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This is to certify that the
dissertation entitled

PLASTICITY IN THE CIRCADIAN SYSTEM: CHANGING
RHYTHMS IN REPRODUCING FEMALE RODENTS

Ph.D.

presented by

JESSICA ANNE SCHRADER

has been accepted towards fulﬁllment
of the requirements for the

degree in Zoology and Ecology,
Evolutionary Biology and
Behavior

 

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PLASTICITY IN THE CIRCADIAN SYSTEM: CHANGING RHYTHMS IN
REPRODUCING FEMALE RODENTS

By

Jessica Anne Schrader

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Zoology and Ecology, Evolutionary Biology, and Behavior

2009

ABSTRACT

PLASTICITY IN THE CIRCADIAN SYSTEM: CHANGING RHYTHMS IN
REPRODUCING FEMALE RODENTS

By

Jessica Anne Schrader

Circadian rhythms in behavior and physiology are critical to ensure
successful reproduction, but few studies have focused on how they may change
with transitions in reproductive state. Those that have focused on pregnancy and
lactation have revealed the emergence of new temporal patterns in a wide range
of functions. The ﬁrst part of this dissertation examines the hypothesis that these
changes might be driven by modification of rhythms within the circadian system
of the brain. I ﬁrst characterized differences between non-pregnant and early
pregnant laboratory rats in rhythms in protein expression of a neuronal activity
marker (Fos) and in one component of the molecular oscillator (Per2) in the
suprachiasmatic nucleus (SCN), which houses the primary pacemaker of the
mammalian brain. In these studies I also examined the ventral subparaventricular
zone (VSPZ), which is involved in modulating SCN output signals. Fos rhythms in
one subregion of the SCN, the shell, and in the vSPZ undenivent changes in
waveform, and the peak of the Per2 rhythm in the whole SCN was phase-
advanced during early pregnancy. To determine whether changes in Fos
expression in the SCN shell and vSPZ were light-dependent, | next characterized
differences in light-induced Fos expression in the SCN and vSPZ in non-

pregnant and early pregnant rats housed in constant darkness. The rhythms of

light-responsivity of the SCN shell and vSPZ were altered in early pregnancy, but
in a manner that indicates that changes in these areas in animals kept in a
light/dark cycle were not light-dependent.

l next present evidence that rhythms in Per2 expression in some, but not
all, extra-SCN oscillators within the brain also change in early pregnancy. This
indicates that some aspect of early pregnancy induces modiﬁcations in the
coupling of different elements of the circadian system. This could potentially
cause rhythms in varying aspects of physiology and behavior to change in
different ways. I also show that temporal patterns of Fos expression in some, but
not all, non-circadian regulatory systems within the brain are modiﬁed in early
pregnancy. Therefore, it appears that integration of concurrent changes in
rhythms within and beyond the circadian system leads to a re-organization of
rhythmicity within the brain. This might be part of the mechanism by which
transitions from one reproductive state to another lead to the emergence of a
diverse range of new rhythmic patterns in behavior and physiology.

In my ﬁnal experiment, I characterized rhythms in activity and temperature
in females of a diurnal rodent species, the Nile grass rat (Arvicanthis niloticus),
as they progressed through a series of reproductive states. In contrast to
nocturnal rodents, rhythms in females of this species underwent changes in
amplitude, but not in phase, during pregnancy and lactation. This suggests that
diurnal and nocturnal species may need to modulate their rhythms in different

ways in order to reproduce successfully.

For every teacher and mentor that encouraged me to think critically.

Acknowledgements

First, I would like to thank my advisor, Dr. Laura Smale, for her
encouragement, support, advice, and critical comments throughout my graduate
career. Thanks also to the other members of my guidance committee: Dr.
Antonio Nunez, who served as an unofﬁcial co-advisor, Dr. Kay Holekamp, Dr.
Joseph Lonstein, and Dr. Theresa Lee. Their advice and critiques of my research
and writing have proved invaluable.

Most of this work was the result of a collaborative effort and would not
have been possible without my labmates in the Smale, Nuﬁez , and Yan labs. Jo
Liao, Carmel Martin-Fairey, Dr. Gladys Martinez, Dr. Josh Nixon, Dr.
Chidambaram Ramanthan, Dr. Michael Schwartz, Dorela Shuboni, Nicole Timm,
and Dr. Lily Yan all provided technical assistance and/or advice on experimental
design and data interpretation. Special thanks to Alexandra Castillo-Ruiz, Anna
Baumgras, and Andrea Kaiser, who have been both wonderful colleagues and
amazing friends. I also owe a huge debt of gratitude to the many undergraduates
that assisted me: Erin J. Walaszczyk, who co-authored one chapter of this
dissertation, Amy Campbell, Steve Disler, Nick Graff, Tara Kisnonsky, Elissa
Pastuzyn, Maria Roberts, Molly Skaer, Eric Stanko, Adam Stowie, and Anthony
Yuhas.

l have been lucky to participate in seminars and activies of not only the
Department of Zoology and Program in Ecology, Evolutionary Biology, and
Behavior but also those of the Department of Psychology and Neuroscience

Program. These communities have been exceptionally supportive and

(7’

collaborative, and its members have been important in my professional
development as well. Thanks to Dr. Sharleen Sakai, Ray Figueira, Pam Montalto,
and Jane Venier for technical advice; to Dr. David Weaver of the University of
Massachusetts for providing the Per2 antibody; to Deepa Agganrval of the MSU
CSTAT ofﬁce for statistical advice; and to Constance Montville, Dr. Jean
Gaymer, Dr. Danielle Ferguson, and the rest of the ULAR animal care staff for
their advice concerning my animal subjects and for providing their care.

I have had the opportunity to teach many lab courses at MSU, and I am
thankful to my teaching mentors and colleagues for shaping my development as
an instructor. Special thanks are due to Dr. Barbara Lundrigan, Dr. April Harlan-
Cognato, Sonya Lawrence, Dr. Carrie Scheele, Dr. Kevin Theis, Ryan Taylor,
Emily Morrison, Emily Grman, and many fellow TAs from Bioscience 110. I also
wish to thank my undergraduate teaching assistants: Diana Cerame, Melina
Christoﬁs, Jisoon Kim, Kari Mitsos, Jeremy Phan, Brittany Stanglewicz, Kayla
Stomack, and Gabrielle Yezbick.

Financial support for my research and training came from the National
institutes of Health (R01-MH53433, awarded to LS) and the National Science
Foundation (lBN-O130977, awarded to L8). Additional support was provided by
the Department of Zoology, College of Natural Science, Neuroscience Program,
and the Graduate School at Michigan State University.

Thanks to my family and friends who have supported me over the years,
and thanks especially to my husband, Steve, for his love, support, and unblinking

honesty.

vi

TABLE OF CONTENTS

LIST OF TABLES ............................................................................. x
LIST OF FIGURES ........................................................................... xi
KEY TO ABBREVIATIONS ............................................................ xv
CHAPTER 1 Introduction ................................................................ 1
General introduction .......................................................................................... 1
The mammalian circadian timekeeping system ................................................. 2
Female reproduction and circadian rhythms ..................................................... 5
The circadian system in early pregnancy .......................................................... 8
Overview of the chapters ................................................................................. 12

CHAPTER 2 Changes in and dorsal to the rat suprachiasmatic

nucleus during early pregnancy .................................................. 15
INTRODUCTION ............................................................................................. 15
EXPERIMENTAL PROCEDURES .................................................................. 19

Animals ........................................................................................................ 19
Determination of estrous cycle stages ......................................................... 19
Tissue collection and immunocytochemistry ................................................ 20
Experiment 1: Fos and Per2 rhythms in LD ................................................. 23
Experiment 2: Light-induced Fos expression ............................................... 24
Quantitative and Statistical Analysis ............................................................ 27
RESULTS ........................................................................................................ 29
Experiment 1 ................................................................................................ 29
Experiment 2 ................................................................................................ 33
DISCUSSION .................................................................................................. 36
The SCN core .............................................................................................. 36
The SCN shell .............................................................................................. 37
The SCN core vs. shell ................................................................................ 39
Potential sex differences in the SCN ........................................................... 40
Changes in the vSPZ during early pregnancy .............................................. 41
The role of the SCN and vSPZ during early pregnancy ............................... 44

CHAPTER 3 Differential changes in extra-SCN oscillators during

early pregnancy in the rat ............................................................. 46
INTRODUCTION ............................................................................................. 46
EXPERIMENTAL PROCEDURES .................................................................. 48

Animals ........................................................................................................ 48
Determination of estrous cycle stages ......................................................... 49
Experimental Protocol .................................................................................. 50
Tissue collection and immunocytochemistry ................................................ 50
Quantitative and Statistical Analysis ............................................................ 52

vii

Ci
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nil.

RESULTS ........................................................................................................ 55

Oval nucleus of the bed nucleus of the stria terminalis ................................ 55
Central and basolateral amygdala ............................................................... 57
A14 periventricular hypothalamic dopaminergic neurons ............................ 57
DISCUSSION .................................................................................................. 58
CHAPTER 4 Changing patterns of Fos expression in the rat brain
during early pregnancy ................................................................. 64
INTRODUCTION ............................................................................................. 64
EXPERIMENTAL PROCEDURES .................................................................. 67
Animals ........................................................................................................ 67
Determination of estrous cycle stages ......................................................... 67
Experimental Protocol .................................................................................. 68
Tissue collection and immunocytochemistry ................................................ 68
Quantitative and Statistical Analysis ............................................................ 70
RESULTS ........................................................................................................ 72
The oval nucleus of the bed nucleus of the stria terminalis ......................... 72
The central and basolateral amygdala ......................................................... 74
The hypothalamic paraventricular nucleus .................................................. 74
The medial preoptic area ............................................................................. 75
The periventricular hypothalamus ................................................................ 75
The dorsomedial hypothalamus ................................................................... 75
The ventrolateral preoptic area .................................................................... 77
The basal forebrain ...................................................................................... 77
DISCUSSION .................................................................................................. 79

CHAPTER 5 Changing patterns of daily rhythmicity across
reproductive states in diurnal female Nile grass rats (Arvicanthis

niloticus) ........................................................................................ 86
INTRODUCTION ............................................................................................. 86
MATERIALS AND METHODS ......................................................................... 88

Animals ........................................................................................................ 88
Surgery ........................................................................................................ 89
Experimental Manipulations ......................................................................... 89
Activity and Temperature Data Analysis ...................................................... 91
Video Recordings and Behavioral Analysis of Nesting Females .................. 93
RESULTS ........................................................................................................ 94
Reproductive Output .................................................................................... 94
Activity Rhythms .......................................................................................... 95
Temperature Rhythms ............................................................................... 100
Behavioral Rhythms in Nesting Females ................................................... 107
DISCUSSION ................................................................................................ 108
CONCLUSIONS AND IMPLICATIONS ......................................................... 116

CHAPTER 6 Conclusions ............................................................ 118

Rhythms in the brain in early pregnancy ....................................................... 118

viii

 

Re

Rhythms and reproduction in diurnal versus nocturnal rodents ..................... 121
Implications of the present work .................................................................... 124

References ................................................................................... 1 27

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LIST OF TABLES

Table 5.1. GMEM Results for Type III Tests of Fixed Effects for Activity and
Temperature Rhythms. *CSH = Heterogeneous Compound Symmetry, CS =
Compound Symmetry, see METHODS for model details. n = 5/group. NS = Not
signiﬁcant (P > 0.05). .......................................................................................... 97

LIST OF FIGURES

Images in this dissertation are presented in color.

Figure 1.1. Potential pathways of neural regulation of changes in overt rhythms in
early pregnancy. (A) Some aspect of pregnancy may induce changes within the
SCN (solid arrows), extra-SCN oscillators (dashed arrows), or non-oscillatory
brain regions (dotted arrows) to promote changes in overt rhythms. (B) Effects of
pregnancy may act at multiple levels in the brain in concert to lead to varied
changes in different overt rhythms (combined arrows). Arrows indicate general
pathways, not neural projections ........................................................................... 9

Figure 2.1. Photomicrograph of the SCN and vSPZ of a female lab rat stained for
AVP-ir. The left SCN core and AVP-rich SCN shell are outlined, and the sampling
region used for vSPZ counts is drawn to scale and placed above the left SCN.
ocx, optic chiasm; 3v, third ventricle. Scale bar = 200 um. ................................. 25

Figure 2.2. Representative photomicrographs of protein expression of Fos at ZT
2, 6, and 18 (A) and Per2 at ZT 2, 14, and 18 (B) in the middle SCN and vSPZ of
diestrous (left) and pregnant rats (right) kept in a 12:12 LD cycle. Per2 is only
expressed in the cell nucleus. Larger cells in (B) that are stained in the cytoplasm
are TH-ir cells and are primarily located around the vSPZ and 3v. ocx, optic
chiasm; 3v, third ventricle. Scale bar = 100 um. ................................................. 30

Figure 2.3. Expression of Fos (left column) and Per2 (right column) in the SCN
core (A,B), SCN shell (CD), and vSPZ (E,F) of diestrous and pregnant rats kept
in a 12:12 LD cycle. Expression is measured as the percentage of the maximum
individual value for each protein in each region. *indicates a time point where
expression is significantly elevated relative to at least two other time points within
the same reproductive state, and “indicates a time point at which expression
signiﬁcantly differs between reproductive states (P<0.05, post-hoc LSD (A,B,E,F)
or Mann-Whitney U (C,D) tests). ......................................................................... 31

Figure 2.4. Representative photomicrographs of protein expression of Fos in the
middle SCN and vSPZ of control and light-pulsed diestrous (left) and pregnant
rats (right) at CT 6 (top row) and CT 18 (bottom row). ocx, optic chiasm; 3v, third
ventricle. Scale bar = 100 um. ............................................................................ 34

Figure 2.5. Expression of Fos in the SCN core (A), SCN shell (B), and vSPZ (C)
of control and light-pulsed diestrous and pregnant rats kept in DD for two days.
Expression is measured as the percentage of the maximum individual Fos-ir
count within the region of interest. * indicates signiﬁcantly higher Fos expression
between light-pulsed and control females (P<0.05, post-hoc LSD (C) or Mann-
Whitney U (A,B) tests). ....................................................................................... 35

xi

Figure 3.1. Line drawings depicting location of the BnST-ov (A), PHDA (B), CEA,
and BLA (C). Sampling boxes (black squares) were used for cell counts in the
PHDA, CEA, and BLA. Anatomical boundaries are based on Paxinos and
Watson (1997). 3v: third ventricle; AC: anterior commissure; CPu: caudate
putamen; cx: cerebral cortex; ec: external capsule; f: fornix; LV: lateral ventricle;
opt: optic tract; Pe: periventricular hypothalamic nucleus; RCh: retrochiasmatic
area ..................................................................................................................... 54

- Figure 3.2. Expression of Per2 in the BnST-ov (A,B), CEA (CD), and BLA (E,F)
of diestrous and pregnant rats kept in a 12:12 LD cycle. (A,C,E): * indicates a
time point where Per2 expression is signiﬁcantly elevated relative to at least one
other time point within the same reproductive state, and ** indicates a time point
at which expression signiﬁcantly differs between reproductive states (P<0.05,
post-hoc Mann-Whitney U tests). (B,D,F): Representative photomicrographs
depicting between group differences in Per2 expression in the BnST-ov at ZT 6
(B), and within group variation at ZT 6 in the CEA (D) and at ZT 22 in the BLA
(F). Scale bars= 200 pm. .................................................................................... 56

Figure 3.3. Expression of Per2 in TH-expressing neurons in the PHDA of
diestrous and pregnant rats kept in a 12:12 LD cycle (A). There was no
signiﬁcant interaction between reproductive state and ZT, and asterisks (*)
indicate time points where Per2 expression in TH cells is signiﬁcantly elevated
relative to at least two other time points in the combined (pregnant and diestrous)
rhythm. (B) Representative photomicrographs depicting Per2/TH-ir (left:
purple/brown) and TH-ir (right: brown) cells in the PHDA in two different
individuals (each row). Scale bars = 10 pm. ....................................................... 59

Figure 4.1. Line drawings depicting location of the sampling regions in the MS,
VDB, and HDB (A); BnST-ov (B); VLPO and MPA (C); PVN and PeVN (D); CEA
and BLA (E), and DMH (F). Sampling boxes (black squares) were used for cell
counts in all regions except the BnST-ov (entire area in black). Anatomical
boundaries are based on Paxinos and Watson (1997). 3v: third ventricle; AC:
anterior commissure; ARC: arcuate nucleus; CPu: caudate putamen; cx: cerebral
cortex; ec: external capsule; f: fornix; LV: lateral ventricle; ocx: optic chiasm; opt:
optic tract; Pe: periventricular hypothalamic nucleus; RCh: retrochiasmatic area;
VMPO: ventromedial preoptic area. .................................................................... 71

Figure 4.2. Expression of Fos in the BnST-ov (A,B), CEA (CD), and PVN (E,F) of
diestrous and pregnant rats kept in a 12:12 LD cycle. (A,C,E): Main effects of
reproductive state (RS) and ZT are indicated in text on each graph (A, E: two-
way ANOVA; C: non-parametric tests). There was no signiﬁcant interaction of ZT
and reproductive state in the BnST-ov or PVN. NS: not signiﬁcant (P>0.05). *
indicates that Fos expression was signiﬁcantly elevated relative to at least one
other time point within the same reproductive state, and ** indicates a signiﬁcant
difference between reproductive states (P<0.05, post-hoc Mann-Whitney U
tests). (B,D,F): Representative photomicrographs of Fos-labelled cells in each
region at ZT 6. LV: lateral ventricle; 3v: third ventricle. Scale bars = 200 um. ....73

xii

 

 

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Figure 4.3. Expression of Fos in the MPA (A,B), PeVN (CD), and DMH (E,F) of
diestrous and pregnant rats kept in a 12:12 LD cycle. (A,C,E): Main effects of
reproductive state (RS) and ZT are indicated in text on each graph (A: non-
parametric tests; C,E: two-way ANOVA). There was a signiﬁcant interaction of ZT
and reproductive state in the PeVN but not the DMH. Notation is the same as in
Fig. 4.2 (post-hoc Mann-Whitney U tests (A) or LSD tests (C)). (B,D,F):
Representative photomicrographs depicting between group differences in Fos
expression in each region at ZT 18. 3v: third ventricle; f: fornix. Scale bars = 200
um. ......................................................... - ............................................................. 76

Figure 4.4. Expression of Fos in the VLPO (A,B), MS (CD), and VDB (E,F) of
diestrous and pregnant rats kept in a 12:12 LD cycle. (A,C,E): Main effects of
reproductive state (RS) and ZT are indicated in text on each graph (A,E: non-
parametric tests; C: two—way ANOVA). There was no signiﬁcant interaction of ZT
and reproductive state in the MS. Notation is the same as in Fig. 4.2 (post-hoc
Mann-Whitney U tests). (B,D,F): Representative photomicrographs depicting
between group differences in Fos expression in each region at ZT 22. ocx: optic
chiasm. Scale bars = 200 pm .............................................................................. 78

Figure 5.1. Averaged general Iocomotor activity rhythms in the reproductive (left)
and control (right) groups (n = 5/group) under a 12h: 12h light (open bars)/dark
(black bars) cycle for pregnancy, pregnancy plus lactation, and lactation (or
equivalents). For each individual, the average activity of each half hour was
standardized by dividing the raw value for that half hour on the day of interest by
the average total daily activity of that female on the pre-reproductive day. Plotted
values are means of these standardized values for each treatment group for
each half hour. Standard errors were removed for ease of comparing the
rhythms. .............................................................................................................. 96

Figure 5.2. (A) Average daily activity across reproductive days (or equivalents) in
the two treatment groups (n = 5/group). The average daily activity was
standardized by dividing the average activity of a female for each day of interest
by the average activity of that female on the pre-reproductive (or equivalent) day.
Values are means 1: SE. *Signiﬁcant differences between the control and
reproductive groups (p < 0.05; LSD test). The data for the reproductive group are
re-plotted in (B) for comparisons of pregnancy and lactation to the combined
state. :tSigniﬁcant differences between lactation and the combined (PL) state (p <
0.05; LSD test). ................................................................................................... 99

Figure 5.3. (A) Ratio of activity in the dark versus light phase across reproductive
days (or equivalents) in the two treatment groups (n = 5/group). Values are
means :I: SE. Notation is the same as in Fig. 5.2A. The data for the reproductive
group are re-plotted in (B) for comparisons of pregnancy and lactation to the
combined state .................................................................................................. 101

Figure 5.4. Averaged Tb rhythms in the reproductive (left) and control (n’ght)
groups (n = 5/group) under a 12h: 12h light (open bars)/dark (black bars) cycle

xiii

for pregnancy, pregnancy plus lactation, and lactation (or equivalents: equiv.).
Values are means for each treatment group for each half hour. Standard errors
were removed for ease of comparing the rhythms. ........................................... 102

Figure 5.5. (A) Daily amplitude of the Tb rhythm across reproductive days (or
equivalents) in the two treatment groups (n = 5/group). Values are means :t SE.
Notation is the same as in Fig. 5.2A. The data for the reproductive group are re-
plotted in (B) for comparisons of pregnancy and lactation to the combined state.
TSigniﬁcant differences between pregnancy and the combined (PL) state (p <
0.05; LSD test). :tSigniﬁcant differences between lactation and the combined
(PL) state (p < 0.05; LSD test). ......................................................................... 104

Figure 5.6. (A and B) Average Tb during the midday (ZT 3 to 9, A) and midnight
(ZT 15 to 21, B) periods across reproductive days (or equivalents) in the two
treatment groups (n = 5/group). Values are means t SE. Notation is the same as
in Fig. 5.2A. The data for the reproductive group are re-plotted in (C) and (D) for
comparisons of pregnancy and lactation to the combined state during the midday
(C) and midnight (D) periods. Notation is the same as in Fig. 5.53. ................. 106

Figure 5.7. Frequencies of off-nest (A) and drinking (B) behaviors for each hour
of the light (open bars, top): dark (black bar, top) cycle during lactation in a
subset of the reproductive group (n = 3). Values are pooled means :I: SE for days
2, 10, and 19 of lactation in 3 lactating dams. *Peaks and 1'troughs in each
rhythm as explained in RESULTS ..................................................................... 109

Figure 6.1. Proposed network model of the pathways (arrows) by which
pregnancy affects rhythms in circadian and non-circadian systems of the brain to
lead to changes in overt rhythms. Some aspect of pregnancy induces changes
within the SCN and vSPZ (solid arrows), some extra-SCN oscillators (dashed
arrows), and some non-oscillatory brain regions (dotted arrows) to promote
changes in overt rhythms. These effects are likely to be integrated at multiple
levels (converging arrows leading to combined arrows). In addition, rhythms in
some brain regions are unaffected (*blocked arrows indicating resistance to
upstream inﬂuences of pregnancy). Arrows indicate general pathways, not neural
projections. ....................................................................................................... 1 19

xiv

KEY TO ABBREVIATIONS

Abbreviation Deﬁnition

3v .......... ' ..... ‘..;..;...th"i.a“;.aaa.

ABC ........................................................ avidin-biotin peroxidase complex

AC ........................................................... anterior commissure

ACTH ...................................................... corticotropin

ANOVA ................................................... analysis of variance

ARC ........................................................ arcuate nucleus

AVP ......................................................... arginine vasopressin

BLA ......................................................... basolateral amygdala

BnST-ov .................................................. oval nucleus of the bed nucleus of the

stria terminalis

CEA ........................................................ central amygdala

CPu ......................................................... caudate putamen

CRF ........................................................ corticotropin releasing factor
CS ........................................................... compound symmetry

CSH ........................................................ heterogeneous compound symmetry
CT ........................................................... circadian time

cx ............................................................ cerebral cortex

DAB ........................................................ diaminobenzidine

DD ........................................................... constant darkness

dm ........................................................... dorsomedial

DMH ........................................................ dorsomedial hypothalamus
ec ............................................................ external capsule

XV

 

 

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GMEM-..
GRP .......

HDB .......

Abbreviation " Deﬁnition

—‘M~—p-—_m--d——_g.-.-. ...,....~.. .-...-.-.‘....‘- ... . ....4 - ... .~-..»-A"..w-m..w.-.ve—Mm .nvm—w-«J‘V—cvﬂcu

DER-[3 ........................................................ estrogen-receptor beta , -.. . I” i '-
f ............................................................... fornix

FRA ......................................................... Fos-related antigens

GMEM ..................................................... general mixed effects model
GRP ........................................................ gastrin-releasing peptide

HDB ........................................................ horizontal diagonal band of Broca
ir .............................................................. immunoreactive

L, L# ........................................................ lactation, lactation day #

LD ........................................................... light/dark

LSD ......................................................... least signiﬁcant difference

LV ........................................................... lateral ventricle

MPA ........................................................ medial preoptic area

MS .......................................................... medial septum

NGS ........................................................ normal goat serum

NHS ........................................................ normal horse serum

NREMS ................................................... non-rapid eye movement sleep
NS ........................................................... not significant

ocx .......................................................... optic chiasm

opt ........................................................... optic tract

OVX ........................................................ ovariectomized

xvi

Abbreviati
P, P# ........

PB ...........

...........

Abbreviation Deﬁnition

 

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PB ........................................................... 0.1 M phosphate buffer

PBS ......................................................... 0.01 M phosphate-buffered saline

PB-TX ..................................................... 0.2% TX in 0.2 M phosphate buffer

Pe, PeVN ................................................ periventricular hypothalamus

Per .......................................................... period

PHDA ...................................................... A14 periventricular hypothalamic
dopaminergic neurons

PL, PL# ................................................... pregnancy plus lactation,
pregnancy plus lactation day #

Post ......................................................... post-reproductive

Pre ................ . ......................................... pre-reproductive

PVN ........................................................ paraventricular hypothalamus

RCh ......................................................... retrochiasmatic area

REMS ..................................................... rapid eye movement sleep

RS ........................................................... reproductive state

SCN ........................................................ suprachiasmatic nucleus

SE ........................................................... standard error

SPZ ......................................................... subparaventricular zone

Tb ............................................................ core body temperature

TH ........................................................... tyrosine hydroxylase

THDA ...................................................... A12 tuberohypophyseal dopaminergic
neurons

xvii

aha

 

Abbrevia

ilDA ......

