Florida State University Libraries
Electronic Theses, Treatises and Dissertations The Graduate School
2005
Circadian Rhythms in the NeuroendocrineDopaminergic Neurons Regulating ProlactinSecretionMichael Timothy Sellix
Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected]
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
13. ESTRADIOL AND PROGESTERONE AFFECT THE MAGNITUDE, BUT NOT
THE TIMING OF THE CIRCADIAN RHYTHMS OF DA TURNOVER IN THE
INTERMEDIATE LOBE ................................................................................................ 54
14. ESTRADIOL AND PROGESTERONE AFFECT THE MAGNITUDE, BUT NOT
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
AND CONSTANT DARKNESS .................................................................................... 84
23. PER2 EXPRESSION IN NDNS UNDER A STANDARD 12:12 L:D CYCLE
AND CONSTANT DARKNESS .................................................................................... 86
24. CLOCK EXPRESSION IN NDNS UNDER A STANDARD 12:12 L:D CYCLE
AND CONSTANT DARKNESS .................................................................................... 89
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
period of approximately 24 hours under constant conditions such as constant darkness (DD) or
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.
Summary and Conclusions
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
63
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
64
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.
67
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
68
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.
74
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.
75
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
within the DMARN, RARN and PeVN, respectively (arrowhead; Fig.23A,C,E). Generally, more
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
within the DMARN, RARN and PeVN, respectively (arrowhead; Fig.24A,C,E). Generally, more
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
94
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.
Summary and Conclusions
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).
95
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
96
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
97
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
98
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
99
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
100
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
101
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
102
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
103
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-
104
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
105
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.)
106
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
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 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.
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 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.
Acute knockdown of PER1, PER2 and CLOCK protein expression in the SCN affects light-
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.
Summary and Conclusions
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
period of approximately 24 hours under constant conditions such as constant darkness (DD) or
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
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
REFERENCES
1. Freeman ME, Kanyicska B, Lerant A, Nagy G 2000 Prolactin: structure, function, and
regulation of secretion. Physiol Rev 80:1523-1631
2. Riddle O, Bates RW, Dykshorn SW 1933 The preparation, identification and assay of
prolactin-a hormone of anterior pituitary. Am J Physiol 105:191-216
3. Riddle O, Braucher PF 1931 Control of the special secretion of the crop-gland in
pigeons by an anterior pituitary hormone. Am J Physiol 97:617-625
4. Ben-Jonathan N 1985 Dopamine: a prolactin-inhibiting hormone. Endocr Rev 6:564-
589
5. Ben-Jonathan N, Arbogast LA, Hyde JF 1989 Neuroendrocrine regulation of prolactin
release. Prog Neurobiol 33:399-447
6. Ben-Jonathan N 1994 Regulation of Prolactin Secretion. In: Imura H (ed). The Pituitary
Gland.Raven Press, New York:261-283
7. Pan JT 1996 Neuroendocrine functions of dopamine. In: Stone TW (ed). CNS
neurotransmitters and neuromodulators: dopamine.CRC press, Boca Raton, FL.:213-232
8. Arimura A, Dunn JD, Schally AV 1972 Effect of infusion of hypothlamic extracts on
serum prolactin levels in rats treated with Nembutal, CNS depressant or bearing
hypothalamic lesions. Endocrinology 90:378-383
9. Murai I, Garris PA, Ben-Jonathan N 1989 Time-dependent increase in plasma
prolactin after pituitary stalk section: role of posterior pituitary dopamine. Endocrinology
124:2343-2349
10. Samuels MH, Henry P, Kleinschmidt-Demasters B, Lillehei K, Ridgway EC 1991
Pulsatile prolactin secretion in hyperprolactinemia due to presumed pituitary stalk
interruption. J Clin Endocr Metab 73:1289-1293
11. Ambach G, Palkovits M, Szentágothai J 1976 Blood supply of the rat hypothalamus.
IV. Retrochiasmatic area, median eminence, arcuate nucleus. Acta Morphol Acad Sci
Hung 24:93-119
12. Palkovits M 1998 Micro- and macroscopic structure, innervation, and vasculature of the
hypothalamus. In: Conn PM, Freeman ME (eds). Neuroendocrinology in physiology and
medicine.Humana Press, Totowa, NJ:23-40
138
13. Mezey E, Palkovits M 1982 Two-way transport in the hypothalamo-hypophyseal
system. In: Ganong WF, Martini L (eds). Front Neuroendo. Raven Press, New York:1-29
14. Bazan JF 1990 Haemopoietic receptors and helical cytokines. Immunology Today
11:350-354
15. Bazan JF 1990 Structural design and molecular evolution of a cytokine receptor
superfamily. Proc Natl Acad Sci U S A 87:6934-6938
16. Kelly PA, Edery M, Finidori J, Postel-Vinay M-C, Gougon L, Ali S, Dinerstein H,
Sotiropoulos A, Lochnan H, Ferrag F, Lebrun J-J, Ormandy C, Buteau H, Esposito
N, Vincent V, Möldrup A 1994 Receptor domains involved in signal transduction of
prolactin and growth hormone. Proc Soc Exp Bio Med 206:280-283
17. Lebrun J-J, Ali S, Sofer L, Ullrich A, Kelly PA 1994 Prolactin-induced proliferation of
Nb2 cells involves tyrosine phosphorylation of the prolactin receptor and its associated
tyrosine kinase JAK2. J Biol Chem 269:14021-14026
18. Lebrun JJ, Ali S, Goffin V, Ullrich A, Kelly PA 1995 A single phosphotyrosine residue
of the prolactin receptor is responsible for activation of gene transcription. Proc Natl
Acad Sci U S A 92:4031-4035
19. Kazansky AV, Kabotyanski EB, Wyszomierski SL, Mancini MA, Rosen JM 1999
Differential effects of prolactin and src/abl kinases on the nuclear translocation of
STAT5B and STAT5A. J Biol Chem 274:22484-22492
20. Jahn GA, Daniel N, Jolivet G, Belair L, Bole-Feysot C, Kelly PA, Djiane J 1997 In
vivo study of prolactin (PRL) intracellular signalling during lactogenesis in the rat:
JAK/STAT pathway is activated by PRL in the mammary gland but not in the liver. Biol
Reprod 57:894-900
21. Pezet A, Buteau H, Kelly PA, Edery M 1997 The last proline of Box 1 is essential for
association with JAK2 and functional activation of the prolactin receptor. Mol Cell
Endocrinology 129:199-208
22. Goudreau JL, Lindley SE, Lookingland KJ, Moore KE 1992 Evidence that
hypothalamic periventricular dopamine neurons innervate the intermediate lobe of the rat
pituitary. Neuroendocrinology 56:100-105
23. Goudreau JL, Falls WM, Lookingland KJ, Moore KE 1995 Periventricular-
hypophysial dopaminergic neurons innervate the intermediate but not the neural lobe of
the rat pituitary gland. Neuroendocrinology 62:147-154
139
24. Mai LM, Shieh KR, Pan JT 1994 Circadian changes of serum prolactin levels and
tuberoinfundibular dopaminergic neuron activities in ovariectomized rats treated with or
without estrogen: The role of the suprachiasmatic nuclei. Neuroendocrinology 60:520-
526
25. DeMaria JE, Zelena D, Vecsernyes M, Nagy GM, Freeman ME 1998 The effect of
neurointermediate lobe denervation on hypothalamic neuroendocrine dopaminergic
neurons. Brain Res 806:89-94
26. Murai I, Ben-Jonathan N 1990 Acute stimulation of prolactin release by estradiol:
Mediation by the posterior pituitary. Endocrinology 126:3179-3184
27. Murai I, Ben-Jonathan N 1986 Chronic posterior pituitary lobectomy: Prolonged
elevation of plasma prolactin and interruption of cyclicity. Neuroendocrinology 43:453-
458
28. Neill JD, Smith MS 1974 Pituitary-ovarian interrelationships in the rat. In: James VHT,
Martini L (eds). Cur Top Exp Endocr .Academic Press, New York:73-106
29. Bethea CL, Neill JD 1979 Prolactin secretion after cervical stimulation of rats
maintained in constant dark or constant light. Endocrinology 104:870-876
30. Mai LM, Pan JT 1995 Bombesin acts in the suprachiasmatic nucleus to affect circadian
changes in tuberoinfundibular dopaminergic neuron activity and prolactin secretion.
Endocrinology 136:4163-4167
31. DeMaria JE, Lerant AA, Freeman ME 1999 Prolactin activates all three populations of
hypothalamic neuroendocrine dopaminergic neurons in ovariectomized rats. Brain Res
