadian and semi-circadian rhythms of DA release, which dictate the

timing of the ovarian steroid induced PRL surge on the afternoon of proestrus, are facilitated by

autonomous rhythms of clock gene expression within the NDN. As in previous experiments (see

Chapter 1,2), I have monitored fluid intake to verify the adequate and consistent function of the

central circadian oscillator. In nocturnal rodents, drinking behavior displays a distinct free-

running and light entrained circadian rhythms (66,81,82). Prior to intracardiac perfusion, OVX

rats were placed in our lickometer device in order to measure drinking activity under both a

standard L:D cycle and constant darkness. As in previous experiments, animals displayed clear

light-entrained and free-running rhythms of drinking behavior characterized by a free-running

τ~24.2+2h. CT 12 was determined for each animal, regardless of lighting condition and utilized

as a reference point for perfusion and tissue collection. These data agree with similar analysis in

published reports from my laboratory (see Chapter 1, 2).

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PER1-IR nuclei are clearly labeled within TH-IR neurons in the DMARN, RARN and

PeVN. Generally, PER1-IR was limited to the nucleus and is readily distinguishable from TH-

IR cytoplasmic staining. Only those neurons expressing a clear PER1-IR nucleus were

considered PER1-IR cells. PER-IR nuclei are distinguished by condensed labeling and often

absent staining within the nucleolus. While only TH-IR and PER/TH-double labeled cells were

counted in the current experiment, I observed numerous PER1-IR nuclei within the analyzed

regions that were not TH-IR. Under a standard L:D cycle, PER1-IR within TIDA neurons

displayed a light-entrained diurnal rhythm with a peak late in the subjective day. In addition,

PER2 expression displayed a light-entrained rhythm of expression with significant peaks at CT6

and CT12. I have previously reported a light-entrained, diurnal rhythm of DA turnover within

TIDA neurons with a biphasic pattern, defined by peaks at CT6 and CT12 (Chapter 1). PER1-IR

peaked within TIDA neurons approximately 12 hours before (or after) the peak of DA turnover.

In addition, PER2 expression peaked in parallel with the time of peak DA turnover within these

neurons. Surprisingly, I failed to detect a free-running circadian rhythm of PER1 or PER2

expression within TIDA neurons. These data suggest that period gene expression does not play a

distinct role in driving the free-running rhythm of DA turnover in the ME (see Chapter 1). Like

the SCN, TIDA neurons express CLOCK-IR in a constitutive manner under both L:D and DD

conditions. Based on these data, I can assume that TIDA neurons act as dampened or slave

oscillators, unable to maintain endogenous free-running rhythms of gene expression as reported

in the SCN. Recent literature agree with this conclusion, suggesting that neurons within the

ARN are unable to express free-running rhythms of PER expression in isolated cell culture

(263,264).

Unlike TIDA neurons, THDA neurons failed to exhibit a light-entrained rhythm of PER1

expression, but did display a diurnal rhythm of PER2 expression with a significant peak at CT6.

Like TIDA neurons, THDA exhibit a light-entrained, diurnal rhythm of DA turnover with a

biphasic pattern, defined by peaks at CT6 and CT12 (Chapter 1, Figs. 6,7). Therefore, the

rhythm of PER2 expression, but not PER1 expression, within THDA neurons corresponds to the

rhythm of DA turnover observed for these neurons under a standard L:D cycle. Although

significant, it is difficult to identify a mechanism whereby PER2 expression drives cellular

activity in the absence of PER1 expression. Regardless, my data suggest a potential mechanism

for PER2 expression within THDA neurons. Like TIDA neurons, THDA neurons express

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CLOCK-IR in a constitutive manner under both L:D and DD conditions. In contrast with TIDA

neurons, THDA neurons do not display free-running rhythms of DA turnover (see Chapter 1).

Thus, the absence of a free-running rhythm of PER1 or PER2 expression within THDA neurons

is considerably less surprising.

In PHDA neurons, PER1 expression displayed a light-entrained rhythm with a significant

peak at CT12. Further, PER2 expression peaks in THDA neurons at CT6. I have shown that DA

turnover within the IL peaks between CT6 and CT10, followed by a trough at CT18 (Chapter 1,

Fig. 6). In agreement with data from both TIDA and THDA neurons, PER1 expression patterns

within PHDA neurons correspond to the timing of DA turnover within the IL. As with TIDA

and THDA neurons, I also failed to observe a free-running rhythm of PER1 or PER2 expression

within PHDA neurons. Like both TIDA and THDA neurons, PHDA neurons express CLOCK in

a constitutive manner. Therefore, arrhythmic period gene expression under DD conditions

indicates that, like TIDA and THDA neurons, PHDA neurons are not free-running, autonomous

circadian oscillators.

The SCN can be divided along its rostrocaudal extent into a dorsomedial shell and a

ventrolateral core. In agreement with the literature, I observed a light-entrained diurnal rhythm

of PER1 and PER2 expression within the SCN shell and core (210). I did not detect a free-

running rhythm of PER1 or PER2 expression within the SCN in either region (210,289,295). In

contrast with previous studies (210,289), I observed a slightly advanced peak of PER1

expression at CT6, but not CT12. Further, I observed a peak of PER2 expression at CT18,

instead of CT12-CT14 as previously reported (210). As with previous experiments, I failed to

detect a significant light-entrained or free-running rhythm of CLOCK expression within the SCN

(for review see (204,206,207,293,296)). SCN expression of CLOCK-IR nuclei remained

constitutive throughout the subjective day. In previous reports, investigators allowed animals to

remain in constant conditions for only one 24-hour cycle prior to perfusion and tissue collection.

In the current experiment, I allowed animals to remain in DD conditions for 5 days. In general,

animals displayed a free-running τ~24h. Further, animals were perfused according to circadian

time, which should eliminate potential phase variability as a factor for within sampling time

variability. Therefore, it is very difficult to understand why I was unable to detect a free-running

rhythm of PER1 or PER2 expression within the SCN of our female rats. However, to our

knowledge, I am the first to examine the rhythmic expression of PER proteins within the SCN of

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the female rat. Moreover, I am certainly the first to examine the rhythmic expression of these

proteins in the OVX adult. Several experiments suggest that ovarian steroid receptors are

expressed in SCN neurons and that steroids exert distinct effects on gene expression and activity

within the SCN (111-113,265,266,297). Therefore, it is difficult to predict what effect OVX

would have on clock gene expression with the SCN. I can assume that light-entrained rhythms

of PER expression, which are highly dependant on neural input from the retina, are likely less

dependant on the influence of ovarian steroids.

PER1, PER2 and CLOCK protein expression was analyzed within zona incerta DA

neurons as a positive control for TH expression and a negative control for DA neurons within the

hypothalamus not involved in the regulation of PRL secretion (298-301). Surprisingly, I

detected a significant diurnal rhythm of PER2 expression within the ZI, but failed to detect

significant light-entrained rhythms of PER1 or CLOCK. Moreover, I failed to observe a

significant free-running rhythm of PER1, PER2 or CLOCK expression within the ZI. The ZI is

an incredibly diverse and mysterious structure (298). To date, the ZI has been linked with nearly

every region of the neuroaxis from the spinal column to the frontal lobes. Moreover, the ZI has

been shown to express over 20 different neurotransmitters, including DA. The ZI has been

implicated in the regulation of arousal, attention, visceral activity, posture and locomotor activity

(298). The so-called “zone of uncertainty” remains an elusive neural structure, without a clear

function. Interestingly, the ZI projects to the hypothalamus and the posterior pituitary gland

(298). Albeit novel, I cannot postulate a significant role for PER2 expression within the ZI.

As I have mentioned, I was unable to detect a light-entrained diurnal rhythm of PER1

expression within THDA neurons. The absence of a free-running PER1 or PER2 expression

rhythm within TIDA and PHDA neurons was surprising, considering the distinct free-running

rhythm of DA turnover within these neurons. However, the absence of a light-entrained rhythm

of PER1 or PER2 expression within THDA neurons agrees with the lack of a free-running

rhythm of DA turnover in the NL that I have previously reported. Recently, Kriegsfeld and

colleagues (277) reported a diurnal rhythm of PER1 expression in the female mouse, using the

same primary antiserum, with a significantly greater number of PER1/TH double labeled cells at

CT10 than CT22. These data suggest that PER1 expression peaks in the latter portion of the

subjective day and reaches a nadir at or near the middle of the subjective night (CT18-22;(277)).

Further, experiments conducted by Bae and colleagues (210) revealed that PER1-IR peaked

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within the SCN at or near CT12 (210,295,302). Thus, these data would suggest that PER1-IR

within NDNs occurs approximately 12 hours after the peak of PER-IR within the SCN.

Experiments indicate that PER1 expression in peripheral tissues peaks approximately 6-12 hours

after PER1/2 expression within the SCN. My data show that PER1-IR in NDNs peaks at CT18,

approximately 6-12 hours after the peak of PER1 expression myself and others have observed

within the SCN (210,295,302). Given the absence of a free-running rhythm of PER1 expression

within NDNs, several hypotheses could be offered to explain the function of the light-entrained

rhythm I have observed. I have determined in previous experiments that NDNs express VPAC2

receptors that are affected by the level of circulating ovarian steroid hormones (172). Further, I

have shown that disruption of VIP peptide expression within the SCN affects the activity of

NDNs under a standard 12:12 L:D cycle (188). Numerous studies have shown that VIP peptide

displays a light-entrained rhythm of expression within the SCN characterized by a significant

increase in VIP expression in the late subjective night between CT18 and CT22 (176,178,303-

309). Further, several of these studies suggest that VIP mRNA and protein does not display a

free-running rhythm of expression. I can conclude from these studies that VIP release from SCN

neurons entrains the activity of NDNs in the late subjective night. Moreover, additional evidence

suggests VIP induces PER1 and PER2 expression in the SCN during the late subjective night

(310). Therefore, I can assume that VIP, released from SCN efferents within the DMARN,

RARN and PeVN, binds to VIP type-2 receptors and activates PER1 and PER2 expression

through increased intracellular cAMP and CREB mediated signaling (311). Although lacking in

evidence, I cannot rule out a role for arginine vasopressin of SCN origin in my model. Further

experiments are necessary to delineate the precise role of both VIP and AVP in the activation

and maintenance of clock gene expression within NDNs. I can conclude, therefore, from these

studies and others that NDNs are dampened or slave oscillators, expressing clock genes in a L:D

cycle under the direct influence of VIPergic input from the SCN. These inputs, along with the

SCN oscillators they originate from, are directly responsive to fluctuating ovarian steroid

hormone levels and therefore highly receptive to the physiological status of the animal

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CHAPTER 4

EFFECTS OF ACUTE PER1, PER2 AND CLOCK GENE KNOCKDOWN IN THE

SUPRACHIASMATIC NUCLEUS ON THE CIRCADIAN RHYTHMS OF DA

TURNOVER IN NEUROENDOCRINE DOPAMINERGIC NEURONS

Introduction

Circadian rhythms are an evolutionary advantage dictated by the earth’s axial rotation.