Abbreviation Deﬁnition

fi‘o‘A’fffiﬁ.'.’.'.I'.'...'.'.’.........g....................'..;.....ix"1'2'iiibéiaiﬁr’u.{ai'Buiéiﬁéﬁéiﬁiﬁéigit:‘
neurons

TX ........................................................... Triton-X 100

VDB ........................................................ vertical diagonal band of Broca

VIP .......................................................... vasoactive intestinal peptide

vl ............................................................. ventrolateral

VLPO ...................................................... ventrolateral preoptic area

VMPO ..................................................... ventromedial preoptic area

vSPZ ....................................................... ventral subparaventricular zone

ZT ........................................................... zeitgeber time

xviii

CHAPTER 1

Introduction

General introduction

Circadian rhythms in behavior and physiology, which have a period of
roughly 24 hours and are generated endogenously, have been documented in
most clades of life, including cyanobacteria, fungi, plants, and animals (Bell-
Pedersen et al., 2005; Dunlap, 1999). When synchronized to the external
environment, these rhythms allow organisms to anticipate predictable daily
ﬂuctuations in the environment and appropriately time behavioral and
physiological events to anticipate those changes (Moore-Ede et al., 1982). The
neural and molecular regulation of rhythms in mammals has been extensively
studied, but this has been done primarily with adult males of nocturnal rodent
species, particularly rats (Rattus norvegicus), hamsters (Mesocn'cetus auratus),
and mice (Mus musculus). Few studies have addressed questions of whether or
how transitions from one reproductive stage to the next inﬂuence rhythms in
behavior and physiology or the underlying mechanisms that govern them. This
leaves major gaps in our understanding of the circadian system, as all mammals,
especially females, progress through a series of signiﬁcant changes in their
reproductive status. These transitions require plasticity in circadian systems that
regulate a diversity of functions. In this introductory chapter, I will ﬁrst review the
neural and molecular mechanisms of the mammalian circadian timekeeping
system. I will follow this with a discussion of our current understanding of how

circadian rhythms change as female mammals transition from one reproductive

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state to another. I will then propose hypotheses that might account for how the
circadian timekeeping system may be involved in promoting these changes.
Finally I will present a summary of the research questions addressed in each

chapter.

The mammalian circadian timekeeping system

Circadian rhythms, which each have a period that is close to, but not
exactly, 24 hours, are revealed when animals are maintained in constant
environmental conditions. However, they are ordinarily entrained by
environmental stimuli, referred to as zeitgebers, or “time-givers” in German. The
neural mechanisms controlling circadian rhythms in mammals have been well-
characterized in males, and it is ﬁrmly established that the suprachiasmatic
nucleus (SCN), located bilaterally in the anterior hypothalamus, houses the
primary pacemaker (Moore and Eichler, 1972; Ralph et al., 1990; Rusak, 1977;
Stephan and Zucker, 1972). This pacemaker is responsible for generating
endogenous rhythms in constant conditions as well as entraining these rhythms
to environmental signals. Light is the primary entraining agent, and photic
information is conveyed to the SCN from the eyes directly via the
retinohypothalamic tract (Johnson et al., 1988; Levine et al., 1991; Moore and
Lenn, 1972) and indirectly via the geniculohypothalamic tract (Rusak et al.,
1989)

The SCN is functionally and neurochemically heterogeneous, and it may
be divided into two main subregions, referred to by some as the “core" and the

“shell” and by others as the ventrolateral (vl-) and dorsomedial (dm-) SCN (Morin,

.l

 

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2007). In this dissertation, I use the terms core and shell (as in Moore et al.,
2002), even when referring to reports in which the authors used the terms vl-SCN
and dm-SCN to differentiate between the two subregions (e.g. Lee et al., 1998;
Cutrera et al., 2002). In laboratory rats, the retinohypothalamic tract innervates
the core, which also receives non-photic information through other pathways
(Johnson et al., 1988; Levine et al., 1991; Moore and Lenn, 1972; Moore et al.,
2002). The shell relies on inputs from the core for photic information, and it
receives non-photic information via the core as well as other brain regions (Antle
and Silver, 2005; Daikoku et al., 1992; lbata et al., 1993; lbata et al., 1997;
Moore et al., 2002). The SCN shell integrates these inputs with endogenous
circadian processes and provides the majority of SCN output signals (Moore et
al., 2002). Its primary target is the subparaventricular zone (SPZ; Leak and
Moore, 2001; Watts and Swanson, 1987; Watts et al., 1987; Watts, 1991), which
projects to many of the same targets as the SCN and may process circadian
signals and homeostatic drive (Abrahamson and Moore, 2006; Kriegsfeld et al.,
2004; Lu et al., 2001; Morin et al., 1994; Saper et al., 2005; Schwartz et al.,
2004; Watts and Swanson, 1987; Watts et al., 1987). The ventral region of the
SP2 (vSPZ), lies immediately dorsal to the SCN shell and is important in the
Control of locomotor activity rhythms (Abrahamson and Moore, 2006; Lu et al.,
2 00 1; Schwartz et al., 2009).
Although cells in both the core and shell of the rat SCN express the
in h i bitory neurotransmitter, GABA (Moore and Speh, 1993), the two subdivisions

of the nucleus differ with respect to other peptides and transmitters. Cells in the

 

 

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SCN shell primarily express arginine vasopressin (AVP), whereas the primary
core output signals are vasoactive intestinal polypeptide (VIP) and gastrin-
releasing peptide (GRP; Antle and Silver, 2005; Moore et al., 2002; Romijn et al.,
1998). VIP and GRP neurons in the core of the rat SCN have axonal projections
that innervate the entire SCN and also project beyond the nucleus (Daikoku et
al., 1992; lbata et al., 1993; lbata et al., 1997; Moore et al., 2002). Both
subregions contain oscillator cells, each of which expresses a molecular
transcription/translation loop that takes roughly 24 hours to complete. This
“molecular oscillator” involves multiple “clock” proteins, including positive
elements (Clock and BMAL) and negative elements (two cryptochrome and three
period (Per) proteins), which also serve as transcription factors for other genes,
called clock-controlled genes (Bell-Pedersen et al., 2005; Dunlap, 1999; Reppert
and Weaver, 2002). Cells expressing this feedback loop are more prevalent in
the SCN shell, where they co-express AVP, but some are in the core where they
co-express VIP (Dardente et al., 2002).
The molecular oscillator is also expressed in cells outside the SCN.
Populations of these cells, referred to as extra-SCN oscillators, have been found
within a number of brain regions and throughout the body’s periphery (Guilding
and Piggins, 2007; Hastings et al., 2003; Reppert and Weaver, 2002; Weinert,
2005). The SCN is necessary to synchronize rhythms in most of these regions
with one another, but there are conflicting data regarding the extent to which
these oscillators require the SCN to maintain their rhythmicity, as some are less

dependent on it than others (Yamazaki et al., 2000; Yoo et al., 2004). It has been

 

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proposed that the function of extra-SCN oscillators may be to integrate light- '
entrained circadian signals originating in the SCN with processes that vary
among cell populations to meet site-speciﬁc needs (Guilding and Piggins, 2007;

Herzog and Tosini, 2001; Weinert, 2005; Yoo et al., 2004).

Female reproduction and circadian rhythms

Circadian rhythms are especially critical to reproduction. In some
mammals, including many rodents, the timing of ovulation is regulated by the
SCN (for review, see de la lglesia and Schwartz, 2006), and rhythms in mating
behavior have been observed in multiple species (Beach and Levinson, 1949;
Dobson and Michener, 1995; Gilbert et al., 1985; Hansen et al., 1979; Harlan et
al., 1980; Mahoney and Smale, 2005). This probably optimizes the likelihood of
successful fertilization. However, in all therian mammals, fertilization is followed
by gestation (pregnancy) and lactation, which must also be carried out
successfully. Circadian rhythms play important roles during these reproductive
states as well, although signiﬁcantly less work has focused on this.

The best-characterized animal model of circadian processes operating
during pregnancy is the nocturnal lab rat. During early pregnancy in this species,
locomotor activity generally decreases and becomes less rhythmic
(Rosenwasser et al., 1987), whereas body temperature rhythms show an
advance in their rising phase and a reduction in amplitude attributable to
increases in the daily temperature minimum (Kittrell and Satinoff, 1988). Sleep
patterns are altered, and the total amount of time spent in both non-rapid eye

movement sleep (NREMS) and rapid eye movement sleep (REMS) during the

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dark phase increases during early pregnancy, as does the number of REMS
bouts in the dark phase (Kimura et al., 1996). Rhythms in hormone secretion also
change. The amplitudes of the rhythms of circulating corticotropin (ACTH) and
corticosterone decrease due to a reduction in the peak values, and the rising
phase of the rhythm in ACTH is advanced (Atkinson and Waddell, 1995).

The most thoroughly-studied circadian rhythm of early pregnancy in the rat
is that of prolactin secretion, which emerges after mating or vaginocervical
stimulation and exhibits twice-daily surges around dawn and dusk (Butcher et al.,
1972). This pattern promotes progesterone secretion by the corpora lutea, which
is necessary for maintenance of the uterine endometrium to support pregnancy
(for review, see Freeman et al., 2000). Similar rhythms have been found in other
rodents (Edwards et al., 1995; McMillan and Wynne-Edwards, 1999; Talamantes
et al., 1984), and this pattern can also be induced via cervical stimulation of intact
or ovariectomized (OVX) females, a procedure that leads to pseudopregnancy
(for review, see Erskine, 1995; Erskine et al., 2004). This rhythm is a true
circadian one, as each prolactin surge free-runs with a period of approximately
24 hours in both blinded females and intact females in constant conditions
(Bethea and Neill, 1979; Pieper and Gala, 1979; but see Yogev and Terkel,
1980). I

Multiple lines of evidence support a role for the circadian system and the
SCN in the regulation of the two prolactin surges during early pregnancy. First,
the circadian phase at which they occur is unaffected by the timing of cervical

stimulation that induces them (Smith and Neill, 1976). Second, both

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retrochiasmatic knife cuts and lesions of the SCN abolish the surges (Bethea and
Neill, 1980; Freeman et al., 1974; Yogev and Terkel, 1980). Furthermore,
transgenic mice lacking the Clock gene, which is a component of the molecular
oscillator, exhibit higher rates of spontaneous abortion throughout gestation,
display a decrease in mid-term progesterone secretion, and have shorter
pseudopregnancies than wild-type females (Miller et al., 2004). This indicates
that functional clock gene expression is vital for normal secretion of prolactin
during early pregnancy and for successful gestation.

Hypothalamic dopamine serves as the primary regulator of prolactin
release through its inhibitory effects on the pituitary gland (Ben-Jonathan, 1985),
and an endogenous rhythm in dopamine secretion has been well-characterized
in rats (for review, see Freeman et al., 2000). The prolactin surges of early
pregnancy are also promoted by stimulation from various prolactin-releasing
factors. VIP and serotonin may stimulate each peak by promoting oxytocin
release, which is sufﬁcient to induce the rhythm of prolactin secretion seen in
early pregnancy (Arey and Freeman, 1989; Arey and Freeman, 1990; Arey and
Freeman, 1992a; Arey and Freeman, 1992b; Egli et al., 2004; Egli et al., 2006;
Kennett et al., 2008). VIP may also promote prolactin release by direct inhibition
of dopaminergic neurons in the arcuate nucleus (Gerhold et al., 2001), since
inhibition of transcription of VIP mRNA increases the expression of Fos-related

antigens in these dopaminergic neurons (Gerhold et al., 2002).

 

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The circadian system in early pregnancy

Although changes in rhythms from a non-pregnant state to early
pregnancy have been recognized for years, very little is known about how or
whether the circadian system promotes them. One hypothesis is that the SCN is
responsible (Fig. 1.1, solid arrow pathways). This could potentially occur via
many mechanisms, such as a re-setting of the phase of the master pacemaker
relative to the light/dark cycle or a change in coupling between the molecular
oscillator network and output signals, such as VIP or AVP, within the SCN.
Alternatively, changes within extra-SCN oscillators might be the primary cause of
modiﬁcations in overt rhythms, and, for example, these extra-SCN oscillators
might respond differently to the SCN (Fig. 1.1A, dashed arrow pathway). It is also
possible that pregnancy acts at the level of both the SCN and downstream
components of the circadian system (Fig. 1.18). Another hypothesis is that the
circadian system may not change at all, but its influence may be modulated by
other physiological and behavioral systems that are modiﬁed as a female
transitions from one reproductive condition to another (Fig. 1.1A, dotted arrow
pathway). Finally, the physiological changes might originate within both circadian
and non-circadian regulatory systems (Fig. 1.18).

In order for any process, within or beyond the circadian system, to play a
role in modifying rhythms during early pregnancy, it must be inﬂuenced by
reproductive state. Presumably, some aspect of mating, fertilization, implantation,
or ﬂuctuations in reproductive hormones alter these systems. Considerable
evidence indicates that circadian mechanisms in the brain are responsive to

changes that accompany pregnancy. For example, in the SCN core, the

8

 

 

 

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Figure 1.1. Potential pathways of neural regulation of changes in overt rhythms in early preg—
nancy. (A) Some aspect of pregnancy may induce changes within the SCN (solid arrows),
extra-SCN oscillators (dashed arrows), or non-oscillatory brain regions (dotted arrows) to
promote changes in overt rhythms. (B) Effects of pregnancy may act at multiple levels in the
brain in concert to lead to varied changes in different overt rhythms (combined arrows). Arrows
indicate general pathways, not neural projections.

 

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expression pattern of Fos, a protein encoded by the immediate early gene cfos
that often is expressed after neuronal activation (Kovacs, 2008), changes from
early to late pregnancy in rats (Lee et al., 1998). Knockout studies and
application of antisense oligonucleotides to cfos mRNA prevent normal
entrainment to phase shifts in the light/dark (LD) cycle, indicating that Fos
expression in the SCN is an important component of circadian regulation
(Honrado et al., 1996; Wollnik et al., 1995). In addition, rhythms in expression of
Per2, a component of the molecular oscillator, in regions of the limbic forebrain
change across the estrous cycle of rats (Perrin et al., 2006). This might occur
because these extra-SCN oscillators respond differently to signals originating in
the SCN or because the SCN emits these signals in a different manner on the
different days of the estrous cycle.

Reproductive hormones may be responsible for changes in the circadian
system that occur as a function of reproductive state. Richards (1966) ﬁrst
described variations in the amount of wheel running across the estrous cycle in
golden hamsters and found that females are more active on proestrus and estrus
than during diestrus. In addition, activity rhythms display a “scalloping” pattern
across the estrous cycle due to a phase advance in activity onset during
proestrus and estrus (Morin et al., 1977), and this “scalloping” pattern persists in
rats housed in constant dim light (Albers et al., 1981). These data led
researchers to hypothesize that changes in secretion of gonadal hormones
inﬂuence endogenous rhythms. This was found to be the case, as estradiol

treatment of OVX female golden hamsters shortens the periods of their free-

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running activity rhythms (Morin et al., 1977). Progesterone administration blocks
this effect and decreases the amount of activity, whereas progesterone given
alone to OVX females does not affect rhythms (Takahashi and Menaker, 1980).
Similar responses to ovarian hormones have been demonstrated in rats (Albers,
1981; Gerall et al., 1973; Thomas and Armstrong, 1989).

Estrogenic effects on activity rhythms probably act via receptors within the
SCN or via inputs to this nucleus. Estrogen receptor-containing neurons in
multiple brain regions, particularly the preoptic area, project to the SCN in female
hamsters (de la lglesia et al., 1999). In both men and women, estrogen,
progesterone, and androgen receptors have been found within the SCN itself
(Kruijver and Swaab, 2002), and estrogen receptor- 6 (ER-B) is expressed in the
SCN of female rats (Shughrue et al., 1997). Binding of hormones to these
receptors may alter SCN function. This is suggested by the fact that, in OVX rats,
the peak of the rhythm of mRNA for Per2 in the SCN is advanced by estradiol
(Nakamura et al., 2005), and estradiol treatment can lead to increased Fos
expression in the SCN in an LD cycle (Abizaid et al., 2004; Peterﬁ et al., 2004).
Prolactin may also change the responsivity of the SCN to light. Pituitary grafts
that increase circulating PRL ten-fold in male hamsters cause a heightened Fos
response to light during the dark phase in both the SCN core and shell (Cutrera
et al., 2002). These data indicate that pituitary and ovarian hormones may
modulate how the SCN responds to light and that estradiol, at least, may reset

the phase of the molecular oscillator. These changes could partially explain how

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new rhythms in behavior and physiology emerge as female mammals transition

from one reproductive state to another.

Overview of the chapters

In this dissertation, I present experiments that were designed to extend
our understanding of (1) how the circadian system might be involved in the
modiﬁcation of rhythms that occur as females progress through transitions in
reproductive condition and (2) how such modiﬁcations might differ between
nocturnal and diurnal species. To address the ﬁrst issue, I sought to identify
changes during early pregnancy in the mechanisms that regulate rhythmic
functions (Chapters 2-4). In this series of studies, I compared patterns of change
in protein expression throughout the day in circadian and non-circadian systems
of the brain in laboratory rats (Rattus norvegicus) during diestrus and early
pregnancy. This served as an ideal model for my research because the rat has
been best characterized in terms of how circadian rhythms in various behaviors
and physiological events change with reproductive state, particularly from a virgin
state to early pregnancy.

In Chapter 2, | used the diestrus/early pregnant rat model system to
evaluate the hypothesis that the primary pacemaker of the brain, which is housed
in the SCN, might promote changes in overt rhythms in early pregnancy. Another
question that l addressed is whether a major target of the SCN, the vSPZ, might
be altered during early pregnancy, as signals from this region converge on other
targets of the SCN and may therefore affect how they respond to SCN signals.

For the purposes of this study, I compared rhythms between diestrus and early

12

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pregnant rats in two aspects of SCN and vSPZ function: neuronal activity (as
measured by expression of Fos) and the molecular oscillator (as measured by
expression of Per2). I also compared patterns of light-induced Fos expression in
the SCN and vSPZ between diestrous and pregnant females housed in constant
darkness.

In Chapter 3, I address the hypothesis that extra-SCN oscillators in the
brain may be involved in driving changes in circadian rhythms during early
pregnancy. If this hypothesis is correct, then rhythms in clock gene expression
should differ between non-pregnant and early pregnant females. In this study, I
used tissue from the same animals used in Chapter 2 to characterize rhythms in
Per2 expression in extra-SCN oscillators within dopaminergic neurons that inhibit
prolactin secretion and in portions of the extended amygdala, which regulate
emotional state, anxiety, and the stress axis (Cardinal et al., 2002; Davis et al.,
1997; Dong et al., 2001; Erb et al., 2001; Szafarczyk et al., 1983).

In Chapter 4, I examine how non-circadian regulatory systems of the brain
might contribute to changes in temporal patterns of functions associated with
early pregnancy. If they do contribute, then we should see changes in rhythms of
their neuronal activity, which can often be assessed by examination of patterns of
Fos expression. I therefore monitored Fos in regions of the brain involved in the
regulation of sleep, arousal, thermal homeostasis, and hormone secretion. In this
study, I used tissue from the same animals as in Chapters 2 and 3.

To address the second major issue of this dissertation, that of how diurnal

and nocturnal species differ with respect to how overt rhythms are altered during

13

 

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transitions in reproductive state, I utilized a diurnal murid rodent model, the Nile
grass rat (Arvicanthis niloticus), to determine how pregnancy, lactation, and the
combination of the two affect daily rhythms in activity and core body temperature
(Chapter 5). I compared these rhythms to those described in published reports of
nocturnal models to determine whether multiple circadian strategies have
evolved to cope with reproductive challenges in different chronotypes.

Finally, I conclude this dissertation with a discussion of how rhythms in the
brain may be re-organized in early pregnancy to produce changes in daily
rhythms, how this re-organization might differ in nocturnal and diurnal species,
and the potential implications of these issues on mammalian conservation and
biology (Chapter 6).

While I served as the primary investigator for the experiments presented in
Chapters 2 through 5, these studies were collaborative efforts, and Chapter 5
has been published with co-authors. Therefore, in these chapters, l have elected
to use the term “we” rather than “I” to indicate that this research was a

collaborative effort.

14

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CHAPTER 2
Changes in and dorsal to the rat suprachiasmatic nucleus

during early pregnancy

INTRODUCTION

Endogenously driven circadian rhythms that are entrained to external cues
allow organisms to predict daily changes in the environment and appropriately
time behavioral and physiological events to anticipate them (Moore-Ede et al.,
1982). The circadian timekeeping system plays an important role in the
coordination of a variety of physiological and behavioral processes essential for
mammalian reproduction. Most of the research on these issues has focused on
the timing of copulation and ovulation. However, in all therian mammals,
fertilization is followed by gestation (pregnancy) and lactation, and the circadian
system plays a critical role during these reproductive states as well.

During pregnancy, locomotor activity generally decreases and becomes
less rhythmic in rats (Rosenwasser et al., 1987), while the peak of the body
temperature rhythm advances (Kittrell and Satinoff, 1988). Pregnant rats also
display more rapid eye movement (REMS) and non-rapid eye movement sleep
(NREMS) during the dark phase than cycling females (Kimura et al., 1996). A
novel prolactin rhythm, with peaks around dawn and dusk, emerges in early
pregnancy and promotes progesterone secretion by the corpora lutea (Butcher et
al., 1972; Freeman et al., 2000). Rhythms in the hypothalamic-pituitary-adrenal
axis also change, with a reduction in the daily peak of secretion of both

corticotropin (ACTH) and corticosterone (Atkinson and Waddell, 1995). While
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these changes have been established for some time, the neural mechanisms
responsible for changes in all but the prolactin rhythm have received little
aﬂenﬁon.

The neural mechanisms controlling circadian rhythms in mammals have
been well-characterized in males and nulliparous females, and it is ﬁrmly
established that the suprachiasmatic nucleus (SCN), located bilaterally in the
anterior hypothalamus, houses the primary pacemaker that generates these
rhythms (Moore and Eichler, 1972; Ralph et al., 1990; Rusak, 1977; Stephan and
Zucker, 1972). The SCN entrains to a light/dark (LD) cycle via the
retinohypothalamic tract, which innervates a subregion of the SCN termed the
“core” (Johnson et al., 1988; Levine et al., 1991; Moore and Lenn, 1972; Moore
et al., 2002). This region also receives non-photic input, and it conveys the two
kinds of information to the SCN “shell”, which also receives non-photic inputs
from other brain regions (Antle and Silver, 2005; Daikoku et al., 1992; lbata et al.,
1993; lbata et al., 1997; Moore et al., 2002). The SCN shell integrates these
signals and provides the major output pathways from the nucleus to other brain
regions (Moore et al., 2002). Its primary target is the subparaventricular zone
(SPZ; Leak and Moore, 2001; Watts and Swanson, 1987; Watts et al., 1987;
Watts, 1991). The ventral portion of the SP2 (vSPZ), immediately dorsal to the
SCN shell, plays a role in the integration of circadian and homeostatic signals to
regulate various functions, such as locomotor activity rhythms (Abrahamson and
Moore, 2006; Lu et al., 2001; Saper et al., 2005; Schwartz et al., 2004; Schwartz

etaL,2009)

16

Although cells in both the core and shell of the rat SCN express the
inhibitory neurotransmitter, GABA (Moore and Speh, 1993), the two subdivisions
differ with respect to other peptides and transmitters they contain. Cells in the
SCN shell primarily express arginine vasopressin (AVP), whereas the primary
core output signals are vasoactive intestinal polypeptide (VIP) and gastrin-
releasing peptide (GRP; Antle and Silver, 2005; Moore et al., 2002; Romijn et al.,
1998). VIP and GRP neurons in the rat SCN core innervate the entire SCN and
also project beyond the nucleus (Daikoku et al., 1992; lbata et al., 1993; lbata et
al., 1997; Moore et al., 2002). Both subregions contain oscillator cells, which
each have a molecular transcription/translation loop that takes roughly 24 hours
to complete. This “molecular oscillator” involves a transcription-translation
feedback loop comprised of multiple “clock” genes and their protein products.
These include the period (Per) proteins, which are themselves transcription
factors for other genes, referred to as clock-controlled genes (Bell-Pedersen et
al., 2005; Dunlap, 1999; Reppert and Weaver, 2002). Cells expressing this
feedback loop are more prevalent in the SCN shell, where they co-express AVP,
but some are in the core and co-express VIP (Dardente et al., 2002). Populations
of cells, called extra-SCN oscillators, express the same transcription-translation
feedback loop. These populations have been found within the brain and
throughout the periphery and, together with the SCN, comprise the circadian
system (Guilding and Piggins, 2007; Hastings et al., 2003; Reppert and Weaver,

2002; Weinert, 2005).