837:236-241
32. Lerant A, Kanyicska B, Freeman ME 2001 Nuclear translocation of STAT5 and
increased expression of Fos related antigens (FRAs) in hypothalamic dopaminergic
neurons after prolactin administration. Brain Res 904:259-269
33. Freeman ME 1988 The ovarian cycle of the rat. In: Ernst Knobil, Jimmy D.Neill (eds).
The Physiology of Reproduction.Raven Press, New York:1893-1928
34. Neill JD 1972 Sexual differences in the hypothalamic regulation of prolactin secretion.
Endocrinology 90:1154-1159
35. Pasqualini C, Lenoir V, Abed AE, Kerdelhue B 1984 Anterior pituitary dopamine
receptors during the rat estrous cycle. Neuroendocrinology 38:39-44
140
36. Pasqualini C, Bojda F, Gaudoux F, Guibert B, Leviel B, Teissier E, Rips R,
Kerdelhue B 1988 Changes in tuberoinfundibular dopaminergic neuron activity during
the rat estrous cycle in relation to the prolactin surge: alteration by a mammary
carcinogen. Neuroendocrinology 48:320
37. Pasqualini C, Bojda F, Kerdelhue B 1986 Direct effect of estradiol on the number of
dopamine receptors in the anterior pituitary of ovariectomized rats. Endocrinology
119:2484-2489
38. Arbogast LA, Voogt JL 2002 Progesterone induces dephosphorylation and inactivation
of tyrosine hydroxylase in rat hypothalamic dopaminergic neurons. Neuroendocrinology
75:273-281
39. Arbogast LA, Voogt JL 1993 Progesterone reverses the estradiol-induced decrease in
tyrosine hydroxylase mRNA levels in the arcuate nucleus. Neuroendocrinology 58:501-
510
40. Arbogast LA, Voogt JL 1994 Progesterone suppresses tyrosine hydroxylase messenger
ribonucleic acid levels in the arcuate nucleus on proestrus. Endocrinology 135:343-350
41. Yen SH, Pan JT 1998 Progesterone advances the diurnal rhythm of tuberoinfundibular
dopaminergic neuronal activity and the prolactin surge in ovariectomized, estrogen-
primed rats and in intact proestrous rats. Endocrinology 139:1602-1609
42. Witcher JA, Freeman ME 1985 The proestrous surge of prolactin enhances sexual
receptivity in the rat. Biol Reprod 32:834-839
43. Carr LA, Voogt JL 1980 Catecholamine synthesizing enzymes in the hypothalamus
during the estrous cycle. Brain Res 196:437-445
44. Demarest KT, Johnston CA, Moore KE 1981 Biochemical indices of
catecholaminergic neuronal activity in the median eminence during the estrous cycle of
the rat. Neuroendocrinology 32:24-27
45. DeMaria JE, Livingstone JD, Freeman ME 1998 Characterization of the dopaminergic
input to the pituitary gland throughout the estrous cycle of the rat. Neuroendocrinology
67:377-383
46. Horn AM, Fraser HM, Fink G 1985 Effects of antiserum to thyrotrophin-releasing
hormone on the concentrations of plasma prolactin, thyrotrophin and LH in the pro-
oestrous rat. J Endocr 104:205-209
141
47. Vijayan E, Samson WK, Said SI, McCann SM 1979 Vasoactive intestinal peptide:
evidence for a hypothalamic site of action to release growth hormone, luteinizing
hormone, and prolactin in conscious ovariectomized rats. Endocrinology 104:53-57
48. Shaar CJ, Clemens JA, Dininger NB 1979 Effect of vasoactive intestinal polypeptide
on prolactin release in vitro. Life Sciences 25:2071-2074
49. Samson WK, Freeman ME 1991 Vasoactive intestinal peptide: A neural modulator of
endocrine function. In: Johnston CA, Barnes CD (eds). Brain-Gut Peptides and
Reproductive Function.CRC Press, Boca Raton:85-103
50. Samson WK, Lumpkin MD, McCann SM 1986 Evidence for a physiological role for
oxytocin in the control of prolactin. Endocrinology 119:554-561
51. Johnston CA, Negro-Vilar A 1988 Role of oxytocin on prolactin secretion during
proestrus and in different physiological or pharmacological paradigms. Endocrinology
122:341-350
52. Arey BJ, Freeman ME 1992 Activity of vasoactive intestinal peptide and serotonin in
the paraventricular nucleus reflects the periodicity of the endogenous stimulatory rhythm
regulating prolactin secretion. Endocrinology 131:736-742
53. Arey BJ, Averill RL, Freeman ME 1989 A sex-specific endogenous stimulatory
rhythm regulating prolactin secretion. Endocrinology 124:119-123
54. Arey BJ, Freeman ME 1992 Activity of oxytocinergic neurons in the paraventricular
nucleus mirrors the periodicity of the endogenous stimulatory rhythm regulating prolactin
secretion. Endocrinology 130:126-132
55. Arey BJ, Kanyicska B, Freeman ME 1991 The endogenous stimulatory rhythm
regulating prolactin secretion is present in the lactating rat. Neuroendocrinology 53:35-40
56. Kacsoh B, Nagy GY 1983 Circadian rhythms in plasma prolactin, luteinizing hormone
and hypophyseal prolactin levels in lactating rats. Endocrinologica Experimentalis
17:301-310
57. Gunnet JW, Freeman ME 1983 The mating-induced release of prolactin: a unique
neuroendocrine response. Endocr Rev 4:44-60
58. Egli M, Bertram R, Sellix MT, Freeman ME 2004 Rhythmic Secretion of Prolactin in
Rats: Action of Oxytocin Coordinated by Vasoactive Intestinal Polypeptide of
Suprachiasmatic Nucleus Origin. Endocrinology
142
59. Arey BJ, Freeman ME 1989 Hypothalamic factors involved in the endogenous
stimulatory rhythm regulating prolactin secretion. Endocrinology 124:878-883
60. Arey BJ, Freeman ME 1990 Oxytocin, vasoactive intestinal peptide and serotonin
regulate the mating-induced surges of prolactin secretion in the rat. Endocrinology
126:279-284
61. Lerant A, Herman ME, Freeman ME 1996 Dopaminergic neurons of periventricular
and arcuate nuclei of pseudopregnant rats: Semicircadian rhythm in fos-related antigens
immunoreactivities and in dopamine concentration. Endocrinology 137:3621-3628
62. Bethea CL, Neill JD 1980 Lesions of the suprachiasmatic nuclei abolish the cervically
stimulated prolactin surges in the rat. Endocrinology 107:1-5
63. Stephan FK, Zucker I 1972 Circadian rhythms in drinking behavior and locomotor
activity of rats are eliminated by hypothalamic lesions. Proc Natl Acad Sci U S A
69:1583-1586
64. Moore RY, Eichler VB 1972 Loss of a circadian adrenal corticosterone rhythm
following suprachiasmatic lesions in the rat. Brain Res 42:201-206
65. Van den Pol AN 1991 The suprachiasmatic nucleus: morphological and cytochemical
substrates for cellular interaction. In: Klein DC, Moore RY, Reppert SM, Klein DF (eds).
Oxford University Press, New York:17-50
66. Moore-Ede M, Sulzman FM, Fuller CA 1982 Characteristics of circadian clocks. In:
Moore-Ede MC, Sulzman FM, Fuller CA (eds). The clocks that time us. Harvard
University Press, Cambridge:30-112
67. Honma S, Katsuno Y, Shinohara K, Abe H, Honma K 1996 Circadian rhythm and
response to light of extracellular glutamate and aspartate in rat suprachiasmatic nucleus.
Am J Phys: Reg Integr Comp Physiol 271:R579-R585
68. De Vries MJ, Treep JA, De Pauw ESD, Meijer JH 1994 The effects of electrical
stimulation of the optic nerves and anterior optic chiasm on the circadian activity rhythm
of the Syrian hamster: Involvement of excitatory amino acids. Brain Res 642:206-212
69. Rusak B 1979 Neural mechanisms for entrainment and generation of mammalian
circadian rhythms. Fed Proc 38:2589-2595
70. Hannibal J 2002 Neurotransmitters of the retino-hypothalamic tract. Cell Tissue Res
309:73-88
143
71. Hannibal J, Moller M, Ottersen OP, Fahrenkrug J 2000 PACAP and glutamate are
co-stored in the retinohypothalamic tract. J Comp Neurol 418:147-155
72. Speh JC, Moore RY 1993 Retinohypothalamic tract development in the hamster and rat.
Developmental Brain Res 76:171-181
73. Moore RY, Speh JC, Card JP 1995 The retinohypothalamic tract originates from a
distinct subset of retinal ganglion cells. J Comp Neuro 352:351-366
74. Hurst WJ, Mitchell JW, Gillette MU 2002 Synchronization and phase-resetting by
glutamate of an immortalized SCN cell line. Biochem Biophys Res Commun 298:133-
143
75. Welsh DK, Logothetis DE, Meister M, Reppert SM 1995 Individual neurons
dissociated from rat suprachiasmatic nucleus express independently phased circadian
firing rhythms. Neuron 14:697-706
76. Aschoff J 1998 Circadian parameters as individual characteristics. J Biol Rhythms
13:123-131
77. Aschoff J 1965 Circadian rhythms in man. Science 148:1427-1432
78. Pittendrigh CS, Daan S 1976 Functional-analysis of circadian pacemakers in nocturnal
rodents. 1. stability and lability of spontaneous frequency. J Comp Physiol 106:223-252
79. Daan S, Pittendrigh CS 1976 Functional-analysis of circadian pacemakers in nocturnal
rodents. 2. Variability of phase response curves. J Comp Physiol 106:253-266
80. Daan S, Pittendrigh CS 1976 Functional-analysis of circadian pacemakers in nocturnal
rodents. 3. Heavy-water and constant light - homeostasis of frequency. J Comp Physiol
106:267-290
81. Pittendrigh CS, Daan S 1976 Functional-analysis of circadian pacemakers in nocturnal
rodents. 4. Entrainment - pacemaker as clock. J Comp Physiol 106:291-331
82. Pittendrigh CS, Daan S 1976 Functional-analysis of circadian pacemakers in nocturnal
rodents. 5. Pacemaker structure - clock for all seasons. J Comp Physiol 106:333-355
83. Palmer JD 2000 The clocks controlling the tide-associated rhythms of intertidal animals.
BioEssays 22:32-37
84. Roenneberg T, Merrow M 2002 Life before the clock: modeling circadian evolution. J
Biol Rhythms 17:495-505
144
85. Roenneberg T, Wirz-Justice A, Merrow M 2003 Life between clocks: daily temporal
patterns of human chronotypes. J Biol Rhythms 18:80-90
86. Czeisler CA, Duffy JF, Shanahan TL, Brown EN, Mitchell JF, Rimmer DW, Ronda
JM, Silva EJ, Allan JS, Emens JS, Dijk DJ, Kronauer RE 1999 Stability, precision,
and near-24-hour period of the human circadian pacemaker. Science 284:2177-2181
87. Czeisler CA, Klerman EB 1999 Circadian and sleep-dependent regulation of hormone
release in humans. Recent Prog Horm Res 54:97-130
88. Satinoff E, Prosser RA 1988 Suprachiasmatic nuclear lesions eliminate circadian
rhythms of drinking and activity, but not of body temperature, in male rats. J Biol