The ability of an organism to entrain its behaviors to the transition between night and day

provides a distinct advantage. The biological clock in mammals is located in the

suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Photic cues are detected by the

retina, most likely following activation of a novel retinal ganglion cell (RGC)-specific opsin

(312,313). These specialized RGCs send glutamatergic efferents to the ventral portion (core) of

the SCN (312,314). The SCN contains several thousand individual autonomous oscillators

shown to express circadian rhythms of gene expression and electrical activity that are robustly

entrained by light and neurotransmitters derived from retinal afferent input (74,204,315-317).

As previously discussed, a growing body of literature suggests that the molecular substrate for

these sustained endogenous rhythms is a tightly regulated transcriptional/translational negative

feedback loop of clock gene transcription factors (see Fig. 3 and introduction for detail).

Mutations affecting these genes have dramatic and varied effects on the activity of the animal

(208,210,295,318).

Neural targets of the SCN, the primary circadian oscillator, express the putative clock

genes with a circadian rhythm (245,263,264,278,279). These tissues appear to function as

“slave” oscillators that are both entrained by photic cues transduced by the SCN and actively

stimulated to maintain rhythmicity via SCN efferents (263). Therefore, in the absence of SCN

input, these tissues cannot sustain a circadian rhythm for more than 3-5 days. These areas

include the arcuate nucleus (ARN), paraventricular nucleus (PVN), pineal and pituitary glands

(263). A notable exception to this pattern is the olfactory bulb, which appears to maintain free-

running oscillations of PER1 expression in SCN lesioned rats (279, 378). Recent studies suggest

that clock gene expression in rat NIH 3T3 fibroblasts may also express sustained, free-running

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circadian rhythms of rev-erbα, PER2 and BMAL1 expression. Novel clock genes, such as the

CLOCK binding non-SCN neuron-specific per-arnt-sim (PAS) domain containing protein

NPAS2, have been implicated in diverse physiological functions including neuronal cellular

metabolism, sleep-wake cycle regulation and mood disorders (319-323). Recent reports have

shown that the neuroendocrine DAergic neurons within the ARN express PER1 protein with a

diurnal rhythm (154,277). As I have revealed in Chapter 3, the rhythm of PER1 and PER2

expression in NDN corresponds to the light-entrained rhythm of DA turnover within all three

populations of NDNs. However, PER1 and PER2 did not display rhythmic expression within

any of the NDNs in constant conditions (see Chapter 3). Therefore, I hypothesize that short

duration knockdown of clock gene expression may have a significant effect on the timing and/or

amplitude of the light-entrained rhythms of NDN activity. I have determined the effects of short-

duration knockdown of per1, per2 and clock mRNA on the light-entrained and free-running

rhythms of NDN activity, serum PRL and serum CORT secretion in the OVX rat.

Materials and Methods

Animals

As outlined in Chapter 1, all experiments used adult female Sprague-Dawley rats (> 60

days of age) weighing 250-300g (Charles River Labs inc., Wilmington, MA) that were housed

under varying lighting conditions in constant temperature (25C) and humidity with standard rat

chow and water available ad libitum. The room was illuminated with four 40 W fluorescent

bulbs, producing a minimum illumination of 100 lux at cage level. For animals housed under

DD all maintenance was performed in dim red light (< 1 lux) or with the aid of infrared goggles

(Unitec Inc, Night Vision Optics, Huntington Beach, CA). Under both L:D and DD conditions

maintenance was performed every third day between 0900h and 1400h (the first half of the 12-

hour light phase) to avoid potential entrainment to non-photic stimuli by disrupting the animals

during the inactive period (232). Animals housed under DD conditions were sacrificed in dim

red light (<1 lux). All experimental protocols were approved by the Florida State University

Animal Care and Use Committee (ACUC).

Bilateral Ovariectomy and Analysis of Drinking Rhythm

Animals were anesthetized with Halothane and OVX bilaterally. All animals were

placed under a standard L:D cycle (lights on 0600h-1800h) for 5 days for habituation to the

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home cage. In the rat, measurement of feeding and drinking patterns are established methods for

determining circadian time, a subjective measure based on the activity of the animal,

independent of the L:D cycle (63,64,234). In constant conditions (DD or LL) the rhythm of

drinking activity free-runs with a period of approximately 24.5 hours (63,235,236). Drinking

was measured over the 24-hour day with an automated device (Dilog Instruments, Tallahassee,

FL.) counting individual licks in 30-second bins over 24 hrs and Circadian Time 12 (CT12; onset

of subjective activity period) was calculated as previously described (see Chapter1, 2). CT12

was used as a reference for tissue collection regardless of lighting condition. Double plotted

actograms of drinking activity (12-hour moving average of drinking activity) were produced with

Circadia software (ver. 2.1.16; Behavioral Cybernetics, Inc., Tallahassee, FL.)

Stereotaxic implantation of Bilateral SCN Cannulae and Intra-SCN Injection of

Deoxyoligonucleotides (ODN)

Five days after OVX, rats were anesthetized (100µ l/ 100 g weight) with ketamine

(49mg/ml)/ xylezine (1.8mg/ml) cocktail and implanted stereotaxically with bilateral stainless

steel guide tubes (1.5mm apart; 9.5mm in length; 27 gauge) whose tips were placed at the dorsal

border of the SCN (0.8 mm posterior to bregma; 7.9 mm ventral to the dorsal surface of the dura

mater. Bilateral solid steel mandrils (33 gauge, 10.5 mm length, 1 mm extension) were inserted

into the guide tubes, animals were allowed to recover on a heated pad and then returned to their

home-cage for 2-3 days. After thorough recovery animals were transferred, during the light

portion of the L:D cycle between 1000h and 1400h, to the lickometer device and allowed to

habituate for a minimum of 24h. Eight adult female OVX rats of the Sprague-Dawley strain

were placed in the Lickometer device in a 12:12 L:D cycle with lights on at 0600h for 5 days. On

day 6, animals either remained in a 12:12 L:D cycle, or were placed in constant darkness (DD)

for five days. On the fifth day under their respective lighting condition animals were injected

intra-SCN with clock gene antisense cocktail. Briefly, animals were anesthetized with halothane

and the bilateral solid steel mandrils were removed. Bilateral internal cannulae (33 gauge, 10.5

mm length, 1 mm extension) were inserted into the guide tubes and 800 nl of AS-ODN or RS-

ODN were injected at 200 nl/min with two 1µl Hamilton syringes attached to a automated

microinfusion pump (Kd scientific, Fisher Scientific, Fair Lawn, NJ). Antisense ODN were

generated against the 5’ transcription start site (5’INI) and 3’ cap site of per1, per2 and clock

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Table 1. Antisense and random sequence deoxyoligonucleotide sequences

Antisense Sequence

Per1 - 5’INI CCT*TCTAGGGGACCACT*CAT

Per1 – 3’CAP GGT*GCTGTTTTCTTCTG*CAG

Per2 – 5’INI TAT*CCATTCATGTCGGG*CTC

Per2 – 3’CAP GAC*ACAAGCAGTCAAC*AAA

Clock – 5’INI CAG*CTTTACGGTAAACAA*CAT

Clock – 3’CAP AAG*GGTCAGTCAGGCT*GTC

Random Sequence

Per1 – RS GCT*CTGGTCTAGTACC*CTA

Per2 – RS ATC*TGCTACTAGGTTC*GTC

Clock – RS ACC*GTACTACTTCGGCT*GTC

* Indicates phosphotioate linkage within the oligonucleotide sequence.

mRNA. Sequences for mper1 (Genebank accession number: NM 011065) and mper2 (Genebank

accession number: NM 011066) AS-ODN were slightly modified from those used by Akiyama

and colleagues (324,325) in order to slightly increase the GC content of the oligonucleotide.

Clock AS-ODNs were developed independently in my laboratory using the known sequence of

clock mRNA (Genebank accession number: NM 007715). Per1, per2 and clock AS-ODN and

RS-ODN sequences are listed in Table 1. According to the protocol developed by Akiyama et al,

I verified both time and dose dependent effects of AS-ODN on PER1, PER2 and CLOCK

expression in the SCN with immunoblots for each clock protein (Fig. 26).

Preliminary experiments verified a significant knockdown of PER1, PER2 and CLOCK

expression in the SCN of more than 60% within 6h of injection at a dose of 3 nmoles (2.5 µg/µl,

800 nl injection volume; see representative clock staining in Fig. 26). Per1, per2 and clock

mRNA levels recovered to control values by 12h post-injection and remained at normal levels at

36h and 48h after infusion of AS-ODN. Therefore, in order to verify the acute effects of clock

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gene mRNA knockdown all experimental animals were injected 6h before sacrifice. Control

animals were injected with random-sequence ODNs with the same nucleotide content (%AGCT)

as the AS-ODN but are not complementary to clock gene mRNA sequences (verified with

Primer 3.1, MIT; RS-ODN sequences listed in table 1). Additional controls included animals in

which AS injections failed to reduce PER1, PER2 or CLOCK and are assumed to be the result of

misplaced guide tubes (missed injection, MI). Additional animals received sham injections and

were pooled with MI-ODN. Immunoblotting for PER1, PER2 and CLOCK was used to quantify

the effects of AS-ODN and RS–ODN in the SCN. Optical density for each gene was normalized

to β-actin loading control for each sample and densitometry was carried out on a Bio-rad gel

documentation system (Bio-rad, Hercules, CA). Optical density (ODu/mm2) of PER1, PER2,

CLOCK and actin loading controls were determined and used to calculate relative abundance of

each protein. Data represent the average relative protein abundance of all AS-ODN, RS-ODN

and MI-ODN animals used for DA turnover analysis.