17

 

the qua
imlue
behind

clock g

Pythr
prote1
acting

was r

The various changes in rhythms that occur during early pregnancy raise
the question of whether and how the circadian timekeeping system might be
involved in promoting them. One possibility is that the SCN is the driving force
behind these changes. If so, changes in SCN rhythms in peptide expression,
clock gene oscillations, and/or neuronal ﬁring should be apparent during this
phase of reproduction. Alternatively, the SCN might remain the same, while
extra-SCN oscillators and/or non-circadian systems are altered, perhaps in a
site-speciﬁc manner. The purpose of this study was to examine the possibility
that rhythms in the SCN, and its primary circadian target, the vSPZ, differ
between non—pregnant and early pregnant rats. To do this, we characterized
rhythms in Per2, a key component of the molecular oscillator, and Fos, the
protein product of the immediate early gene cfos that often rises after neuronal
activation (Kovacs, 2008), in pregnant and non-pregnant rats. The ﬁrst group
was examined on day 6 of pregnancy, as the changes in overt rhythms described
above are all established by that time. Females on day 1 of diestrus, when
circulating gonadal hormones are lowest, served as our non-pregnant controls.
This initial experiment revealed differences in Fos expression in the SCN shell
and vSPZ between the two groups of females in the mid-light and/or mid-dark
phases of the LD cycle. We therefore conducted a second experiment to
determine whether rhythms in light-induced Fos expression in the SCN and vSPZ

could account for these patterns.

18

ExPERll

Animals

An
obtained 1
cats OT 9"
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serpents
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EXPERIMENTAL PROCEDURES

Animals

Animals were adult female and male Sprague-Dawley laboratory rats
obtained from Harlan Laboratories (Indianapolis, IN, USA). Males were housed in
pairs or groups of three when not paired with females. Females were housed in
groups of three or four for a habituation period of at least two weeks and were
subsequently separated for single-housing. All subjects were housed in
polypropylene cages (48x27x20 cm) with ad Iibitum access to food (Teklad 8640
rodent diet, Harlan) and water under a 12h:12h light/dark (LD) cycle (unless
othenivise noted) with lights on at Zeitgeber Time (ZT) 0 and off at ZT 12. A dim
red light (<5 lux) remained on constantly for animal care purposes. All
experiments were performed in compliance with guidelines established by the
Michigan State University Institutional Animal Care and Use Committee and the
National Institutes of Health Guide for the Care and Use of Laboratory Animals.
All efforts were made to keep the number of animals used and their discomfort at

a minimum.

Determination of estrous cycle stages

Once singly housed, females were subjected to daily vaginal smears
between ZT 0 and ZT 3 to track their estrous cycles. A cotton-tipped applicator
soaked in sterile physiological saline was inserted into the vaginal opening,
gently rotated, and withdrawn. The applicator was immediately rolled onto a

Glass slide and allowed to air dry. The dried slides were soaked in 0.25%

methylene blue dye for ﬁve minutes, rinsed with distilled water, and allowed to

19

onrpno'
epineie
shew
cen;es
(tenses
aster!

tenses

ﬁssue

I"1.0505
Phase
DEﬁUSj
38.“,st
3C O‘e’e

3'3 lJrn

dry prior to examination under a light microscope for the presence of nucleated
epithelial cells, corniﬁed epithelial cells, leukocytes, and mucus. Estrous cycle
stage was determined as follows: proestrus: predominantly nucleated epithelial
cells; estrus: predominantly to exclusively corniﬁed epithelial cells; diestrus
(between 2 to 3 consecutive days): predominantly leukocytes, some corniﬁed
and/or nucleated epithelial cells and mucus present. At least three estrous cycles

were established for each female prior to further experimental manipulations.

Tissue collection and immunocytochemistry

At the time of perfusion, animals were given an overdose of sodium
pentobarbital and perfused transcardially with 0.01 M phosphate-buffered saline
(PBS), pH 7.2, followed by 4% paraformaldehyde (Sigma, St. Louis, MO) in 0.1
M phosphate buffer, unless otherwise noted. Animals perfused during the dark
phase or under constant darkness were ﬁtted with a light-tight hood prior to
perfusion to prevent acute exposure to light. Brains were post-ﬁxed for 4 hours,
transferred to 20% sucrose solution overnight, and stored in cryoprotectant at 4
°C overnight then at -20 °C until sectioning. Brains were sectioned coronally at
30 pm into three alternate series using either a freezing microtome or cryostat,
and sections were placed in cryoprotectant for further storage at -20 °C.

For tissue series that were processed for immunocytochemical staining to
detect Fos protein expression in the cell nucleus, free ﬂoating sections were
rinsed in 0.01 M PBS and then incubated in 5% normal goat serum (NGS; Vector
Laboratories, Burlingame, CA, USA) in PBS with 0.3% Triton X-100 (TX) for 1 h

at room temperature. After a 10 minute rinse in PBS, sections were incubated

20

TX'rnP!
mennn
amped

tempen

with a primary anti-cFos antibody (made in rabbit, Santa Cruz Biochemistry,
Santa Cruz, CA, USA; 1225,000, unless othenivise noted) in 3% NGS and 0.3%
TX in PBS on a rotator at 4°C for 48h, unless othenNise noted. Sections were
then rinsed in PBS and incubated with a biotinylated secondary goat-anti-rabbit
antibody Nectar; 1:200) in 3% NGS and 0.3% TX in PBS for 1 h at room
temperature, rinsed again, and then incubated with an avidin-biotin peroxidase
complex (0.9% each avidin and biotin solutions; ABC Vectastain kit; Vector) in
0.3% TX with PBS for 1 h at room temperature. Next, sections were rinsed in
0.125 M acetate buffer, pH 7.2, then reacted with diaminobenzidine (DAB; 0.25
mg/mL, Sigma), nickel sulfate (25 mg/mL, Sigma), and hydrogen peroxide (0.825
uL 0.3% H202/mL buffer) to yield a blue-black reaction product.

The series of sections processed for Per2 was double labeled for
detection of tyrosine hydroxylase (TH) for the analyses of extra-SCN oscillators
described in Chapter 3. This tissue was processed in the same manner as for
Fos except with a primary anti-Per2 antibody (mPER2 # 38, made in rabbit,
125,000, 24 h incubation; a generous gift from Dr. David Weaver, University of
Massachusetts, MA, USA). After the reaction for Per2 staining, to process the
tissue for TH expression, the same sections were rinsed ﬁrst in 0.1 M phosphate
buffer (PB) and then in 0.2% TX in 0.2 M PB (PB-TX), and they were then
incubated in 5% normal horse serum (NHS; Vector) in PB-TX for 30 min at room
temperature. After rinses in PB-TX, sections were incubated with a primary anti-
TH antibody (made in mouse, Immunostar, Hudson, WI, USA; 1:20,000) in 3%

NHS and PB-TX on a rotator at 4°C for 24 h. Sections were then rinsed in PB-TX

21

and m

ids-not;
NEW
a one
name

950;):

00Verso

and incubated with a biotinylated secondary horse-anti-mouse antibody (Vector;
1:200) in 3% NHS and PB-TX for 1 h at room temperature, rinsed again, and
then incubated with an avidin-biotin peroxidase complex (0.9% each avidin and
biotin solutions; ABC Vectastain kit; Vector) in PB-TX for 1 h at room
temperature. Next, sections were rinsed in Trizma buffer (pH 7.2, Sigma) then
reacted with DAB (0.2 mg/mL, Sigma) and hydrogen peroxide (0.35 pL 30%
H202 ImL buffer) to yield a brown reaction product.

Some sections were stained for arginine vasopressin (AVP) in order to
identify the boundary between the shell and core; these sections initially
undenivent processing for Fos, as described above, but that reaction failed due to
a dilution error. After the Fos reaction, the tissue was processed in the same
manner as for Fos with the following exceptions: (1) with a primary anti-
vasopressin antibody (made in guinea pig, Peninsula Laboratories, Belmont, CA,
USA; 130,000; 24 h incubation); (2) with a secondary goat-anti-guinea pig
antibody (Vector; 1:200); and (3) after incubation with the avidin-biotin
peroxidase complex, the tissue was rinsed in Trizma buffer (pH 7.2, Sigma) then
reacted with DAB (0.2 mg/mL, Sigma) and hydrogen peroxide (0.35 pL 30%
HzOzlmL buffer) to yield a brown reaction product. Trial immunocytochemical
reactions with the reagents used for each reaction above were performed with
tissue from females that did not have easily determined estrous cycles or had
mated but were not pregnant to ensure speciﬁcity of the primary antibodies (data
not shown). All sections were mounted on clean slides, dehydrated, and

coverslipped.

22

998?:
Seat:
Immu
lmmu

but 'rt

Experiment 1: Fos and Per2 rhythms in LB

Animals were acclimated for at least two weeks to a 12h:12h LD cycle,
and then females were singly housed, divided into one of two experimental
groups, and subjected to daily vaginal smears to determine the estrous cycle of
each. Rats in the ﬁrst group were perfused at 4 h intervals (ZT 2, 6, 10, 14, 18,
and 22) on day one of diestrus as described above, except that 1.3% lysine and
4% sodium periodate were added to the 4% paraformaldehyde. In the second
group, a male was placed in the female’s cage on the morning of proestrus
(between ZT 0 and 3). Males were removed the next morning, and a vaginal
smear was taken from the female to check for the presence of sperm to verify
mating had occurred. If none were detected, the female was re-paired on the
morning of her next proestrus period. Once mating had been veriﬁed, vaginal
smears were discontinued, and the day of conﬁrmation was designated as day 0
of pregnancy. Six days later, females were perfused in the same manner as the
diestrous females. Pregnancy was conﬁrmed by visual inspection of the uterus.
Sections from one alternate series of tissue were processed for Fos
immunoreactivity, and sections from another series were processed for Per2/T H
immunoreactivity. The TH reaction was unnecessary for the present experiment,
but it was included for use in Chapter 3.

Photographs of the SCN/vSPZ were taken from three sections, bilaterally,
for each tissue series using a CCD video camera (CX900, MBF Bioscience,
Williston, VT, USA) attached to a light microscope (Zeiss, Gdttingen, Germany).
These images were processed using Adobe Photoshop 7 and imported to Adobe

Illustrator CS 4 (Adobe Systems, Mountain View, CA, USA) to draw boundaries
23

immedra
egress
three 56
CE'JﬂiS I

petust

Expenr

EQUIVE

1. t’Eb‘V I

for the SCN core and shell and the vSPZ. The borders of the SCN core and shell
were based upon the distribution of AVP in the rat SCN (Moore et al., 2002). The
vSPZ was sampled by counting cells within a 215 pm x 160 pm rectangle placed
immediately dorsal to the SCN and lateral to the third ventricle (Fig. 2.1). Cells
expressing Fos or Per2 were then counted bilaterally in these three regions for all
three sections using the NIH lmageJ program (NIH, Bethesda, MD, USA). All
counts were made by an investigator unaware of the reproductive state or time of

perfusion of each animal.

Experiment 2: Light-induced Fos expression

Animals were acclimated for three weeks to a 12h:12h LD cycle with ZT 0
(lights on) at 1300 h. The females were then singly housed, divided into one of
two experimental groups, and subjected to daily vaginal smears to determine the
estrous cycle of each individual. Rats in the ﬁrst group (diestrous) were moved to
a separate room in constant darkness (DD) on the morning of proestrus
(between ZT 0 and 3). Daily vaginal smears were taken from them in DD to
ensure that they were still cycling. On the ﬁrst day of diestrus (two days after
entering DD), experimental females were transferred to a separate room for 30
min in DD and subsequently exposed to a one hour light pulse beginning at
either circadian time (CT) 5 or CT 17. Animals were perfused immediately after
the light pulse ended at CT 6 or CT 18. These circadian times were the
equivalents of the Us from the previous LD cycle. In the second group, a male
was placed in the female’s cage on the morning of the last day of diestrus

(between ZT 0 and 3). Males were removed the morning of estrus, and a vaginal

24

 

Figure 2.1. Photomicrograph of the SCN and vSPZ of a female lab rat stained for AVP-ir. The
left SCN core and AVP-rich SCN shell are outlined, and the sampling region used for vSPZ
counts is drawn to scale and placed above the left SCN. ocx, optic chiasm; 3v, third ventricle.
Scale bar = 200 pm.

25

smear was taken from the female on both days of pairing to check for the
presence of sperm to verify mating had occurred. We paired them one day earlier
than in experiment 1 with the expectation that it would increase the likelihood of
mating during the ﬁrst pairing, which it did. If no sperm was detected, the female
was re-paired as before during her next estrous cycle. Once mating had been
veriﬁed, vaginal smears were discontinued, and the day of conﬁrmation was
designated as day 0 of pregnancy. Four days later, females were moved to DD,
as above, and, after two days (i.e. day 6 of pregnancy), they were subjected to
the same light pulse/perfusion paradigm as females in the diestrous group.
Pregnancy was conﬁrmed by visual inspection of the uterus. For both the
pregnant and diestrous groups, control females were subjected to the same
treatments but did not experience a light pulse in the hour prior to perfusion.
Sections from one series of tissue (every third section) were processed for
Fos immunoreactivity (Fos primary concentration of 125,000; incubated for 24 h).
We counted the number of Fos-positive cells in the SCN core, SCN shell, and
vSPZ bilaterally in each section. This was done with a light microscope equipped
with a drawing tube (Leitz, Laborlux S, Wetzlar, Germany), since this method
was more sensitive to moderately-stained nuclei than the lmageJ method used in
Experiment 1 and the Fos antibody used for this tissue (from a different lot) had
a weaker afﬁnity. The borders of the SCN core and shell were based upon the
distribution of AVP in adjacent sections, and the vSPZ was delineated by a 215

pm x 160 pm rectangle dorsal to the SCN as described above (Fig. 2.1). All

26

cpJﬁS‘

pefusﬁ

Quanh
|

, r
iITYClJQT
V

pater

counts were made by an investigator unaware of the reproductive state, time of

perfusion, or light treatment of each animal.

Quantitative and Statistical Analysis

In each experiment, three sections (one rostral, middle, and caudal)
through the SCN and vSPZ were selected from each animal for analysis of
protein expression. In Experiment 1, sections chosen for analysis of Per2
expression were adjacent to those selected for analysis of Fos. For each region
of interest (SCN core, SCN shell, and vSPZ), counts of labeled cells from all
sections from an individual were summed. We then converted that sum for each
region in each individual to a percent of maximum value by dividing the sum by
the value from the individual with the highest number of immunoreactive cells in
the region in question. For all ﬁgures, these percentages, without further
transformation, are presented as means :I: SE.

The data for expression of each protein in each region were subjected to
Levene’s test for homogeneity of variances and then arcsine-transformed if they
did not meet the criteria of that test. If arcsine transformation did not equalize the
variances, then nonparametric tests (detailed below) were used to analyze the
original percent of maximum values for that region/protein combination. All data
analyses were conducted with SPSS 17 software (SPSS Inc., Chicago, IL, USA).
All differences were considered signiﬁcant when P<0.05. Statistical details are
omitted from the text when they are presented in ﬁgure legends.

Experiment 1. A total of 57 females were used in the ﬁnal analysis (n=28

diestrous, n=29 pregnant, n=4-6 per time point for each reproductive state). For

27

tea the
tensor

tartano

state it

“when E

6an
We

Ir
I399

data that met the homogeneity of variance criteria (vSPZ Fos, arcsine
transformations of SCN core Fos and Per2 and vSPZ Per2), a 2-way analysis of
variance (ANOVA) was performed, with reproductive state (pregnant or
diestrous) and time of perfusion (ZT) as between-subjects factors. Relevant post-
hoc least signiﬁcant difference (LSD) tests were conducted with these data when
any signiﬁcant interactions were found. For data that did not meet the
homogeneity of variance criteria even when transformed (SCN shell Fos and
Per2), nonparametric analyses were conducted to compare reproductive states
at each ZT (Mann-Whitney U tests) and to compare ZTs within each reproductive
state (Kruskal-Wallis tests, followed by post-hoc pairwise Mann-Whitney U tests
when signiﬁcant effects were found).

Experiment 2. A total of 47 females were used in the final analysis (n=23
diestrous, n=24 pregnant, n=5—7 per light treatment/CT for diestrous rats and n=6
per light treatment/CT for pregnant rats). For data that met the homogeneity of
variance criteria (vSPZ), a 3-way ANOVA was performed, with reproductive state
(pregnant or diestrous), time of perfusion (CT), and light treatment (pulse or
control) as between-subjects factors. Relevant post-hoc LSD tests were
conducted with these data when any signiﬁcant interactions were found. For data
that did not meet the homogeneity of variance criteria even when transformed
(SCN core and shell), nonparametric analyses were conducted to compare
reproductive states (pulsed and control separately) and effects oflight pulses

(pregnant and diestrous separately and combined) at each CT (Mann-Whitney U

28

narrc

$80:

M
U

ENG c
to .
hOVJe

Sign

tests) and to compare CTs within each reproductive state (pulsed and control

separately, Mann-Whitney U tests).

RESULTS

Experiment 1

SCN core. Representative photomicrographs depicting patterns of F os
and Per2 expression are shown in Fig. 2.2. A two-way ANOVA of Fos expression
in the core revealed a signiﬁcant main effect of ZT, with peak expression at ZT 2
(Fig. 2.3A; F=27.034, df=5, P<0.001). A non-signiﬁcant trend for higher Fos
expression in pregnant than diestrous females was also found (F=3.623, df=1,
P<0.065), but there was no interaction between reproductive state and ZT
(F=0.808, df=5, P>0.5). A two-way ANOVA of Per2 expression revealed a
signiﬁcant interaction between reproductive state and ZT (F=3.316, df=5, P<0.02)
as well as a signiﬁcant main effect of ZT (F=4.146, df=5, P<0.005). In contrast to
Fos expression in the SCN core, the rhythm of Per2 expression here differed as
a function of reproductive state (Fig. 2.33). The peak in diestrous females was
narrow and occurred at ZT 18. In pregnant rats, the phase of elevated Per2 was
longer, and expression was highest at ZT 14, at which point there was a
signiﬁcant difference between the two groups of females.

SCN shell. As in the core, Fos expression in the SCN shell peaked at ZT 2
and dropped to basal levels from ZT 6 through ZT 22 in the diestrous females
(Fig. 2.30; ZT: X2=11.918, df=5, P<0.04, Kruskal—Wallis test). In pregnant rats,
however, expression remained high throughout the light phase and did not reach

signiﬁcantly lower levels from ZT 2 or 6 until ZT 18 (ZT: X2=13.791, df=5, P<0.02,

29

In. Up

We

          

“B. Diestrous

Pregnant
2T2 N i I A 3"”
J ' T‘r. ' I. .

.u, .

 

Flgure 2.2. Representative photomicrographs of protein expression of Fos at ZT 2, 6, and 18 .
(A) and Per2 at ZT 2, 14, and 18 (B) in the middle SCN and vSPZ of diestrous (left) and preg-
nant rats (n'ght) kept in a 12:12 LD cycle. Per2 is only expressed in the cell nucleus. Larger cells
in (B) that are stained in the cytoplasm are TH-Ir cells and are primarily located around the
vSPZ and 3v. ocx, optic chiasm; 3v, third ventricle. Scale bar = 100 um.

30

Is ‘ ‘
n a .l
l a .1.

n r...
ah-
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15

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Id
C

A. SCN core B. SCN core

30 * + Diestrous 80 + Diestrous
' Pregnant **

-0- Pregnant

% of maximum Fos-ir
A
O

% of maximum Per2-ir

 

 

U 2' 6' 1i)
C. SCN shell

80

 

80

 

0')
O

% of maximum Per2-ir
N h

% of maximum Fos-ir

 

 

 

% of maximum Fos-ir
% of maximum Per2-ir

 

Zeitgeber Time Zeitgeber Time

Figure 2.3. Expression of Fos (left column) and Per2 (right column) in the SCN core (A,B), SCN
shell (CD), and vSPZ (E,F) of diestrous and pregnant rats kept in a 12:12 LD cycle. Expression
is measured as the percentage of the maximum individual value for each protein in each region.
*indicates a time point where expression is signiﬁcantly elevated relative to at least two other
time points within the same reproductive state, and “indicates a time point at which expression
signiﬁcantly differs between reproductive states (P<0.05, post-hoc LSD (A,B,E,F) or Mann-
Whitney U (C,D) tests).

31

Kruskal-
r'ran 138
5313 :'
F5332.

"FIF‘: -a
. cult

Kruskal-Wallis test). At ZT 6, expression was also signiﬁcantly higher in pregnant
than diestrous rats. As in the SCN core, the rhythm of Per2 expression in the
SCN shell of diestrous rats peaked at ZT 18 (Fig. 2.30; ZT: X2=13.372, df=5,
P5002, Kruskal-Wallis test), but the rhythm in pregnant females was of a lower
amplitude with a broader peak from ZT 10 to ZT 18 (ZT: X2=12.420, df=5,
P<0.03, Kruskal-Wallis test). Also, as in the core, expression in the shell of
pregnant rats was higher than that of diestrous females at ZT 14. Although
interactions cannot be directly assessed with nonparametric tests, these patterns
suggest that an interaction between reproductive state and ZT may exist for both
Fos and Per2 expression in the SCN shell.

vSPZ. A two-way ANOVA of Fos expression in the vSPZ revealed a
signiﬁcant interaction between reproductive state and ZT (F=3.459, dk5, P<0.02)
as well as a signiﬁcant main effect of ZT (F=5.203, dﬁ5, P<0.002). In diestrous
rats, there was a bimodal rhythm in Fos expression in the vSPZ, with peaks at ZT
2 and ZT 18 (Fig. 2.3E), but no rhythm existed in the vSPZ of pregnant rats
(F=1.724, dﬁ5, P>0.15, post-hoc one-way ANOVA for ZT effects). Expression in
the vSPZ of the two groups differed at ZT 6 (more in pregnant females) and at ZT
18 (more in diestrous females). A two-way ANOVA on Per2 expression in the
vSPZ revealed a signiﬁcant interaction between reproductive state and ZT
(F=2.822, df=5, P<0.03). There was no rhythm in Per2 expression in the vSPZ in
pregnant rats (Fig. 2.3F; F=0.932, df=5,P>0.4, post-hoc one-way ANOVA), but a
non-signiﬁcant trend for a rhythm with highest expression in the mid to late dark

phase was found in diestrous rats (F=2.191, df=5, P<0.095, post-hoc one-way

32

‘ﬁ‘i I '
,s'tr‘to’ u A

l
.

TI’_I
a)
(If)

u)
(I:

ins time

Experin

S
paused
IrESiTUL
18, with
m the c

Stntar‘

ANOVA). Expression differed between the two reproductive states only at ZT 14,
as pregnant females again had higher Per2 expression than diestrous females at
this time.

Experiment 2

SCN core. Representative photomicrographs depicting patterns of light-
induced Fos expression are shown in Fig. 2.4. Light-pulsed pregnant and
diestrous rats expressed more Fos in the core than controls at both CT 6 and CT
18, with signiﬁcantly more light-induced expression evident at CT 18 than at CT 6
in the core of pregnant rats (Fig. 2.5A; U=3,P<0.02, Mann-Whitney U test). A
similar trend was seen in diestrous females (U=5.5,P<0.085, Mann-Whitney U
test). There was no signiﬁcant difference, however, between diestrous and
pregnant rats in Fos responses to light in the SCN core at either CT 6 or CT 18
(U=17.5 and 8, P>0.9 and 0.2, respectively, Mann-Whitney U tests). Although we
could not directly test interactions with nonparametric tests, these data suggest
that neither light treatment nor time of perfusion interacted with reproductive
state, although they did appear to interact with one another.

SCN shell. A light pulse at CT 6 increased Fos in the SCN shell of
diestrous, but not pregnant, females (Fig. 2.5B; U=17, P>0.9, Mann-Whitney U
test). At CT 18, however, a light pulse led to higher Fos expression in both
groups of rats, and there was a trend for higher light-induced expression in
pregnant than in diestrous females (U=5.5, P<0.085, Mann-Whitney U test).

Although we could not directly test interactions with nonparametric tests, these

33

Co
0.6 .

FIgurl
88d V5
and C

Diestrous Pregnant
Control Light-Pulsed Control Light-Pulsed

, s y'- ~I I .2“
A .. ‘l‘f

    

   

Figure 2.4. Representative photomicrographs of protein expression of Fos in the middle SCN
and vSPZ of control and light-pulsed diestrous (left) and pregnant rats (right) at CT 6 (top row)
and CT 18 (bottom row). ocx, optic chiasm; 3v, third ventricle. Scale bar = 100 um.

34

A. SCN core

80' - Control
D Light-Pulsed

60‘

40¢

20‘

% of maximum Fos-ir

 

 

 

 

Diestrous Pregnant Diestrous Pregnant

B. SCN shell
1007

80- *
60-
40$
20-

 

 

% of maximum Fos-ir

 

 

 

 

Diestrous Pregnant Diestrous Pregnant

C.vSPZ

80-

 

% of maximum Fos-ir

 

 

 

 

 

 

 

Diestrous Pregnant Diestrous Pregnant
CT6 CT18

Figure 2.5. Expression of Fos in the SCN core (A), SCN shell (B), and vSPZ (C) of control and
light-pulsed diestrous and pregnant rats kept in DD for two days. Expression is measured as the
percentage of the maximum individual Fos-ir count within the region of interest. * indicates
signiﬁcantly higher Fos expression between light-pulsed and control females (P<0.05, post-hoc
LSD (C) or Mann-Whitney U (A,B) tests).

35

data so;

interact.

{reprodt
main of
rats exh
oestrou
light-pul

l8 lFrg.

DlSCU
l

imperti

ti=€Se it

The SC

data suggest that a three-way reproductive state x light treatment x CT
interaction may exist.

vSPZ. A three-way ANOVA revealed a signiﬁcant three-way interaction
(reproductive state x light treatment x CT, F=4.374, df=1, P<0.05) as well as a
main effect of light treatment (F=17.252, df=1, P<0.001). Diestrous and pregnant
rats exhibited responses to light at opposite times of day, with light-pulsed
diestrous females expressing more Fos in the vSPZ than controls at CT 6 and
light-pulsed pregnant rats expressing more Fos in the vSPZ than controls at CT

18 (Fig. 2.50).

DISCUSSION

The patterns of results seen in these two studies reveal that functional
properties of the SCN core, SCN shell, and vSPZ, and the relationships amongst

these regions, are substantially re-organized during early pregnancy.

