MASKING: THE ACUTE EFFECTS OF LIGHT ON THE BRAIN AND
BEHAVIOR
By
Dorela Doris Shuboni

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Psychology – Doctor of Philosophy
2013

ABSTRACT
MASKING: THE ACUTE EFFECTS OF LIGHT ON THE BRAIN AND
BEHAVIOR
By
Dorela Doris Shuboni

Masking of behavior by external stimuli works with the circadian system to
ensure that animals are active during the correct time-of-day. Light for diurnal
and nocturnal species produces different masking responses, enhancing activity
for diurnal species and suppressing activity for nocturnal species. Few studies
have examined the neural mechanisms of masking; none these experiments use
animals active during the day. The first experiment of this dissertation uses the
protein of the immediate-early gene cFOS to compare activation of brain regions
to light between nocturnal mice and diurnal grass rats during a time-point where
they showed a distinct behavioral dichotomy in response. Grass rats showed a
consistent increase in activation in areas that receive retinal innervation or were
related to sleep/arousal, while mice showed either no difference or a decrease in
activation with the exception of the SCN. This study demonstrates the differences
in behavioral and neurological responses to masking pulse of light between a
nocturnal and diurnal rodent species.
We next examined the functional role of the ventral subparaventricular
zone (vSPZ) on masking to light in the grass rat. The vSPZ uniquely responded
to light in diurnal grass rats, receives direct retinal innervation from the eye and
after ablation showed a possible alteration in masking. Schwartz et al. (2009)

showed that grass rats with damage to the vSPZ had bouts of arrhythmia in LD
conditions, indicating that the masking system may be altered. In Chapter 3, we
tested the functional for of the region by bilateral lesioning the vSPZ and directly
testing the effects of light on masking. Animals with complete ablations still
increased activity in response to light in two different masking protocols. In
Chapter 4, we examined another brain structure that could have played a role in
masking, the pineal gland. Melatonin is a hormone produced by the pineal gland
that plays a role in circadian rhythms and seasonality. The expression of
melatonin is sensitive to light exposure, additionally, the presence of the
hormone also feedbacks and influences the effects of light on the brain.
Additionally, pinealectomy in rats alters the behavior profile of activity in LD
conditions. Removal of the pineal gland in the grass rats did not influence the
ability of animals to mask to light or the animal’s circadian rhythm of activity.
In summary, the work presented in this dissertation demonstrates the
immediately impact of light on behavior and the possible brain regions that may
play a functional role in the masking response. We established that two neural
structures when ablated or removed do not impact the grass rat’s ability to
respond to light with an increase in activity. Here we broaden the scope of
research into the neural mechanisms of masking to include a diurnal species,
beginning the journey toward understanding the neural changes needed for a
species to transition between temporal niches.

Copyright by
DORELA DORIS SHUBONI
2013

This dissertation is dedicated to my family whose love and support inspired me to pursue
my scientific dreams; and to my advisor, Dr. Laura Smale for allowing me to accomplish my
goals with her invaluable guidance and encouragement.

v

ACKNOWLEDGEMENTS

I would like to thank Dr. Laura Smale for giving me the opportunity to become a
scientist. She has been an excellent advisor, her creativity and passion has inspired my
research and allowed me to grow immensely. I would also like to thank the rest of my
dissertation committee, Dr. Antonio Nunez, Dr. Weiming Li and Dr. Lily Yan, for their
guidance and critical comments.
All the members of the SYN lab have helped me immensely with my work,
providing invaluable comments, technical assistance, and friendship. I’d first like to
thank the Post-docs that I have worked with Dr. Chidambaram Ramanathan and Dr.
Andrew Gall. They have both been wonderful mentors and great friends. I would also
like to thank Dr. Alexandra Castillo-Ruiz, as the senior graduate student in our
laboratory she took me under her wing and taught me the ins-and-outs of research at
MSU. Additionally, I’d like to thank my fellow SYN graduate students, Carmel MartinFairey, Jennifer Langel, and Sean Deats, together we have been though many ICC
transfers and grass rats. Our laboratory technicians, Adam Stowie, Amna Aghna, Molly
Skaer, and Thomas Groves, have been a wonderful help over the years. Finally, I would
like to thank the undergraduates that I worked with over the years, Nicolas Anderson,
Josh Loeb, and Shannon Cramm.
I would also like to thank the Behavioral Neuroscience faculty and staff that has
helped guided me over the years. Dr. Lily Yan has been a major influence on my ability
to execute scientific techniques; she has improved my efficiency and skill in the lab and
played a major role in perfecting the surgery in Chapter 4. Thank you to Dr. Joe