Tissue Preparation and Serum Collection

Six hours after AS or RS –ODN injection animals were briefly sedated by inducing

hypercapnia (50% CO2: O2) and then rapidly decapitated. Trunk blood was collected. Serum

samples were frozen at –20C until assayed for PRL and corticosterone (CORT) concentrations

by RIA. The brain and pituitary gland were quickly removed, placed on ice, and the median

eminence, as well as neural and intermediate lobes of the pituitary gland were carefully

dissected, placed in homogenization buffer (0.2 N perchlorate with 50 µM EGTA) and rapidly

(~30 sec.) frozen in an ArticIce tube transport block (USA Scientific Inc., Ocala FL.). Tissue

samples were stored at -80C until assayed for DA and DOPAC. On the day of analysis for

catecholamines, tissue samples were thawed and processed for HPLC-EC analysis as previously

described (see Chapter 1, 2).

Measurement of Dopamine (DA) and Dihydroxyphenylacetate (DOPAC) by High

Performance Liquid Chromatography with Electrochemical Detection (HPLC-EC)

The HPLC-EC technique has been well established in my laboratory (114). The

concentrations of DA and DOPAC, a primary metabolite of DA, were measured in tissue extracts

from the pituitary gland and mediobasal hypothalamus as previously described (Chapters 1, 2).

The amount of catecholamine in each sample was estimated by direct comparison to the area

under each peak for known amounts of catecholamine. The amount of 3,4-

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dihydroxybenzylamine (DHBA, RT = 6.5 min) recovered was compared to the amount of DHBA

added as internal standard and corrected for sample loss (usually < 5%). The assay detects 30 pg

of DA and 15 pg of DOPAC. DA turnover is defined as the exocytotic release of DA from

neuroendocrine DAergic nerve terminals, DA re-uptake, and the degradation of DA to DOPAC

by monoamine oxidase (MAO) in the presynaptic terminal (242).

Western blotting, Immunodetection and Densitometry of clock gene products in

neuroendocrine tissues following AS-ODN, RS-ODN and MI-ODN -treatment

As described in Chapter 3, tissue extracts containing the SCN, ARN and piriform cortex

were analyzed by Western blot for PER1, PER2 and CLOCK protein. Densitometry was carried

out on a Bio-rad gel documentation system. Optical density (ODu/mm2) of PER1, PER2,

CLOCK and actin loading controls were determined and used to calculate relative protein

abundance ratios. An inclusion threshold of approximately 50-60% reduction in PER1, PER2

and CLOCK proteins was used when considering the success of AS-ODN injections(188).

Protein Assay

The amount of protein in samples for HPLC-EC and Western blot analysis were

measured using a micro-modified form of the Pierce Bichonchoninic Acid (BCA) Protein Assay

Kit (Pierce, Rockford, IL) as previously described (Chapter 1, 2). Assay sensitivity was 1 µg

protein and the intra-assay coefficient of variation was 5-10%.

Radioimmunoassay

The concentration of PRL in serum was determined by radioimmunoassay (RIA)

using NIDDK materials supplied through the National Pituitary Hormone Distribution Program

(A.F. Parlow) and Protein-A as previously described (31). Serum corticosterone (CORT)

concentration was determined using the commercially available Coat-a-Count®

rat corticosterone

RIA kit (Diagnostic Products Corp., Los Angeles, CA) according to the manufacturer’s

specifications.

Experimental Design

Animals under 12:12 L:D or constant dark (DD) conditions were injected with antisense

ODN (AS-ODN; MI-ODN) or random sequence ODN (RS-ODN) and samples obtained at CT

0,6,9,12,15 and 18 for measurement of DA turnover in the ME, NL, IL, serum PRL and serum

CORT concentrations. Four adult female Sprague-Dawley rats per timepoint were OVX and

housed individually in cages attached to the automated drinking device under L:D conditions for

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5 days. On day 6, animals remained under L:D conditions for 5 more days, or were placed in

DD for five days. On the fifth day under their respective lighting condition animals were

injected 6 hours before sacrifice with AS-ODN, RS-ODN or sham AS-ODN. Six hours after

injection animals were sacrificed and tissue was collected for HPLC-EC and Western blot

analysis as described in methods. In addition, serum was collected to determine serum PRL and

CORT by RIA. Animals that received AS-ODN injections but showed no reduction in PER1,

PER2 or CLOCK protein in the SCN or animals given sham injections were pooled, designated

as misses and considered controls (missed injection of ODN; MI-ODN).

Data Analysis

Serum PRL, serum CORT and DA turnover are expressed as mean (ng/ml, ng/ml and

DOPAC:DA ratio, respectively) + SEM of 4 animals, presented as a function of circadian time

and double plotted to emphasize rhythms (see above). Although they exhibit a distinct rhythm,

all of my data do not conform to a sine/cosine wave function, which prohibits a non-linear

regression analysis to present the data as a function of time and lighting condition. Moreover, as

samples were obtained by decapitation at individual time points over a 24 h period (CT0, 6, 9,

12, 15, 18), it is difficult to extrapolate accurate phase and period measures. While it is

preferable when performing circadian studies to collect serial samples of individual animals,

analyses of recovered tissue preclude such an approach in my experiments. To facilitate direct

comparisons, all data points regardless of lighting condition were aligned by circadian time.

Data were analyzed with two-way ANOVA for (A) time of day effects, ODN effects and the

interaction between time and ODN treatment, followed by Bonferroni paired post-hoc statistical

tests. Relative protein abundance in the SCN and PC from AS-ODN, RS-ODN and MI-ODN

injected animals were compared with two-way ANOVA and Bonferroni post-hoc tests..

Significant differences were considered at P<0.05. ANOVA were performed and graphs were

created with Graph-pad Prism software (Graphpad Software Inc., San Diego, CA.)

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Results

Acute knockdown of per1, per2 and clock mRNA expression disrupts circadian rhythms of

drinking behavior

To verify a functional circadian clock and to determine a reference point for AS-ODN

injection and tissue collection, animals were placed in our lickometer device following

stereotaxic surgery. The beginning of the 12-hour activity period, identified as CT12, was

determined on the two days prior to tissue collection for each animal and averaged to predict the

onset of activity on the following day. CT12 was predicted under entrained and free-running

conditions with an assumed error of 10-15 minutes, given a variance in activity onset among my

rats (generally 10-15 minutes from cycle to cycle), which I consider acceptable with a sampling

frequency of 2-4 hours. In L:D rats (Fig. 27A), CT12 was approximately 1730+0.2h. Five days

after the transition to DD, CT12 was delayed approximately 2 hours to 1930+ 0.2h, resulting in

an approximate free-running period (τ) of 24.4 hours (Fig. 27B). In order to determine the

effects of AS-ODN on the rhythm of drinking activity, several animals were placed in a 12:12

L:D or DD for a single day, followed by injection of AS-ODN or RS-ODN on the second day

under either condition. Animals were allowed to remain in the lickometer device for several

days following AS-ODN or RS-ODN injection. AS-ODN appeared to eliminate the free-running

rhythm of drinking activity when compared with a MI-ODN animal, which recovered after

approximately 72h (Fig. 27B). Drinking behavior dropped to a minimum during this period,

marked by an average number of water bottle licks below 10 licks/10 minute bin. These data

suggest that AS-ODN cocktail transiently disrupted the function of the central molecular

oscillator within the SCN.

AS-ODN cocktail against per1, per2 and clock mRNA reduces PER1, PER2 and CLOCK

protein expression in the SCN

Two-factor analysis of PER1, PER2 and CLOCK expression within SCN tissue extracts

as a function of lighting condition and circadian time avows a significant main effect of lighting

condition (F=17.52, p<0.01) and treatment (F=57.19, p<0.01), but not a significant interaction

between lighting x treatment (F=3.75, p>0.05). Densitometry of Western blots containing SCN

samples from animals injected with AS-ODN or RS-ODN revealed significant decrease in PER1,

107

Figure 27. Injection of clock gene AS-ODN disrupts circadian rhythms of

drinking behavior. Injection of RS-ODN into the SCN or MI-ODN failed to

disrupt light-entrained or free-running rhythms of drinking activity. Following

AS-ODN injection into the SCN (grey arrowhead) light-entrained and free-running

drinking activity became arrhythmic for up to 72 hours, marked by a constitutively

low amount of drinking. In all figures, black arrowheads indicate approximate

CT12. After the period of arrhythmia, drinking behavior, regardless of lighting

condition, displayed a transient phase advance. For these experiments, animals

were placed in DD for 1 day prior to antisense (AS), random sequence (RS) or

missed injection (MI) –treatment (black circle).

108

Figure 28. Injection of clock gene AS-ODN reduces PER1, PER2 and

CLOCK expression within the SCN. Injection of AS-ODN significantly

reduced (A,C) PER1, (A,D) PER2 and (A,E) CLOCK expression within the

SCN compared to RS-ODN and MI-ODN –treated controls regardless of

lighting condition. (B,F) SCN injection of AS-ODN failed to reduce CLOCK

gene expression within the piriform cortex (PC). Similar results were

observed for PER1 and PER2 (data not shown). In A and B, arrows indicate

non-specific binding of CLOCK primary antibody, while arrowheads indicate

CLOCK specific staining. In C-F, differing letters indicate significant effects

of lighting condition within ODN treatment groups (antisense (AS), random

sequence (RS) and missed injection (MI)) and # indicates significant

effects of ODN-treatment within lighting condition.

109

PER2 and CLOCK protein levels within the SCN following AS-ODN injection (see Fig. 28A).

As shown in Figure 28C-E, SCN tissue samples from AS-ODN injected rats contained

significantly less PER1 (p<0.01; Fig. 28C), PER2 (p<0.01; Fig. 28D) and CLOCK (p<0.01; Fig.

28E) protein than RS-ODN or MI-ODN injected controls. Tissue samples from the piriform

cortex, a brain region known to express PER1, PER2 and CLOCK and located well outside of

the hypothalamus, were collected and analyzed for CLOCK protein expression as a positive

control. Two-factor analysis of CLOCK expression as a function of lighting condition and

treatment failed to reveal a significant effect of treatment (F=6.04, p>0.05), but no effect of

lighting condition (F=2.89, p>0.05) or an interaction between lighting x time (F=0.91, p>0.05).