The SCN core

Expression of Fos in the SCN core did not differ between pregnant and
diestrous rats. In both groups, Fos peaked in this region at ZT 2 and reached
baseline levels by ZT 6, a pattern that mirrors those reported previously in rats
(Guido et al., 1999; Jac et al., 2000; Schwartz et al., 2004; Schwartz et al., 1994;
Sumova et al., 2000; Sumova and Illnerova, 2005). It is likely that the peak at ZT
2 reﬂects the induction of Fos by lights-on (Schwartz et al., 1994). Light-induced
Fos expression in the SCN core, which occurred at both CT 6 and CT 18 but was

higher at CT 18, was also the same in pregnant and diestrous rats.

36

Although no differences in Fos expression were seen in the SCN core,
rhythms in Per2 in this region did vary as a function of reproductive state. In
diestrous rats, Per2 peaked at ZT 18 and dropped by ZT 22. Previous reports
have also shown peaks in Per2 expression in the dark phase in male and cycling
female Wistar rats, although at an earlier time than in diestrous rats in this study
(Amir et al., 2004; Perrin et al., 2006). In pregnant rats, however, the peak of the
Per2 rhythm in the core occurred four hours earlier (Fig. 2.3B). This led to a new
pattern of coupling between Fos and Per2 rhythms. This might reﬂect functionally
distinct cell populations, or differences within cells. For example, transcriptional
pathways driving Fos and Per2 expression may be differentially affected by

pregnancy.

The SCN shell
In the SCN shell of animals kept in an LD cycle, the rhythm of Fos

expression mirrored that in the core in diestrous females, with Fos rising sharply
from ZT 22 to ZT 2 and then dropping by ZT 6. In pregnant rats, Fos also rose
sharply from ZT 22 to ZT 2. However, in this case, high levels were maintained
throughout the light phase. The heightened Fos expression in the SCN shell at
ZT 6 was not due to an increase in responsivity to photic cues, as light did not
induce Fos in pregnant rats at this time but did in diestrous ones (Fig. 2.5B).
Interestingly, at CT 18 there was a trend for a higher response to light in
the SCN shell in pregnant females than in diestrous ones. This may be driven by
elevated levels of circulating prolactin. The SCN expresses mRNA for the short

form of the prolactin receptor on day 2 of pregnancy (Bakowska and Morrell,

37

2003), and it may do so throughout pregnancy, although no one has examined
this. Furthermore, although the effects of prolactin on the SCN of rats are not
known, in male hamsters, prolactin increases light-induced Fos in the SCN
during the mid-dark phase of the LD cycle (Cutrera et al., 2002). Perhaps
prolactin has a similar effect in the SCN shell in pregnant rats. It is unclear,
however, whether effects of prolactin are different at mid-day than at mid-night.
Circulating prolactin is nearing its peak at ZT 18 and reaches trough levels by ZT
6 on day 6 of pregnancy (Butcher et al., 1972; Freeman et al., 2000), which may
be why the SCN shell is unresponsive at CT 6 and hyper-responsive at CT 18.
As was the case in the core of the SCN, the peak in the rhythm of Per2
expression in the shell advanced during early pregnancy. However, whereas the
Per2 rhythm in the shell mirrored that in the core in diestrous rats, in pregnant
rats, the phase of elevated Per2 in this region appeared to be broader, and levels
were lower than in diestrous females. Although the Per2 peak appeared lower,
the peak of Fos expression in the shell did not change in magnitude. However,
the peaks of expression of both proteins were broadened in the shell of pregnant
rats, and these trends could be linked. The shell is less heterogeneous in cell
phenotype than the core, so perhaps Fos and Per2 are more often co-expressed ‘
in the shell, most likely in AVP cells (Dardente et al., 2002). This could explain
why the changes their rhythms undergo in pregnancy are similar, as they may

both reﬂect altered patterns of activity of AVPergic outputs from the shell.

38

The SCN core vs. shell

The difference between the SCN core and shell with respect to the
plasticity of their rhythms in Fos expression during early pregnancy is intriguing.
In a previous study, Lee et al. (1998) found that Fos rhythms in the SCN core,
but not the shell, differed between early and late pregnant rats. Thus, whereas
the transition from diestrus to early pregnancy is associated with changes in Fos
rhythms in the shell but not the core (Fig. 2.3), the opposite is the case during the
transition from early to late pregnancy. When we compare our data from early
pregnancy to those of Lee et al. (1998) on the same day, we see similar trends in
the two subregions. However, patterns in the core were somewhat different in the
two studies because animals were not sampled at the same times of day. Lee et
al. (1998), who did not sample between ZT 0 and ZT 4, may have missed the
peak that occurred around ZT 2 (Schwartz et al., 1994). Given the similarities
between the two studies on day 6 of pregnancy, it appears that, during late
pregnancy, both subregions of the SCN should exhibit different Fos rhythms from
those in a non-pregnant female, since changes in Fos expression in the shell
during early pregnancy are conserved in late pregnancy, and the rhythm of Fos
expression in the core changes from early to late pregnancy. Therefore, we
cannot conclude that Fos expression in the core is resistant to changes in
reproductive state in females, as might be deduced from our data alone, but
rather that Fos expression is affected differently in the SCN core and shell at
different stages of pregnancy.

Although Fos expression was differentially affected in the SCN core and

shell during early pregnancy, the peaks of Per2 rhythms in both regions were

39

advanced. '
secretions.
orariectorn
from ZT 12
they did no
prolactin ar
estrogens .
phase-adv.
phase of b
Morin et al
early pregr
0n activity
Plagnant r
ﬁne, and l'

‘t tem of

Potential

Altl
emlesslm
the SCN i:
Guido e1 E
2200). H o
Tore F03

a.SDOHSIVI

advanced. These advances may be due to changes in ovarian hormone
secretions. Nakamura et al. (2005) demonstrated that estradiol treatment of
ovariectomized (OVX) rats phase-advances the peak of Per2 mRNA expression
from ZT 12 to ZT 8 and also reduces the amplitude of this rhythm in the SCN, but
they did not examine the core and shell separately. Although progesterone and
prolactin are the predominant reproductive hormones during early pregnancy,
estrogens are also slightly elevated relative to diestrus. If estrogens can lead to a
phase-advance in Per2 expression, this might explain how they advance the
phase of behavioral rhythms of OVX females (Albers, 1981; Gerall et al., 1973;
Morin et al., 1977; Thomas and Armstrong, 1989). This effect may not occur in
early pregnancy, however, as progesterone counteracts the effects of estradiol
on activity rhythms in OVX females. However, the physiological milieu of a
pregnant rat is fundamentally different from that of an OVX, hormone-treated
one, and individual hormones may induce different effects on the circadian

system of females in the two reproductive states.

Potential sex differences in the SCN

Although male rats have repeatedly shown strong induction of Fos
expression after a light pulse in the subjective night, it has long been thought that
the SCN is non-responsive during the subjective day (Edelstein et al., 2000;
Guido et al., 1999; Rusak et al., 1990; Schwartz et al., 1994; Schwartz et al.,
2000). However, in our study, both diestrous and pregnant rats clearly expressed
more Fos in the SCN core after a light pulse at CT 6, and the shell was also

responsive in the diestrous rats at this time. These results therefore suggest that

40

aetdﬂe:
”nolknc
nsenes
ihzade
estgenr
attsgil
renounr
imeeshn
andtnna
emaesr
hedayr

tnetoh

Change

F
tmabs
ethese
tiialsee
0i ihe s
Washk
honxm

In This |

a sex difference may exist in how the SCN responds to light in this species. We
do not know if this difference is dependent on gonadal hormones, but estradiol
increases Fos expression in the SCN of female rats during the light phase
(Abizaid et al., 2004; Peterfi et al., 2004). This action may be direct, since
estrogen receptor- 6 (ER-B) is expressed in the SCN of female rats (Shughrue et
al., 1997), and this may explain why the SCN in our females was responsive to
light during the subjective day, while those of males in previous studies were not.
Interestingly, the rhythm of SCN VIP mRNA transcription is out of phase in male
and female rats, peaking in the dark phase in males and the light phase in
females (Krajnak et al., 1998). Therefore, the high expression of VIP mRNA in
the day may either lead to or be a result of our observed Fos responsivity in the

core to light in the mid-subjective day in non-pregnant females.

Changes in the vSPZ during early pregnancy

Fos expression in the vSPZ was quite different in diestrous and pregnant
females. The vSPZ of diestrous rats exhibited a clear bimodal rhythm of Fos
expression, with peaks at ZT 2 and again at ZT 18. This pattern contrasts with
that seen in the SCN shell, confirming that the vSPZ is not a functional extension
of the shell. The second peak was lost during early pregnancy, when expression
was highest from ZT 2 to ZT 6. The vSPZ of laboratory rats has not been
thoroughly described, but, in males, there is a unimodal, light-dependant rhythm
in this region, and it is very similar to that seen here in pregnant females
(Schwartz et al., 2004). The disappearance of the rhythm in DD in the males of

that study suggests that the peak at ZT 1 is stimulated by light onset (Schwartz et

41

at . 2304i V
hams
oeshous b
been seen
enmeoa
Fos in the

tsnhn

Ur
in the uS
Wise inc
emahs,
the diffe
hat Tats
at tile C
Di rep“
Poise (I
tats ke
males

SUQQe:

VSPZ

al., 2004), which is also likely to be the case for the peak at ZT 2 in both groups
of females in the current study. The second peak, at ZT 18, which is seen in
diestrous but not pregnant females, is more difﬁcult to explain. This rise has not
been seen in male rats, although it is possible that it would if they had been
sampled at ZT 18. This seems unlikely, however, as males have low levels of
Fos in the vSPZ at both ZT 17 and ZT 20 (Schwartz et al., 2004). The causes of
this potential difference between the sexes, and of the change from a diestrous
to a pregnant state, are unclear but may be related to changes in communication
between the SCN and the SPZ.

Under DD conditions, there were no time of day effects on Fos expression
in the vSPZ in either group of females. However, in diestrous females, a light
pulse induced Fos in the vSPZ at CT 6 but not CT 18, whereas in pregnant
females, the opposite pattern was observed (Fig. 2.5C). lntriguingly, this parallels
the differences in Fos expression at ZT 6 and ZT 18 in an LD cycle (Fig. 2.3E), in
that rats of both groups exhibited a response to light at a time when Fos was low
at the corresponding time in LD. In DD, however, no variation due to time of day
or reproductive state was apparent in Fos expression in the absence of a light
pulse (Fig. 2.5C). This suggests that the Fos rhythm seen in the vSPZ of female
rats kept on an LD cycle may not be endogenous, which is also the case for
males (Schwartz et al., 2004). However, the patterns of response to light pulses
suggest that an endogenous rhythm in responsiveness to light may exist in the

vSPZ and that this rhythm is very different in pregnant and diestrous females.

42

Na 5
pregnant or
ferales 1P
ion in the \
pregnant r.
Tris lndlcz
Ithence
r: the uSl
ISPZ of I
heuSPZ
do so.

Tl
ntegrat:
{Saperg

363835;

No signiﬁcant rhythm in Per2 expression was found in the vSPZ of our
pregnant or diestrous rats, but there was a trend in the vSPZ of diestrous
females (P < 0.095), with highest expression at ZT 18 and 22, when Per2 was
low in the vSPZ of pregnant rats. Additionally, Per2 was higher in the vSPZ of
pregnant rats at ZT 14, which coincided with peak Per2 expression in the SCN.
This indicates that Per2 expression in the vSPZ, while not signiﬁcantly rhythmic,
is likely to be inﬂuenced either by the SCN or by the same mechanisms that
inﬂuence Per2 in the SCN. Also, given that Fos expression is distinctly rhythmic
in the vSPZ of diestrous females and that light can induce Fos expression in the
vSPZ of both diestrous and pregnant rats, it is unlikely that Per2 expression in
the vSPZ drives Fos expression in this region. Instead, inputs from the SCN may
do so.

The vSPZ appears to serve as a modulator of SCN output signals and
integrator of these with other internally driven processes and environmental cues
(Saper et al., 2005; Schwartz et al., 2004; Schwartz et al., 2009). It is also
necessary for the generation of activity rhythms in rats (Abrahamson and Moore,
2006; Lu et al., 2001). During early pregnancy, when Iocomotor rhythms are lost
in DD (Rosenwasser et al., 1987), neither Fos nor Per2 expression in the vSPZ
were rhythmic. This is especially intriguing because core body temperature,
which is normally correlated with activity (Reﬁnetti, 1997; Reﬁnetti, 1999),
remains robustly rhythmic (Kittrell and Satinoff, 1988). The loss of rhythms in the

vSPZ reported here might therefore be responsible for this dissociation between

43

The role of
The
changes In
men?
seen in 0U
sash as th
'httnms.

atrancec
the SCN.
iC’IlE‘TEd l
SCN she
between
prolactrr

189:8). it

and Pa

related

body temperature and activity rhythms and the apparent loss of locomotor activity

rhythms.

The role of the SCN and vSPZ during early pregnancy

The changes in Fos and Per2 rhythms seen in this study may contribute to
changes in physiological and behavioral rhythms that occur during this critical
phase of pregnancy. This is supported by some comparisons between patterns
seen in our data and those from previous reports of rhythms in early pregnancy,
such as that noted above between the loss of locomotor activity and vSPZ
rhythms. Similarly, the peak of the core body temperature rhythm is phase-
advanced by a few hours (Kittrell and Satinoff, 1988), as is Per2 expression in
the SCN, and the peaks of circulating ACTH and corticosterone rhythms are
lowered (Atkinson and Waddell, 1995), as is the peak of Per2 expression in the
SCN shell. Although there are no immediately apparent direct correlations
between patterns seen in our data and the emergence of bimodal rhythms in
prolactin or in the increase in nighttime sleep (Butcher et al., 1972; Kimura et al.,
1996), it is still possible that the changes we observed contribute to these effects.

One important question raised by our results is how the changes in Fos
and Per2 expression in the core, shell, and vSPZ associated with pregnancy are
related to each other. One possibility is that the advance in phase of the Per2
rhythm in the core leads to changes in signals that it sends to the shell and to the
vSPZ, where input from the core and shell converge (Antle and Silver, 2005;
Daikoku et al., 1992; lbata et al., 1993; lbata et al., 1997; Leak and Moore, 2001;

Moore et al., 2002; Watts and Swanson, 1987; Watts et al., 1987; Watts, 1991).

44

This model would require only a change in how the core communicates to the
shell and vSPZ to account for the suite of changes in the rhythms that emerge
during pregnancy. It predicts that pregnancy should be accompanied by changes
in rhythms in core output signals, such as VIP and GRP, that are mirrored by
altered rhythms in shell outputs, such as AVP, and output signals from the vSPZ,
which have not yet been characterized.

Changes in processes seen here are likely to reﬂect only a few of the
mechanisms responsible for the varied effects of pregnancy on overt rhythms.
Other components of the circadian system, either within or beyond the SCN and
vSPZ, and regulatory networks outside of the circadian system altogether are
probably involved as well. Further examination of how these components change
will be necessary to determine how overt rhythms are altered during early

pregnancy.

45

CHAPTER 3
Differential changes in extra-SCN oscillators during early

pregnancy in the rat

INTRODUCTION

Circadian rhythms allow organisms to predict changes in the environment
and appropriately time behavioral and physiological events to anticipate them
(Moore-Ede et al., 1982), and they are critical to the regulation of mammalian
reproduction. During pregnancy, many overt rhythms in physiology and behavior
are altered in various ways. For example, in laboratory rats, activity rhythms
dampen while sleep increases during the dark phase (Kimura et al., 1996;
Rosenwasser et al., 1987), and the peak of the body temperature rhythm
advances (Kittrell and Satinoff, 1988). In these animals, a novel prolactin rhythm,
with peaks around dawn and dusk, emerges in early pregnancy and promotes
progesterone secretion by the corpora lutea (Butcher et al., 1972; Freeman et al.,
2000). Rhythms in the hypothalamic-pituitary-adrenal axis also change, with a
reduction in peak secretion of both corticotropin (ACTH) and corticosterone
(Atkinson and Waddell, 1995). While the establishment of the prolactin rhythm
has been well-studied (Erskine, 1995; Erskine et al., 2004; Freeman et al., 2000;
Lee et al., 1998), the neural mechanisms mediating changes in other rhythms
have received little attention.

Most daily rhythms are generated by endogenous circadian mechanisms
in the brain, and changes in them during early pregnancy may be promoted by

changes in these mechanisms. The suprachiasmatic nucleus (SCN), located

46

bilaterally in the anterior hypothalamus, houses the primary pacemaker in
mammals (Moore and Eichler, 1972; Ralph et al., 1990; Rusak, 1977; Stephan
and Zucker, 1972). The SCN contains oscillator cells, which each express a
molecular transcription/translation feedback loop that takes roughly 24 hours to
complete. This “molecular oscillator” involves multiple “clock” proteins, including
positive and negative elements, such as the period (Per) proteins, which are
themselves transcription factors for other genes, called clock-controlled genes
(Bell-Pedersen et al., 2005; Dunlap, 1999; Reppert and Weaver, 2002). Extra-
SCN oscillators, in which the same molecular feedback loops are expressed,
have been found within the brain and throughout the periphery and, together with
the SCN, comprise the circadian system (Guilding and Piggins, 2007; Hastings et
al., 2003; Reppert and Weaver, 2002; Weinert, 2005). The SCN is necessary to
synchronize most of these regions with one another, but there are conﬂicting
data on the extent to which extra-SCN oscillators within these regions require the
SCN to maintain internal synchrony, with some being less dependent on it than
others (Yamazaki et al., 2000; Yoo et al., 2004). It has been suggested that the
function of extra-SCN oscillators may be to integrate non-photic stimuli with
signals from the SCN to coordinate intracellular processes that vary from one
brain region or peripheral tissue to another (Guilding and Piggins, 2007; Herzog
and Tosini, 2001; Weinert, 2005; Yoo et al., 2004).

In Chapter 2, we demonstrated that both Fos and Per2 rhythms in the
SCN are altered during early pregnancy and argued that this may be part of the

mechanism driving changes in overt rhythms. However, other components of the

47

circadian timekeeping system, such as extra-SCN oscillators, may also play a
role. This seems particularly likely given that different overt rhythms are changing
in varied ways. In this study, we sought to evaluate this hypothesis by examining
Per2 expression patterns in oscillators within regions that regulate functions that
change during early pregnancy. We evaluated rhythms in Per2 expression in
diestrous and early pregnant rats ﬁrst in three portions of the extended
amygdala: the oval nucleus of the bed nucleus of the stria terminalis (BnST-ov),
central amygdala (CEA), and basolateral amygdala (BLA). The extended
amygdala is involved in regulating motivation and emotional state as well as
stress responses (Cardinal et al., 2002; Davis et al., 1997; Dong et al., 2001; Erb
et al., 2001; Szafarczyk et al., 1983), including insomnia (Cano et al., 2008).
During early pregnancy, female rats may exhibit reduced levels of anxiety
(Macbeth et al., 2008; but see Picazo and Fernandez-Guasti, 1993; Neumann et
al., 1998), and the amplitudes of both the ACTH and cortisol rhythms are
reduced (Atkinson and Waddell, 1995). We also examined patterns of Per2
expression in neuroendocrine dopaminergic cells that tonically inhibit prolactin
secretion (Ben-Jonathan, 1985; Freeman et al., 2000), which exhibits a novel

semicircadian rhythm in early pregnancy (Butcher et al., 1972).

EXPERIMENTAL PROCEDURES

Animals

Animals were the adult female and male Sprague-Dawley laboratory rats
used in Chapter 2 and obtained from Harlan Laboratories (Indianapolis, IN,

USA). Males were housed in pairs or groups of three when not paired with

48

females. Females were housed in groups of three or four for a habituation period
of at least two weeks and were subsequently separated for single-housing. All
subjects were housed in polypropylene cages (48x27x20 cm) with ad libitum
access to food (Teklad 8640 rodent diet, Harlan) and water under a 12h:12h
light/dark (LD) cycle with lights on at Zeitgeber Time (ZT) 0 and off at ZT 12. A
dim red light (<5 lux) remained on constantly for animal care purposes. All
experiments were performed in compliance with guidelines established by the
Michigan State University Institutional Animal Care and Use Committee and the
National Institutes of Health Guide for the Care and Use of Laboratory Animals.
All efforts were made to keep the number of animals used and their discomfort at

a minimum.

Determination of estrous cycle stages

Once singly housed, females were subjected to daily vaginal smears
between ZT 0 and ZT 3 to track their estrous cycles. A cotton-tipped applicator
soaked in sterile physiological saline was inserted into the vaginal opening,
gently rotated, and withdrawn. The applicator was immediately rolled onto a
glass slide and allowed to air dry. The dried slides were soaked in 0.25%
methylene blue dye for ﬁve minutes, rinsed with distilled water, and allowed to
dry prior to examination under a light microscope for the presence of nucleated
epithelial cells, corniﬁed epithelial cells, leukocytes, and mucus. Estrous cycle
stage was determined as follows: proestrus: predominantly nucleated epithelial
cells; estrus: predominantly to exclusively corniﬁed epithelial cells; diestrus

(between 2 to 3 consecutive days): predominantly leukocytes, some corniﬁed

49

and/or nucleated epithelial cells and mucus present. At least three estrous cycles

were established for each female.

Experimental Protocol

One group of females was perfused (see below for details) on day one of
diestrus, and another on day 6 of pregnancy. To obtain the second group, a male
was placed in the female’s cage on the morning of proestrus (between ZT 0 and
3). Males were removed the next morning, and a vaginal smear was taken to
check for the presence of sperm. If none were detected, the female was re-
paired on the morning of her next proestrus period. Once mating had been
veriﬁed, vaginal smears were discontinued, and the day of conﬁrmation was
designated as day 0 of pregnancy. Females were perfused 6 days later, and

pregnancy was confirmed by visual inspection of the uterus.

Tissue collection and immunocytochemistry

Tissue for this study was collected and processed in a previous
experiment (Chapter 2). Groups of animals from each reproductive state were
perfused at ZT 2, 6, 10, 14, 18, or 22. Each animal was given an overdose of
sodium pentobarbital, and perfused transcardially with 0.01 M phosphate-
buffered saline (PBS), pH 7.2, followed by 4% paraformaldehyde (Sigma, St.
Louis, MO) in 0.1 M phosphate buffer with 1.3% lysine and 4% sodium periodate.
Animals perfused during the dark phase were ﬁtted with a light-tight. hood prior to
perfusion to prevent acute exposure to light. Brains were post-ﬁxed for 4 hours,
transferred to 20% sucrose solution overnight, and stored in cryoprotectant at 4

°C overnight then at -20 °C until sectioning. Brains were sectioned coronally at

50

Tum IIIIOI

"‘3‘ iii‘l C
h it

One

Ir r a,

tease .a.

were nose

sector La

second;
938 for
avidin-t
‘t’eetas‘
Section
diamin
and hi
TEagnc
e)‘IJT‘es
and th
”(Irma

AIiETr

30 um into three alternate series using a freezing microtome, and sections were
placed in cryoprotectant for further storage at -20 °C.

One series of tissue was processed for immunocytochemical staining for
double labeling of Per2 and tyrosine hydroxylase (TH). Free ﬂoating sections
were rinsed in 0.01 M PBS and then incubated in 5% normal goat serum (NGS;
Vector Laboratories, Burlingame, CA, USA) in PBS with 0.3% Triton X-100 (TX)
for 1 h at room temperature. After a 10 minute rinse in PBS, sections were
incubated with a primary anti-Per2 antibody (mPER2 # 38, made in rabbit,
1:5,000, 24 h incubation; a generous gift from Dr. 'David Weaver, University of
Massachusetts, MA, USA) in 3% NGS and 0.3% TX in PBS on a rotator at 4°C
for 48 h. Sections were then rinsed in PBS and incubated with a biotinylated
secondary goat-anti-rabbit antibody (Vector; 1:200) in 3% NGS and 0.3% TX in
PBS for 1 h at room temperature, rinsed again, and then incubated with an
avidin-biotin peroxidase complex (0.9% each avidin and biotin solutions; ABC
Vectastain kit; Vector) in 0.3% TX with PBS for 1 h at room temperature. Next,
sections were rinsed in 0.125 M acetate buffer, pH 7.2, then reacted with
diaminobenzidine (DAB; 0.25 mg/mL, Sigma), nickel sulfate (25 mg/mL, Sigma),
and hydrogen peroxide (0.825 pL 0.3% H202 ImL buffer) to yield a blue-black
reaction product. After the reaction for Per2 staining, to process the tissue for TH
expression, the same sections were rinsed ﬁrst in 0.1 M phosphate buffer (PB)
and then in 0.2% TX in 0.2 M PB (PB-TX), and they were then incubated in 5%
normal horse serum (NHS; Vector) in PB-TX for 30 min at room temperature.

After rinses in PB-TX, sections were incubated with a primary anti-TH antibody

51

(made in mouse, Immunostar, Hudson, WI, USA; 1:20.000) in 3% NHS and PB-
TX on a rotator at 4°C for 24 h. Sections were then rinsed in PB-TX and
incubated with a biotinylated secondary horse-anti-mouse antibody (Vector;
1:200) in 3% NHS and PB-TX for 1 h at room temperature, rinsed again, and
then incubated with an avidin-biotin peroxidase complex (0.9% each avidin and
biotin solutions; ABC Vectastain kit; Vector) in PB-TX for 1 h at room
temperature. Next, sections were rinsed in Trizma buffer (pH 7.2, Sigma) then
reacted with DAB (0.2 mg/mL, Sigma) and hydrogen peroxide (0.35 uL 30%
H202 lmL buffer) to yield a brown reaction product. Trial immunocytochemical
reactions with the reagents used for this reaction were performed with tissue
from females that did not have easily determined estrous cycles or had mated
but were not pregnant to ensure speciﬁcity of the primary antibodies (data not
shown). All sections were mounted on clean slides, dehydrated, and

coverslipped.