Rhythms 3:1-22
89. Bartness TJ, Goldman BD, Bittman EL 1991 SCN lesions block responses to systemic
melatonin infusions in Siberian hamsters. Am J Physiol Regul Integr Comp Physiol
260:R102-R112
90. LeSauter J, Silver R 1999 Localization of a suprachiasmatic nucleus subregion
regulating locomotor rhythmicity. J Neurosci 19:5574-5585
91. Devlin PF, Kay SA 2001 Circadian photoreception. Ann Rev Physiol 63:677-694
92. Stephan FK 2002 The "other" circadian system: food as a Zeitgeber. J Biol Rhythms
17:284-292
93. Stephan FK 1986 The role of period and phase in interactions between feeding- and
light-entrainable circadian rhythms. Physiol Behav 36:151-158
94. Pittendrigh CS 1993 Temporal organization: reflections of a Darwinian clock-watcher.
Annu Rev Physiol 55:16-54
95. Stephan FK 1983 Circadian rhythms in the rat: constant darkness, entrainment to T
cycles and to skeleton photoperiods. Physiol Behav 30:451-462
96. Sharma VK, Daan S 2002 Circadian phase and period responses to light stimuli in two
nocturnal rodents. Chronobiol Int 19:659-670
97. Evans JA, Elliott JA, Gorman MR 2004 Photoperiod differentially modulates photic
and nonphotic phase response curves of hamsters. Am J Physiol Regul Integr Comp
Physiol 286:R539-R546
145
98. Aschoff J, Daan S 1997 Human time perception in temporal isolation: effects of
illumination intensity. Chronobiol Int 14:585-596
99. Rusak B, Groos G 1982 Suprachiasmatic stimulation phase-shifts rodent circadian-
rhythms. Science 215:1407-1409
100. Hastings MH 1991 Neuroendocrine rhythms. Pharmacol Ther 50:35-71
101. Turek FW 1994 Circadian rhythms. Recent Prog Horm Res 49:43-90
102. Piggins HD 2000 Neuroendocrinology briefings 10: biological timekeeping. J
Neuroendocrinol 12:935-936
103. Vitaterna MH, Takahashi JS, Turek FW 2001 Overview of circadian rhythms.
Alcohol Res Health 25:85-93
104. Everett JW, Sawyer CH 1950 A 24-hour periodicity in the "LH-release apparatus" of
female rats, disclosed by barbiturate sedation. Endocrinology 47:198-218
105. Neill JD, Freeman ME, Tillson SA 1971 Control of the proestrus surge of prolactin and
luteinizing hormone secretion by estrogens in the rat. Endocrinology 89:1148-1453
106. Freeman ME, Reichert LE, Jr., Neill JD 1972 Regulation of the proestrus surge of
prolactin secretion by gonadotropin and estrogens in the rat. Endocrinology 90:323-328
107. Legan SJ, Karsch FJ 1975 A daily signal for the LH surge in the rat. Endocrinology
96:57-62
108. Legan SJ, Coon GA, Karsch FJ 1975 Role of estrogen as initiator of daily LH surges in
the ovariectomized rat. Endocrinology 96:50-56
109. Shughrue PJ, Lane MV, Merchenthaler I 1997 Comparative distribution of estrogen
receptor-alpha and -beta mRNA in the rat central nervous system. J Comp Neurol
388:507-525
110. Su JD, Qiu J, Zhong YP, Chen YZ 2001 Expression of estrogen receptor -alpha and -
beta immunoreactivity in the cultured neonatal suprachiasmatic nucleus: with special
attention to GABAergic neurons. NeuroReport 12:1955-1959
111. Nakamura TJ, Shinohara K, Funabashi T, Kimura F 2001 Effect of estrogen on the
expression of Cry1 and Cry2 mRNAs in the suprachiasmatic nucleus of female rats.
Neurosci Res 41:251-255
146
112. Shinohara K, Funabashi T, Mitushima D, Kimura F 2000 Effects of estrogen on the
expression of connexin32 and connexin43 mRNAs in the suprachiasmatic nucleus of
female rats. Neurosci Lett 286:107-110
113. Shinohara K, Funabashi T, Nakamura TJ, Kimura F 2001 Effects of estrogen and
progesterone on the expression of connexin-36 mRNA in the suprachiasmatic nucleus of
female rats. Neurosci Lett 309:37-40
114. DeMaria JE, Livingstone JD, Freeman ME 2000 Ovarian steroids influence the
activity of neuroendocrine dopaminergic neurons. Brain Res 879:139-147
115. Veldhuis JD 2000 The neuroendocrine control of ultradian rhythms. In: Conn PM,
Freeman ME (eds). Neuroendocrinology in physiology and medicine.Humana Press,
Totowa, NJ:453-472
116. Goldman BD 1999 The circadian timing system and reproduction in mammals. Steroids
64:679-685
117. Bartness TJ, Goldman BD 1988 Effects of melatonin on long-day responses in short-
day housed adult Siberian hamsters. Am J Physiol 255:R823-R830
118. Steger RW, Bartke A, Goldman BD, Soares MJ, Talamantes F 1983 Effects of short
photoperiod on the ability of golden hamster pituitaries to secrete prolactin and
gonadotropins in vitro. Biol Reprod 29:872-878
119. Goldman BD, Darrow JM, Yogev L 1984 Effects of timed melatonin infusions on
reproductive development in the Djungarian hamster (Phodopus sungorus).
Endocrinology 114:2074-2083
120. Tamarkin L, Reppert SM, Klein DC 1979 Regulation of pineal melatonin in the Syrian
hamster. Endocrinology 104:385-389
121. Tamarkin L, Reppert SM, Klein DC, Pratt B, Goldman BD 1980 Studies on the daily
pattern of pineal melatonin in the Syrian hamster. Endocrinology 107:1525-1529
122. Moore RY 1991 The suprachiasmatic nucleus and the circadian timing system. In:
Suprachiasmatic nucleus: the mind's clock. Klein DC, Moore RY, Reppert SM (eds).
Oxford Press, New York 13-15
123. Kennaway DJ, Wright H 2002 Melatonin and circadian rhythms. Curr Top Med Chem
2:199-209
147
124. Teclemariam-Mesbah R, Ter Horst GJ, Postema F, Wortel J, Buijs RM 1999
Anatomical demonstration of the suprachiasmatic nucleus-pineal pathway. J Comp Neuro
406:171-182
125. Bittman EL, Bartness TJ, Goldman BD, deVries GJ 1991 Suprachiasmatic and
paraventricular control of photoperiodism in Siberian hamsters. Am J Physiol 260:R90-
101
126. Goldman BD 2001 Mammalian photoperiodic system: formal properties and
neuroendocrine mechanisms of photoperiodic time measurement. J Biol Rhythms 16:283-
301
127. Bartness TJ, Song CK, Demas GE 2001 SCN efferents to peripheral tissues:
implications for biological rhythms. J Biol Rhythms 16:196-204
128. Isobe Y, Fujioi J, Nishino H 2001 Circadian rhythm of melatonin release in pineal gland
culture: arg-vasopressin inhibits melatonin release. Brain Res 918:67-73
129. Isobe Y, Nishino H 2004 Signal transmission from the suprachiasmatic nucleus to the
pineal gland via the paraventricular nucleus: analysed from arg-vasopressin peptide,
rPer2 mRNA and AVP mRNA changes and pineal AA-NAT mRNA after the melatonin
injection during light and dark periods. Brain Res 1013:204-211
130. Kalsbeek A, Garidou ML, Palm IF, van d, V, Simonneaux V, Pevet P, Buijs RM
2000 Melatonin sees the light: blocking GABA-ergic transmission in the paraventricular
nucleus induces daytime secretion of melatonin. Euro J Neurosci 12:3146-3154
131. Barassin S, Kalsbeek A, Saboureau M, Bothorel B, Vivien-Roels B, Malan A, Buijs
RM, Pevet P 2000 Potentiation effect of vasopressin on melatonin secretion as
determined by trans-pineal microdialysis in the Rat. J Neuroendocrinol 12:61-68
132. Hunt AE, Al Ghoul WM, Gillette MU, Dubocovich ML 2001 Activation of MT (2)
melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian clock.
Am J Physiol Cell Physiol 280:C110-C118
133. Sugden D, Mcarthur AJ, Ajpru S, Duniec K, Piggins HD 1999 Expression of mt(1)
melatonin receptor subtype mRNA in the entrained rat suprachiasmatic nucleus: a
quantitative RT-PCR study across the diurnal cycle. Brain Res Mol Brain Res 72:176-
182
134. Weaver DR, Reppert SM 1996 The Mel1a melatonin receptor gene is expressed in
human suprachiasmatic nuclei. NeuroReport 8:109-112
148
135. Dubocovich ML, Benloucif S, Masana MI 1996 Melatonin receptors in the mammalian
suprachiasmatic nucleus. Behavioural Brain Res 73:141-147
136. Wittkowski W, Bockmann J, Kreutz MR, Böckers TM 1999 Cell and molecular
biology of the pars tuberalis of the pituitary. Interl Rev Cytol 185:157-194
137. Böckers TM, Bockmann J, Fauteck JD, Wittkowski W, Sabel BA, Kreutz MR 1996
Evidence for gene transcription of adenohypophyseal hormones in the ovine pars
tuberalis. Neuroendocrinology 63:16-27
138. Williams LM, Hannah LT, Hastings MH, Maywood ES 1995 Melatonin receptors in
the rat brain and pituitary. J Pineal Res 19:173-177
139. Guerra M, Rodriguez EM 2001 Identification, cellular and subcellular distribution of
21 and 72 kDa proteins (tuberalins?) secreted by specific cells of the pars tuberalis. J
Endocr 168:363-379
140. Thompson SA 1982 Localization of immunoreactive prolactin in ependyma and
circumventricular organs of rat brain. Cell Tissue Res 225:79-93
141. Pelletier J, Counis R, de Reviers M-M, Tillet Y 1992 Localization of luteinizing
hormone β-mRNA by in situ hybridization in the sheep pars tuberalis. Cell Tissue Res
267:301-306
142. Gross DS, Turgeon JL, Waring DW 1984 The Ovine Pars Tuberalis: A Naturally
Occurring Source of Partially Purified Gonadotropes which Secrete Luteinizing Hormone
in Vitro. Endocrinology 114:2084-2091
143. Morgan PJ, Webster CA, Mercer JG, Ross AW, Hazlerigg DG, MacLean A, Barrett
P 1996 The ovine pars tuberalis secretes a factor(s) that regulates gene expression in both
lactotropic and nonlactotropic pituitary cells. Endocrinology 137:4018-4026
144. Morgan PJ 2000 The pars tuberalis: the missing link in the photoperiodic regulation of
prolactin secretion? J Neuroendocrinol 12:287-295
145. Pevet P, Pitrosky B, Vuillez P, Jacob N, Teclemariam-Mesbah R, Kirsch R, Vivien-
Roels B, Lakhdar-Ghazal N, Canguilhem B, Masson-Pévet M 1996 The
suprachiasmatic nucleus: the biological clock of all seasons. In: Buijs RM, Kalsbeek A,
Romijn HJ, Pennartz CMA, Mirmiran M (eds). Progress in Brain Res 111:369-384
146. Moore-Ede MC, Moline ML 1985 Circadian rhythms and photoperiodism. Ciba Found
Symp 117:23-37
149
147. Lincoln GA, Andersson H, Loudon A 2003 Clock genes in calendar cells as the basis of
annual timekeeping in mammals--a unifying hypothesis. J Endocr 179:1-13
148. Legan SJ, Karsch FJ 1983 Importance of Retinal Photoreceptors to the Photoperiodic
Control of Seasonal Breeding in the Ewe. Biol Reprod 29:316-325
149. Lincoln GA, Clarke IJ 2002 Noradrenaline and dopamine regulation of prolactin
secretion in sheep: Role in prolactin homeostasis but not photoperiodism. Journal of
Neuroendocrinology 14:36-44
150. Bittman EL, Karsch FJ, Hopkins JW 1983 Role of the pineal gland in ovine
photoperiodism: regulation of seasonal breeding and negative feedback effects of
estradiol upon luteinizing hormone secretion. Endocrinology 113:329-336
151. Seamark RF, Kennaway DJ, Matthews CD, Fellenberg AJ, Phillipou G, Kotaras P,
McIntosh JE, Dunstan E, Obst JM 1981 The role of the pineal gland in seasonality. J
Reprod Fertil Suppl 30:15-21
152. Okano T, Fukada Y 2003 Chicktacking pineal clock. J Biochem (Tokyo) 134:791-797
153. Karolczak M, Burbach GJ, Sties G, Korf HW, Stehle JH 2004 Clock gene mRNA and
protein rhythms in the pineal gland of mice. Euro J Neurosci 19:3382-3388
154. Shieh KR 2003 Distribution of the rhythm-related genes rPERIOD1, rPERIOD2, and
rCLOCK, in the rat brain. Neurosci 118:831-843
155. Von Gall C, Garabette ML, Kell CA, Frenzel S, Dehghani F, Schumm-Draeger PM,
Weaver DR, Korf HW, Hastings MH, Stehle JH 2002 Rhythmic gene expression in
pituitary depends on heterologous sensitization by the neurohormone melatonin. Nat
Neurosci 5:234-238
156. Nelson RJ, Moffatt CA, Goldman BD 1994 Reproductive and nonreproductive
responsiveness to photoperiod in laboratory rats. J Pineal Res 17:123-131
157. Freeman ME, Smith MS, Nazian SJ, Neill JD 1974 Ovarian and hypothalamic control
of the daily surges of prolactin secretion during pseudopregnancy in the rat.