As shown on figure 28B, I found no significant difference in CLOCK expression within the

piriform cortex between AS-ODN injected and RS-ODN or MI-ODN-treated controls at any

time (p>0.05). However, I did observe a small, but significant, difference between RS-ODN and

MI-ODN –treated controls under a standard 12:12 L:D cycle (p<0.05; Fig. 28B). Similar

observations were found for PER1 and PER2 expression within the piriform cortex (data not

shown). These data support a localized and specific reduction of PER1, PER2 and CLOCK

expression within the SCN following AS-ODN injection

Acute knockdown of PER1, PER2 and CLOCK protein expression in the SCN

differentially affects light-entrained and free-running rhythms of PRL and CORT

secretion

Two-factor analysis of serum PRL levels in AS-ODN, RS-ODN and MI-ODN –treated

animals maintained under a standard L:D cycle as a function of time and treatment failed to

reveal a significant main effect of time (F= 0.84, p>0.05), treatment (F=1.949, p>0.05) or an

interaction between time x treatment (F=0.71, p>0.05). In agreement with previous reports from

my laboratory and others, I did not observe a significant diurnal rhythm of serum PRL in RS-

ODN or MI-ODN injected OVX rats. Moreover, treatment with AS-ODN failed to affect the

timing or magnitude of PRL secretion in RS-ODN or MI-ODN under a standard L:D cycle (Fig.

29A,C). Analysis of serum PRL levels in AS-ODN, RS-ODN and MI-ODN –treated animals

maintained under DD as a function of time and treatment exposed a significant effect of time

(F=3.721, p<0.01), but not treatment (F=0.95, p>0.05) or their interaction (F=0.76, p>0.05).

Unlike previous experiments, I failed to detect a significant free-running rhythm of PRL

110

Figure 29. Clock gene knockdown does not disrupt light-entrained or free-

running rhythms of PRL secretion. Serum PRL did not display a significant

diurnal rhythm in either (A) RS-ODN or (C) MI animals maintained under a 12:12

L:D cycle. However, I did observe a significant rhythm of PRL secretion in (D) MI

animals under DD. Treatment with AS-ODN induced a significant rhythm of PRL

secretion in animals under (B,D) DD conditions, but failed to affect PRL secretion

in animals under L:D conditions. I did not observe a significant difference at any

time between AS, RS or MI –ODN injected animals. Data from RS (dashed line)

and MI (dashed line) animals are double-plotted, while data from AS animals (solid

line) are single plotted on the right. Differing letters within treatment indicate

significant effects of time (P<0.05). * indicate significant peaks above baseline

within treatment regardless of adjacent differences. # indicates significant

differences between ODN treatments.

111

secretion in RS-ODN–treated rats (Fig. 29B, D). However, MI-ODN control rats did display a

significant rhythm of PRL secretion with an acrophase above basal levels at CT24 (p<0.05). AS-

ODN treatment induced a rhythm of PRL secretion in animals under DD characterized by a

significant increase above basal levels between CT18 and CT24 (p<0.01), followed by a trough

throughout the remainder of the subjective day (Fig. 29B,D). Although a significant rhythm, this

effect did not represent a significant difference, within time of day, above either control group

(Fig. 29B,D). In fact, the rhythm induced by AS-ODN under DD appears identical to the rhythm

in MI-ODN controls. Given my inability to detect a significant light-entrained or free-running

rhythm of PRL secretion in the OVX rat, I cannot conclude from these data that AS-ODN

disruption of the molecular oscillator exerted a significant effect on PRL secretion in the OVX

rat.

Twin-factor analysis of serum CORT levels in AS-ODN, RS-ODN and MI-ODN –treated

animals maintained under a standard L:D cycle as a function of time and treatment revealed a

significant effect of time (F=3.04, p<0.05), treatment (F=7.48, p<0.01) but not an interaction

between time x treatment (F=1.12, p>0.1). Individual comparisons within RS-ODN-treated

animals under a 12:12 L:D cycle established a significant rhythm of CORT secretion with a rise

to peak level above baseline at CT24 (p<0.05 when compared with basal levels at CT6 and CT9;

Fig. 30A). Treatment with AS-ODN eliminated this light-entrained rhythm of CORT secretion

(p>0.05 across time within AS-ODN-treated animals under DD). Unlike RS-ODN injected rats,

MI-ODN control animals failed to display a significant CORT rhythm under a standard L:D.

Therefore, treatment with AS-ODN, which results in arrhythmic CORT secretion when

compared with RS-ODN animals, did not show a similar response when compared with MI-

ODN rats. These data suggest that AS-ODN disrupt the light-entrained rhythm of CORT

secretion in the OVX rat. However, this conclusion is weakened somewhat by the lack of a

significant difference, with respect to treatment, between MI-ODN and AS-ODN –treated

animals. Two-factor analysis of serum CORT levels in AS-ODN, RS-ODN and MI-ODN –

treated animals maintained under DD as a function of time and treatment revealed a significant

effect of treatment (F=3.90, p<0.05), but not an effect of time (F=0.59, p>0.05) or an interaction

between time x treatment (F=1.606, p>0.05). Pairwise comparisons show that serum CORT in

RS-ODN and MI-ODN controls failed to display a strong free-running rhythm (Fig. 30B,D).

Treatment with AS-ODN resulted in a substantial change in the shape of the free-running rhythm

112

Figure 30. Clock gene knockdown disrupts the light-entrained, but not free-

running, rhythm of CORT secretion. Serum CORT exhibited a significant diurnal

rhythm in (A) RS-ODN-treated rats with a rise to peak at CT0 that was abolished by

(A) AS-ODN treatment. MI-ODN controls failed to display a significant (C) light-

entrained rhythm of CORT secretion and were therefore not affected by treatment

with AS-ODN. (C,D) Neither RS-ODN nor MI-ODN controls showed a free-running

rhythm of CORT secretion under DD, precluding a significant difference between

arrhytmic AS-ODN-treated rats and controls. Data from RS (dashed line) and MI

(dashed line) animals are double-plotted, while data from AS animals (solid line) are

single plotted on the right. Differing letters within treatment indicate significant

effects of time (P<0.05). * indicate significant peaks above baseline within treatment

regardless of adjacent differences. # indicates significant differences between ODN

treatments.

113

of CORT secretion. However, this change did not produce a statistically significant difference

within time across treatments (Fig. 30B,D). I can conclude that AS-ODN, while able to blunt

light-entrained rhythms of CORT secretion, failed to significantly disrupt CORT secretion under

DD. I cannot conclude that AS-ODN in the SCN failed to disrupt free-running CORT secretion,

as I was unable to detect a free-running rhythm of CORT in either RS-ODN or MI-ODN

controls.

Acute knockdown of PER1, PER2 and CLOCK protein expression in the SCN affects light-

entrained, but not free-running, rhythms of DA turnover in the ME.

Two-way ANOVA of DA turnover within the ME of animals maintained under a 12:12

L:D cycle as a function of time and ODN treatment revealed a significant effect of time

(F=12.97, p<0.001) and treatment (F=20.25, p<0.001), but not the interaction between time x

treatment (F=0.88, p>0.05). Individual comparisons within RS-ODN-treated rats under a

standard L:D cycle revealed a significant biphasic light-entrained rhythm of DA turnover defined

by peaks at CT15 (p<0.01) and CT24 (p<0.05) above basal levels at CT9 and CT18 (Fig. 31A).

Treatment with AS-ODN abolished the peak at CT15 (p<0.05 when compared with RS-ODN

rats), resulting in a U-shaped rhythm with a single peak above baseline at CT24 (p<0.05; Fig.

31A). Pairwise comparisons within MI-ODN control animals maintained under a 12:12 L:D

cycle as a function of time exposed a significant rhythm of DA turnover with an acrophase at

CT24 (p<0.05) that was not significantly affected by treatment with AS-ODN (Fig. 31C). Thus,

AS-ODN modified the magnitude of the light-entrained rhythm of DA turnover in the ME when

compared with RS-ODN, but not MI-ODN controls.

Two-factor analysis of DA turnover within the ME of animals maintained under DD as a

function of time and ODN treatment did not delineate a significant effect of time (F=2.125,

p=0.08), treatment (F=1.39, p>0.05) or an interaction between time x treatment (F=1.24,

p>0.05). In contrast with my previous results, I failed to observe a significant free-running

rhythm of DA turnover in both RS-ODN and MI-ODN controls (no effect of time, p>0.05; Fig.

31B). Comparison between RS-ODN, MI-ODN and AS-ODN-treated animals failed to reveal a

significant effect of AS-ODN treatment at individual times as a function of treatment (Fig.

31B,D). Therefore, I can conclude that AS-ODN injection into the SCN failed to significantly

disrupt DA turnover within the ME in animals in a constant environment.

114

Figure 31. Clock gene knockdown disrupts light-entrained, but not free-running

rhythms of DA turnover in the ME. DA turnover within the ME displayed a

significant light-entrained circadian rhythm in (A) RS-ODN and (C) MI-ODN

controls. (A,C) AS-ODN injection affected the light-entrained rhythm of DA turnover

in RS-ODN, but not MI-ODN controls. (B,D) None of the experimental groups

displayed significant free-running rhythm of DA turnover. (B,D) AS-ODN failed to

disrupt DA turnover rhythms under DD conditions. Data from RS and MI animals are

double-plotted, while data from AS animals are single plotted on the right. Differing

letters within treatment indicate significant effects of time (P<0.05). * indicates

significant peaks within treatment regardless of adjacent differences. # indicates

significant differences between ODN treatments.

115


entrained, but not free-running, rhythms of DA turnover in the NL.

Two-factor analysis of DA turnover within the NL of animals maintained under a

standard L:D cycle as a function of time and ODN treatment did not show a significant effect of

time (F=1.76, p>0.05) and treatment (F=1.86, p>0.05), or a significant interaction between time

x treatment (F=1.11, p>0.05). In contrast with my prior results, pairwise comparisons within RS-

ODN and MI-ODN treated rats maintained in a standard 12:12 L:D cycle failed to reveal

significant light-entrained, diurnal rhythms of DA turnover in the NL (Fig. 32A,C). However,

the shape of the rhythm in RS-ODN treated animals appears very similar to the significant

diurnal rhythm I have previously seen in the OVX rat (Fig. 32A). Moreover, animals treated

with AS-ODN also failed to display a significant diurnal rhythm of DA turnover in the NL (Fig.

32A,C). Thus, AS-ODN injection into the SCN failed to disrupt the timing or magnitude of DA

turnover in the NL. Two-factor analysis of DA turnover within the NL of animals maintained

under DD as a function of time and ODN treatment revealed a significant effect of time (F=2.78,

p<0.05), but not treatment (F=1.06, p>0.05) or the interaction between time x treatment (F=0.91,

p>0.05). Individual comparisons show that neither RS-ODN nor MI-ODN controls displayed

significant free-running rhythms of DA turnover in the NL (Fig. 32B,D). These data are in

agreement with previous data (see Chapter 1) showing that THDA neurons fail to exhibit free-

running rhythms of DA release in DD. AS-ODN resulted in a significant increase in DA

turnover within the NL, characterized by an acrophase at CT9 surrounded by basal levels at CT6

(p<0.05) and CT12 (p<0.01;Fig. 32B). Therefore, AS-ODN-treatment, while failing to affect the

light-entrained rhythm of DA turnover in the NL, was able to induce a significant free-running

rhythm in DA turnover not seen in either control group.


entrained, but not free-running, rhythms of DA turnover in the IL.