Quantitative and Statistical Analysis
For single-label Per2 counts in the BnST-ov, CEA, and BLA, bilateral

images were taken from three sections of each region using a CCD video
camera (CX900, MBF Bioscience, Williston, VT, USA) attached to a light
microscope (Zeiss, Gdttingen, Germany). These images were processed using
Adobe Photoshop 7 (Adobe Systems, Mountain View, CA, USA). Per2-
immunoreactive (-ir) cells were then counted in these images with the NIH

ImageJ program (NIH, Bethesda, MD, USA). For the CEA and BLA, a 400 um x

52

400 um box was used to count Per2 cells, while the entire area of the BnST-ov
was counted (Fig. 3.1).

Preliminary examination of the three neuroendocrine dopaminergic cell
populations that inhibit prolactin secretion by the pituitary gland revealed that
double-labeled staining in the A12 tuberoinfundibular (TIDA) and
tuberohypophyseal (THDA) dopaminergic groups of the arcuate nucleus could
not be reliably identiﬁed. However, this was not the case in the A14 dopaminergic
cells of the periventricular hypothalamus (PHDA). Therefore, we examined co-
expression only in these cells. PHDA neurons exhibit the same Per2 rhythm as
the TIDA and THDA in ovariectomized (OVX) rats (Sellix et al., 2006) and the
same rhythm in expression of Fos-related antigens (FRA) as the other two cell
groups during pseudopregnancy in OVX females (Lerant et al., 1996). A 250 um
x 250 pm box was used to count the number of PHDA cells expressing TH and
the number of cells co- expressing TH and Per2 bilaterally through three sections
with a light microscope equipped with a drawing tube. These boxes were placed
adjacent to the third ventricle, just ventral to the midpoint of its length (Fig. 3.1).
All counts were made by an investigator unaware of the reproductive state or
time of perfusion of each animal.

A total of 57 females were used in the ﬁnal analysis (n=28 diestrous, n=29
pregnant, n=4-6 per time point for each reproductive state). A two-way analysis
of variance (ANOVA) was performed on the number of Per2-ir cells in the CEA
with reproductive state (pregnant or diestrous) and time of perfusion (ZT) as

between-subjects factors. Nonparametric analyses were performed on data from

53

 

 

 

LV 3v

  
  

BnST—ov

CPu

 

AC RCh

 

 

 

 

 

 

Figure 3.1. Line drawings depicting location of the BnST—ov (A), PHDA (B), CEA, and BLA (C).
Sampling boxes (black squares) were used for cell counts in the PHDA, CEA, and BLA.
Anatomical boundaries are based on Paxinos and Watson (1997). 3v: third ventricle; AC:
anterior commissure; CPu: caudate putamen; cx: cerebral cortex; ec: external capsule; f: fornix;
LV: lateral ventricle; opt: optic tract; Pe: periventricular hypothalamic nucleus; RCh: retrochias-
matic area.

54

 

the BnST-ov and BLA, as these datasets did not meet homogeneity of variance
criteria, even with square-root transformation. We compared the effects of
reproductive state at each ZT (Mann-Whitney U tests) and of ZT within each
reproductive state (Kruskal-Wallis tests, followed by post-hoc pairwise Mann-
Whitney U tests if signiﬁcant effects were found). We also examined the main
effects of ZT and reproductive state for the entire dataset using the same
statistical tests. To examine whether rhythms in Per2 expression in TH-ir cells in
the PHDA differed between diestrous and pregnant rats, we calculated the
proportion of TH-ir cells that co-expressed Per2 for each female and
subsequently arcsine-transformed these data. We then performed a two-way
ANOVA on the transformed data with reproductive state and ZT as between-
subjects factors. We performed a similar two-way ANOVA on the total number of
TH-ir cells to determine whether this varied across time or between reproductive
states. Post-hoc least signiﬁcant differences (LSD) tests were conducted when
signiﬁcant main effects were found. All data analyses were conducted with SPSS
17 software (SPSS Inc., Chicago, IL, USA). All differences were considered
signiﬁcant when P<0.05. Statistical details are omitted from the text when they

are presented in ﬁgure legends. Data are presented as means 1: SE.

RESULTS

Oval nucleus of the bed nucleus of the stria terminalis

The expression of Per2 changed as a function of time in diestrous females
(ZT: X2=11.832, dﬁS, P<0.04, Kruskal-Wallis test), with peak values occurring

from ZT 14 to 18, and trough values at ZT 22 (Fig. 3.2A). Per2 also changed as a

55

A. BnSt-ov B. Diestrous Pregnant

 

 

# Per2-ir cells
0' ._
O

O

 

 

10 14 18 22
Zeitgeber Time

Flgure 3.2. Expression of Per2 in the BnST-ov (A,B), CEA (CD), and BLA (E,F) of diestrous
and pregnant rats kept in a 12:12 LD cycle. (A,C,E): ‘indicates a time point where Per2 expres-
sion is signiﬁcantly elevated relative to at least one other time point within the same reproduc-
tive state, and '* indicates a time point at which expression signiﬁcantly differs between repro-
ductive states (P<0.05, post-hoc Mann-Whitney U tests). (B,D,F): Representative photomicro-
graphs depicting between group differences in Per2 expression in the BnST—ov at ZT 6 (B), and
within group variation at ZT 6 in the CEA (D) and at ZT 22 in the BLA (F). Scale bars= 200 um.

56

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function of time in pregnant females, but in this case, peak numbers of labeled
cells were present at ZT 6, at which time levels were signiﬁcantly higher than in
diestrous females (Fig. 3.2A and B; ZT: X2=13.391, df=5, P<0.02, Kruskal-Wallis
test). Thus, although interactions cannot be directly assessed with nonparametric
tests, these results reveal that rhythms in Per2 expression in the BnST-ov are

quite different in pregnant and diestrous females.

Central and basolateral amygdala

A two-way ANOVA revealed no signiﬁcant effect of ZT (F=1.274, df=5,
P>0.2) and no interaction between ZT and reproductive state (F=0.362, df=5,
P>0.8) on Per2 expression in the CEA (Fig. 3.2C). A non-signiﬁcant trend for a
main effect of reproductive state, with higher expression in pregnant than
diestrous rats, was found (pregnant: 129.4117.1 cells; diestrous: 89.41125 cells;
F=3.416, df=1, P<0.075), and expression in both groups was highly variable (Fig.
3.2D).

The density of Per2 labeled cells was much lower in the BLA than the
CEA (Fig. 3.2E). Neither ZT nor reproductive state had any effect on the number
of labeled cells in the BLA (ZT: X2=9.338, dﬁ5, P>0.09, Kruskal-Wallis test;
reproductive state: U=356, P>0.15, Mann-Whitney U tests). As in the CEA, Per2

expression in both groups was highly variable (Fig. 3.2F).

A14 periventricular hypothalamic dopaminergic neurons

The total number of TH-ir neurons sampled in the PHDA varied from 31 to
81 cells (mean 50.9113 cells), but the expression of TH did not differ between

reproductive states and was not rhythmic (Reproductive state: F=0.009, dﬁ1,

57

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P>0.9; ZT: F=0.572, dﬁ-5, P>0.7; Interaction: F=1.044, df=5, P>0.4, two-way
ANOVA). The expression of Per2 in these neurons, however, was rhythmic and
did differ as a function of reproductive state, although the effects of these
variables did not signiﬁcantly interact (Fig. 3.3; Reproductive state: F=5.508,
dk1, P<0.025; ZT: F=2.917, df=5, P<0.025; Interaction: F=1.111, df=5, P>0.3,
two-way ANOVA). Overall, pregnant rats expressed Per2 in a higher percentage
of TH-ir cells (36.612.6%) than diestrous females (27.9:2.7%), and the rhythm in
expression in the combined groups peaked from ZT 6 to ZT 10, with lowest
expression occurring at ZT 18 and 22. While no signiﬁcant interaction was found,
examination of the data indicates that the peak at ZT 6 was primarily due to
elevated Per2 expression in the pregnant rats, and a rise appears to have
occurred between ZT 6 and ZT 10 in diestrous but not pregnant females (Fig.

3.3A).

DISCUSSION

The current data reveal the emergence of new patterns of relationships
amongst extra-SCN oscillators during early pregnancy. The BnST-ov, CEA, and
BLA are part of the extended amygdala and play critical roles in the regulation of
fear, anxiety, and stress responses (Cardinal et al., 2002; Davis et al., 1997;
Dong et al., 2001; Erb et al., 2001; Szafarczyk et al., 1983). In diestrous rats,
Per2 expression was not rhythmic in either the CEA or BLA, and the rhythm of
Per2 expression in the BnST-ov was blunted, with highest expression occurring
in the early to mid-dark phase. The patterns in the CEA and BnST-ov seen here

are similar to those reported by Perrin et al. (2006) from cycling rats on metestrus

58

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% of TH-ir cells expressing Per2 .>
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Figure 3.3. Expression of Per2 in TH-expressing neurons in the PHDA of diestrous and pregnant
rats kept in a 12:12 LD cycle (A). There was no signiﬁcant interaction between reproductive state
and Zl', and asterisks (*) indicate time points where Per2 expression in TH cells is signiﬁcantly
elevated relative to at least two other time points in the combined (pregnant and diestrous)
rhythm. (B) Representative photomicrographs depicting Per2/TH-ir (left: purple/brown) and TH-ir
(right: brown) cells in the PHDA in two different individuals (each row). Scale bars = 10 um.

59

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(the equivalent of day 1 of diestrus). However, Per2 expression in the BLA in that
study was distinctly rhythmic, with peak expression occurring in the early light
phase, which was not the case in our diestrous rats. The reasons for the
discrepancy between their results and ours are unclear, but it may be due to the
use of different Per2 antibodies.

Although Per2 expression in the CEA and BLA did not signiﬁcantly change
during early pregnancy, the peak of the rhythm of expression in the BnST-ov was
higher and occurred roughly 8 to 12 hours earlier in pregnant rats than in
diestrous ones. Speciﬁcally, expression peaked at ZT 6 in pregnant females,
whereas in diestrous rats, it peaked from ZT 14 through 18 (Fig. 3.2A). A similar
increase in peak expression of Per2 in the BnST-ov occurs from diestrus to
proestrus and estrus, but on the latter days, it occurs around ZT 13 and is also
evident in Per2 expression in the CEA (Perrin et al., 2006). The dissociation
between Per2 expression in the CEA and BnST-ov has not, to our knowledge,
been found under any of the many experimental conditions in which Per2 has
been examined. Speciﬁcally, Per2 expression in these two nuclei responds in the
same manner to ﬂuctuations in various hormones, lesions of the SCN, removal of
the adrenal glands, dopaminergic manipulation, and food restriction (Amir and
Stewart, 2009a; Amir and Stewart, 2009b). The current data raise the question of
what might cause such a dissociation to occur in early pregnancy. The CEA
projects to the BnST-ov; thus it is possible that either signals sent from the CEA

or responses to those signals in the BnST-ov are altered during early pregnancy.

60

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Corticotropin-releasing factor (CRF) and enkephalin are both expressed in
cells within the BnST-ov and the CEA, including some that also contain
neurotensin (Day et al., 1999; Dong et al., 2001; Honkaniemi et al., 1992). Per2
is expressed in the enkephalin-containing cells of the BnST-ov of male rats (Amir
et al., 2004; Lamont et al., 2005), where the rhythm in its expression is inﬂuenced
by the CRF neurons (Amir and Stewart, 2009a; Amir and Stewart, 2009b;
Bhargava et al., 2004). We do not know whether CRF expression is different in
the BnST-ov on day 6 of pregnancy. However, at this time, rhythms in ACTH and
corticosterone secretion have reduced amplitudes, and ACTH secretion rises
earlier and remains elevated longer than in diestrous females (Atkinson and
Waddell, 1995). Although this advance is less dramatic than that in peak Per2
expression in the BnST-ov, it may also be, in part, a result of changes in CRF
expression in the BnST—ov. Another possibility is that changes in Per2 expression
in enkephalin cells could drive altered functioning of the CRF cells and lead to
downstream effects on stress hormone secretion and behavioral responses to
stress and anxiety.

Pregnancy is a reproductive state accompanied by a decline in stress
hormone secretion and a potential reduction in anxiety (Atkinson and Waddell,
1995; Macbeth et al., 2008; but see Picazo and Fernandez-Guasti, 1993;
Neumann et al., 1998), both of which are regulated by the BnST-ov (Davis et al.,
1997; Dong et al., 2001; Erb et al., 2001; Szafarczyk et al., 1983). The inverse
relationships between pregnancy-related changes in stress/anxiety and Per2

expression in the BnST-ov are most likely linked, as rhythmic expression of Per2

61

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in this region is dependent upon rhythmic exposure to glucocorticoids (Segall et
al., 2006). Therefore, our observed increase in peak Per2 expression in the
BnST-ov may reﬂect an increased sensitivity to feedback from adrenal
honnones.

As in the BnST—ov, Per2 expression in the PHDA cells was elevated in
pregnant relative to diestrous rats. This difference was driven by heightened
expression in the early to mid-light phase in pregnant rats. Examination of the
data (Fig. 3.3) suggests that the rising phase of the rhythm may have been
advanced during early pregnancy. Changes in rhythms in neuroendocrine
dopaminergic neurons may play a role in the emergence of the bimodal prolactin
rhythm that occurs in this stage of pregnancy (Butcher et al., 1972; Freeman et
al., 2000). In pseudopregnant rats, expression of FRA drops roughly 3 hours
before each peak in the PHDA, as well as in the TIDA and THDA neurons, and
dopamine concentrations in the PHDA follow this pattern of FRA expression
(Lerant et al., 1996). In OVX rats, the temporal pattern of Per2 in the PHDA
parallels that of dopamine turnover in the intermediate lobe of the pituitary gland
(Sellix et al., 2006), to which the PHDA exclusively projects (Goudreau et al.,
1995). We do not know if this relationship is maintained in early pregnancy, but
the apparent advance in the rise of Per2 expression in the PHDA seen here may
be necessary to allow FRA and dopamine synthesis in the PHDA to decline prior
to the prolactin surge at ZT 12. It is unclear whether this is necessary for the
establishment of the prolactin rhythm by vaginocervical stimulation. Prolactin

inhibits its own secretion by activating these cells (Hentschel et al., 2000a;

62

Hentschel et al., 2000b; Moore et al., 1980), and changes in Per2 expression in
them may therefore occur after the establishment of the prolactin rhythm and
serve only to maintain it (Bertram et al., 2006; Helena et al., 2009).

Taken together, our data suggest that changes in rhythms in the SCN,
such as those seen in Chapter 2, are not the only factors responsible for changes
in physiological and behavioral rhythms that occur during early pregnancy. The
fact that Per2 rhythms were modiﬁed in some extra-SCN oscillators, but not
others, suggests that the larger circadian timekeeping system is re-organized
during early pregnancy. The mechanisms responsible are uncertain, though
vaginocervical stimulation during mating, or the prolactin surges induced by this
stimulation, are very likely to play a role, perhaps inﬂuencing the SCN and extra-
SCN oscillators in different ways. However, changes in the SCN may still
contribute to those in extra-SCN oscillators. Rhythms in Per2 expression in these
regions are dependent upon the SCN (Lamont et al., 2005; Sellix et al., 2006),
and, although their magnitudes vary, phase advances in peak Per2 expression
appear to occur in the SCN, BnST-ov, and potentially the PHDA. Overall, the
data presented here support the hypothesis that extra-SCN oscillators serve to
integrate information concerning reproductive state with photically entrained
circadian signals from the SCN to differentially affect overt rhythms in a site-

speciﬁc manner as females undergo natural transitions in reproductive state.

63

CHAPTER 4
Changing patterns of Fos expression in the rat brain during

early pregnancy

INTRODUCTION

Circadian rhythms allow individuals to appropriately time behavioral and
physiological events to predictable changes in the environment (Moore-Ede et
al., 1982). These rhythms are essential to the regulation of mammalian
reproduction and may be subject to change as individuals progress through
various reproductive states. For example, during early pregnancy in laboratory
rats, locomotor activity is reduced and arrhythmic (Rosenwasser et al., 1987),
and sleep increases during the dark phase of the day (Kimura et al., 1996), a
time when non-pregnant rats are most active. Whereas the sleep-wake cycle
becomes less rhythmic, the core body temperature rhythm is robust but
experiences an advance in the phase of its peak (Kittrell and Satinoff, 1988).
Rhythms of both circulating corticotropin (ACTH) and corticosterone also persist,
although their amplitude is reduced compared to the non-pregnant state
(Atkinson and Waddell, 1995). Additionally, a new rhythm in prolactin secretion
emerges during early pregnancy, with daily surges around dawn and dusk that
serve to promote progesterone secretion by the corpora lutea (Butcher et al.,
1972; Freeman et al., 2000). The neural mechanisms underlying the emergence
of this rhythm have been partially identiﬁed (Erskine, 1995; Erskine et al., 2004;

Freeman et al., 2000; Lee et al., 1998), but those leading to changes in other

64

rhythms as a female progresses from a non-pregnant state to early pregnancy
have not been examined.

The circadian system includes a primary circadian pacemaker located in
the suprachiasmatic nucleus (SCN) of the hypothalamus (Moore and Eichler,
1972; Ralph et al., 1990; Rusak, 1977; Stephan and Zucker, 1972), as well as
various extra-SCN oscillators (Guilding and Piggins, 2007; Hastings et al., 2003;
Reppert and Weaver, 2002; Weinert, 2005). This system undergoes some
changes during early pregnancy (see Chapters 2 and 3), and these changes may

drive altered functioning of non-circadian systems to lead to different rhythms in

 

early pregnancy. However, non-circadian systems may also be inﬂuenced by
transitions in reproductive state and respond to signals from the circadian system
in different ways from one another to further modify overt rhythms. The fact that
rhythms in different functions are modiﬁed in multiple ways suggests that
changes in a more central circadian timekeeping system are unlikely to account
entirely for the patterns that emerge in early pregnancy. In this study, we address
this issue by comparing rhythms in expression of Fos in various brain regions
that regulate these functions between early pregnant and diestrous rats. The Fos
protein is encoded by the immediate early gene cfos that is often expressed after
neuronal activation (Kovacs, 2008).

Of the brain regions we examined in this study, some regulate more than
one behavioral or physiological system that is altered in early pregnancy, and
others serve more speciﬁc functions. The medial preoptic area (MPA), for

instance, plays a role in the regulation of core body temperature (Satinoff et al.,

65

 

1982), the initiation of prolactin surges in pregnancy (Freeman and Banks, 1980;
Gunnet and Freeman, 1984), and the maintenance of sleep, as well as sexual
and parental behaviors (for reviews, see Boulant, 2000; Kumar et al., 2007;
Numan, 2007; Sakuma, 2008). The paraventricular hypothalamus (PVN) and
dorsomedial hypothalamus (DMH) have both been implicated in the regulation of
the prolactin surges in early pregnancy and are also involved in the regulation of
the stress axis (Arey and Freeman, 1992a; Arey and Freeman, 1992b; Bernardis
and Bellinger, 1998; Freeman and Banks, 1980; Gunnet et al., 1981; Herman et
al., 2003). In contrast, the ventrolateral preoptic area (VLPO) and cholinergic
basal forebrain have more narrowly deﬁned functions. The VLPO is a sleep—
active brain region (Gaus et al., 2002; Sherin et al., 1996; Szymusiak et al.,
1998), whereas wakefulness is promoted by parts of the basal forebrain: the
medial septum (MS) and vertical and horizontal diagonal bands of Broca (VDB
and HDB, respectively; Greco et al., 2000; Jones, 2008).

We also examined rhythms in Fos expression in regions that contain
oscillator cells, such as the three regions of the extended amygdala studied in
Chapter 3: the central and basolateral amygdala (CEA and BLA, respectively)
and the oval nucleus of the bed nucleus of the stria terminalis (BnST-ov). These
regions are involved in regulating motivation and emotional state as well as
stress responses (Cardinal et al., 2002; Davis et al., 1997; Dong et al., 2001; Erb
et al., 2001; Szafarczyk et al., 1983), including insomnia (Cano et al., 2008). We
also characterized patterns of Fos expression in the periventricular hypothalamus

(PeVN), which houses the periventricular dopaminergic neurons, studied in

66

Chapter 3, that inhibit prolactin secretion. This region also contains oxytocinergic

neurons that may promote prolactin secretion (Kennett et al., 2008).

EXPERIMENTAL PROCEDURES

Animals

Animals were the adult female and male Sprague-Dawley laboratory rats
used in Chapters 2 and 3 and obtained from Harlan Laboratories (Indianapolis,
IN, USA). Males were housed in pairs or groups of three when not paired with
females. Females were housed in groups of three or four for a habituation period
of at least two weeks and were subsequently separated for single-housing. All
subjects were housed in polypropylene cages (48x27x20 cm) with ad libitum
access to food (Teklad 8640 rodent diet, Harlan) and water under a 12h:12h
light/dark (LD) cycle with lights on at Zeitgeber Time (ZT) 0 and off at ZT 12. A
dim red light (<5 qu) remained on constantly for animal care purposes. All
experiments were performed in compliance with guidelines established by the
Michigan State University Institutional Animal Care and Use Committee and the
National Institutes of Health Guide for the Care and Use of Laboratory Animals.
All efforts were made to keep the number of animals used and their discomfort at

a minimum.

Determination of estrous cycle stages

Once singly housed, females were subjected to daily vaginal smears
between ZT 0 and ZT 3 to track their estrous cycles. A cotton-tipped applicator
soaked in sterile physiological saline was inserted into the vaginal opening,

gently rotated, and withdrawn. The applicator was immediately rolled onto a

67

glass slide and allowed to air dry. The dried slides were soaked in 0.25%
methylene blue dye for ﬁve minutes, rinsed with distilled water, and allowed to
dry prior to examination under a light microscope for the presence of nucleated
epithelial cells, corniﬁed epithelial cells, leukocytes, and mucus. Estrous cycle
stage was determined as follows: proestrus: predominantly nucleated epithelial
cells; estrus: predominantly to exclusively corniﬁed epithelial cells; diestrus
(between 2 to 3 consecutive days): predominantly leukocytes, some corniﬁed
and/or nucleated epithelial cells and mucus present. At least three estrous cycles

were established for each female prior to further experimental manipulations.

Experimental Protocol

One group of females was perfused (see below for details) on day one of
diestrus, and another on day 6 of pregnancy. To obtain the second group, a male
was placed in the female’s cage on the morning of proestrus (between ZT 0 and
3). Males were removed the next morning, and a vaginal smear was taken to
check for the presence of sperm. If none were detected, the female was re-
paired on the morning of her next proestrus period. Once mating had been
veriﬁed, vaginal smears were discontinued, and the day of conﬁrmation was
designated as day 0 of pregnancy. Females were perfused 6 days later, and

pregnancy was conﬁrmed by visual inspection of the uterus.

Tissue collection and immunocytochemistry

Tissue for this study was collected and processed in a previous
experiment (Chapter 2). Groups of animals from each reproductive state were

perfused at ZT 2, 6, 10, 14, 18, or 22. Each animal was given an overdose of

68

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sodium pentobarbital, and perfused transcardially with 0.01 M phosphate-
buffered saline (PBS), pH 7.2, followed by 4% paraformaldehyde (Sigma, St.
Louis, MO) in 0.1 M phosphate buffer with 1.3% lysine and 4% sodium periodate.
Animals perfused during the dark phase were ﬁtted with a light-tight hood prior to
perfusion to prevent acute exposure to light. Brains were post-ﬁxed for 4 hours,
transferred to 20% sucrose solution overnight, and stored in cryoprotectant at 4
°C overnight then at -20 °C until sectioning. Brains were sectioned coronally at
30 um into three alternate series using a freezing microtome, and sections were
placed in cryoprotectant for further storage at -20 °C.

One series of tissue was processed for immunocytochemical staining to
detect Fos protein expression in the cell nucleus. Free ﬂoating sections were
rinsed in 0.01 M PBS and then incubated in 5% normal goat serum (NGS; Vector
Laboratories, Burlingame, CA, USA) in PBS with 0.3% Triton X-100 (TX) for 1 h
at room temperature. After a 10 minute rinse in PBS, sections were incubated
with a primary anti-cFos antibody (made in rabbit, Santa Cruz Biochemistry,
Santa Cruz, CA, USA; 125,000) in 3% NGS and 0.3% TX in PBS on a rotator at
4°C for 48h. Sections were then rinsed in PBS and incubated with a biotinylated
secondary goat-anti-rabbit antibody (Vector; 1:200) in 3% NGS and 0.3% TX in
PBS for 1 h at room temperature, rinsed again, and then incubated with an
avidin-biotin peroxidase complex (0.9% each avidin and biotin solutions; ABC
Vectastain kit; Vector) in 0.3% TX with PBS for 1 h at room temperature. Next,
sections were rinsed in 0.125 M acetate buffer, pH 7.2, then reacted with

diaminobenzidine (DAB; 0.25 mg/mL, Sigma), nickel sulfate (25 mg/mL, Sigma),

69

and hydrogen peroxide (0.825 uL 0.3% H202 lmL buffer) to yield a blue-black
reaction product. Trial immunocytochemical reactions with the reagents used for
this reaction were performed with tissue from females that did not have easily
determined estrous cycles or had mated but were not pregnant to ensure
specificity of the primary antibodies (data not shown). All sections were mounted

on clean slides, dehydrated, and coverslipped.