Endocrinology 94:875-882
158. Smith MS, Freeman ME, Neill JD 1975 The control of progesterone secretion during
the estrous cycle and early pseudopregnancy in the rat: Prolactin, gonadotropin and
steroid levels associated with rescue of the corpus luteum of pseudopregnancy.
Endocrinology 96:219-226
150
159. Arey BJ, Freeman ME 1991 Ontogeny of the endogenous stimulatory rhythm regulating
prolactin secretion in immature female rats. Endocrinology 128:1481-1484
160. Hoffman GE, Smith MS, Verbalis JG 1993 c-Fos and related immediate early gene
products as markers of activity in neuroendocrine systems. Front Neuroendocrinol
14:173-213
161. Hoffman GE, Lee WS, Smith MS, Abbud R, Roberts MM, Robinson AG, Verbalis
JG 1993 c-Fos and Fos-related antigens as markers for neuronal activity: perspectives
from neuroendocrine systems. NIDA Res Monogr 125:117-133
162. Lerant A, Freeman ME 1997 Dopaminergic neurons in periventricular and arcuate
nuclei of proestrous and ovariectomized rats: Endogenous diurnal rhythm of Fos-related
antigens expression. Neuroendocrinology 65:436-445
163. Mai LM, Shieh KR, Pan JT 1994 Circadian changes of serum prolactin levels and
tuberoinfundibular dopaminergic neuron activities in ovariectomized rats treated with or
without estrogen: the role of the suprachiasmatic nuclei. Neuroendocrinology 60:520-526
164. Shieh KR, Pan JT 1995 An endogenous cholinergic rhythm may be involved in the
circadian changes of tuberoinfundibular dopaminergic neuron activity in ovariectomized
rats treated with or without estrogen. Endocrinology 136:2383-2388
165. Shieh KR, Chu YS, Pan JT 1997 Circadian change of dopaminergic neuron activity:
Effects of constant light and melatonin. NeuroReport 8:2283-2287
166. Silver R, LeSauter J, Tresco PA, Lehman MN 1996 A diffusible coupling signal from
the transplanted suprachiasmatic nucleus controlling circadian locomotor rhythms.
Nature 382:810-813
167. Lehman MN, Silver R, Gladstone WR, Kahn RM, Gibson M, Bittman EL 1987
Circadian rhythmicity restored by neural transplant. Immunocytochemicl characterization
of the graft and its integration with the host brain. J Neurosci 7:1626-1638
168. Meyer-Bernstein EL, Jetton AE, Matsumoto SI, Markuns JF, Lehman MN, Bittman
EL 1999 Effects of suprachiasmatic transplants on circadian rhythms of neuroendocrine
function in golden hamsters. Endocrinology 140:207-218
169. Kalsbeek A, Teclemariam-Mesbah R, Pévet P 1993 Efferent projections of the
suprachiasmatic nucleus in the golden hamster (Mesocricetus auratus). J Comp Neuro
332:293-314
151
170. Kalsbeek A, Buijs RM 2002 Output pathways of the mammalian suprachiasmatic
nucleus: coding circadian time by transmitter selection and specific targeting. Cell Tissue
Res 309:109-118
171. Horvath TL 1997 Suprachiasmatic efferents avoid phenestrated capillaries but innervate
neuroendocrine cells, including those producing dopamine. Endocrinology 138:1312-
1320
172. Gerhold L.M., Horvath TL, Freeman ME 2001 Vasoactive intestinal peptide fibers
innervate neuroendocrine dopaminergic neurons. Brain Res 919:48-56
173. Duncan MJ, Cheng XR, Heller KS 1995 Photoperiodic exposure and time of day
modulate the expression of arginine vasopressin mRNA and vasoactive intestinal peptide
mRNA in the suprachiasmatic nuclei of Siberian hamsters. Mol Brain Res 32:181-186
174. Duncan MJ, Herron JM, Hill SA 2001 Aging selectively suppresses vasoactive
intestinal peptide messenger RNA expression in the suprachiasmatic nucleus of the
Syrian hamster. Mol Brain Res 87:196-203
175. Inouye ST, Shibata S 1994 Neurochemical organization of circadian rhythm in the
suprachiasmatic nucleus. Neurosci Res 20:109-130
176. Isobe Y, Muramatsu K 1995 Day-night differences in the contents of vasoactive
intestinal peptide, gastrin-releasing peptide and Arg-vasopressin in the suprachiasmatic
nucleus of rat pups during postnatal development. Neurosci Lett 188:45-48
177. Okamura H, Kawakami F, Tamada Y, Geffard M, Nishiwaki T, Ibata Y, Inouye S-
IT 1995 Circadian change of VIP mRNA in the rat suprachiasmatic nucleus following p-
chlorophenylalanine (PCPA) treatment in constant darkness. Mol Brain Res 29:358-364
178. Shinohara K, Funabashi T, Kimura F 1999 Temporal profiles of vasoactive intestinal
polypeptide precursor mRNA and its receptor mRNA in the rat suprachiasmatic nucleus.
Brain Res Mol Brain Res 63:262-267
179. Krajnak K, Kashon ML, Rosewell KL, Wise PM 1998 Sex differences in the daily
rhythm of vasoactive intestinal polypeptide but not arginine vasopressin messenger
ribonucleic acid in the suprachiasmatic nuclei. Endocrinology 139:4189-4196
180. Krajnak K, Kashon ML, Rosewell KL, Wise PM 1998 Aging alters the rhythmic
expression of vasoactive intestinal polypeptide mRNA but not arginine vasopressin
mRNA in the suprachiasmatic nuclei of female rats. J Neurosci 18:4767-4774
152
181. Kriegsfeld LJ, Silver R, Gore AC, Crews D 2002 Vasoactive intestinal polypeptide
contacts on gonadotropin-releasing hormone neurones increase following puberty in
female rats. J Neuroendocrinol 14:685-690
182. Sheward WJ, Lutz EM, Harmar AJ 1995 The distribution of vasoactive intestinal
peptide2 receptor messenger RNA in the rat brain and pituitary gland as assessed by in
situ hybridization. Neurosci 67:409-418
183. Kalamatianos T, Kallo I, Piggins HD, Coen CW 2004 Expression of VIP and/or
PACAP receptor mRNA in peptide synthesizing cells within the suprachiasmatic nucleus
of the rat and in its efferent target sites. J Comp Neurol 475:19-35
184. Kallo I, Kalamatianos T, Wiltshire N, Shen S, Sheward WJ, Harmar AJ, Coen CW
2004 Transgenic approach reveals expression of the VPAC2 receptor in phenotypically
defined neurons in the mouse suprachiasmatic nucleus and in its efferent target sites.
Euro J Neurosci 19:2201-2211
185. Saeb-Parsy K, Lombardelli S, Khan FZ, McDowall K, Au-Yong IT, Dyball RE 2000
Neural connections of hypothalamic neuroendocrine nuclei in the rat. J Neuroendocrinol
12:635-648
186. Ben Jonathan N, Hnasko R 2001 Dopamine as a prolactin (PRL) inhibitor. Endocr Rev
22:724-763
187. Harney JP, Scarbrough K, Rosewell KL, Wise PM 1996 In vivo antisense antagonism
of vasoactive intestinal peptide in the suprachiasmatic nuclei causes aging-like changes in
the estradiol-induced luteinizing hormone and prolactin surges. Endocrinology 137:3696-
3701
188. Gerhold LM, Sellix MT, Freeman ME 2002 Antagonism of vasoactive intestinal
peptide mRNA in the suprachiasmatic nucleus disrupts the rhythm of FRAs expression in
neuroendocrine dopaminergic neurons. J Comp Neurol 450:135-143
189. Harmar T, Lutz E 1994 Multiple receptors for PACAP and VIP. Trends Pharmacol Sci
15:97-99
190. Laburthe M, Couvineau A, Marie JC 2002 VPAC receptors for VIP and PACAP.
Receptors Channels 8:137-153
191. Kalsbeek A, Van Heerikhuize JJ, Wortel J, Buijs RM 1996 A diurnal rhythm of
stimulatory input to the hypothalamo-pituitary-adrenal system as revealed by timed
intrahypothalamic administration of the vasopressin V1 antagonist. J Neurosci 16:5555-
5565
153
192. Hermes ML, Ruijter JM, Klop A, Buijs RM, Renaud LP 2000 Vasopressin increases
GABAergic inhibition of rat hypothalamic paraventricular nucleus neurons in vitro. J
Neurophys 83:705-711
193. Turek FW, Pinto LH, Vitaterna MH, Penev PD, Zee PC, Takahashi JS 1995
Pharmacological and genetic approaches for the study of circadian rhythms in mammals.