Two-way ANOVA of DA turnover within the IL of animals maintained under a 12:12

L:D cycle as a function of time and ODN treatment revealed a significant effect of treatment

(F=3.90, p<0.05) and an interaction between time and treatment (F=4.10, p<0.001), but no main

effect of time (F=2.15, p=0.07). Pairwise comparisons within RS-ODN and MI-ODN-treated

controls under a standard 12:12 L:D revealed a significant diurnal rhythm of DA turnover in the

NL of RS-ODN animals, but not MI-ODN treated rats (Fig. 33A,C). DA turnover within the NL

116

Figure 32. Clock gene knockdown failed to disrupt light-entrained or

free-running rhythms of DA turnover in the NL. DA turnover within the

NL did not display a significant light-entrained circadian rhythm in (A) RS-

ODN, (C) MI-ODN or (A,C) AS-ODN injected rats. DA turnover in the NL

did not exhibit a significant circadian rhythm under DD conditions in either

(B) RS-ODN or (D) MI-ODN animals. (B,D) AS-ODN injection produced a

significant free-running rhythm of DA turnover, defined by a peak at CT9,

followed by a trough at CT12. Differing letters within treatment indicate

significant effects of time (P<0.05). Data from RS and MI animals are double-

plotted, while data from AS animals are single plotted on the right. * indicate

significant peaks above baseline within treatment regardless of adjacent

differences. # indicates significant differences between ODN treatments.

117

Figure 33. Clock gene knockdown disrupts light-entrained, but not free-running,

rhythms of DA turnover in the IL. (A,C) DA turnover within the NL displayed a

light-entrained circadian rhythm in (C) RS-ODN-treated controls but not MS-ODN

animals. (A,C) AS-ODN-treatment induced a significant rhythm of PRL secretion

with a singlur peak at CT0, surrounded by basal levels throughout the remainder of the

subjective day. (B,D) RS-ODN, MI-ODN and AS-ODN failed to display significant

free-running rhythms of DA turnover in the IL. Data from RS and MI animals are

double-plotted, while data from AS animals are single plotted on the right. Differing

letters within treatment indicate significant effects of time (P<0.05). * indicate

significant peaks above baseline within treatment regardless of adjacent differences. #

indicates significant differences between ODN treatments.

118

of RS-ODN controls under a 12:12 L:D cycle exhibits an acrophase at CT24 (p<0.05), compared

with a nadir at CT15. AS-ODN-treatment significantly adjusted the shape of this rhythm by

increasing DA turnover within the IL at CT24 (p<0.001, AS-ODN vs. RS-ODN) and decreasing

DA turnover at CT6 (p<0.05, AS-ODN vs. RS-ODN; Fig. 33A,C). Thus, AS-ODN affected the

magnitude and timing of the light-entrained rhythm of DA turnover within the IL of OVX rats by

advancing the peak of DA turnover from CT6 to CT0. Two-factor analysis of DA turnover

within the IL of animals maintained under DD as a function of time and ODN treatment revealed

a significant effect of treatment (F=4.240, p<0.05), but not time (F=0.48, p>0.05), or the

interaction between time and treatment (F=1.02, p>0.05). Pairwise comparisons show that DA

turnover within the IL of RS-ODN and MI-ODN controls failed to exhibit a free-running

circadian rhythm (Fig. 33B,D). Further, comparisons reveal that AS-ODN did not significantly

affect the magnitude or timing of DA turnover in the IL of animals maintained in constant

conditions. From these data, I can conclude that AS-ODN treatment modulates light-entrained,

but not free-running, rhythms of DA turnover within the IL. These data agree with the results for

DA turnover from the ME presented above. As these two populations displayed both light-

entrained and free-running rhythms of DA turnover in previous experiments, it is not surprising

that they exhibit similar responses with respect to AS-ODN treatment. As observed for the ME,

I cannot exclude a potential effect of AS-ODN on the free-running rhythm of DA turnover in the

IL, but must assume a negative affect based on my inability to detect significant free-running

rhythms of DA turnover in the ME and IL of RS-ODN and MI-ODN-treated controls.


entrained, but not free-running, rhythms of DA concentration in the AL.

Two-factor analysis of DA concentration within the AL of animals maintained under a

standard 12:12 L:D cycle as a function of time and ODN treatment reveled a significant effect of

time (F=2.80, p<0.05) and treatment (F=8.84, p<0.001), but not an interaction between time x

treatment (F=1.18, p>0.05). Comparisons as a function of time within RS-ODN-treated rats

under a standard L:D cycle failed to reveal a significant diurnal rhythm of DA concentration

within the AL (Fig. 34A). AS-ODN injection into the SCN induced a significant diurnal rhythm

of DA concentration in the AL with a significant acrophase between CT6 and CT9 (p<0.05),

119

Figure 34. Clock gene knockdown disrupts light-entrained, but not free-running,

rhythms of DA concentration in the AL. Under both (A) a standard 12:12 L:D cycle

and (B) DD, DA concentration in the AL of RS-ODN treated animals failed to exhibit a

significant circadian rhythm. Treatment with (A,C) AS-ODN induced a circadian rhythm

of DA in the anterior lobe under a 12:12 L:D cycle with a peak between CT6 and CT12.

(D) AS-ODN treatment failed to affect DA levels in the AL under DD. Data from RS and

MI animals are double-plotted, while data from AS animals are single plotted on the right.

Differing letters within treatment indicate significant effects of time (P<0.05). * indicate

significant peaks above baseline within treatment regardless of adjacent differences. #

indicate significant differences between ODN treatments.

120

when compared with a nadir at CT24 (Fig. 34A). The diurnal rhythm of DA concentration in

the AL induced by AS-ODN-treatment represents a significant increase above RS-ODN controls

at both CT6 (p<0.05) and CT9 (p<0.01). MI-ODN-treated animals also displayed a significant

diurnal rhythm, with an acrophase at CT6 (p<0.01) compared with a nadir at CT24 (Fig. 34C).

Two-factor analysis of DA concentration within the AL of animals maintained under DD as a

function of time and treatment revealed a significant effect of treatment (F=4.70, p<0.05), but

not time (F=0.32, p>0.05) or an interaction between time x treatment (F=1.28, p>0.05).

Individual comparisons as a function of time within RS-ODN and MI-ODN-injected rats under

DD failed to indicate a free-running rhythm of DA concentration in the AL (Fig. 34B,D). AS-

ODN-treatment did not affect DA concentration within the AL in animals housed in DD. Thus,

my data support a role for a functional molecular clock in the timing of DA concentration within

the anterior lobe of animals entrained to a 12:12 L:D cycle, but not in animals in a constant

environment.


In the present study I have determined the effects of transient per1, per2 and clock

mRNA knockdown on the light-entrained and free-running rhythms of PRL secretion, CORT

secretion, DA concentration within the anterior lobe of the pituitary gland and DA turnover

within the terminal regions of TIDA, THDA and PHDA neurons. I have attempted to ascertain

the influence of clock gene-controlled activity within the SCN on the rhythm of DA release from

NDNs. Using a cocktail of per1, per2 and clock mRNA AS-ODN, I generated a temporary

“molecular” lesion of the central circadian oscillator. Based on data presented in Chapter 1, I

hypothesized that NDNs may continue to oscillate with a free-running rhythm in the absence of

rhythmic cues from the SCN. These rhythms would in fact be maintained by rhythmic clock

gene expression within the NDN. However, I have determined (in Chapter 3) that NDNs do not

display free-running rhythms of PER1 or PER2 expression. Moreover, results from these

experiments conclude that clock genes play a role in light-entrained, but not free-running,

rhythms of DA turnover within NDN terminal regions. In addition, I have seen similar results

for serum CORT levels in adult OVX rats.

121

Like previous experiments, I failed to detect a light-entrained or free-running rhythm of

PRL secretion in the OVX rat (114,162). My inability to detect significant PRL secretory

rhythms, regardless of lighting condition, precludes my ability to determine the effects of AS-

ODN treatment on PRL secretion. Additional experiments, using both steroid-primed and

normal cycling rats, could provide additional insight into the role of clock genes within the SCN

in the control of PRL secretion. Several experiments suggest a direct effect of hypothalamic or

intrapituitary VIP in the regulation of PRL secretion (326-331). For example, I cannot rule out

the potential effects of clock gene knockdown in the SCN on the release of VIP or other signals

from PVN or SON efferents terminating directly on hypothalamo-hypophyseal portal vessels

within the median eminence (185,332). Serum CORT exhibited a light-entrained diurnal rhythm

in RS-ODN-treated controls that was disrupted by AS-ODN treatment. Data from numerous

experiments suggest that AVPergic afferents of SCN origin terminate on CRH neurons within

the medial parvicellular paraventricular nucleus (191,333-338). Further evidence suggest that

AVP and CRH mRNA are synthesized in PVN neurons with a distinct circadian rhythms (339).

Moreover, data indicate that AVP released from neurons within the PVN and supraoptic nucleus

directly into hypophyseal portal blood supply potentiates CRF-stimulated ACTH secretion

(340,341). However, unlike my previous experiments (see Chapter1, 2), I was unable to detect

free-running rhythms of serum CORT secretion in AS-ODN, RS-ODN or MI-ODN –treated rats.

Several possible effects may explain my inability to detect free-running rhythms of CORT

secretion. Perhaps CORT secretion, which is clearly influenced by stress, reflects the response

of the animal to the stress of halothane anesthesia and/or ODN injection. Further, it is possible

that AVPergic afferents, which travel dorsomedially from the SCN to the PVN, may have been

in some way compressed or damaged by my surgical procedure. Evidence suggests that SCN

afferents inhibit CRH release during the day (342) and damage to these fibers could lead to

increased ACTH and CORT secretion, therefore abolishing the free-running rhythm of CORT

secretion. Regardless, my inability to measure the effects of AS-ODN on the free-running

rhythm of CORT secretion in the OVX obviates any clear interpretation of my data with respect

to circadian control of CORT secretion in a constant environment.