Quantitative and Statistical Analysis

To count the number of Fos-immunoreactive (-ir) cells in each brain
region, photographs were taken from three sections of each region, on both sides
of the brain or at the midline (MS and VDB), using a CCD video camera (CX900,
MBF Bioscience, Williston, VT, USA) attached to a light microscope (Zeiss,
Gottingen, Germany). These images were processed using Adobe Photoshop 7
(Adobe Systems, Mountain View, CA, USA). Cells expressing Fos were then
counted in the images with the NIH lmageJ program (NIH, Bethesda, MD, USA).
The entire area of the BnST—ov was counted (Fig. 4.1), but sampling boxes of the
following sizes were used to count Fos-ir cells in the other regions of interest:
400 pm x 400 pm in the CEA, BLA, MPA, and DMH; 300 um x 300 pm in the
VLPO and PVN; 300 um x 600 pm in the MS, VDB, and HDB; 250 um x 250 pm
in the PeVN (see Fig. 4.1 for placement). All counts were made by an
investigator unaware of the reproductive state or time of perfusion of each
animal.

A total of 57 females were used in the ﬁnal analysis (n=28 diestrous, n=29

pregnant, n=4-6 per time point for each reproductive state). A two-way analysis

70

 

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Figure 4.1. Line drawings depicting location of the sampling regions in the MS, VDB, and HDB
(A); BnST—ov (B); VLPO and MPA (C); PVN and PeVN (D); CEA and BLA (E), and DMH (F).
Sampling boxes (black squares) were used for cell counts in all regions except the BnST—ov
(entire area in black). Anatomical boundaries are based on Paxinos and Watson (1997). 3v:
third ventricle; AC: anterior commissure; ARC: arcuate nucleus; CPu: caudate putamen; cx:
cerebral cortex; ec: external capsule; f: fornix; LV: lateral ventricle; ocx: optic chiasm; opt: optic
tract; Pe: periventricular hypothalamic nucleus; RCh: retrochiasmatic area; VMPO: ventromedial

preoptic area.

71

of variance (ANOVA), with reproductive state (pregnant or diestrous) and time of
perfusion (ZT) as between-subjects factors, was performed on the number of
Fos-ir cells in the PeVN, PVN, and DMH, and on square-root transformed counts
in the BnST—ov and MS. These transformations were used to equalize variances
amongst treatment groups. Post-hoc least signiﬁcant differences (LSD) tests
were conducted on these data when signiﬁcant main effects were found.
Nonparametric analyses were performed on data from regions in which square
root transformations failed to equalize the variances: the CEA, BLA, VLPO, VDB,
HDB, and MPA. In these cases, we compared the effects of reproductive state at
each ZT and for all ZTs combined (Mann-Whitney U tests) and of ZT within each
reproductive state and for the two reproductive states combined (Kruskal-Wallis
tests, followed by post-hoc pairwise Mann-Whitney U tests if signiﬁcant effects
were found). All data analyses were conducted with SPSS 17 software (SPSS
Inc, Chicago, IL, USA). All differences were considered signiﬁcant when P<0.05.
Details of statistics are omitted from the text when they are presented in ﬁgure

legends. Data are presented as means :t SE.

RESULTS
The oval nucleus of the bed nucleus of the stria terminalis

Fos expression in the BnST-ov was signiﬁcantly affected by time (Fig.
4.2A; F=8.853, df=5, P<0.001, two-way ANOVA) and by reproductive state
(F=16.785, df=1, P<0.001), but there was no interaction between these variables

(F=1.568, df=5, P> 0.15). Pregnant females expressed more Fos in the BnST-ov

72

-o- Diestrous

A. BnST-ov . B. Diestrous Pregnant

 

 

 

 

 

 

.9.
250 RS: P < 0.001 Pregnant
g 200 ZT:P<0.001
O
3 150
2 100
LL
* 50
C. CEA D-
RS: P < 0.01 4‘ 1" .,
ZT:P<0.05 diestrous only 2T6 . , '-*::
g 150 "53
O
0
{g 100
u.
*t . .
. ‘ _ . 5
,, ‘ --.- ...ia'
E. PVN
80
RS: NS
g 60 ZT:P<0.05
0
0
LL
a: 20

 

<3

 

Zeitgeber Time

Figure 4.2. Expression of Fos in the BnST—ov (A,B), CEA (CD), and PVN (E,F) of diestrous and
pregnant rats kept in a 12:12 LD cycle. (A,C,E): Main effects of reproductive state (RS) and ZT
are indicated in text on each graph (A, E: two-way ANOVA; C: non-parametric tests). There was
no signiﬁcant interaction of ZT and reproductive state in the BnST-ov or PVN. NS: not signiﬁcant
(P>0.05). * indicates that Fos expression was signiﬁcantly elevated relative to at least one other
time point within the same reproductive state, and ** indicates a signiﬁcant difference between
reproductive states (P<0. 05, post-hoc Mann- Whitney U tests) (B D ,:F) Representative photomi-
crographs of Fos-labelled cells' in each region at ZT 6. LV: lateral ventricle; 3v: third ventricle.
Scale bars= 200 um.

73

than diestrous females, but the rhythm was the same in the two groups (Fig. 4.2A

and B). It peaked at ZT 2 and reached trough values at ZT 10.

The central and basolateral amygdala

In the CEA, a low amplitude rhythm with a peak during the dark phase
was seen in diestrous females (Fig. 4.2C; ZT: X2=11.304, dﬁ5, P<0.05. Kruskal-
Wallis test), but there'was no effect of time in pregnant females (ZT: X2=8.646,
dﬁ5, P>0.1, Kruskal-Wallis test). In addition, Fos was higher in pregnant than in
diestrous females (main effect: U=243, P<0.01, Mann-Whitney U-test),
particularly at ZT 6 (Fig. 4.2C and D). Although interactions cannot be directly
assessed with nonparametric tests, these patterns suggest that the transition to
the pregnant state eliminates the rhythm in Fos within the CEA due to rises in its
expression. In contrast, in the BLA, Fos expression was low (6.51:1:1 .74 cells, all
animals) and did not change over time among either pregnant or diestrous rats
(pregnant ZT: x2=5.210, df=5, P>0.3; diestrous ZT: X2=5.885, df=5, P>0.3 ,
Kruskal-Wallis tests), nor did it differ as a function of reproductive state (U=329,

P>0.2, Mann-Whitney U test).

The hypothalamic paraventricular nucleus

Fos expression in the PVN was affected by time (F=3.104, df=5, P<0.02,
two-way ANOVA) but not reproductive state (F=1.652, df=1, P>0.2) or an
interaction between these variables (Fig. 4.2E and F; F=1.872; df=5, P>0.15‘).
The pattern was bimodal with peak levels of expression occurring at ZT 2 and
again at ZT 14 and troughs occurring in the mid to late light phase and again at

ZT 22.

74

The medial preoptic area

In the MPA of diestrous rats, Fos expression changed as a function of
time of day (Fig. 4.3A; X2=14.434, dﬁ5, P<0.02, Kruskal-Wallis test), and peak
values occurred from the early dark phase through the early light phase, with
signiﬁcantly elevated expression at ZT 2 and 18. In pregnant rats, Fos
expression in the MPA, which was tonically low, was arrhythmic (ZT: X2=8.812,
df=5, P>0.1, Kruskal-Wallis test). Fos was signiﬁcantly lower in pregnant rats
than in diestrous ones at ZT 2, 10, 18, and 22 (Fig. 4.3A and B). These patterns
suggest that there is likely an interaction between the effects of reproductive

state and ZT on Fos expression in this area.

The periventricular hypothalamus

Patterns of Fos expression in the PeVN were significantly affected by time
of day (Fig. 4.3C; F=3.567, dﬁ5, P<0.01, two-way ANOVA) and by an interaction
between time of day and reproductive state (F=3.063, df=5, P<0.02). A rhythm
was seen in diestrous females with peak values occurring at ZT 2 and again at
ZT 18, but the effect of time on expression in the PeVN of pregnant females did
not quite reach statistical signiﬁcance (F=2.58, df=5, P<0.06, post-hoc one-way
ANOVA). Expression was also signiﬁcantly higher in the PeVN of diestrous rats

at ZT 18 than in pregnant females (Fig. 4.3C and D).

The dorsomedial hypothalamus

In the DMH, Fos expression was affected by time (Fig. 4.3E; F=3.269;
df=5, P<0.02, two-way ANOVA) but not by reproductive state (Fig. 4.3E and F;

F=0.08, df=1, P>0.75), and there was no interaction between these variables

75

A‘ MPA -°- Diesmus B. Diestrous Pregnant
300 RS:P<0.001 *‘P'egﬂgnt 21;ij .. ‘ — :3
** ZT: P < 0.05 diestrous only ', '. I

§

# Fos-ir cells

   

    

# Fos-ir cells

I“
a
guess
:r:
.11

 

-
Q-
d
O
—l
A
—I
or:
R3

 

1m ‘. .". ’_ .. “ .‘ ‘ 'V I .1
E 60 ' .v 3V Ti?
_: . _ _ '..,-

' .' - . ‘ . “I v
t to _ . , .
LL - ..."
3t 20 '.x’.". W 3' -. "i"

 

C3

 

I I ﬁt ﬂ l l
2 6 1o 14 1a 22
Zeitgeber 'llme

Figure 4.3. Expression of Fos in the MPA (A,B), PeVN (CD), and DMH (E,F) of diestrous and
pregnant rats kept in a 12:12 LD cycle. (A,C,E): Main effects of reproductive state (RS) and ZT
are indicated in text on each graph (A: non-parametric tests; C,E: two-way ANOVA). There was
a signiﬁcant interaction of ZT and reproductive state in the PeVN but not the DMH. Notation is
the same as in Fig. 4.2 (post-hoe Mann-Whitney U tests (A) or LSD tests (C)). (B,D,F): Repre-
sentative photomicrographs depicting between group differences in Fos expression in each
region at ZT 18. 3v: third ventricle; f: fornix. Scale bars = 200 um.

76

(F=0.91, (II

1112 early Ill

The ventr

Fos
diestrous
in pregna
df=5, P<I
reproduc
expressi
ones (m
suggesl

ZT on I
The be
female

not aﬁ

also n

were

 

(F=0.91, df=5, P>0.45). The rhythm peaked from the early dark phase through

the early light phase, with highest expression at ZT 2, 14, and 18.

The ventrolateral preoptic area

Fos expression in the VLPO did not change as a function of time in
diestrous rats (Fig. 4.4A; X2=6.399, df=5, P>0.25, Kruskal-Wallis test), but it did
in pregnant ones, in which peak values occurred from ZT 22 to 2 (X2=12.525,
df=5, P<0.03, Kruskal-Wallis test). Expression did not differ between the two
reproductive states at any time point (Fig. 4.4A and B), but there was a trend for
expression to be higher in the VLPO of diestrous females compared to pregnant
ones (main effect: U=289.5, P<0.1, Mann-Whitney U-test). These patterns
suggest that an interaction may exist between effects of reproductive state and

ZT on Fos expression in this area.

The basal forebrain

Fos expression was higher in the MS in diestrous rats than pregnant
females (Fig. 4.4C and D; F=13.574, dﬁ1, P<0.002, two-way ANOVA), but it was
not affected by time of day in either group (F=1.638, df=5, P>0.15). There was
also no interaction between these variables (F=0.948, df=5, P>0.45). Patterns
were somewhat similar in the VDB, but here a rhythm of Fos expression was
detected in diestrous rats, in which Fos was elevated throughout the dark phase
(Fig. 4.4E; X2=11.052, df=5, P<0.05, Kruskal-Wallis test). In contrast, Fos
expression in the VDB of pregnant rats was tonically low and was not affected by
time of day (X2=4.929, df=5, P>0.4, Kruskal-Wallis test). Expression was

signiﬁcantly higher in diestrous than pregnant females (main effect:

77

+ Diestrous . .
60 RS: NS '9' Pregnant 2T 22
ZT: P < 0.05 pregnant only '. ,

A. VLPO B. Diestrous Pregnant

# Fos-ir cells

   

 

# Fos-ir cells

 

 

E. VDB F.
50- RS: P < 0.005 2123?; i ' .f - .3; .
ZT: P < 0.05 diestrous only 3 . - ,j ".5 ." ‘ -
-1 ** 3 ' ‘ H ,3 w.
2 40 ' _.
g .
h 30-

em-
“ 10-1

 

 

 

0 u u
2 6 10 14 18 22
ZeitgeberTime

Figure 4.4. Expression of Fos in the VLPO (A,B), MS (CD), and VDB (E,F) of diestrous and
pregnant rats kept in a 12:12 LD cycle. (A,C,E): Main effects of reproductive state (RS) and ZT
are indicated in text on each graph (A,E: non-parametric tests; C: two-way ANOVA). There was
no signiﬁcant interaction of ZT and reproductive state in the MS. Notation is the same as in Fig.
4.2 (post-hoc Mann-Whitney U tests). (B,D,F): Representative photomicrographs depicting
between group differences in Fos expression in each region at ZT 22. ocx: optic chiasm. Scale
bars = 200 um.

78

Uzlil.
These
reprod
expres
afunc
ﬂiﬁOf
Ipreg'

Krusl

[NESC

nnnf
inste
and
\HDE
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ind'
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rea

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bra

U=177.5, P<0.003, Mann-Whitney U-test), particularly at ZT 22 (Fig. 4.4E and F).
These patterns suggest that there is likely an interaction between the effects of
reproductive state and ZT on Fos expression in the VDB. In contrast, Fos
expression in the HDB was low (8.64:1.36 cells, all animals) and did not differ as
a function of reproductive state (U=290.5, P>0.1, Mann-Whitney U test). In this
region, Fos was not affected by time of day in either pregnant or diestrous rats
(pregnant ZT: x2=2.437, df=5, P>0.75; diestrous ZT: x2=4.442, dﬁ5, P>0.45,

Kruskal-Wallis tests).

DISCUSSION

The current data reveal that reproductive state affects light-entrained
rhythms in Fos expression in various brain regions in site-speciﬁc ways. For
instance, pregnancy led to increased expression of Fos in the BnST-ov and CEA
and a decrease in Fos expression and lack of rhythmicity in the MPA, MS, and
VDB, whereas there were no changes in the rhythms in the PVN or DMH.
Additionally, pregnancy led to the emergence of a significant rhythm in Fos
expression in the VLPO and the loss of a rhythm in the PeVN without affecting
the total amount of Fos expressed in the day in either area. This variation
indicates that the changes associated with pregnancy cannot be entirely
explained by those in the SCN/vSPZ described in Chapter 2. For the same
reason, all of these effects of reproductive state cannot be due entirely to
modulation of the circadian system as a whole.

Some salient patterns seen in this study may reﬂect altered functioning of

brain regions that could directly lead to changes in overt rhythms that occur

79

during early pregnancy. For instance, pregnancy—induced modulation of the
BnST-ov and CEA might alter stress hormone secretion and anxiety (Cardinal et
al., 2002; Davis et al., 1997; Dong et al., 2001; Erb et al., 2001; Szafarczyk et al.,
1983), both of which are reduced in early pregnancy in rats (Atkinson and
Waddell, 1995; Macbeth et al., 2008; but see Picazo and Fernandez-Guasti,
1993; Neumann et al., 1998). The increase in Fos in these two brain regions,
which contain glucocorticoid receptors (Honkaniemi et al., 1992; Lechner and
Valentino, 1999), might reflect an increased sensitivity to stress hormone
feedback. The lack of any effect on Fos expression in the PVN, however, which
more directly regulates corticosterone and ACTH secretion, may indicate that
changes in the BnST-ov and CEA are not directly inﬂuencing changes in the
stress axis. Another possibility is that these changes are indicative of how
pregnancy alters the regulation of anxiety by stress hormones (Roozendaal et al.,
~ 2009)

In the PeVN, which plays a role in the regulation of prolactin secretion
(Ben-Jonathan, 1985; Freeman et al., 2000; Kennett et al., 2008), Fos
expression was signiﬁcantly lower in pregnant rats at ZT 18, a time when Per2
expression was also signiﬁcantly lower within periventricular tyrosine hydroxylase
(TH) cells of pregnant rats (Fig. 3.3A). The reduced Fos expression at ZT 18 in
the PeVN occurs roughly two hours before the second daily surge in prolactin
(Butcher et al., 1972; Freeman et al., 2000), a pattern suggesting that it might
reﬂect activity of prolactin-inhibiting (dopaminergic) neurons. lntriguingly, the

patterns of Fos expression in the PeVN of both diestrous and pregnant rats

80

appear almost identical to those in Fos expression in the vSPZ (Fig. 2.3E), which
lies adjacent to and projects sparsely to the PeVN (Kriegsfeld et al., 2004; Morin
et al., 1994; Watts and Swanson, 1987; Watts et al., 1987). Perhaps a loss of
rhythmicity in the vSPZ contributes to that occurring in the PeVN of pregnant
females.

The MPA plays a role in the regulation of multiple behaviors and
physiological parameters, including sexual and parental behaviors, sleep, and
core body temperature (for reviews, see Boulant, 2000; Kumar et al., 2007;
Numan, 2007; Sakuma, 2008). Fos expression in this region was strikingly
different between pregnant and diestrous rats, being arrhythmic and tonically low
in the former and high and rhythmic in the latter. These patterns contrast with
those reported by Lee et al. (1998), who found a signiﬁcant rhythm in Fos
expression in the MPA on day 6 of pregnancy in the rat. The reason for the
discrepancy is unclear, though perhaps it is due to the fact that Lee et al. (1998)
sampled a more caudal portion of the MPA than we did. The change that our
animals underwent from diestrus to pregnancy may be related to the absence of
proceptive sexual behaviors, which are dependant on the MPA (Guarraci et al.,
2004; Hoshina et al., 1994; Whitney, 1986; Yang and Clemens, 2000). The MPA
also plays a critical role in the coordination of sleep and the associated decrease
in core body temperature (Kumar et al., 2007). During early pregnancy, the
sleep/wake cycle and rhythms in core body temperature are dissociated (Kimura
et al., 1996; Kittrell and Satinoff, 1988; Rosenwasser et al., 1987). Therefore, the

reduction of Fos expression in the MPA may reﬂect a dissociation between cells

81

in this reg
body Iem‘
Pe
forebrain
ptomotin
2000'. Jc
not see
the basi
the tact
states.
moven
dark pl
bouts i
amNe
1987)
I0 Che
the ‘0
VDB.
alous
Sires
sugg
SYSle

aIOU

in this region that coordinate sleep/wake behavior and those that inﬂuence core
body temperature.

Perhaps the most interesting ﬁndings in this study are those in the basal
forebrain (the MS and VDB) and VLPO, which are wakefulness- and sleep-
promoting regions of the brain, respectively (Gaus et al., 2002; Greco et al.,
2000; Jones, 2008; Sherin et al., 1996; Szymusiak et al., 1998). Although we did
not see a signiﬁcant rhythm in Fos expression in the VLPO of diestrous females,
the basic pattern of expression was similar to that in pregnant females despite
the fact that sleep patterns are distinctly different between these two reproductive
states. More speciﬁcally, the total amount of time spent in both non-rapid eye
movement sleep (NREMS) and rapid eye movement sleep (REMS) during the
dark phase is signiﬁcantly higher in early pregnancy, as is the number of REMS
bouts in the dark phase (Kimura et al., 1996). Additionally, pregnant rats are less
active throughout the day, especially during the dark phase (Rosenwasser et al.,
1987). Our data indicate that these changes in sleep and activity may not be due
to changes in the sleep-promoting cells in the VLPO but may, instead, be due to
the loss of drive from arousal-promoting regions of the brain, such as the MS and
VDB. A similar dissociation between activation of brain mechanisms that promote
arousal and those leading to increases in sleep (VLPO) is also apparent during
stress-induced insomnia in the rat (Cano et al., 2008). Together, these patterns
suggest that plasticity in sleep/wake patterns is primarily driven by arousal
systems of the brain. Further exploration of how activity in other sleep and

arousal promoting regions change during early pregnancy would be useful in

82

testing this hypothesis at a more general level. Patterns of activity in the arousal
promoting orexinergic cells are of particular interest, as they project directly to the
basal forebrain and modulate activity in this region (Saper et al., 2001; Wu et al.,
2004).

The patterns of change in some, but not all, regions of the brain examined
in this chapter highlight the fact that effects of pregnancy in these areas are time-
of-day dependent. It is common practice to use Fos to examine effects of
experimental manipulations on specific brain regions, but often these are done at
only one, or perhaps two, time(s) of day. This could lead to a misunderstanding
of the processes under consideration. For instance, if we compared Fos
expression in the MPA of diestrous and pregnant rats at only 2T 6 (Fig. 4.4A), we
would ﬁnd no difference, which could lead to the erroneous conclusion that
functions associated with Fos in this region are the same in the two reproductive
states.

The mechanisms leading to the different effects of pregnancy on temporal
patterns of Fos expression in various brain regions are unclear. Some effects
may be driven by changes in the circadian system, such as those demonstrated
in Chapters 2 and 3. However, others may be brought about more directly by
changes in systems mediating aspects of physiology and behavior that are
altered during early pregnancy. For example, the elimination of the rhythm in the
VDB could be due to changes in inhibitory processes that do not originate in the
circadian system. This issue is related to the question of which aspects of early

pregnancy, such as modiﬁed patterns of hormone secretion or stimuli from

83

mating. tertil
may vary ar
data is how
regions oft

daily rhythr

 

mating, fertilization, or implantation, contribute to these changes and how this
may vary amongst brain regions. Another important question raised be these
data is how the different patterns of change in Fos expression in these myriad
regions of the brain might contribute to modification of the temporal patterning of

daily rhythms in behavior and physiology that occurs during early pregnancy.

84

Chapter 5

Schrader, J.A., E.J. Walasczcyk,. and L. Smale (2009). "Changing patterns of
daily rhythmicity across reproductive states in diurnal female Nile grass rats
(Arvicanthis niloticus)" thsiologv & Behavior 98(5): 547-556.

85

CHAPTER 5
Changing patterns of daily rhythmicity across reproductive

states in diurnal female Nile grass rats (Arvicanthis niloticus)

INTRODUCTION

Circadian rhythms allow organisms to predict ﬂuctuations in the
environment and appropriately time behavioral and physiological events to
anticipate those changes (Moore-Ede et al., 1982). In mammals, the
mechanisms governing these rhythms have been studied primarily in nocturnal
rodents, particularly rats (Rattus norvegicus), hamsters (Mesocn'cetus auratus),
and mice (Mus musculus), which exhibit behavioral and physiological rhythms
that are roughly twelve hours out of phase with those of diurnal species
(Reﬁnetti, 2008). Circadian rhythms in females of these nocturnal species
change across the estrous cycle (Morin et al., 1977) and throughout pregnancy
and lactation (Kittrell and Satinoff, 1988; Scribner and Wynne-Edwards, 1994a).
The inﬂuences of ovarian hormones responsible for changes in non-pregnant
females have been examined extensively (Albers, 1981; Gerall et al., 1973;
Labyak and Lee, 1995; Morin et al., 1977; Takahashi and Menaker, 1980;
Thomas and Armstrong, 1989), but little is known about how pregnancy and
lactation change circadian rhythms despite the fact that wild adult female
mammals spend signiﬁcantly more time in these states than they do undergoing
estrous cycles.

To meet the demands of pregnancy and lactation, females must change

many parameters of their behavior and physiology. Females of many species

86

increase food intake or mobilize fat stores to sustain fetal and neonatal growth
(Gittleman and Thompson, 1988; Speakman, 2008; Wade and Schneider, 1992),
and hypertherrnia during lactation is almost universal (Speakman, 2008). In many
species, activity levels are low throughout pregnancy and lactation (Albers et al.,
1981; Richards, 1966; Rosenwasser et al., 1987; Scribner and Wynne-Edwards
1994a; Speakman, 2008), and sleep disruption is more common (Kimura et al.,
1996; Speakman, 2008). Although these general trends are well-established,
relatively little is known about how circadian rhythms in these physiological and
behavioral phenomena change (for an exception see Kittrell and Satinoff, 1988).
Most studies of the effects of pregnancy and lactation on circadian
rhythms have focused on locomotor activity and core body temperature (Tb) in
nocturnal rodents and have found changes in the phase and/or the amplitude of
these rhythms. Lab rats and various hamster species are less active, and that
activity is less rhythmic, when they are pregnant or pseudopregnant than when
they are non-reproductive (Albers et al., 1981; Richards, 1966; Rosenwasser et
al., 1987; Scribner and Wynne-Edwards 1994a). The peak of the Tb rhythm in lab
rats is phase-advanced by several hours during both pregnancy and lactation,
and the amplitude of the rhythm decreases during gestation and increases during
lactation (Kittrell and Satinoff, 1988). We do not know whether any of these
changes occur in diurnal rodents. One might expect them to be similar, as
pregnancy and lactation impose major metabolic challenges regardless of an
animal’s temporal niche (Gittleman and Thompson, 1988; Speakman, 2008).

However, animals that are active during the warmest time of the day may not be

87

able to change their activity and Th rhythms in the same manner as those that
are most active during the coldest phase of the day-night cycle.

The current study used a diurnal species, the Nile grass rat (Arvicanthis
niloticus), a murid rodent from sub-Saharan Africa (Rosevear, 1969), to examine
two questions about how rhythms are modulated by reproductive state. The ﬁrst
was whether pregnancy and lactation affect circadian rhythms in activity and Th in
diurnal rodents in the same manner as they do in nocturnal ones. The second
was whether the effects of pregnancy and lactation on circadian rhythms are
additive or instead interact in more complex ways. To address these questions,
we monitored Tb and activity of female grass rats as they progressed through a
series of reproductive states: virgin, pregnant, pregnant and lactating, lactating

only, and post-weaning.