Front Neuroendocrinol 16:191-223
194. Konopka RJ, Benzer S 1971 Clock mutants of Drosophila melanogaster. Proc Natl
Acad Sci U S A 68:2112-2116
195. Frisch B, Hardin PE, Hamblen-Coyle MJ, Rosbash M, Hall JC 1994 A promoterless
period gene mediates behavioral rhythmicity and cyclical per expression in a restricted
subset of the Drosophila nervous system. Neuron 12:555-570
196. Zwiebel LJ, Hardin PE, Liu X, Hall JC, Rosbash M 1991 A post-transcriptional
mechanism contributes to circadian cycling of a per-beta-galactosidase fusion protein.
Proc Natl Acad Sci U S A 88:3882-3886
197. Zwiebel LJ, Hardin PE, Hall JC, Rosbash M 1991 Circadian oscillations in protein
and mRNA levels of the period gene of Drosophila melanogaster. Biochem Soc Trans
19:533-537
198. King DP, Zhao Y, Sangoram AM, Wilsbacher LD, Tanaka M, Antoch MP, Steeves
TD, Vitaterna MH, Kornhauser JM, Lowrey PL, Turek FW, Takahashi JS 1997
Positional cloning of the mouse circadian clock gene. Cell 89:641-653
199. King DP, Takahashi JS 2000 Molecular genetics of circadian rhythms in mammals.
Annu Rev Neurosci 23:713-742
200. Steeves TDL, King DP, Zhao YL, Sangoram AM, Du FH, Bowcock AM, Moore RY,
Takahashi JS 1999 Molecular cloning and characterization of the human clock gene:
Expression in the suprachiasmatic nuclei. Genomics 57:189-200
201. Ralph MR, Menaker M 1988 A mutation of the circadian system in golden hamsters.
Science 241:1225-1227
202. Chang DC, Reppert SM 2001 The circadian clocks of mice and men. Neuron 29:555-
558
203. Reppert SM, Weaver DR 2000 Comparing clockworks: mouse versus fly. J Biol
Rhythms 15:357-364
154
204. Reppert SM, Weaver DR 2002 Coordination of circadian timing in mammals. Nature
418:935-941
205. Shearman LP, Sriram S, Weaver DR, Maywood ES, Chaves I, Zheng B, Kume K,
Lee CC, van der Horst GT, Hastings MH, Reppert SM 2000 Interacting molecular
loops in the mammalian circadian clock. Science 288:1013-1019
206. Reppert SM, Weaver DR 2001 Molecular analysis of mammalian circadian rhythms.
Annu Rev Physiol 63:647-676
207. Emery P, Reppert SM 2004 A rhythmic Ror. Neuron 43:443-446
208. Kume K, Zylka MJ, Sriram S, Shearman LP, Weaver DR, Jin X, Maywood ES,
Hastings MH, Reppert SM 1999 mCRY1 and mCRY2 are essential components of the
negative limb of the circadian clock feedback loop. Cell 98:193-205
209. Shearman LP, Zylka MJ, Weaver DR, Kolakowski LF, Jr., Reppert SM 1997 Two
period homologs: circadian expression and photic regulation in the suprachiasmatic
nuclei. Neuron 19:1261-1269
210. Bae K, Jin XW, Maywood ES, Hastings MH, Reppert SM, Weaver DR 2001
Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock. Neuron
30:525-536
211. Nakajima Y, Ikeda M, Kimura T, Honma S, Ohmiya Y, Honma K 2004 Bidirectional
role of orphan nuclear receptor RORalpha in clock gene transcriptions demonstrated by a
novel reporter assay system. FEBS Lett 565:122-126
212. Sato TK, Panda S, Miraglia LJ, Reyes TM, Rudic RD, McNamara P, Naik KA,
FitzGerald GA, Kay SA, Hogenesch JB 2004 A functional genomics strategy reveals
Rora as a component of the mammalian circadian clock. Neuron 43:527-537
213. Smolen P, Hardin PE, Lo BS, Baxter DA, Byrne JH 2004 Simulation of Drosophila
circadian oscillations, mutations, and light responses by a model with VRI, PDP-1, and
CLK. Biophys J 86:2786-2802
214. Preitner N, Damiola F, Lopez-Molina L, Zakany J, Duboule D, Albrecht U, Schibler
U 2002 The orphan nuclear receptor REV-ERBalpha controls circadian transcription
within the positive limb of the mammalian circadian oscillator. Cell 110:251-260
215. Kawamoto T, Noshiro M, Sato F, Maemura K, Takeda N, Nagai R, Iwata T,
Fujimoto K, Furukawa M, Miyazaki K, Honma S, Honma K, Kato Y 2004 A novel
155
autofeedback loop of Dec1 transcription involved in circadian rhythm regulation.
Biochem Biophys Res Commun 313:117-124
216. Bao S, Rihel J, Bjes E, Fan JY, Price JL 2001 The Drosophila double-times mutation
delays the nuclear accumulation of period protein and affects the feedback regulation of
period mRNA. J Neurosci 21:7117-7126
217. Eide EJ, Vielhaber EL, Hinz WA, Virshup DM 2002 The circadian regulatory proteins
BMAL1 and cryptochromes are substrates of casein kinase Iepsilon. J Biol Chem
277:17248-17254
218. Eide EJ, Virshup DM 2001 Casein kinase I: another cog in the circadian clockworks.
Chronobiol Int 18:389-398
219. Kloss B, Price JL, Saez L, Blau J, Rothenfluh A, Wesley CS, Young MW 1998 The
Drosophila clock gene double-time encodes a protein closely related to human casein
kinase Iepsilon. Cell 94:97-107
220. Lee C, Weaver DR, Reppert SM 2004 Direct association between mouse PERIOD and
CKIepsilon is critical for a functioning circadian clock. Mol Cell Biol 24:584-594
221. Lee C, Etchegaray JP, Cagampang FR, Loudon AS, Reppert SM 2001
Posttranslational mechanisms regulate the mammalian circadian clock. Cell 107:855-867
222. Lowrey PL, Shimomura K, Antoch MP, Yamazaki S, Zemenides PD, Ralph MR,
Menaker M, Takahashi JS 2000 Positional syntenic cloning and functional
characterization of the mammalian circadian mutation tau. Science 288:483-492
223. Vielhaber E, Eide E, Rivers A, Gao ZH, Virshup DM 2000 Nuclear entry of the
circadian regulator mPER1 is controlled by mammalian casein kinase I epsilon. Mol Cell
Biol 20:4888-4899
224. Vielhaber E, Virshup DM 2001 Casein kinase I: from obscurity to center stage. IUBMB
Life 51:73-78
225. Whitmore D, Cermakian N, Crosio C, Foulkes NS, Pando MP, Travnickova Z,
Sassone-Corsi P 2000 A clockwork organ. Bio Chem 381:793-800
226. Keesler GA, Camacho F, Guo Y, Virshup D, Mondadori C, Yao Z 2000
Phosphorylation and destabilization of human period I clock protein by human casein
kinase I epsilon. NeuroReport 11:951-955
156
227. Takano A, Uchiyama M, Kajimura N, Mishima K, Inoue Y, Kamei Y, Kitajima T,
Shibui K, Katoh M, Watanabe T, Hashimotodani Y, Nakajima T, Ozeki Y, Hori T,
Yamada N, Toyoshima R, Ozaki N, Okawa M, Nagai K, Takahashi K, Isojima Y,
Yamauchi T, Ebisawa T 2004 A missense variation in human casein kinase I epsilon
gene that induces functional alteration and shows an inverse association with circadian
rhythm sleep disorders. Neuropsychopharm 29:1901-9.
228. Toh KL, Jones CR, He Y, Eide EJ, Hinz WA, Virshup DM, Ptacek LJ, Fu YH 2001
An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome.
Science 291:1040-1043
229. Pan JT, Gala RR 1985 Central nervous system regions involved in the estrogen-induced
afternoon prolactin surge. I. Lesion studies. Endocrinology 117:382-387
230. Pittendrigh CS, Daan S 1974 Circadian oscillations in rodents: a systematic increase of
their frequency with age. Science 186:548-550
231. Mikkelsen JD, Vrang N, Mrosovsky N 1998 Expression of Fos in the circadian system
following nonphotic stimulation. Brain Res Bull 47:367-376
232. Mrosovsky N 1999 Critical assessment of methods and concepts in nonphotic phase
shifting. Bio Rhythm Res 30:135-148
233. Freeman ME, Sterman JR 1978 Ovarian steroid modulation of prolactin surges in
cervically-stimulated ovariectomized rats. Endocrinology 102:1915-1920
234. Eichler VB, Moore RY 1971 Pineal hydroxyindole-O-methyltransferase and gonadal
responses to blinding or continuous darkness blocked by pineal denervation in the male
hamster. Neuroendocrinology 8:81-85
235. Stephan FK, Zucker I 1972 Rat drinking rhythms: central visual pathways and
endocrine factors mediating responsiveness to environmental illumination. Physiol Behav
8:315-326
236. Zucker I, Stephan FK 1973 Light-dark rhythms in hamster eating, drinking and
locomotor behaviors. Physiol Behav 11:239-250
237. Stephan FK, Zucker I 1974 Endocrine and neural mediation of the effects of constant
light on water intake of rats. Neuroendocrinology 14:44-60
238. Stephan FK, Nunez AA 1977 Elimination of circadian rhythms in drinking, activity,
sleep, and temperature by isolation of the suprachiasmatic nuclei. Behav Biol 20:1-61
157
239. Stephan FK 1983 Circadian rhythm dissociation induced by periodic feeding in rats with
suprachiasmatic lesions. Behav Brain Res 7:81-98
240. Kochavi D, Davis JD, Smith GP 2001 Corticotropin-releasing factor decreases meal
size by decreasing cluster number in Koletsky (LA/N) rats with and without a null
mutation of the leptin receptor. Physiol Behav 74:645-651
241. Clarke SN, Bernstein IL 2001 NaCl preference increases during pregnancy and
lactation: assessment using brief access tests. Pharmacol Biochem Behav 68:555-563
242. Lindley SE, Gunnet JW, Lookingland KJ, Moore KE 1990 3,4-
Dihydroxyphenylacetic acid concentrations in the intermediate lobe and neural lobe of
the posterior pituitary gland as an index of tuberohypophysial dopaminergic neuronal