In agreement with my previous experiments, TIDA and PHDA neurons displayed light-

entrained diurnal rhythms of DA turnover with significant peaks in the early subjective day (CT0

in TIDA and PHDA neurons) and early subjective night (CT15 in TIDA neurons). Treatment

122

with AS-ODN against per1/2 and clock significantly adjusted the magnitude of DA turnover at

specific times, leading to a significant change in the shape of the rhythm. Treatment with AS-

ODN eliminated the second peak of DA turnover within TIDA neurons that occurred at CT15

(approximately 2030h) but failed to affect the peak of DA turnover at CT24. AS-ODN treatment

advanced the acrophase of DA turnover in the IL from CT6 to CT24. Moreover, AS-ODN

treatment increased DA turnover at CT15, such that it no longer represents the absolute nadir of

DA turnover, as it does in RS-ODN controls. These data suggest that AS-ODN treatment

influences the magnitude of DA release but fails to completely eliminate the diurnal rhythm.

Nonetheless, this effect correlated with my previous experiments, suggesting that treatment with

AS-ODN against VIP affected the pattern of immediate early gene expression within NDNs.

Experiments with VIP AS-ODN suggest that NDN activity, indicated by fos-related antigen

expression, declines throughout the subjective day and reaches a nadir in the early night, near

1900h. My data support this finding, but repeated experiments indicate a secondary surge of DA

release between CT12 and CT15 (~1730h and 2030h under a standard 12:12 L:D cycle). My

data suggest that clock gene antisense treatment at CT9 eliminates the increase in DA turnover at

CT15. Although the experimental designs for these two experiments are considerably different

(VIP AS-ODN were injected 36h prior to sacrifice), I cannot ignore the parallel between my

data. Of course, an increase in FRAS expression and a decrease in DA turnover do not appear

congruent, my incomplete understanding of the relationship between FRAS expression and DA

turnover in the ME allows us to speculate freely on this potential relationship. Several studies

suggest that AVP expression within neurons of the SCN shell or dorsomedial SCN displays a

free-running endogenous rhythm under the direct control of CLOCK:BMAL1 enhancers, while

VIP expression exhibits a light-entrained, but not free-running, diurnal rhythm (305,343).

Although I cannot rule out the potential influence of AVP from the SCN shell, I can assume that,

if VIP is in the primary neurotransmitter of the SCN-NDN tract, that VIP release is light-induced

and therefore responsible for light-induced rhythms of DA release from NDNs.

I failed to detect significant free-running or light-entrained rhythms of DA turnover

within THDA neurons. These data are surprising, given my previous report showing that THDA

neurons exhibit clear diurnal rhythms with significant peaks near CT6 and CT12. However, I

did not detect a significant free-running rhythm of DA turnover in the NL in previous

experiments (see Chapter 1). Therefore, my inability to detect a free-running rhythm in the

123

current experiment is not unexpected. However, my inability to detect a light-entrained or free-

running rhythm of DA turnover within the NL prevents us from making any concise conclusions

regarding the function of clock gene expression within these neurons.

Within the AL of the pituitary gland, I failed to detect a significant free-running or light-

entrained circadian rhythm of DA concentration within RS-ODN or MI-ODN control animals.

However, AS-ODN injection induced a significant diurnal rhythm with peaks between CT6 and

CT9. Unfortunately this response does not appear to agree with the rhythms of DA turnover

within the ME, IL and NL of AS-ODN treated animals. However, this response is not expected,

given the generally low level of DA release from all three populations in the OVX rat observed

in previous experiments (114). Variation in the rhythmic release of DA from each individual

population, as a result of ovarian steroid hormone withdrawal and the absence of a significant

PRL surge may result in the distinct, albeit dissociated rhythm I have detected here.

From these results, while admittedly inconclusive in some regards, I can make some clear

conclusions. My data show that AS-ODN treatment significantly reduced PER1, PER2 and

CLOCK expression within NDNs and affected light-entrained rhythm of NDN activity and

CORT secretion. Previous experiments in my laboratory suggest that NDNs receive VIPergic

afferent input from the SCN that is affected by varying titers of ovarian steroids. Evidence from

the literature suggest that VIP mRNA and protein synthesis within the SCN exhibits light-

entrained diurnal rhythms but fail to maintain free-running, circadian rhythms in constant

conditions. Therefore, I can assume, based on my data and previous evidence, that the light-

entrained rhythm of DA release in TIDA, THDA and PHDA neurons are dependent on rhythmic

release of VIP from SCN efferents. Disruption of VIP synthesis, either directly via VIP AS-

OSN or indirectly through disruption of clock-controlled transcription, leads to arrhythmic DA

release. Further experiments are needed to strengthen the potential relationship between VIP

synthesis in the SCN and the activity of NDNs.

Discussion

In order to be considered an endogenous circadian rhythm, a cyclic phenomenon such as

DA turnover, PRL secretion or gene expression must possess three attributes: (1) the rhythm

must have a period of approximately 24 hours, (2) it should continue to cycle with a free-running


124

constant light (LL) and (3) it should be entrained to the environmental light:dark cycle

(66,76,94,230). For over 25 years investigators have attempted to better understand the neural

mechanism by which the central biological clock drives physiological rhythms (122). Even now,

we lack a complete understanding of the mechanism by which the central circadian clock drives

rhythms within the neuroendocrine and endocrine systems. Perhaps best understood, the

mechanism driving seasonal reproduction or photoneuroendocrine system remains a veritable

treasure trove of questions. Although our understanding of the mechanism driving endogenous

rhythms of cellular activity has grown with the cloning of the molecular clock (204,206,207),

new evidence continues to question established dogma (278-280,282,344). Without question, I

can rely on numerous experiments showing that ablation of the central biological clock results in

disruption of endocrine rhythms, including corticosterone secretion, PRL secretion under various

states and luteinizing hormone secretion (88,229,262,345-350). Moreover, I must assume that

maintenance of these circadian rhythms requires direct neuronal input from the SCN (Fig. 35).

Evidence from transplant studies suggest that removal of the SCN from the anterior

hypothalamus followed by placement in an ectopic location restores locomotor activity and other

behavioral rhythms, but fails to restore endocrine and neuroendocrine rhythms (166,351-353).

Although compelling, earlier studies on the mechanism of the LH pulse generator suggest that

the mechanism for GnRH pulse generation resides within the medial pre-optic area, as

transplants of the region to the third ventricle of GnRH deficient mutant mice induced surges, in

the relative absence of SCN afferents (for review see(100)). Thus, variation within

neuroendocrine systems with regard to initiation and maintenance of circadian rhythms requires

further study to gain new insight.

My initial experiments revealed that NDNs display light-entrained and free-running

circadian rhythms of DA release in the OVX rat. Under a standard L:D cycle, DA turnover

within the ME, NL and IL peaked early in the subjective day, declined to a nadir at the time of

the expected PRL increase (around 1600h-1800h; ~CT10-12), and returned to peak levels early

125

Figure 35. The photo-neuroendocrine system regulating PRL secretion in the

adult female rat. PRL secretion from the anterior pituitary gland is coordinated by

inhibitory input from NDNs (blue line) and stimulatory input from oxytocinergic

neurons within the paraventricular nucleus (red line, PVN). My experiments

suggest that NDNs and oxytocin neurons are influenced by timing signals

originating in the central circadian oscillator within the SCN and mediated by VIP

(yellow line). Experiments also indicate the influence of midbrain serotonergic (5-

HT) afferents in the regulation of oxytocinergic neurons in the PVN.

Abbreviations: optic chiasm, OC; median eminence, ME; arcuate nucleus, ARN;

anterior commisure, AC; fornix, F. (schematic courtesy of M. Egli)

126

in the subjective night. In the absence of ovarian steroids the rhythm was notably small in

amplitude and often displayed multiphasic patterns. Under DD, only the ME and IL displayed

clear free-running rhythms of DA turnover. Treatment with exogenous ovarian steroids affected

the magnitude and timing of these DA turnover rhythms, as well the rhythms of PRL and CORT

secretion. Thus, ovarian steroids may modulate circadian rhythms of DA turnover through

genomic or non-genomic effects both locally, at the level of the DA neuron, and/or indirectly

through actions at the SCN. Based on these data, I hypothesized that NDNs express clock genes

with a pattern similar to the SCN and act as either semi-autonomous slave oscillators or self-

sustained circadian oscillators, independent of the SCN.

However compelling, recent evidence suggests that regions of the CNS and periphery

that receive neural or humoral input from the SCN, also retain the ability to express clock genes

with a distinct circadian rhythm when isolated from the SCN (245,264,354-356). In fact, recent

studies indicate that olfactory bulb neurons express endogenous, free-running rhythms of PER1

expression in the absence of input from the SCN (278,279). Given the multitude of data

suggesting that neural targets of the SCN, like the mediobasal hypothalamus, may express

functional clock genes, I hypothesized that clock gene expression within NDNs facilitate

endogenous circadian rhythms of neuronal activity. Results from these experiments suggest a

minor role for clock gene expression within NDNs with respect to free-running rhythms of DA

turnover. I was able to localize PER1, PER2 and CLOCK protein and mRNA to the NDNs

within the ARN and PeVN. Moreover, I observed light-entrained diurnal rhythms of PER1 and

PER2 expression within all three populations. Interestingly I was unable to detect free-running

rhythms of PER1 or PER2 expression in NDNs. Moreover, disruption of clock gene expression

within the SCN exerted significant effects on light-entrained rhythms of DA turnover within

TIDA and PHDA neurons, both of which displayed significant free-running rhythms of DA

turnover in previous experiments. Regardless, my inability to observe significant free-running

rhythms of DA turnover and my failure to detect free-running rhythms of clock protein

expression in the current experiments dampens my enthusiasm for local control of autonomous

DA synthesis and release within NDNs in a constant environment. However, these data support

numerous reports indicating robust control of diurnal rhythms of serum PRL and NDN activity

by the biological clock in the SCN (30,41,58,163,165,188,229,253,262,350,357). I observed a

significant response of the light-entrained rhythms of DA turnover in both TIDA and PHDA

127

neurons, as well as a significant response of the serum CORT rhythms, to treatment with AS-

ODN against per1/2 and clock mRNA. I have developed several hypotheses to explain the subtle

effects I have observed following clock gene AS-ODN treatment. Of course, I cannot rule out

potential rebound effects following AS-ODN injection or threshold variance for adequate

function among different mRNAs. Therefore, the choice of a 40-50% level of protein expression,

while obviously slightly lower than the level generally seen in heterozygotic mutants, may still

be adequate to allow for proper oscillator function. In fact, experiments using heterozygotic

per1/2 mutant mice suggest that single copy mutations fail to prevent free-running rhythms of

activity (210,344). However, a high amount of variability exists with respect to the incidence of

free-running activity in these animals. These data would suggest that single copy mutation of the

per1 and per2 genes would have similar effects on DA turnover, serum PRL and serum CORT

rhythms. Currently, a triple per1/2 and clock gene mutant does not exist. However, data from

per1/2 heterozygotes and clock mutants, as well as data from my own experiments, suggest that