MATERIALS AND METHODS

Animals

Animals were adult (>60 days) female and male Nile grass rats,
Arvicanthis niloticus, obtained from a breeding colony at Michigan State
University (Katona and Smale, 1997). All animals were housed in polycarbonate
cages (48 x 28 x 16 cm) and given red transparent plastic huts (18 x 6 x 6 cm) for
optional shelter. For singly housed animals and lactating females (see below),
only one hut was provided, but mating couples were given two huts. Food (PMI
Nutrition Prolab, RMH 2000, Brentwood, MD) and water were provided ad
Iibitum. Animals were kept in a 12 h:12 h light/dark cycle (~250 lux during the

light phase), with the lights on from Zeitgeber Time (ZT) 0 to ZT 12, and a red

88

light (<5 lux) was on continuously to allow for visual observations and video
recording during the dark phase. All experiments were performed in compliance
with guidelines established by the Michigan State University Institutional Animal
Care & Use Committee (04/03-053-00) and in accordance with the standard in
the National Research Council Guide for Care and Use of Laboratory Animals

(National Academy of Science).

Surgery
Calibrated temperature/activity transmitters (PDT ER-4000 G e-mitters,

Minimitter, Bend, OR) were implanted intra-peritoneally into adult female grass
rats under anesthesia (2-5 % isoﬂuorane), and females were given pre-surgery
subcutaneous injections of the analgesic, buprenorphine hydrochloride
(Buprenex, 0.06 mg/kg of body weight). A single 1 cm incision was made along
the midline of the abdomen and muscle wall, and a transmitter was inserted into
the peritoneal cavity. The muscle wall was sutured with nylon sutures, and the
skin was sutured subcutaneously with dissolvable chromic gut sutures, which
were reinforced with autoclips. Animals were then given sterile saline (2.0 cc,
0.9% NaCl) subcutaneously, removed from anesthesia, and monitored until they
regained normal motor capacity. They received injections of Buprenex (same

dose as above) every 12 hours for the next two days.

Experimental Manipulations

Implanted females were housed singly in cages atop receiver bases (ER-
4000, Minimitter, Bend, OR), which conveyed transmitter readings to a computer

in an adjacent room that was equipped with Vitalview 4.0 software (Minimitter,

89

Bend, OR). Gross motor activity and body temperature readings were collected
and stored every ﬁve minutes, and the data ﬁle was exported once weekly, which
required stopping the data collection for a ﬁve minute period. Once all the
females had healed from surgery, recordings were taken for two weeks prior to
splitting them into two treatment groups (n = 5 per group): reproductive females
and age-matched virgin controls. Each female in the reproductive group was
paired with an adult virgin male grass rat, while the controls were kept singly-
housed throughout the experiment. Virgin females of this species do not exhibit a
clear estrous cycle and appear to be in an anestrous state (McElhinny, 1996), but
female grass rats do come into a reliable post-partum estrus (McElhinny et al.,
1997). Therefore, we could not assess the onset of pregnancy until the ﬁrst litter
was born. The mating couples were checked daily for the presence of their ﬁrst
litter, and the day that litter was discovered was designated day 0 of pregnancy
and lactation (PLO, beginning at ZT 12 prior to birth) since a post-partum mating
had occurred. Gestation in this species lasts approximately 25 days (McElhinny
et al., 1997), and the day of conception of the ﬁrst litter (pregnancy day 0 = P0)
was calculated as 25 days prior to day PLO. At 21 days of age (PL21), the ﬁrst
litter was separated from the mother, and the male was also removed. The
female’s cage was checked daily for the birth of the second litter, and the day of
birth was designated as day 0 of lactation (L0). The second litter was separated
from the mother at day L21, and recordings were taken from the female for at
least two more weeks after weaning. Readings were taken from all of the control

females until recording from the last reproductive female was complete.

90

Activity and Temperature Data Analysis

To determine how circadian rhythms in activity and body temperature
varied among reproductive states, we sampled data from days 2, 10, and 19 of
pregnancy (P), pregnancy plus lactation (PL), and lactation (L) from the
reproductive females. These days were chosen because both pregnancy and
lactation are dynamic states, and we wished to compare early, mid, and late days
within these states without the confounds of other transitions, such as parturition
(day P0), implantation (likely around P4 to P6), and weaning (L21). We also
sampled on a pre-reproductive baseline day (Pre: four days before pairing) and a
post-reproductive day (Post: 11 days after the second litter was weaned). Since
the control females did not go through these reproductive states, we determined
equivalent days for this group by sampling the data from each control female
from the same calendar dates as we had sampled from a randomly pair-matched
reproductive female so that ﬁve reproductive/control pairs of data were date-
matched.

To examine how activity rhythms were altered by reproductive state, we
generated actograms from the raw data in ClockLab v. 2.61 (Actimetrics,
Wilmette, IL). For each day of interest, we used the program to determine both
onset and offset of activity. Occasionally, the Clocklab algorithm designated the
onset or offset to be at a time that was not consistent with visual inspection of the
actogram. In these cases, the onset or offset was estimated instead by eye. To
assess effects on the magnitude of general activity, we had to standardize the
activity counts within each individual, since the activity outputs of the transmitters

were not calibrated to one another. Therefore, to determine whether the total

91

daily activity changed within individuals over the course of the study, we
calculated the average daily activity for each reproductive day of interest (P2
through Post or control equivalent) as the ratio of the average daily activity to the
average daily activity of the pre-reproductive day. We also calculated the dark:
light ratio of total activity for each day of interest, including the pre-reproductive
day.

The temperature readings were calibrated for each transmitter by
Minimitter (Bend, OR), and these readings could be directly compared between
individuals. For each day of interest, Tb measurements were averaged for each
half hour. The amplitude of the daily rhythm was calculated as the difference
between the maximum and minimum half hour Tb. To examine how the phase of
the rhythm changed, the start of the half hour with the minimum Tb was
determined. This served as the best phase marker as the timing of the daily
maximum was much more variable amongst and within individuals (data not
shown). A preliminary examination of the half hour averages revealed that the
time periods exhibiting the most noticeable and consistent changes were the
midday (ZT 3 to 9) and midnight (ZT 15 to 21) periods. The average Tb for each
of these two six hour periods was therefore determined for each day of interest.

Since individual animals were randomly assigned to the two treatment
groups and repeated measures were taken from each individual, a general mixed
effects model (GMEM) was most appropriate to examine the inﬂuences of
reproductive days on activity and Tb rhythms. For each dependent variable

(rhythm parameter) described above, a GMEM was generated in SAS v. 9.1

92

(SAS Institute, Inc., Cary, NC) with reproductive day (or equivalent), treatment
group, and the interaction between the two as ﬁxed factors and reproductive day
(or equivalent) as a repeated measure within the random effect of the individual
within a group. In the GMEM, the covariance structure of the data had to be
speciﬁed. To determine the best covariance structure for each dependent
variable, the model was run with each of four different structures (compound
symmetry, heterogeneous compound symmetry, autoregressive, and
unstructured), and the Akaike’s Information Criterion value for each model was
used to determine which model best ﬁt the data. Post-hoc least signiﬁcant
difference tests were performed for any signiﬁcant factors identified by the
GMEM with the best covariance structure for pair-wise comparisons of means

between groups within a reproductive day and between days within a group.

Video Recordings and Behavioral Analysis of Nesting Females

In order to determine whether rhythms in nesting females might change
across lactation and also to examine how core body temperatures might mediate
nesting, we examined nesting and drinking rhythms in a subset of non-pregnant,
lactating females (n = 3). Dams were videotaped with a low-light lens CCTV
camera connected to a time-lapse video recorder that condensed a 24 hour
period onto two hours of videotape. For each female, days 2, 10, and 19 of
lactation were scored by two different individuals. All individuals involved in
video scoring performed preliminary test scoring to ensure 94% or higher
agreement between all scorers. This agreement threshold was deﬁned as having

94% of all time bins scored the same by all scorers. Each video was scored for

93

off-nest and drinking behaviors. Off-nest behavior was deﬁned as the female
leaving contact with the pups; drinking, eating, and general locomotion occurred
during this time. Drinking behavior was deﬁned as the female placing her tongue
or snout against the ball bearing in the water bottle spout. It usually occurred
during off-nest bouts, but some females could rear up while still on the nest and
reach the water bottles. The twenty four hours of each video were split into ﬁve
minute bins, and for each bin, each of the two scorers recorded in an all-or-none
fashion which of the behaviors occurred. For each hour, the frequency for each
behavior was calculated by dividing the number of bins scored positive for that
behavior by both scorers by the total number of bins the scorers agreed upon
within that hour (thus excluding any bins with discrepant scores).

For analysis of these frequencies, since all females were subjected to the
same treatment, a two-way repeated measures ANOVA was performed for each
behavior with the frequency for that behavior as the dependent variable and day
of lactation (day 2, 10, or 19) and ZT (24 total hours) as ﬁxed effects in SPSS v.
15 (SPSS, Inc., Chicago, IL). Least signiﬁcant difference post-hoc tests were
performed when signiﬁcant effects were found. Differences were considered
signiﬁcant when p < 0.05. Data are given as means :I: SE unless otherwise

stated .

RESULTS

Reproductive Output

For all reproductive females, the ﬁrst litters were born within 27 to 32 days

of pairing with males (mean 29.8 :I: 1.2 days). These initial litters ranged in size

94

from 4 to 7 pups (mean 5.8 :l: 0.5 pups), and their sex ratios varied from twice as
many males as females to the opposite ratio (mean 1.05 :l: 0.25 males/females).
Second litters were born 25 to 26 days after births of ﬁrst litters (mean 25.8 :I: 0.2
days) and ranged in size from 3 to 9 pups (mean 5.6 :t 1.1 pups). Their sex ratios
varied from no males to 3 males for every 2 females (mean 1.23 :I: 0.52

males/females).

Activity Rhythms
As seen in previous laboratory work with this species (Katona and Smale,

1997; McElhinny et al., 1997), female grass rats in the current study exhibited

 

diurnal activity rhythms with crepuscular tendencies (Fig. 5.1). This pattern is
somewhat different from that seen in a natural setting where activity peaks in the
middle of the day and there are no peaks around dawn or dusk (Blanchong and
Smale, 2000). Although females were diurnal in the current study, the amount of
activity and its distribution across the day varied among reproductive states. We
measured the effects of the reproductive days (Pre through Post or equivalent
days for the control group to account for the effects of age) on the phases of
activity onset and offset, the average daily activity (relative to the pre-
reproductive day), and the darkzlight activity ratio. Table 5.1 contains a summary
of the type III tests of ﬁxed effects from the GMEM analyses for these variables.
The phases of activity onset and offset were not signiﬁcantly different across
reproductive days (or equivalents) in either group, and they did not differ between

groups (Table 5.1). Activity onsets occurred roughly 1.5 hours before lights on

95

Reproductive Females Control Females

 

 

 

 

 

 

 

 

 

5.‘ + P2 _._ P2 equivalent
5 t‘ 0 P10 77 0 P10 equivalent
1| ""‘ P19 ': t‘ "T" P19 equivalent
II
0' .11. ,\ ‘ . -.’
o '8 ‘0 W ' V 0
VI _._ PL2 __._ PL2 equivalent
I" 0.. puo , -~---~o PL10 equivalent
1| -+- pug 1r r x “P PL19equivalent
|
l

 

 

 

 

 

 

 

61 L2 _._ L2 equivalent

5 0 L10 0 L10equivalent
' -+- L19 "*‘ L19 equivalent

4_9

3. l

2.

 

 

 

 

 

Standardized General Activity Standardized General Activity Standardized General Activity

12 15 18 21 O 3 6 9 12 15 18 21 0 3 6 9
ZeitgeberTime ZeitgeberTime

Figure 5.1. Averaged general locomotor activity rhythms in the reproductive (left) and control
(right) groups (n = 5/group) under a 12h: 12h light (open bars)/dark (black bars) cycle for
pregnancy, pregnancy plus lactation, and lactation (or equivalents). For each individual, the
average activity of each half hour was standardized by dividing the raw value for that half hour
on the day of interest by the average total daily activity of that female on the pre-reproductive
day. Plotted values are means of these standardized values for each treatment group for each

96

 

Reproductive

 

 

 

 

 

 

 

 

Rhythm Covariance Day Treatment 'Eepmdgct've
Parameter Structure* (or Equivalent) Group Effect lay x LOUD
Effect nteractlon
Phase of CSH F(10, 26.6) = 1.25 F(1, 7.94) = 0.76 F(10, 26.6) = 1.24
Activity Offset NS NS NS
Phase of CSH F(10, 27.9) = 1.50 F(1, 8.28) = 5.17 F(10, 27.9) = 0.61
Activity Onset NS NS NS
Average Daily CSH F(9, 27.6) = 3.51 F(1, 8.01) = 5.11 F(9, 27.6) = 2.74
Activity P < 0.01 NS P < 0.05
DarkzLight CSH F(10, 25.7) = 2.08 F(1, 8.08) = 0.26 F(10, 25.7) = 2.67
Activity Ratio NS NS P < 0.05
Amplitude of Tb cs F(10, 80) = 10.60 F(1, 8) = 9.73 F(10, 80) = 4.01
Rhythm P < 0.0001 P < 0.05 P < 0.0005
Phase of CSH F(10, 26.3) = 1.27 F(1, 10.4)= 6.69 F(10, 26.3) = 0.90
Minimum Tb NS P < 0.05 NS
Average Midday CS F(10, 80) = 12.90 F(1, 8) = 10.71 F(10, 80) = 12.53
Tb P < 0.0001 P < 0.05 P < 0.0001
Average cs F(10, 80) = 14.36 F(1, 8) = 18.73 F(10, 80) = 14.79
Midnight Tb P < 0.0001 P < 0.005 P < 0.0001

Fri

 

 

Table 5.1. GMEM Results for Type III Tests of Fixed Effects for Activity and

Temperature Rhythms. *CSH = Heterogeneous Compound Symmetry, CS =
Compound Symmetry, see METHODS for model details. n = 5/group. NS = Not
signiﬁcant (P > 0.05).

97

(ZT 22.58 :1: 0.10 hours), and the offsets occurred around 1.75 hours after lights
off (ZT 13.76 :I: 0.17 hours).

Locomotor activity increased from early to late pregnancy, regardless of
whether or not females were also lactating (Fig. 5.1). The average daily activity
was signiﬁcantly affected by the interaction between reproductive days (or
equivalents) and treatment group and by the main effect of reproductive days
(Table 5.1). In the reproductive group, the average daily activity was signiﬁcantly
higher than the post-reproductive day from day P19 through PL19 (Fig. 5.2A; p <
0.05; post-hoc pair-wise least signiﬁcant difference (LSD) tests). Daily activity
levels of the reproductive females appeared to return to pre-reproductive levels
by the post-reproductive day (daily activity: 1.02 :l: 0.23, with a value of 1
indicating that activity levels were the same as on the pre-reproductive day).
Therefore, females were also more active on days P19 through PL19 than during
the pre-reproductive period. Additionally, reproductive females were more active
on days P19, PL10, and PL19 than controls on the equivalent days (Fig. 5.2A; p
<0.05; LSD tests). Within pregnancy, activity levels signiﬁcantly increased from
day 2 to day 19 (Fig. 5.2B; p < 0.05; LSD test). A similar trend was apparent
during the period of pregnancy plus lactation, but there were no signiﬁcant
differences across days in this state (Fig. 5.2B). During lactation this trend was
not seen, and females on day L19 had signiﬁcantly lower levels of activity than
on PL19 (Fig. 5.2B; p < 0.05; LSD test). The average daily activity did not vary
signiﬁcantly among any of the days of interest in the control group (Fig. 5.2A;

mean for all days: 0.955 :I: 0.25; F(9, 7.73) = 0.44, p > 0.5; post-hoc GMEM).

98

 

 

 

>

t" .N I" 9’ 5"
U" o 01 o U"
1 1 1 1 1

.5
o
1

 

Standardized Average Daily Activity

+ Reproductive Females

—O— Control Females
*

{3t-

 

 

 

P
01

:17

9°
(TI
1

I" I" I" 9°
01 O 01 O
1 1 L L

.L
o
1

 

Standardized Average Daily Activity

P2 P10 P19 PL2 PL10 PL19 L2 L10 L19 Post
Reproductive Day or Control Equivalent

+ Pregnancy
—£i— Pregnancy and Lactation
+ Lactation

A Post-reproductive

 

 

 

.°
01

the control and

I T r

Day 2 Day 10 Day 19
Day of Reproductive State

Figure 5.2. (A) Average daily activity across reproductive days (or equivalents) in the two treat-
ment groups (n = 5/group). The average daily activity was standardized by dividing the average
activity of a female for each day of interest by the average activity of that female on the
ore-reproductive (or equivalent) day. Values are means :I: SE. *Signiﬁcant differences between
reproductive groups (p < 0.05; LSD test). The data for the reproductive group are
re-plotted in (B) for comparisons of pregnancy and lactation to the combined state. tSigniﬁcant
differences between lactation and the combined (PL) state (p < 0.05; LSD test).

99

 

The distribution of activity across the light/dark cycle was highly labile (Fig.
5.3A), but this distribution was affected by the interaction between reproductive
days (or equivalents) and treatment group (Table 5.1). The reproductive females
had a signiﬁcantly lower darkzlight activity ratio on PL19 (more diurnal) and a
significantly higher ratio on L2 (more nocturnal) than controls (p < 0.05; LSD
tests), but the ratio did not differ signiﬁcantly from the pre- or post-reproductive
day for any day examined in either group (Fig. 5.3A). Lactating females were
more nocturnal during early than mid-lactation, regardless of whether or not they

were pregnant, and the ratio was signiﬁcantly higher on day PL2 than PL10 or

 

PL19 and on day L2 than on L10 (Fig. 5.3B; p < 0.05; LSD tests). Within the
control group, the ratio of activity in the night versus the day did not change
across the days of interest, and females were more active in the light than the
dark (Fig. 5.3A; dark: light ratio: 0.714 :1: 0.070; post-hoc GMEM; F(10, 9.14) =

0.53, p > 0.8).

Temperature Rhythms

Females were hyperthermic during much of pregnancy and lactation, with
the most noticeable rises in Tb occurring during the mid-dark (major trough) and
mid-light (minor trough) phases of the day, while rhythms in control females did
not noticeably change over time (Fig. 5.4). Peaks occurred around the light/dark
transitions. GMEM analysis of the amplitude of the Tb rhythms and midday and
midnight Tb averages all revealed signiﬁcant main effects of reproductive days (or
equivalents) and treatment group and a signiﬁcant interaction between the two

(Table 5.1). However, reproductive state did not alter the phase of the rhythm as

100

 

 

 

 

1.2 1 + Reproductive Females
9.. —O— Control Females *
.c
.9 ..
-' 1.0 .
as
> a
x __
a e-
: O
.— l O * .
3. .r . ‘ ._
2.; 0.6 1 e .. 1
o -L , ‘—
<
H—
o I
.9 0.4 -
*5
0:

0.2 I l I 1 ﬂ I f l I I F

 

 

Pre P2 P10 P19 PL2 PL10PL19 L2 L10 L19 Post
Reproductive Day or Control Equivalent

w

+ Pre-reproductive

 

 

"2 _ + Pregnancy
E -—D— Pregnancy and Lactation
5’ + Lactation
<15 1-0 ‘ A Post-reproductive
>
.5 *r
‘0“ 0.8 ~
.5 A
.é‘
.5 0.6 -
o __
<
u...
o
.9 0.4 -
115'
CE
0.2 , .

 

 

Day 2 Day 10 Day 19
Day of Reproductive State

Figure 5.3. (A) Ratio of activity in the dark versus light phase across reproductive days (or
equivalents) in the two treatment groups (n = 5/group). Values are means :1: SE. Notation is the
same as in Fig. 5.2A. The data for the reproductive group are re-plotted in (B) for comparisons of
pregnancy and lactation to the combined state.

101

 

Reproductive Females Control Females

_._ P2 equiv.
0 P10 equiv.
—4— P19 equiv.

 

 

 

  

    

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

121514321 6 5 69' 121518 210 3 6 9
Zeitgeber Time Zeitgeber Time

 

Figure 5.4. Averaged Tb rhythms in the reproductive (left) and control (right) groups (n =
5/group) under a 12h: 12h light (open bars)/dark (black bars) cycle for pregnancy, pregnancy
plus lactation, and lactation (or equivalents: equiv.) Values are means for each treatment
group for each half hour. Standard errors were removed for ease of comparing the rhythms.

102

 

indicated by the half-hour interval with the lowest average Tb (Table 5.1). There
was a signiﬁcant effect of treatment group, however, with control females
exhibiting a slightly later minimum Tb (ZT 18.19 :1: 0.34) than reproductive females
(ZT 17.09 :1: 0.34) throughout the study, including prior to pairing the reproductive
females with their mates.

The amplitude of the Tb rhythm (based on half hour intervals) changed
across time in both groups of animals but in different ways (Fig. 5.5A). Within the
control group, the amplitude significantly increased toward the end of the study,
from the PL19 and L2 equivalents to the post-reproductive equivalent (Fig. 5.5A;
p < 0.05; LSD tests). In the reproductive group, the amplitude was also highest
on the post-reproductive day, and it did not differ from that of controls at that
point (Fig. 5.5A). These data indicate that amplitude increases with age.
However, in the reproductive females, the amplitude of the Tb rhythm dropped
from mid-pregnancy through mid-lactation and was signiﬁcantly lower from days
P10 through PL10 as well as on L2 and L10 than on the pre-reproductive day
(Fig. 5.5A; p < 0.05; LSD tests). The amplitude from days P10 through PL10 was
also signiﬁcantly lower than in controls on the equivalent days (Fig. 5.5A; p <
0.05; LSD tests). Pregnancy and lactation inﬂuenced the rhythm amplitude
differently during the combined state, with one masking the effects of the other
on different days (Fig. 5.5B). The amplitude on PL10 was the same as on L10,
but it was signiﬁcantly higher than on P10 (Fig. 5.5B; p < 0.01; LSD test),
suggesting that pregnancy has no effect on the rhythm amplitude in females that

are 10 days into lactation. In contrast, the amplitude was the same on PL19 as

103

 

+ Reproductive Females
—-O— Control Females

 

 

W

.N N .N .N .N .w .w
0 N «b 0) 00 O N
l L l l l J

1.8 -

Daily Tb Amplitude (°C)

1.6 4
1.4 -

 

I

| 1 I r l T 1 1 I 1

Pre P2 P10 P19 PL2 PL10PL19 L2 L10 L19 Post

+

+ Pregnancy

Pre-reproductive

+ Pregnancy. and Lactation %

A

_ + Lactation

Post-reproductive

 

 

1.2

I I 7

Day 2 Day 10 Day 19

Day of Reproductive State

Figure 5.5. (A) Daily amplitude of the Tb rhythm across reproductive days (or equivalents) in the
two treatment groups (n = 5/group). Values are means 1 SE. Notation is the same as in Fig.
5.2A. The data for the reproductive group are re-plotted in (B) for comparisons of pregnancy and
lactation to the combined state. TSigniﬁcant differences between pregnancy and the combined
(PL) state (p < 0.05; LSD test). :tSigniﬁcant differences between lactation and the combined (PL)
state (p < 0.05; LSD test).

104

on P19 but was lower on PL19 than on L19 (Fig. 5.5B; p < 0.01: LSD test),
indicating that by 19 days, lactation no longer had an effect on the rhythm
amplitude, regardless of whether or not females were pregnant.

Drops in rhythm amplitude appear to be due to rises in the average body
temperatures of females during the midnight period in all three reproductive
states relative to the pre-reproductive period and to controls. Midday
temperatures rose in a similar fashion (Fig. 5.6A and B). During the midday
period, Tb values did not change in the control group (Fig. 5.6A; F(10, 40) = 0.83,
p > 0.5; post-hoc GMEM), and during the midnight, Tb actually dropped in
controls from the P19 equivalent to the post-reproductive equivalent day (Fig.
5.68; p < 0.05; LSD test), which is the probable cause of the rise in rhythm
amplitude seen at this time (Fig. 5.5A). In the reproductive group, however, the
midday Tb averages increased from the pre-reproductive day for all days of
pregnancy and lactation and, except on P19, were also higher on these days
than on the post-reproductive day and than controls on the equivalent days (Fig.
5.6A; p < 0.05; LSD tests). Similar deviations from the pre- and post-
reproductive days were seen for the midnight Tb averages (Fig. 5.6B). The mean
midnight Tb was signiﬁcantly higher than on the pre-reproductive day from P2
through L10 and than on the post-reproductive day from P10 through L10;
reproductive females had signiﬁcantly higher midnight Tb means than controls on
these days as well (Fig. 5.6B; p < 0.05; LSD tests). '

Midday and midnight Tb values were highest in early to mid lactation,

whether or not females were also pregnant. During the midday period, the

105

A Midday Midnight B

Re roductive Females
39.0- . : Co trol Females r 39.0

 

 

 

36.0

 

 

Pre 2 10 1'9 2 10 19 2 1'0 19 Post Pre 2 10 1'9 2 10 1'9 2 10 19 Post
P PL L P PL L
Reproductive Day or Control Equivalent Reproductive Day or Control Equivalent

 

 

 

 

C + PFtee-rrsgrquuctive D
A 39-01 1' —0— Pregnancy and Lactation ‘390
o T + Lac ation , g
E; 38.5- A Post-reproductive .335 g3
|_ _.
a) 38.0- t .3803
g g i
'9 37.5. .37,5§
< t g
a; 37.01 I370 r:
"O
E 36.5. 1 § .3653;

o
36.0 . . e - . . . - 360"
Day 2 Day 10 Day 19 Day 2 Day 10 Day 19
Day of Reproductive State Day of Reproductive State

Figure 5.6. (A and 8) Average Tb during the midday (ZT 3 to 9, A) and midnight (ZT 15 to 21, B)
periods across reproductive days (or equivalents) in the two treatment groups (n = 5/group).
Values are means :1: SE. Notation is the same as in Fig. 5.2A. The data for the reproductive
group are re-plotted in (C) and (D) for comparisons of pregnancy and lactation to the combined
state during the midday (C) and midnight (D) periods. Notation is the same as in Fig. 5.58

106

average Tb values during days 2 and 10 of pregnancy were signiﬁcantly lower
than the corresponding days of the combined pregnant/lactating state (Fig. 5.6C;
p < 0.01; LSD tests), and midday Tb did not change throughout pregnancy (Fig.
5.6C). However, the midday Tb signiﬁcantly dropped from day 10 to 19 in both
lactation and the combined pregnant/lactating state (Fig. 5.6C; p < 0.005; LSD
tests). Similar trends were evident in the midnight period with some differences
(Fig. 5.6D). The midnight Tb average was signiﬁcantly lower on P2 than on PL2,
and that on L19 was signiﬁcantly lower than on PL19, again demonstrating
differential effects of pregnancy and lactation during the combined state (Fig.
5.6D; p < 0.05; LSD tests). Midnight Tb means peaked on both P10 and PL10
and were signiﬁcantly higher than on days 2 and 19 of the same states (Fig.
5.6D; p < 0.05; LSD tests). Similarly, the mean midnight Tb on L10 was
significantly higher than on L19 (p < 0.005; LSD test) but did not differ from that
on L2 (Fig. 5.6D).