activity. Brain Res 506:133-138
243. Moore RY, Eichler VB 1976 Central neural mechanisms in diurnal rhythm regulation
and neuroendocrine responses to light. Psychoneuroendocrinology 1:265-279
244. DeMaria JE, Nagy GM, Freeman ME 2000 Immunoneutralization of prolactin
prevents stimulatory feedback of prolactin on hypothalamic neuroendocrine
dopaminergic neurons. Endocrine 12:333-337
245. Abe M, Herzog ED, Yamazaki S, Straume M, Tei H, Sakaki Y, Menaker M, Block
GD 2002 Circadian rhythms in isolated brain regions. J Neurosci 22:350-356
246. Taylor MM, Samson WK 2001 The prolactin releasing peptides: RF-amide peptides.
Cell Mol Life Sci 58:1206-1215
247. Samson WK, Taylor MM, Baker JR 2003 Prolactin-releasing peptides. Regul Pept
114:1-5
248. Toth BE, Homicsko K, Radnai B, Maruyama W, DeMaria JE, Vecsernyes M,
Fekete MI, Fulop F, Naoi M, Freeman ME, Nagy GM 2001 Salsolinol is a putative
endogenous neuro-intermediate lobe prolactin-releasing factor. J Neuroendocrinol
13:1042-1050
249. Lerant AA, DeMaria JE, Freeman ME 2001 Decreased expression of fos-related
antigens (FRAs) in the hypothalamic dopaminergic neurons after immunoneutralization
of endogenous prolactin. Endocrine 16:181-187
250. Kawakami M, Arita J, Yoshioka E 1980 Loss of estrogen-induced daily surges of
prolactin and gonadotropins by suprachiasmatic nucleus lesions in ovariectomized rats.
Endocrinology 106:1087-1092
158
251. Shieh KR, Pan JT 1996 Sexual differences in the diurnal changes of tuberoinfundibular
dopaminergic neuron activity in the rat: role of cholinergic control. Biol Reprod 54:987-
992
252. Yang S-P, Lee Y, Voogt JL 1999 Fos Expression in the female Rat Brain during the
Proestrous Prolactin surge and Following mating. Neuroendocrinology 69:281-289
253. Huang SK, Pan JT 1996 Stimulatory effects of vasoactive intestinal peptide and
pituitary adenylate cyclase-activating peptide on tuberoinfundibular dopaminergic neuron
activity in estrogen- treated ovariectomized rats and their correlation with prolactin
secretion. Neuroendocrinology 64:208-214
254. Lerant A, Freeman ME 1998 Ovarian steroids differentially regulate the expression of
PRL-R in neuroendocrine dopaminergic neuron populations: a double label confocal
microscopic study. Brain Res 802:141-154
255. Lookingland KJ, Jarry HD, Moore KE 1987 The metabolism of dopamine in the
median eminence reflects the activity of tuberoinfundibular neurons. Brain Res 419:303-
310
256. Banks JA, Mick C, Freeman ME 1980 A possible cause for the differing response of
the LH surge mechanism of ovariectomized rats to short-term exposure to estradiol.
Endocrinology 106:1677-1691
257. Shors TJ, Pickett J, Wood G, Paczynski M 1999 Acute stress persistently enhances
estrogen levels in the female rat. Stress 3:163-171
258. Miller MM, Silver J, Billiar RB 1984 Effects of gonadal steroids on the in vivo binding
of [125I]alpha-bungarotoxin to the suprachiasmatic nucleus. Brain Res 290:67-75
259. Shinohara K, Honma S, Katsuno Y, Abe H, Honma K 1995 Two distinct oscillators in
the rat suprachiasmatic nucleus in vitro. Proc Natl Acad Sci U S A 92:7396-7400
260. Arbogast LA, Voogt JL 1991 Mechanisms of tyrosine hydroxylase regulation during
pregnancy: Evidence for protein dephosphorylation during the prolactin surges.
Endocrinology 129:2575-2582
261. Krajnak K, Rosewell KL, Wise PM 2001 Fos-induction in gonadotropin-releasing
hormone neurons receiving vasoactive intestinal polypeptide innervation is reduced in
middle-aged female rats. Biol Reprod 64:1160-1164
159
262. Palm IF, Van der Beek EM, Swarts HJ, van d, V, Wiegant VM, Buijs RM, Kalsbeek
A 2001 Control of the estradiol-induced prolactin surge by the suprachiasmatic nucleus.
Endocrinology 142:2296-2302
263. Yamazaki S, Numano R, Abe M, Hida A, Takahashi R, Ueda M, Block GD, Sakaki
Y, Menaker M, Tei H 2000 Resetting central and peripheral circadian oscillators in
transgenic rats. Science 288:682-685
264. Wilsbacher LD, Yamazaki S, Herzog ED, Song EJ, Radcliffe LA, Abe M, Block G,
Spitznagel E, Menaker M, Takahashi JS 2002 Photic and circadian expression of
luciferase in mPeriod1-luc transgenic mice invivo. Proc Natl Acad Sci U S A 99:489-494
265. Morin LP, Fitzgerald KM, Zucker I 1977 Estradiol shortens the period of hamster
circadian rhythms. Science 196:305-307
266. Morin LP 1980 Effect of ovarian hormones on synchrony of hamster circadian rhythms.
Physiol Behav 24:741-749
267. Gonze D, Halloy J, Leloup JC, Goldbeter A 2003 Stochastic models for circadian
rhythms: effect of molecular noise on periodic and chaotic behaviour. C R Biol 326:189-
203
268. Kennaway DJ, Rowe SA, Ferguson SA 1996 Serotonin agonists mimic the phase
shifting effects of light on the melatonin rhythm in rats. Brain Res 737:301-307
269. Kim DY, Kang HC, Shin HC, Lee KJ, Yoon YW, Han HC, Na HS, Hong SK, Kim
YI 2001 Substance p plays a critical role in photic resetting of the circadian pacemaker in
the rat hypothalamus. J Neurosci 21:4026-4031
270. Meijer JH, Van der Zee EA, Dietz M 1988 Glutamate phase shifts circadian activity
rhythms in hamsters. Neurosci Lett 86:177-183
271. Mintz EM, Marvel CL, Gillespie CF, Price KM, Albers HE 1999 Activation of
NMDA receptors in the suprachiasmatic nucleus produces light-like phase shifts of the
circadian clock in vivo. J Neurosci 19:5124-5130
272. Miyake S, Sumi Y, Yan L, Takekida S, Fukuyama T, Ishida Y, Yamaguchi S, Yagita
K, Okamura H 2000 Phase-dependent responses of Per1 and Per2 genes to a light-
stimulus in the suprachiasmatic nucleus of the rat. Neurosci Lett 294:41-44
273. Van Gelder RN, Gibler TM, Tu D, Embry K, Selby CP, Thompson CL, Sancar A
2002 Pleiotropic effects of cryptochromes 1 and 2 on free-running and light-entrained
murine circadian rhythms. Journal of Neurogenetics 16:181-203
160
274. Wee R, Castrucci AM, Provencio I, Gan L, Van Gelder RN 2002 Loss of photic
entrainment and altered free-running circadian rhythms in math5-/- mice. J Neurosci
22:10427-10433
275. de Groot MH, Rusak B 2002 Entrainment impaired, masking spared: an apparent
genetic abnormality that prevents circadian rhythm entrainment to 24-h lighting cycles in
California mice. Neurosci Lett 327:203-207
276. Boer GJ, Griffioen HA, Duindam H, Van der Woude TP, Rietveld WJ 1993
Light/dark-induced effects on behavioral rhythms in suprachiasmatic nucleus-lesioned
rats irrespective of the presence of functional suprachiasmatic nucleus brain implants. J
Interdis Cycle Res 24:118-136
277. Kriegsfeld LJ, Korets R, Silver R 2003 Expression of the circadian clock gene Period 1
in neuroendocrine cells: an investigation using mice with a Per1::GFP transgene. Eur J
Neurosci 17:212-220
278. Granados-Fuentes D, Saxena MT, Prolo LM, Aton SJ, Herzog ED 2004 Olfactory
bulb neurons express functional, entrainable circadian rhythms. Eur J Neurosci 19:898-
906
279. Granados-Fuentes D, Prolo LM, Abraham U, Herzog ED 2004 The suprachiasmatic
nucleus entrains, but does not sustain, circadian rhythmicity in the olfactory bulb. J
Neurosci 24:615-619
280. Nagoshi E, Saini C, Bauer C, Laroche T, Naef F, Schibler U 2004 Circadian gene
expression in individual fibroblasts; cell-autonomous and self-sustained oscillators pass
time to daughter cells. Cell 119:693-705
281. Travnickova-Bendova Z, Cermakian N, Reppert SM, Sassone-Corsi P 2002 Bimodal
regulation of mPeriod promoters by CREB-dependent signaling and CLOCK/BMAL1
activity. Proc Natl Acad Sci U S A
282. Gachon F, Nagoshi E, Brown SA, Ripperger J, Schibler U 2004 The mammalian
circadian timing system: from gene expression to physiology. Chromosoma 113:103-112
283. Guillaumond F, Sage D, Deprez P, Bosler O, Becquet D, Francois-Bellan AM 2000
Circadian binding activity of AP-1, a regulator of the arylalkylamine N-acetyltransferase
gene in the rat pineal gland, depends on circadian Fra-2, c-Jun, and Jun-D expression and
is regulated by the clock's zeitgebers. J Neurochem 75:1398-1407
161
284. Liu J, Merlie JP, Todd RD, O'Malley KL 1997 Identification of cell type-specific
promoter elements associated with the rat tyrosine hydroxylase gene using transgenic
founder analysis. Brain Res Mol Brain Res 50:33-42
285. Dardente H, Poirel VJ, Klosen P, Pevet P, Masson-Pevet M 2002 Per and