AS-ODN cocktail did affect the molecular oscillator. Data from clk/clk mutant mice reveal a

disrupted neuroendocrine system, marked by abnormal estrous cyclicity (358). Clock mutant

females have extended, irregular cycles, lack a precisely timed luteinizing hormone (LH) surge

on the day of proestrus, and have a high rate of pregnancy failure. Clock mutants also show an

unexpected decline in progesterone levels at mid-pregnancy and a shortened duration of

pseudopregnancy, suggesting that maternal prolactin release may be abnormal. Further the

authors show that clk/clk mutant animals failed to exhibit LH surges in response to estradiol

priming, though they maintained normal levels of serum gonadotrophin-releasing hormone,

pituitary gland LH release and ovarian steroid hormones. These data suggest a deficiency within

the hypothalamus, specifically at the level of the connection between the SCN oscillators and

GnRH neurons within the pre-optic area. The authors failed to detect PRL levels or DA turnover

in NDN terminal regions within their mutants. Additional studies display a more subtle effect of

the clock mutation of reproductive success (359,360). I would hypothesize, based on my current

data, that the clk mutation causes substantial disruption of the proestrous PRL surge and may

abolish rhythmic release of DA and oxytocin from neuroendocrine cells of the hypothalamus.

In my current experiment I utilized OVX females to isolate DA turnover rhythms from

the effects of rhythmic ovarian steroid hormone secretion (24) or surge-level PRL secretion

(31,32,244). Thus, I cannot exclude the potential for a dramatic effect of AS-ODN treatment on

128

the circadian rhythms of PRL secretion and NDN activity I have observed in both normal cycling

or steroid-primed animals. Numerous experiments support the assertion that ovarian steroids

modulate neuronal activity and gene expression with SCN neuronal oscillators

(107,251,265,361-364). Specifically, cryptochrome gene expression was significantly affected

by ovarian steroid hormone treatment, as was gap junction forming connexin mRNA(111-113).

Experiments have shown that disruption of gap junction formation and function disrupts

rhythmic release of both AVP and VIP from cultured SCN (365). Thus, I can assume that

ovarian steroids may exert a significant effect on clock gene expression and rhythmicity

throughout the estrous cycle. Further experiments are necessary to determine the precise role of

ovarian steroids in this system. In contrast with previous experiments, I was unable to detect a

free-running rhythm of DA turnover within TIDA and PHDA neurons in both RS-ODN and MI-

ODN controls. The use of Western blot analysis in order to verify successful gene knockdown

obviates anatomical verification of the injection site. Therefore, I cannot rule out damage to

SCN efferents coursing dorsomedially over the SCN as a result of cannula placement. However,

preliminary experiments suggest that cannula placement, using my protocol, does not disrupt

estrous cyclicity in intact females (M. Poletini, unpublished observation). Therefore, I can likely

rule out any significant effect of cannula placement on damage to the SCN that results in

disruption of adequate reproductive cycles. In addition, I cannot completely rule out the

influence of halothane treatment on my results. As mentioned in Chapter 4, animals were

anesthetized with halothane 6 hours before sacrifice in order to inject ODN into the SCN. Due to

the location of the cannula I required the animal be immobilized in order to deliver the antisense

injection. Halothane has been shown to be an effective gap-junction blocker and has been

implicated in disruption of AVP and VIP release from SCN cultures (365). Further, I cannot

completely rule out effects of arousal during ODN injection, particularly during DD, on the

rhythms of DA release, PRL secretion and CORT secretion seen in the current experiment.

However, my experiments were designed such that the times of ODN injection did not

correspond with circadian times associated with dramatic phase shifts of locomotor activity (79).

Animals were injected at CT0,3,6,9,12 and 18. Light-pulses at each of these times would fail to

significantly phase-shift locomotor activity according to a type-1 phase-response curve. Thus, I

can assume that our manipulation, albeit done in dim red light, would not induce a significant

phase shift or immediate activation of clock gene expression within NDNs or SCN neurons.

129

More difficult to eliminate are any concerns regarding arousal induced phase-shifts during the

subjective day (366). Regardless, animals were sacrificed only 6 hours after the manipulation

and would most likely not exhibit the effects of such a shift until a minimum of one to two 24h

cycles after the manipulation.

In previous experiments, my laboratory and others have determined that VIPergic

afferents of SCN origin terminate on NDNs and that the expression of VIP type-2 receptors

(VPAC2) is influenced by steroid hormone replacement ((171,172) and Fig.35). Further,

experiments reveal that VIP afferents on GnRH neurons increase following puberty, suggesting

significant reorganizing effects of ovarian steroids on the connection between the SCN and its

hypothalamic targets (181). The role of SCN afferents in the control of the LH surge is well

established in the literature (367). Injection of VIP antisense into the SCN has been shown to

disrupt LH secretion and a decline of rhythmic VIP release in aging females has been linked to

reproductive senescence (180,187,261,368-370). Previous experiments in my laboratory reveal

that disruption of VIP expression within the SCN modulates the timing and magnitude of

immediate early gene expression in NDNs (188). In these experiments, fos-related antigen

expression displayed a rhythm with a peak at 0700h, followed by a nadir at 1900h. Following

VIP-AS-ODN treatment, FRAS expression increased at 1900h, indicating that the diurnal rhythm

of activity within DA neurons was abolished (188). In the current experiment, I have disrupted

per1, per2 and clock expression within the SCN and observed a disruption of light-entrained DA

turnover rhythms within both TIDA and PHDA neurons. In fact, TIDA neurons displayed a

specific decrease in DA turnover near CT15 (~2030h). Although interesting, it is unclear

whether the increase in FRAs expression at 1900h in a previous experiment (188)correlates with

the decrease in DA release I have seen following clock gene antisense. Increases in FRAs

expression are generally believed to be associated with increases in cellular activity

(160,161,371). However, our understanding of the role of FRA-1/2, FOS and ∆FOSB (the fos

related antigens) within the NDN is currently incomplete. Therefore, I cannot eliminate any

potential relationship between FRAs expression and DA synthesis and release from NDNs.

Thus, I cannot ignore the obvious importance of VIPergic afferents in the regulation of DA

turnover rhythms and the potential relationship between VIP-AS-ODN treatment and my current

result. Several experiments conducted in my laboratory and others suggest that VIP afferents of

130

RHT

PHDATHDATIDA

VIP RHYTHM

PHDATHDATIDA

RHT

NO VIP RHYTHM

?

?

DA RHYTHM

DISRUPTED DA RHYTHM

RHT

PHDATHDATIDA

VIP RHYTHM

PHDATHDATIDA

RHT

NO VIP RHYTHM

?

?

DA RHYTHM

DISRUPTED DA RHYTHM

Figure 36. Synergy between clock gene expression within VIPergic neurons

of the SCN and NDNs in the regulation of DA turnover rhythms in NDNs.

Under a standard 12:12 L:D cycle, rhythmic VIP release from SCN neurons,

driven by light-activated clock gene expression, initiate and entrain diurnal

rhythms of DA turnover and PRL secretion. In constant darkness, a dampened

VIP rhythm fails to initiate and/or entrain rhythms of clock gene expression

within NDN “slave” oscillators. Gradual arrhythmia of clock gene expression

within NDNs leads to variabile activity within DA neurons and therefore

dampened rhythms of DA release and PRL secretion. Abbreviations:

retinohypothalamic tract, RHT.

131

SCN origin play a significant role in both the endogenous stimulatory rhythm and

pseudopregnant surges of PRL secretion (52,53,58). These experiments also suggest that VIP

release, predominately during the latter portion of the dark phase, plays a significant role in the

entrainment of daily PRL surges (Figs. 36 and 37). Thus, my results, in agreement with a

multitude of previous experiments, indicate that VIP afferents entrain diurnal rhythms of DA

turnover in NDNs during the late evening (Fig. 36). In the absence of photoperiod cues VIP

release dampens and results in downstream dampening of DA release from neuroendocrine DA

neurons (Fig. 36). Future experiments should reveal the precise mechanism by which the

dampening of the endogenous rhythm of DA release occurs. My results indicate that each

population of NDNs display a independent rhythm of clock gene expression under a 12:12 L:D

cycle. Therefore, it appears likely that DA neurons lack the ability to oscillate independent of

the SCN and dampen in the absence of activating and entraining signals transduced by VIP.

Experiments with DAergic neuronal cultures and slice physiology experiments could provide

further insight into this hypothesis. Although evidence reveals that VIP activates PER

expression within SCN neurons, it is unclear whether a similar mechanism exists at the level of

the NDN (Fig. 37 and (310)). Evidence suggests that binding of VIP to the VPAC2 receptors

leads to activation of a stimulatory G-protein mediated pathway, resulting in an increased level

of intracellular cAMP levels (189,372). Increasing levels of intracellular cAMP would most

likely lead to increased phosphorylation of MAP kinases and activation of cAMP-response-

element binding protein (373-375). Recent experiments indicate that period gene expression is

directly influences by CREB binding to a cAMP response element 5’ to the CLOCK:BMAL1

binding E-box in the 5’-promoter region of the gene (281). Thus, PER expression is initiated by

both CLOCK:BMAL enhancer activation (the endogenous pathway) and exogenous activation of

CREB signaling by VIP-receptor activation. I have verified the presence of over 20 variations of

the canonical E-box sequence within the 5’-promoter region of the tyrosine hydroxylase gene

(unpublished observation). As seen in Figure 37, binding of CLOCK:BMAL1 heterodimers to

the TH gene promoter would result in a diurnal rhythm of TH expression. Additional enzymes

for DA synthesis, including L-amino acid decarboxylase, may also contain canonical E-box

sequence and CRE sequences within their 5’ promoter region. VIP-activated PER protein could

interact with CLOCK:BMAL1 heterodimers at the promoter for these various enzymes, resulting

in finely tuned expression patterns within the DAergic neuron (Fig. 37). Evidence suggests that

132

AC

cAMPATP

CREBP

P

VPAC2-R

Gs

per+1 P1

th+1C B

ldcP1

TH

1.

3.

2.

4.