Behavioral Rhythms in Nesting Females

Three of the females from the reproductive group were videotaped on
days 2, 10, and 19 of lactation, and the tapes were scored to assess the
proportion of ﬁve minute intervals each hour (beginning at the designated ZT)
during which the lactating dam was off the nest and/or drinking. A two-way
repeated measures ANOVA revealed no effects of day of lactation (Off-nest: F (2,
4) = 0.459, p > 0.5; Drinking: F(2, 4) = 3.694, p > 0.1) and no interaction between

day of lactation and ZT for either behavior (Off-nest: F(46, 92) = 1.047, p > 0.1;

107

Drinking: F(46, 92) = 0.948, p > 0.5). However, there were signiﬁcant effects of
time of day (ZT) on each behavior (Off-nest: F(23, 46) = 5.897, p < 0.001;
Drinking: F(23, 46) = 2.227, p < 0.05), indicating a daily rhythm in nesting and
drinking that did not change across lactation. These rhythms reﬂected the
crepuscular nature of activity in this species in captivity. Off-nest activity peaked
near the light/dark transitions (ZT 1 and 12), and troughs occurred in the mid-light
and mid-dark phases (ZT 7 and 20; p < 0.05: LSD tests between peaks and
troughs), though off-nest behavior was signiﬁcantly less frequent at ZT 20 than at
ZT 7 (Fig. 5.7A; p < 0.01; LSD test). Drinking was less frequent than off-nest
behavior, but the rhythm looks similar in shape, though not in amplitude, to that
behavior (Fig. 5.7B). There was not one distinct peak for this rhythm, although
the highest frequencies of drinking were around the light/dark transitions (ZT 2
and 11; Fig. 5.7B). The signiﬁcant troughs of the rhythm occurred a few hours
before lights-on (ZT 20 and 21) and again at lights-on (ZT 0; Fig. 5.7B; p < 0.05;

LSD tests between each trough and ZT 1 and 2).

DISCUSSION

Many females spend most of their lives pregnant and/or lactating, which
mandates major changes in various parameters of physiology and behavior that
are rhythmic. In this study, we found that circadian strategies can be modulated
during pregnancy and lactation in quite different ways in diurnal and nocturnal
species. Additionally, we demonstrated that the effects of pregnancy and
lactation are not simply additive during the combined state but rather that the

effects of pregnancy are evident in certain parameters of activity and temperature

108

 

 

 

 

 

 

 

 

 

 

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Figure 5.7. Frequencies of off-nest (A) and drinking (B) behaviors for each hour of the light (open
bars, top): dark (black bar, top) cycle during lactation in a subset of the reproductive group (n =
3). Values are pooled means t SE for days 2, 10, and 19 of lactation in 3 lactating dams. *Peaks

and Ttroughs in each rhythm as explained in RESULTS.

109

rhythms while the effects of lactation are evident in others. We have also found
that rhythms in maternal nesting of grass rats do not change over the ﬁrst 19
days of pup development.

One important issue to consider when interpreting these data is that males
remained with their females during both the pregnant and the combined
pregnant/lactating states but were absent during the lactation-only state and
among control females. Therefore, differences seen within females in the P/PL
states compared to the L condition, as well as differences between P/PL females
and controls, could theoretically be due to the presence/absence of the male. We
were unable to include P and PL conditions without males because grass rats do
not undergo spontaneous estrous cycles, and the postpartum estrus is the only
one that can be reliably predicted. Effects of reproductive state can be evaluated
independently of effects that males might have had on Tb or activity when we
compare P with PL states (both of which occurred when the male was present)
and when we compare L females with non-pregnant controls (neither of which
involved the presence of a male). Both sets of comparisons reveal clear effects
of reproductive state. Furthermore, changes occurring within any given state (P,
PL and L) are likely to begin with changes in the female and/or the pups and
cannot be due to changes in the presence/absence of males. It should also be
noted that all of the comparisons we have made are likely to reﬂect what occurs
in nature, where adult male and female grass rats live together in communal

burrows with one or two generations of their offspring (Blanchong and Smale,

110

2000; Packer, 1983; Senzota, 1982) and where non-pregnant females (whether
they are lactating or not) are unlikely to be living with males.

During pregnancy but not lactation, locomotor activity increased to roughly
2.5 times the level seen in virgin females (Fig. 5.1 and 5.2). However, there were
no changes in the timing of the offset or onset of activity or in the relative amount
of activity in the dark versus light portions of the day. This is entirely different
from patterns observed in nocturnal rodent models, which become less rhythmic
in their activity and show lower levels of both wheel-running and general activity
(Albers et al., 1981; Richards, 1966; Rosenwasser et al., 1987; Scribner and
Wynne-Edwards 1994a). In rats and hamsters, estradiol can consolidate, mildly
phase-advance, and increase nighttime activity (Albers, 1981; Morin et al., 1977;
Takahashi and Menaker, 1980), and, in hamsters, progesterone counteracts
these effects (Takahashi and Menaker, 1980). Takahashi and Menaker (1980)
argued that this might explain why pregnant rats, which have high levels of
circulating progesterone relative to estrogens, are much less active and less
rhythmic in their activity. Although we do not know the effects of these hormones
on activity in grass rats, the patterns we see here suggest that responses to
these hormones may be very different in this species compared to lab rats and
hamsters.

The most dramatic effects of reproductive state on rhythms stemmed from
rises in Tb that occurred during pregnancy, lactation, and when the two states
were combined (Fig. 5.4). The increases were largest during the midday and

midnight phases of the rhythm rather than at the light/dark transitions. This may

111

reﬂect a physiological constraint limiting increases at those transitions, when Tb
was high even in females that were not reproductive. The increases were also
larger at night than during the day (Fig. 5.6), which resulted in a reduction in
rhythm amplitude (Fig. 5.4). During pregnancy, the rises in Tb could have been
partially driven by changes in activity, which, although not necessary for them,
has modulatory effects on rhythms in Tb (Reﬁnetti, 1997; Reﬁnetti, 1999).
However, it is unlikely that activity was solely responsible for the changes since it
increased from early to late pregnancy. An increase in the midnight Tb did occur
from day 2 to day 10 of pregnancy, but if increased activity were inducing
hyperthermia, this would have also been evident in the midday, which it was not.
Various other factors probably contributed to increasing Tb and changes in its
temporal pattern during pregnancy. First, the increased secretion of certain
hormones may have driven hyperthermia. Progesterone and prolactin increase
Tb in rodents (Leon et al., 1978; Woodside et al., 1981), as do birth control pills,
which contain progestagens, in humans (Baker et al., 2001a; Baker et al., 2001b;
Wright and Badia, 1999). During the ﬁrst half of pregnancy there is an
endogenous circadian rhythm in prolactin secretion in many rodents (Butcher et
al., 1972; Edwards et al., 1995; McMillan and Wynne-Edwards, 1999;
Talamantes et al., 1984). The rhythmic release of this hormone might therefore
inﬂuence the rhythmic rises in Tb. Other physiological processes associated with
pregnancy could also contribute to rises in Tb and changes in its temporal
patterning. Since this is an energetically expensive state (Gittleman and

Thompson, 1988; Speakman, 2008), an increase in feeding or metabolic

112

turnover, as has been seen in other rodent species (Biggerstaff and Mann, 1992;
Cripps and Williams, 1975; Quek and Trayhurn, 1990; Wade et al., 1986), could
account for thetime—dependent changes in hyperthermia seen in grass rats.

Hyperthermia was even more pronounced in lactation than in pregnancy,
especially during the energetically expensive early to mid-lactational period (Fig.
5.6). There are several possible explanations for this. Circulating hormones
related to lactation and maternal care could contribute directly to increased Tb
(Leon et al., 1978; Woodside et al., 1981), as may be the case with pregnancy.
However, an increase in metabolic turnover causing hyperthermia is more
consistent with our data. Hyperthermia during the mid-dark phase disappeared
from mid to late lactation, when pups began to incorporate solid food into their
diet, reducing energetic demands of the pups on their mother. Also, the fact that
hyperthermia was more pronounced during lactation than pregnancy, which is
less energetically expensive (Gittleman and Thompson, 1988; Speakman, 2008),
lends support to this hypothesis. An alternative explanation was proposed by
Croskerry et al. (1978) suggesting that contact with pups in the nest induces
maternal hyperthermia and serves as a constraint on the amount of time a dam
may spend on her nest. This hypothesis has received much attention, and
evidence both for and against it has been gathered from various species
(Scribner and Wynne-Edwards, 1994b; Walton and Wynne-Edwards, 1997;
Woodside et al., 1980), but see (Bates et al., 1985; Stern and Lonstein, 1996;
Stern and Azzara, 2002). Our data indicate that contact with pups did not

correlate with peaks in Tb in lactating grass rats. The behavioral data show that

113

females were off the nest least from ZT 15 to 22, when core body temperature
had not yet peaked. If females were overheating on the nest, then they should
have been warmest at these times. However, signiﬁcant increases in Tb did occur
during this mid-dark phase of the day, and maternal-pup thermal transfer may
thus have contributed to some of the increase in Tb, though it certainly did not
appear to constrain maternal care in grass rats. Kittrell and Satinoff (1988)
argued that their data from lab rats were also inconsistent with the hypothesis of
Croskerry et al. (1978). Their females exhibited the highest core body
temperatures when they were most active off of the nest, not after long bouts on
the nest. However, in grass rats, activity also could not account for lactational
hyperthermia, as females were no more active than controls or than they
themselves were before or after the period of reproduction. This would indicate
that metabolic and/or hormonal processes were the most important factors
driving hyperthermia in lactating grass rats.

Patterns of change in the phase of daily rhythms in Tb across reproductive
states were different in grass rats from those seen in nocturnal lab rats.
Speciﬁcally, whereas in lab rats, the phase of the peak of the Tb rhythm
advances during both pregnancy and lactation (Kittrell and Satinoff, 1988), no
changes in phase occurred in the rhythms of grass rats. This difference between
the species may reﬂect their evolutionary histories in environments with different
ecological constraints during the active phase. For instance, it may be that for a
nocturnal animal, it would be more energetically expensive to increase Tb further

during the cold night, when the Tb peak usually occurs, and in order to do so, the

114

phase must be shifted so that it occurs under warmer ambient conditions.
Another possibility is that the species difference is directly related to activity
rhythms. When grass rats became pregnant, the amplitude of their activity
rhythms remained high, and their phase did not change (Fig. 5.1). However, in
pregnant lab rats, activity rhythms break down in constant darkness
(Rosenwasser et al., 1987), and in hamsters, activity becomes less rhythmic
under a light/dark cycle (Albers, 1981). Therefore, a lack of a stable activity
rhythm in these nocturnal species may allow for a change in phase of the Tb
rhythm whereas the presence of a stable activity rhythm may prevent such a
change in grass rats.

When we focus directly on the rhythms and ask how they were affected by
reproductive state, clear time-of-day effects on the nature of the interaction
between pregnancy and lactation emerge. One example is apparent when
considering reproductive females on day 10. Here, the midday Tb was identical in
the two lactating conditions and signiﬁcantly lower when females were just
pregnant (Fig. 5.6C), but midnight Tb was indistinguishable amongst the three
states (Fig. 5.6D). Thus, effects of lactation were considerably higher than those
of pregnancy during the day, but not during the night. Another example is evident
when considering day 19, at which point midday Tb was the same in the three
reproductive states (Fig. 5.6C), but the midnight Tb was considerably lower when
females were lactating but not pregnant (Fig. 5.6D). This pattern suggests that
the return to patterns characteristic of the non-reproductive state, (evident at day

19 of lactation in non-pregnant females) occurs more rapidly for the nighttime

115

than the daytime phase of the rhythm. In neither of these examples, at either time
of day, were effects of pregnancy and lactation on rhythms simply additive. This
could be due to time-of-day effects on some of the processes noted above. For

' example, in some cases (eg. day 10) the non-additive effects could reﬂect a
rhythm in a physiological ceiling on the maximum Tb (compare Fig. 56C and D).
Although pregnancy and lactation are both energetically demanding, they are
nonetheless physiologically different states. Perhaps it should not be surprising,
therefore, that their effects are non-additive, but the intriguing part of the patterns

are that they can be additive at some times of day but not others.

CONCLUSIONS AND IMPLICATIONS

The current data reveal clear patterns of change in daily rhythms of grass
rats as they transition from one reproductive state to another and that these are
not identical to the changes seen in nocturnal rodents. They also show
interacting effects of lactation and pregnancy that could not have been predicted
from either state alone and that these interactions themselves change as a
function of time of day. The patterns observed here highlight the importance of
considering the circadian timekeeping system as a dynamic one whose inﬂuence
on behavioral and physiological processes can change in systematic ways with
different reproductive states. These data raise the question of what neural and
physiological processes might produce changes in temporal organization across
these states. One possibility is that they are driven by modulation of the
mechanisms generating circadian rhythms and transmitting temporal information

from them to other regulatory systems. Alternatively, these basic circadian

116

mechanisms could be buffered against changes in reproductive state, and the
emergence of new patterns of rhythmicity could instead be brought about by
more direct inﬂuences of reproductive processes on systems regulating Tb and
activity. The most likely possibility, however, is that the modulation of rhythms
from one reproductive state to another is driven by changes both within and

beyond the circadian timekeeping system.

117

 

CHAPTER 6

Conclusions

Rhythms in the brain in early pregnancy

In the introductory chapter of this dissertation, I proposed three main
hypotheses concerning whether and how the circadian system of the brain might
promote changes in overt circadian rhythms during early pregnancy in the rat.
The results presented in Chapters 2, 3, and 4 clearly indicate that some aspect of
early pregnancy is inﬂuencing rhythms in the SCN, extra-SCN oscillators, and
non-circadian regulatory areas of the brain. These inﬂuences are most likely
acting at multiple levels of these neural networks (Fig. 6.1), given the variable
effects in regions beyond the SCN (see Chapters 3 and 4).

The research presented in this dissertation provides a foundation for
future research concerning the mechanisms that modulate rhythms as an
individual progresses from one reproductive condition to another. Four main
questions are raised by my data. First, it is unclear what aspect of early
pregnancy leads to the effects seen in Chapters 2 through 4. Some of these
might be caused by mating-related stimuli, such as auditory, tactile, or chemical
cues associated with the male (e.g. pheromones or signals from the ejaculate).
Other possibilities include fertilization- or implantation-related stimuli and
endocrine changes associated with early pregnancy. It is possible that different
stimuli induce changes in different regions of the brain. A serial process of
elimination could be employed to identify which of the stimuli are necessary

and/or sufﬁcient to induce changes in temporal patterns of Fos and Per2

118

 

Extra-SCN

osc1l|ator Extra-SCN
oscillator

Non- Overt
oscillator Rhythms
in
Non- Behavior
oscillator and

I Non-

! .
I . ' osc1l|ator
l

 

‘
Pregnancy

Figure 6.1. Proposed network model of the pathways (arrows) by which pregnancy affects
rhythms in circadian and non-circadian systems of the brain to lead to changes in overt rhythms.
Some aspect of pregnancy induces changes within the SCN and vSPZ (solid arrows), some
extra-SCN oscillators (dashed arrows), and some non-oscillatory brain regions (dotted arrows)
to promote changes in overt rhythms. These effects are likely to be integrated at multiple levels
(converging arrows leading to combined arrows). In addition, rhythms in some brain regions are
unaffected (*blocked arrows indicating resistance to upstream inﬂuences of pregnancy). Arrows
indicate general pathways, not neural projections.

119

expression in each brain region of interest. The effects of non-mechanical
mating stimuli, presence of the ejaculate, fertilization, and implantation could be
discounted if the same changes seen here can be induced in a pseudopregnant
model. This approach could also be used to determine the relative importance of
ovarian and adrenal hormones, as a semicircadian rhythm in prolactin secretion
can be induced both in ovariectomized and in adrenalectomized females
(reviewed in Cameron et al., 2003; Freeman and Sterrnan, 1978). Finally, since
the prolactin rhythm may be initiated with a single administration of either
oxytocin or prolactin (Egli et al., 2006; Helena et al., 2009), we can determine
whether vaginocervical stimulation is required to induce the changes in Per2 and
Fos rhythms in the brain observed here. Additionally, a closer examination of the
time courses of the post-copulation changes in rhythms within the brain could be
informative.

The second major question raised by our data is how altered patterns of
Fos and Per2 expression might lead to changes in overt rhythms. This is a
difﬁcult question to address, but some information bearing on it might be
provided by determination of the phenotypes of the cells expressing Fos and
Per2 and by examination of how rhythms in output signals within these cells,
such as AVP in the SCN, change during early pregnancy.

A third question raised by the patterns observed here is whether and how
these systems and interactions among them might change across other phases
of an individual’s reproductive life. We have demonstrated a major re-

organization of rhythms within the circadian system and non-circadian regulatory

120

systems on day 6 of pregnancy, but this is only one day of the development and
care of a female’s offspring. For a rat, gestation and lactation of one litter last
almost two months, and rhythms in behavior and physiology are changing
throughout this time (Atkinson and Waddell, 1995; Brunton et al., 2008; Freeman
et al., 2000; Kimura et al., 1996; Speakman, 2008). Rhythms within several
regions of the brain have been shown to differ between early and late pregnancy
(Lee et al., 1998), and new patterns are likely to emerge as females progress
beyond that state as well.

Finally, the changes in rhythms in the brain in early pregnancy were only
characterized in one nocturnal rodent model in the studies presented in Chapters
2, 3, and 4. This raises the question of the ways in which the patterns seen here
would, and would not, generalize to other species with different evolutionary
histories or ecologies. The changes could be different, for example, in the brains
of diurnal species or in species that exhibit paternal care. In the latter, it would be
interesting to know if males undergo changes in their circadian systems similar to
those of their mates. Studies of both neural and overt rhythms in a biparental
model system may shed light on how fathers and mothers partition their time to
optimize their reproductiveoutput and whether circadian dysfunction in fathers

might hamper the development of their offspring.

Rhythms and reproduction in diurnal versus nocturnal rodents

The Nile grass rat (Arvicanthis niloticus) is a diurnal murid rodent species
closely related to laboratory rats and mice (Lecompte et al., 2008), and, at least

in captivity, males of this species exhibit paternal care (personal observations). In

121

Chapter 5, I found two primary differences between this species and nocturnal
models with respect to how activity and core body temperature (Tb) rhythms
change as females progress through a series of reproductive states. First, robust
activity rhythms persist throughout pregnancy and lactation in grass rats, with
their amplitude actually increasing in the pregnant state. Second, the peak of the
Tb rhythm is not advanced in phase as it is in lab rats.

The maintenance of a robust rhythm in locomotor activity in the pregnant
grass rat contrasts with the loss of locomotor activity rhythms due to reduced
activity in nocturnal models (Albers et al., 1981; Richards, 1966; Rosenwasser et
al., 1987; Scribner and Wynne-Edwards, 1994a; Speakman, 2008). In rats, the
loss of the locomotor activity rhythm in early pregnancy correlates with a lack of
Fos and Per2 rhythms in the vSPZ (Fig. 2.3E and F) and in Fos rhythms in the
MS and VDB (Fig. 4.4C and E), all regions involved in promoting activity and/or
wakefulness (Abrahamson and Moore, 2006; Greco et al., 2000; Jones, 2008; Lu
et al., 2001; Saper et al., 2005; Schwartz et al., 2004; Schwartz et al., 2009).
Since activity is distinctly rhythmic in the pregnant grass rat, at least in a
light/dark cycle (Fig. 5.1), we might expect to ﬁnd no effect of pregnancy on
rhythms in the vSPZ, MS, or VDB in this species or that they might even increase
in amplitude, as does the rhythm in activity. We might also expect the
maintenance of Fos rhythms in the MPA of grass rats, as this region is involved
in integrating the sleep/wake cycle with Tr, homeostasis (Kumar et al., 2007).

Studies comparing the circadian systems of nocturnal and diurnal species

have found no major differences in the SCN of the two chronotypes (reviewed in

122

Smale et al., 2003; Smale et al., 2008). However, rhythms in Fos and Per1
expression in the vSPZ are different in male lab rats and grass rats (Ramanathan
et al., 2006; Schwartz et al., 2004; Antonio Nunez, personal communication).
Additionally, Fos rhythms in the vSPZ of male grass rats, but not lab rats, are
endogenous and persist in constant darkness (Schwartz et al., 2004). These
differences may contribute to the differential effects of pregnancy on activity
rhythms in lab rats and grass rats. Since the vSPZ is endogenously rhythmic in
the grass rat, its rhythms may persist during pregnancy and thus prevent the loss
of the locomotor activity rhythm. In Chapter 5, I argue that the lack of a phase
change in the Tb rhythm in pregnant grass rats might be due to the maintenance
of the activity rhythm in this species. Therefore, this second major difference
between lab rats and grass rats might also be due to differences in the vSPZ of
these two species. However, Per2 rhythms in the SCN of lab rats advance in
phase during early pregnancy, which also correlates with the advance in the
peak of the Tb rhythm. Although Per2 rhythms in the SCN are similar in male lab
rats and grass rats (Amir et al., 2004; Ramanathan et al., 2006), it is possible
that, during early pregnancy, the Per2 rhythm differs between the two species. If
no advance occurs in the SCN of grass rats, this might also explain why there is
no change in phase in the Tb rhythm of pregnant grass rats.

Further work concerning how circadian rhythms change during pregnancy
in diurnal versus nocturnal species, both in the brain and in overt rhythms, may
shed light on how these two chronotypes acclimate to changes in reproductive

state. However, it may also inform research concerning how diurnality is

123

achieved in species that have evolved from nocturnal ancestors by exposing
those aspects of the circadian system that are more or less plastic in diurnal and

nocturnal species.

Implications of the present work

Female mammals spend the majority of their adult reproductive lives
pregnant and/or lactating, states in which physiological and behavioral demands
are changing in major, but predictable, ways. Many systems make substantial
adjustments, including those that organize the daily patterning of integrative
processes. However, virtually nothing is known about the ultimate reasons for, or
the proximate causes of, the changes in circadian rhythms that pregnant and
lactating females undergo. This dissertation describes the ﬁrst studies designed
to identify ways in which modulation of the mammalian circadian system might
lead to the changes in rhythms that occur between one reproductive state and
the next. It also provides an in-depth description of circadian rhythms in core
body temperature and activity in grass rats during various reproductive states to
demonstrate that diurnal and nocturnal mammals can adopt very different
circadian strategies during pregnancy and lactation, which suggests that more
than one strategy may be adaptive.

Aside from providing information about natural changes in circadian
rhythms in adult mammals, the data from this dissertation indicate that this
knowledge has implications for reproductive medicine and biology. Shift-working
women encounter fertility and pregnancy complications (Boden and Kennaway,

2006). One reasonable assumption about the effect of shift-work is that it is

124

mediated by stress, which can complicate a pregnancy. However, the data from
Chapters 2 through 4 indicate that rhythms in protein expression in both circadian
and non-circadian systems are dramatically re-organized in pregnancy. If these
systems are desynchronized prior to copulation, as can result from shift work, the
re-organization of these systems necessary to optimize pregnancy may not be
properly achieved, resulting in reduced fertility and fecundity.

Circadian rhythms researchers often neglect the relevance of their work to
mammalian conservation and reproduction. However, wild and captive mammals
may experience circadian dysfunction, often due to anthropogenic effects.
Captive zoo animals, domesticated pets, service animals, and animals used in
entertainment, such as the circus, are subject to activity, enrichment, and feeding
schedules determined by their owners and keepers. Certain enrichment items,
such as running wheels (Blanchong et al., 1999; Eikelboom and Lattanzio, 2003),
and food are powerful entraining agents (Mendoza, 2007), and they may lead to
desynchrony amongst certain circadian oscillators (Mendoza, 2007;
Chidambaram Ramanathan, personal communication). This could result in the
inability of an individual to properly re-organize rhythms in the brain to promote
altered overt rhythms during transitions in reproductive state. For those species
which are more reliant upon circadian rhythms to reproduce, these entraining
agents might thus reduce the success of captive breeding efforts. Additionally,
anthropogenic effects on rhythms in wild mammals, which might occur via such
entraining agents as introduced non-natural food items, non-native species, or

artiﬁcial light, may similarly affect the reproductive success of these animals.

125

Applications of my dissertation to shift-working women are equally viable for
these non-human “shift-workers.” Given that diurnal and nocturnal species exhibit
clear differences in h0w their overt rhythms are altered during pregnancy and .
lactation (Chapter 5), a more comparative approach to the interface between
reproduction and circadian biology in the future may allow us to determine how

reproduction in wild and captive mammals is affected by perturbations in

circadian rhythmicity.

126

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