neuropeptide expression in the rat suprachiasmatic nuclei: compartmentalization and
differential cellular induction by light. Brain Res 958:261-271
286. Ibata Y, Okamura H, Tanaka M, Tamada Y, Hayashi S, Iijima N, Matsuda T,
Munekawa K, Takamatsu T, Hisa Y, Shigeyoshi Y, Amaya F 1999 Functional
morphology of the suprachiasmatic nucleus. Front Neuroendocrinol 20:241-268
287. Yan L, Miyake S, Okamura H 2000 Distribution and circadian expression of dbp in
SCN and extra-SCN areas in the mouse brain. J Neurosci Res 59:291-295
288. Watson RE, Wiegand SJ, Clough RW, Hoffman GE 1986 Use of cryoprotectant to
maintain longterm peptide immunoreactivity and tissue morphology. Peptides 7:155-163
289. Field MD, Maywood ES, O'Brien JA, Weaver DR, Reppert SM, Hastings MH 2000
Analysis of clock proteins in mouse SCN demonstrates phylogenetic divergence of the
circadian clockwork and resetting mechanisms. Neuron 25:437-447
290. Moore RY, Silver R 1998 Suprachiasmatic nucleus organization. Chronobiol Int 15:475-
487
291. Vitaterna MH, King DP, Chang A-M, Kornhauser JM, Lowrey PL, McDonald JD,
Dove WF, Pinto LH, Turek FW, Takahashi JS 1994 Mutagenesis and mapping of a
mouse gene, Clock, essential for circadian behavior. Science 264:719-725
292. Honma S, Ikeda M, Abe H, Tanahashi Y, Namihira M, Honma K, Nomura M 1998
Circadian oscillation of BMAL1, a partner of a mammalian clock gene Clock, in rat
suprachiasmatic nucleus. Biochem Biophys Res Commun 250:83-87
293. Maywood ES, O'Brien JA, Hastings MH 2003 Expression of mCLOCK and other
circadian clock-relevant proteins in the mouse suprachiasmatic nuclei. J Neuroendocrinol
15:329-334
294. Isojima Y, Okumura N, Nagai K 2003 Molecular mechanism of mammalian circadian
clock. J Biochem (Tokyo) 134:777-784
295. Hastings MH, Field MD, Maywood ES, Weaver DR, Reppert SM 1999 Differential
regulation of mPER1 and mTIM proteins in the mouse suprachiasmatic nuclei: new
insights into a core clock mechanism. J Neurosci 19:RC11
162
296. Hastings MH, Reddy AB, Garabette M, King VM, Chahad-Ehlers S, O'Brien J,
Maywood ES 2003 Expression of clock gene products in the suprachiasmatic nucleus in
relation to circadian behaviour. Novartis Found Symp 253:203-217
297. Kruijver FP, Swaab DF 2002 Sex hormone receptors are present in the human
suprachiasmatic nucleus. Neuroendocrinology 75:296-305
298. Mitrofanis J, Ashkan K, Wallace BA, Benabid AL 2004 Chemoarchitectonic
heterogeneities in the primate zona incerta: Clinical and functional implications. J
Neurocyto 33:429-440
299. Mitrofanis J 2005 Some certainty for the "zone of uncertainty"? Exploring the function
of the zona incerta. Neurosci 130:1-15
300. Power BD, Mitrofanis J 2002 Ultrastructure of afferents from the zona incerta to the
posterior and parafascicular thalamic nuclei of rats. J Comp Neurol 451:33-44
301. Power BD, Leamey CA, Mitrofanis J 2001 Evidence for a visual subsector within the
zona incerta. Vis Neurosci 18:179-186
302. Field MD, Maywood ES, O'Brien JA, Weaver DR, Reppert SM, Hastings MH 2000
Analysis of clock proteins in mouse SCN demonstrates phylogenetic divergence of the
circadian clockwork and resetting mechanisms. Neuron 25:437-447
303. Okamura H, Kawakami F, Tamada Y, Geffard M, Nishiwaki T, Ibata Y, Inouye ST
1995 Circadian change of VIP mRNA in the rat suprachiasmatic nucleus following p-
chlorophenylalanine (PCPA) treatment in constant darkness. Brain Res Mol Brain Res
29:358-364
304. Ban Y, Shigeyoshi Y, Okamura H 1997 Development of vasoactive intestinal peptide
mRNA rhythm in the rat suprachiasmatic nucleus. J Neurosci 17:3920-3931
305. Piggins HD, Cutler DJ 2003 The roles of vasoactive intestinal polypeptide in the
mammalian circadian clock. J Endocr 177:7-15
306. Yang J, Cagampang FRA, Nakayama Y, Inouye S-IT 1993 Vasoactive intestinal
polypeptide precursor mRNA exhibits diurnal variation in the rat suprachiasmatic nuclei.
Mol Brain Res 20:259-262
307. Shinohara K, Honma S, Katsuno Y, Abe H, Honma K 1994 Circadian rhythms in the
release of vasoactive intestinal polypeptide and arginine-vasopressin in organotypic slice
culture of rat suprachiasmatic nucleus. Neurosci Lett 170:183-186
163
308. Shinohara K, Tominaga K, Inouye SIT 1999 Phase dependent response of vasoactive
intestinal polypeptide to light and darkness in the suprachiasmatic nucleus. Neurosci Res
33:105-110
309. Shinohara K, Tominaga K, Isobe Y, Inouye S-IT 1993 Photic regulation of peptides
located in the ventrolateral subdivision of the suprachiasmatic nucleus of the rat: Daily
variations of vasoactive intestinal polypeptide, gastrin- releasing peptide, and
neuropeptide Y. J Neurosci 13:793-800
310. Nielsen HS, Hannibal J, Fahrenkrug J 2002 Vasoactive intestinal polypeptide induces
per1 and per2 gene expression in the rat suprachiasmatic nucleus late at night. European
J Neurosci 15:570-574
311. Laburthe M, Couvineau A 2002 Molecular pharmacology and structure of VPAC
receptors for VIP and PACAP. Reg Peptides 108:165-173
312. Hannibal J, Fahrenkrug J 2002 Melanopsin: a novel photopigment involved in the
photoentrainment of the brain's biological clock? Ann Med 34:401-407
313. Ruby NF, Brennan TJ, Xie X, Cao V, Franken P, Heller HC, O'Hara BF 2002 Role
of melanopsin in circadian responses to light. Science 298:2211-2213
314. Gooley JJ, Lu J, Chou TC, Scammell TE, Saper CB 2001 Melanopsin in cells of
origin of the retinohypothalamic tract. Nat Neurosci 4:1165
315. Barnes JW, Tischkau SA, Barnes JA, Mitchell JW, Burgoon PW, Hickok JR,
Gillette MU 2003 Requirement of mammalian Timeless for circadian rhythmicity.
Science 302:439-442
316. Hurst WJ, Earnest D, Gillette MU 2002 Immortalized suprachiasmatic nucleus cells
express components of multiple circadian regulatory pathways. Biochem Biophys Res
Comm 292:20-30
317. Prosser RA, Gillette MU 1989 The mammalian circadian clock in the suprachiasmatic
nuclei is reset in vitro by cAMP. J Neurosci 9:1073-1081
318. Shearman LP, Jin X, Lee C, Reppert SM, Weaver DR 2000 Targeted disruption of the
mPer3 gene: subtle effects on circadian clock function. Mol Cell Biol 20:6269-6275
319. Dioum EM, Rutter J, Tuckerman JR, Gonzalez G, Gilles-Gonzalez MA, McKnight
SL 2002 NPAS2: a gas-responsive transcription factor. Science 298:2385-2387
164
320. Dudley CA, Erbel-Sieler C, Estill SJ, Reick M, Franken P, Pitts S, McKnight SL
2003 Altered patterns of sleep and behavioral adaptability in NPAS2-deficient mice.
Science 301:379-383
321. Garcia JA, Zhang D, Estill SJ, Michnoff C, Rutter J, Reick M, Scott K, Diaz-
Arrastia R, McKnight SL 2000 Impaired cued and contextual memory in NPAS2-
deficient mice. Science 288:2226-2230
322. Reick M, Garcia JA, Dudley C, McKnight SL 2001 NPAS2: an analog of clock
operative in the mammalian forebrain. Science 293:506-509
323. Rutter J, Reick M, Wu LC, McKnight SL 2001 Regulation of clock and NPAS2 DNA
binding by the redox state of NAD cofactors. Science 293:510-514
324. Akiyama M, Kouzu Y, Takahashi S, Wakamatsu H, Moriya T, Maetani M,
Watanabe S, Tei H, Sakaki Y, Shibata S 1999 Inhibition of light- or glutamate-induced
mPer1 expression represses the phase shifts into the mouse circadian locomotor and
suprachiasmatic firing rhythms. J Neurosci 19:1115-1121
325. Wakamatsu H, Takahashi S, Moriya T, Inouye ST, Okamura H, Akiyama M,
Shibata S 2001 Additive effect of mPer1 and mPer2 antisense oligonucleotides on light-
induced phase shift. NeuroReport 12:127-131
326. Chew LJ, Seah V, Murphy D, Carter D 1996 Anterior pituitary vasoactive intestinal
peptide mRNA is colocalised with prolactin mRNA in hyperoestrogenised rats. J Mol
Endocr 16:211-220
327. Oliva D, Vallar L, Giannattasio G, Spada A, Nicosia S 1984 Combined Effects of
Vasoactive Intestinal Peptide and Dopamine on Adenylate Cyclase in Prolactin-Secreting
Cells. Peptides 5:1067-1070
328. Köves K, Chen IL, Görcs TJ, Scammell JG, Arimura A 1996 Different ultrastructural
localization of VIP and prolactin in anterior pituitary cells of rats chronically treated with
estrogen. Endocrine 5:219-223
329. Lasaga M, Debeljuk L, Afione S, Aleman IT, Duvilanski B 1989 Effects of passive
immunization against vasoactive intestinal peptide on serum prolactin and LH levels.