TYR

L-DOPA

VIP

ACAC

cAMPATP

CREBP

PCREB

PP

VPAC2-R

Gs

per+1 P1

th+1C B

ldcP1

TH

1.

3.

2.

4.

TYR

L-DOPA4.

TYR

L-DOPA

VIP

Gs

Figure 37. Hypothetical regulation of DA synthesis enzyme gene expression by both

rhythmic VIP activated cAMP response element binding protein (CREB) activation of

period gene expression and endogenous rhythms of CLOCK:BMAL1 driven transcription

in NDNs. Rhythmic expression of DA synthetic enzymes may be driven by both localized

semi-autonomous rhythms of clock gene expression and additional light-entrained activation of

PER expression driven by VIP-mediated initiation of second messengers including MEK

kinase, STATs and CREB. In addition, DA turnover, including both DA metabolism and

release, may be driven by clock gene driven cellular activity. Moreover, clock genes may tune

rhythmic expression and/or activation of ion channels and membrane pumps within NDN

membranes responsible for changes in neuronal excitability, as has been suggested for SCN

oscillators. Modulation of VIP type-2 receptor (VPAC-2) expression on DAergic neuronal

membranes and clock genes within the SCN by ovarian steroids leads to intensified entrainment

of NDN activity. Abbreviations: adenylate cyclase, AC; adenosine triphosphate, ATP;

stimulatory g-protein, Gs; dopa decarboxylase, ldc; PERIOD1, P1; phosphate, P; tyrosine

hydroxylase, TH; tyrosine, TYR; CLOCK, C; BMAL, B. Dashed line = nuclear membrane

133

clock genes may also regulate expression of ion channels, including both membrane Ca2+

and K+

channels that could also be affected by the interacting loops outlined above (376,377). Of

course, numerous additional experiments are needed in order to verify the legitimacy of our

model. However, my results provide a strong foundation for future investigations into the

potential mechanisms by which putative clock genes facilitate the rhythmic activity of

neuroendocrine cells and therefore rhythmic PRL secretion under various physiological states.

134

APPENDIX

COPYRIGHT PERMISSION LETTER

14 February 2005 Our ref: HG/ct/feb 05/J102

Michael Sellix

Dear Mr Sellix

BRAIN RESEARCH, Vol 1005, 2004, pp 164-181, Sellix, “Ovarian steroid hormones …”

As per your letter dated 10 February 2005, we hereby grant you permission to reprint the

aforementioned material at no charge in your thesis subject to the following conditions:

1. If any part of the material to be used (for example, figures) has appeared in our publication

with credit or acknowledgement to another source, permission must also be sought from that

source. If such permission is not obtained then that material may not be included in your

publication/copies.

2. Suitable acknowledgment to the source must be made, either as a footnote or in a reference

list at the end of your publication, as follows:

“Reprinted from Publication title, Vol number, Author(s), Title of article, Pages No.,

Copyright (Year), with permission from Elsevier”.

3. Reproduction of this material is confined to the purpose for which permission is hereby

given.

4. This permission is granted for non-exclusive world English rights only. For other languages

please reapply separately for each one required. Permission excludes use in an electronic

form. Should you have a specific electronic project in mind please reapply for permission.

5. This includes permission for UMI to supply single copies, on demand, of the complete

thesis. Should your thesis be published commercially, please reapply for permission.

Yours sincerely

Helen Gainford

Rights Manager

135

Dear Michael Sellix

Thank you very much for the clarifications in your below e-mail.

Permission is granted herewith to use figures 1, 2, 3, 4 as well as the text

passages from the article:

Sellix, M.T.; Freeman, M.E.: Neuroendocrinology 2003;77:59-70

in your dissertation provided that complete credit is given to the original source and S. Karger

AG, Basel, is mentioned.

I hope that I have been of assistance to you.

Yours sincerely

Isabelle Flückiger

Rights and Permissions

S. Karger AG

Medical and Scientific Publishers

Allschwilerstrasse 10

CH - 4009 Basel

Switzerland

E-mail: [email protected]

Tel. +41 61 306-1475

Fax +41 61 306-1234

**************************************************************

>>> <[email protected]> 01.02.2005 16:22:46 >>>

Ms. Fluckiger,

Thank you for your rapid response. I apologize for the lack of clarity in my email and letter. I

plan to reprint all of the figures and a majority of the text (some minor changes and editing

withstanding) from the aforementioned article. That would include Figures 1,2,3 and 4.

Thank you,

Michael Sellix

-----Original Message-----

From: Permission [mailto:[email protected]]

Sent: Tuesday, February 01, 2005 2:14 AM

To: [email protected]

Subject: Antw: copyright permission

Dear Michael Sellix

Thank you very much for your below e-mail. Before I can process your permission

request, I need to know exactly which data from the article:

Sellix, M.T.; Freeman, M.E.: Neuroendocrinology 2003;77:59-70

136

you would like to use in your dissertation (e.g. figure 1, etc.). As soon as I have this information,

I will be able to get back to you again.

I look forward to hearing from you again regarding the above.

Yours sincerely

Isabelle Flückiger

Rights and Permissions

S. Karger AG

Medical and Scientific Publishers

Allschwilerstrasse 10

CH - 4009 Basel

Switzerland

E-mail: [email protected]

Tel. +41 61 306-1475

Fax +41 61 306-1234

>>> <[email protected]> 31.01.2005 19:32:27 >>>

Dear Ms. Fluckiger,

My name is Michael Sellix and I am preparing my dissertation at The Florida State

University. I would like to request permission to reprint figures from my publication in

Neuroendocrinology entitled "Circadian rhythms of neuroendocrine dopaminergic neuronal

activity in ovariectomized rats". Please see the attached letter requesting permission. Thank you

for your time.

Sincerely,

Michael T. Sellix

Michael Sellix B.S.

Doctoral Candidate

Neuroscience Program

Dept. of Biological Science

The Florida State University

www.neuro.fsu.edu/graduateStudents/sellix

"Outside of a dog, a book is a man's best friend. Inside a dog, it's too

dark to read." -Groucho Marx.

"Eat and drink, for tomorrow we die" (Isaiah 22:13).

"If I did not fall, I could not have arisen; if I had not been in darkness, it would not have been

light for me" (Midrash Tehillim 22).

137

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170

BIOGRAPHICAL SKETCH

MICHAEL T. SELLIX

BORN: 08/18/1976 in Ridgewood, NJ

EDUCATION:

B.S. Psychology, Biology Minor, 1998, Florida State University, Tallahassee,

FL

Graduate Biological Sciences, Program in Neuroscience, 1998-present, Florida State

University, Tallahassee, FL

AWARDS AND MEMBERSHIPS:

Program in Neuroscience Fellowship Award (2001-present)

Bryan Robinson Foundation for Neuroscience Achievement Award (2001, 2002)

College of Arts and Sciences, Florida State University, Dissertation Research Grant (2003-2004)

Society for Neuroscience – Student Member (1999-present)

Endocrine Society – Member (2000-present)

PSI CHI - national honor society in psychology – Member (1997-present)

Society for the Study of Reproduction - Trainee Member (2002-present)

ABSTRACTS:

Reorganization of A Motor Cortical Region During Song Learning in the Zebra Finch. M.T.

Sellix* and F. J ohnson. 29th

Annual Meeting of the Society for Neuroscience, Miami FL. 1999.

Circadian Rhythms of Hypothalamic Neuroendocrine Dopaminergic Neuron Activity in the

Ovariectomized Rat. M.T. Sellix* and M.E. Freeman.

31ST

Annual Meeting of the Society for Neuroscience, San Diego CA. 2001.

Circadian Rhythms of Neuroendocrine Dopaminergic Neuron Activity in OVX and OVX-

Estrogen Primed Rats. M.T. Sellix* and M.E. Freeman

81st Annual Meeting of the Endocrine Society, San Francisco CA. 2002

Steroid Hormones Effect The Rhythm of Tuberoinfundibular Dopaminergic Neuronal Activity in

a Constant Environment. M.T. Sellix* and M.E. Freeman

32nd

Annual Meeting of the Society for Neuroscience, Orlando FL. 2002

Vasoactive intestinal peptide of suprachiasmatic nucleus origin controls prolactin and oxytocin

secretion in pseudopregnant rats M. Egli*, M.T. Sellix and M.E. Freeman 82nd

Annual Meeting

of the Endocrine Society, Philadelphia, PA. 2003

171

Ovarian steroids modulate light-entrained circadian rhythms of neuroendocrine dopaminergic

neuronal activity. M.T. Sellix* and M.E. Freeman. 33rd

Annual Meeting of the Society for

Neuroscience, New Orleans, LA. 2003

Rhythmic hormone secretion in rats: Computational model of the hypothalamic mechanism

controlling prolactin secretion. M. Egli*, R. Bertram, M.T. Sellix, M.E. Freeman. American

Mathematical Society Sectional Meeting, Tallahassee, FL. 2004

MANUSCRIPTS:

Reorganization of a telencephalic motor region during sexual differentiation and vocal learning

in zebra finches. Johnson F., and Sellix M. Brain Res Dev Brain Res. (2000) Jun 30; 121(2):

253-63.

Antagonism of vasoactive intestinal peptide mRNA in the suprachiasmatic nucleus disrupts

neuroendocrine dopaminergic neuron activity. (2002) Gerhold LG, Sellix MT, Freeman ME.

J Comp Neurol. 2002 Aug 19;450(2):135-43.

Autocrine regulation of prolactin secretion by endothelins: a permissive role for estradiol

(2002) Kanyicska B, Sellix MT, Freeman ME. Endocrine. 2001 Nov; 16(2): 133-7.

Autocrine regulation of prolactin secretion by endothelins during the estrous cycle (2002)

Kanyicska B, Sellix MT, Freeman ME. Endocrine Feb-Mar; 20(1-2): 53-8.

Circadian rhythms of neuroendocrine dopaminergic neurons in the ovariectomized rat. (2003)

Sellix MT and Freeman ME. Neuroendocrinology Jan; 77(1): 59-70.

Ovarian steroid hormones modulate circadian rhythms of neuroendocrine dopaminergic neuronal

activity. (2003) Sellix MT, Egli M., Henderson RP and Freeman ME. Brain Res. 2004 Apr

16;1005(1-2):164-81.).

Semicircadian rhythm of OT and VIP: roles for regulating the pattern of PRL secretion during

pseudopregnancy. (2003) Egli M, Bertram R, Sellix MT, Nguyen F, Freeman ME.

Endocrinology. 2004 Jul;145(7):3386-94.

172

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