Neuroendocrinology 49:574-579
330. Samson WK, Said SI, Snyder G, McCann SM 1980 In vitro stimulation of prolactin
release by vasoactive intestinal peptide. Peptides 1:325-332
165
331. Shimatsu A, Kato Y, Ohta H, Toyo K, Kabayama Y, Inoue T, Yanaihara N, Imura
H 1984 Involvement of hypothalamic vasoactive intestinal polypeptide (VIP) in prolactin
secretion induces by serotonin in rats. Proc Soc Exp Bio Med 175:414-416
332. Cui LN, Saeb-Parsy K, Dyball RE 1997 Neurones in the supraoptic nucleus of the rat
are regulated by a projection from the suprachiasmatic nucleus. J Physiol 502:149-159
333. Buijs RM, Wortel J, Van Heerikhuize JJ, Feenstra MG, Ter Horst GJ, Romijn HJ,
Kalsbeek A 1999 Anatomical and functional demonstration of a multisynaptic
suprachiasmatic nucleus adrenal (cortex) pathway. Eur J Neurosci 11:1535-1544
334. Kalsbeek A, van d, V, Buijs RM 1996 Decrease of endogenous vasopressin release
necessary for expression of the circadian rise in plasma corticosterone: a reverse
microdialysis study. J Neuroendocrinol 8:299-307
335. Kalsbeek A, Drijfhout WJ, Westerink BH, Van Heerikhuize JJ, Van der Woude TP,
van d, V, Buijs RM 1996 GABA receptors in the region of the dorsomedial
hypothalamus of rats are implicated in the control of melatonin and corticosterone
release. Neuroendocrinology 63:69-78
336. Sakamoto H, Yasukawa H, Masuhara M, Tanimura S, Sasaki A, Yuge K, Ohtsubo
M, Ohtsuka A, Fujita T, Ohta T, Furukawa Y, Iwase S, Yamada H, Yoshimura A
1998 A Janus kinase inhibitor, JAB, is an interferon-gamma-inducible gene and confers
resistance to interferons. Blood 92:1668-1676
337. Watts AG, Swanson LW, Sanchez-Watts G 1987 Efferent projections of the
suprachiasmatic nucleus: I studies using anterograde transport of Phaseolus vulgaris
leucoagglutinin in the rat. Comp Neurol 258:204-229
338. Watts AG, Swanson LW 1987 Efferent projections of the suprachiasmatic nucleus: II,
studies using retrograde transport of fluorescent dyes and simultaneous peptide
immunohistochemistry in the rat. Comp Neurol 258:230-252
339. Watts AG, Tanimura S, Sanchez-Watts G 2004 Corticotropin-releasing hormone and
arginine vasopressin gene transcription in the hypothalamic paraventricular nucleus of
unstressed rats: daily rhythms and their interactions with corticosterone. Endocrinology
145:529-540
340. Ono N, De Castro JB, Khorram O, McCann SM 1985 Role of Arginine Vasopressin
in control of ACTH and LH release During Stress. Life Sciences 36:1779
341. Bruhn TO, Plotsky PM, Vale WW 1984 Effect of paraventricular lesions on
corticotropin-releasing factor (CRF)-like immunoreactivity in the stalk-median eminence:
166
studies on the adrenocorticotropin response to ether stress and exogenous CRF.
Endocrinology 114:57-62
342. Gomez F, Chapleur M, Fernette B, Burlet C, Nicolas JP, Burlet A 1997 Arginine
vasopressin (AVP) depletion in neurons of the suprachiasmatic nuclei affects the AVP
content of the paraventricular neurons and stimulates adrenocorticotrophic hormone
release. J Neurosci Res 50:565-574
343. Jin X, Shearman LP, Weaver DR, Zylka MJ, De Vries GJ, Reppert SM 1999 A
molecular mechanism regulating rhythmic output from the suprachiasmatic circadian
clock. Cell 96:57-68
344. Bae K, Weaver DR 2003 Light-induced phase shifts in mice lacking mPER1 or mPER2.
J Biol Rhythms 18:123-133
345. Reppert SM, Perlow MJ, Artman HG, Ungerleider LG, Fisher DA, Klein DC 1984
The circadian rhythm of oxytocin in primate cerebrospinal fluid: effects of destruction of
the suprachiasmatic nuclei. Brain Res 307:384-387
346. Ruiter M, La Fleur SE, Van Heijningen C, van d, V, Kalsbeek A, Buijs RM 2003
The daily rhythm in plasma glucagon concentrations in the rat is modulated by the
biological clock and by feeding behavior. Diabetes 52:1709-1715
347. Reppert SM, Perlow MJ, Ungerleider LG, Mishkin M, Tamarkin L, Orloff DG,
Hoffman HJ, Klein DC 1981 Effects of damage to the suprachiasmatic area of the
anterior hypothalamus on the daily melatonin and cortisol rhythms in the rhesus monkey.
J Neurosci 1:1414-1425
348. Kalsbeek A, Fliers E, Franke AN, Wortel J, Buijs RM 2000 Functional connections
between the suprachiasmatic nucleus and the thyroid gland as revealed by lesioning and
viral tracing techniques in the rat. Endocrinology 141:3832-3841
349. Aguilar-Roblero R, Morin LP, Moore RY 1994 Morphological correlates of circadian
rhythm restoration induced by transplantation of the suprachiasmatic nucleus in hamsters.
Exp Neurol 130:250-260
350. Pan JT, Gala RR 1985 Central nervous system regions involved in the estrogen-induced
afternoon prolactin surge. II. Implantation studies. Endocrinology 117:388-395
351. LeSauter J, Romero P, Cascio M, Silver R 1997 Attachment site of grafted SCN
influences precision of restored circadian rhythm. J Biol Rhythms 12:327-338
167
352. Hakim H, DeBernardo AP, Silver R 1991 Circadian locomotor rhythms, but not
photoperiodic responses, survive surgical isolation of the SCN in hamsters. J Biol
Rhythms 6:97-113
353. Lehman MN, Silver R, Gladstone WR, Kahn RM, Gibson M, Bittman EL 1987
Circadian rhythmicity restored by neural transplant. Immunocytochemical
characterization of the graft and its integration with the host brain. J Neurosci 7:1626-
1638
354. Izumo M, Johnson CH, Yamazaki S 2003 Circadian gene expression in mammalian
fibroblasts revealed by real-time luminescence reporting: temperature compensation and
damping. Proc Natl Acad Sci U S A 100:16089-16094
355. Yamazaki S, Straume M, Tei H, Sakaki Y, Menaker M, Block GD 2002 Effects of
aging on central and peripheral mammalian clocks. Proc Natl Acad Sci U S A 99:10801-
10806
356. Kishi M, Takeuchi T, Katayama H, Yamazaki Y, Nishio H, Hata F, Takewaki T
2000 Involvement of cyclic AMP-PKA pathway in VIP-induced, charybdotoxin-sensitive
relaxation of longitudinal muscle of the distal colon of Wistar-ST rats. Br J Pharma
129:140-146
357. Dunn JD, Johnson DC, Castro AJ, Swenson R 1980 Twenty-four hour pattern of
prolactin levels in female rats subjected to transection of the mesencephalic raphe or
ablation of the suprachiasmatic nuclei. Neuroendocrinology 31:85-91
358. Miller BH, Olson SL, Turek FW, Levine JE, Horton TH, Takahashi JS 2004
Circadian clock mutation disrupts estrous cyclicity and maintenance of pregnancy. Cur
Bio 14:1367-1373
359. Kennaway DJ, Voultsios A, Varcoe TJ, Moyer RW 2003 Melatonin and activity
rhythm responses to light pulses in mice with the Clock mutation. Am J Physiol Regul
Integr Comp Physiol 284:R1231-R1240
360. Kennaway DJ 2005 The role of circadian rhythmicity in reproduction. Hum Reprod
Update 11:91-101
361. Pardey-Borrero BM, Tamasy V, Timiras PS 1985 Circadian Pattern of Multiunit
Activity of the Rat Suprachiasmatic Nucleus during the Estrous Cycle.
Neuroendocrinology 40:450-456
362. Labyak SE, Lee TM 1995 Estrus- and steroid-induced changes in circadian rhythms in a
diurnal rodent, Octodon degus. Physiol Behav 58:573-585
168
363. Carmichael MS, Nelson RJ, Zucker I 1981 Hamster activity and estrous cycles: control
by a single versus multiple circadian oscillator(s). Proc Natl Acad Sci U S A 78:7830-
7834
364. Takahashi JS, Menaker M 1980 Interaction of estradiol and progesterone: effects on
circadian locomotor rhythm of female golden hamsters. Am J Physiol 239:R497-R504
365. Shinohara K, Funabashi T, Mitushima D, Kimura F 2000 Effects of gap junction
blocker on vasopressin and vasoactive intestinal polypeptide rhythms in the rat
suprachiasmatic nucleus in vitro. Neurosci Res 38:43-47
366. Mrosovsky N, Reebs SG, Honrado GI, Salmon PA 1989 Behavioural entrainment of
circadian rhythms. Experientia 45:696-702
367. Levine JE 1997 New concepts of the neuroendocrine regulation of gonadotropin surges
in rats. Biol Reprod 56:293-302
368. Krajnak K, Rosewell KL, Duncan MJ, Wise PM 2003 Aging, estradiol and time of
day differentially affect serotonin transporter binding in the central nervous system of
female rats. Brain Res 990:87-94
369. Smith MJ, Jiennes L, Wise PM 2000 Localization of the VIP2 receptor protein on
GnRH neurons in the female rat. Endocrinology 141:4317-4320
370. Wise PM 1997 Neuroendocrine correlates of aging. In: Conn PM, Freeman ME (eds).
Neuroendocrinology in physiology and medicine. Humana Press, 371-387
371. Hoffman GE, Lyo D 2002 Anatomical markers of activity in neuroendocrine systems:
are we all 'fos-ed out'? J Neuroendocrinol 14:259-268
372. Laburthe M, Couvineau A 2002 Molecular pharmacology and structure of VPAC
Receptors for VIP and PACAP. Regul Pept 108:165-173
373. Servillo G, Della Fazia MA, Sassone-Corsi P 2002 Coupling cAMP signaling to
transcription in the liver: pivotal role of CREB and CREM. Exp Cell Res 275:143-154
374. Sassone-Corsi P 1998 Coupling gene expression to cAMP signalling: role of CREB and
CREM. Inter J Biochem Cell Biol 30:27-38
375. Tamai KT, Monaco L, Nantel F, Zazopoulos E, Sassone-Corsi P 1997 Coupling
signalling pathways to transcriptional control: nuclear factors responsive to cAMP.
Recent Prog Horm Res 52:121-139
169
376. Ceriani MF, Hogenesch JB, Yanovsky M, Panda S, Straume M, Kay SA 2002
Genome-wide expression analysis in Drosophila reveals genes controlling circadian
behavior. J Neurosci 22:9305-9319
377. Kuhlman SJ, Silver R, Le Sauter J, Bult-Ito A, McMahon DG 2003 Phase resetting
light pulses induce Per1 and persistent spike activity in a subpopulation of biological
clock neurons. J Neurosci 23:1441-1450
378. Welsh DK, Yoo SH, Liu AC, Takahashi JS, Kay SA 2004 Bioluminescence imaging
of individual fibroblasts reveals persistent, independently phased circadian rhythms of
clock gene expression. Curr Biol 14:2289-2295
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