vi

Lonstein, who provided us with the sleep monitoring program, Dr. Sharleen Sakai, who
provided us with the Cholera Toxin tract tracer, Dr. Cherly Sisk, who greatly improved
my editing abilities, and Dr. Cindy Jordan, who has given me great ICC advice. Also
thanks to Dr. Marc Breedlove for providing me with the financial support for my third
year through the Neuroscience Department’s Training Grant. Finally, I’d like to thank the
wonderful Neuroscience and Psychology office staff and to the best ULAR technicians
(Constance, Darcy, and Chris).
Lastly, I would like to thank my family and friends for supporting me over these
past four years. My parents, Dorel and Doina, have been the greatest inspiration for me.
They worked tirelessly to provide our family with opportunities beyond those they had in
Yugoslavia, I can never repay them for the great sacrifices that they made for my sisters
and me. Both of my parents have also stressed the importance of education and
pushed me to continue my studies, the work ethic they instilled in me allowed me to
complete this dissertation. My sisters, Simona and Christina, my niece, Arrianna, and
my grandparents, Josip and Iconija, have been a great distraction and stress relievers
for me during my writing. Also, thanks to my friends here at MSU and beyond: Lizzy
Reznikov, Lily Wang, Breyanna Cavanaugh, Danielle Hainline, Megan Tatoris, Chieh
Chen, Alex Castillo-Ruiz, and Andy and Heather Gall. Also thank you to the Albion
College Psychology Department, especially Dr. Tammy Jechura who introduced me to
Rhythms Research and started me down the path of academia as her “baby scientist”.
Finally, I would like to thank my boyfriend Tim Mulligan for his love and support.

vii

TABLE OF CONTENTS
LIST OF TABLES…………………………………………………………………......xi
LIST OF FIGURES ………………………………………………………………….xii
KEY TO ABBREVIATIONS…………………………………………….………….xviii
CHAPTER 1 Introduction…………………………………………………….............1
General Introduction………………………………..………………………………..1
Masking Definitions………………………………..…………………………...……3
Neural Mechanisms of Masking…………………….……………………………...6
Overview of the chapters…………………..………………………………………..9
CHAPTER 2 Acute effects of light on the brain and behavior of Arvicanthis
niloticus and Mus Musculus……………………...………………………………...11
ABSTRACT………………………………………………………………….…..….11
INTRODUCTION……………………………………………………………..…….13
METHODS………………………………………………………..……………..…..16
Animals…………………………………………………………………….……...16
Experimental Procedures……………………………………………..…………20
Experiment 1: Mapping of the retinal projections……….………..….……...20
Experiment 2: Behavioral and cFOS response to nighttime light…..……..20
Immunocytochemistry…..…………………………………………………..……21
Experiment 1: Mapping of the retinal projections…………….………..…....21
Experiment 2: Behavioral and cFOS response to nighttime light……..…..22
Data Analysis………..………………………………………………………...….23
Experiment 1: Mapping of the retinal projections……….………..….……...23
Experiment 2: Behavioral and cFOS response to nighttime light…..……..23
RESULTS……………………………………………………………………….…..25
Experiment 1: Mapping of the retinal projections ……………………….…....25
Experiment 2: Behavioral and cFOS response to nighttime light……….......30
Behavior………………………………………………………………………...30
cFOS expression in Retino-Recipent Regions……………….…………….34
cFOS expression in Arousal/Sleep-Related Regions……….…………......37
DISCUSSION………………………………………….……………………………43
cFOS expression in Retino-Recipent Regions ....………………………..…...43
SCN……………………………………………………………………………...45
vSPZ……………………………………………………………………………..47
IGL………………………………………………………………………………..51
LHb………………………………………………………………….……………54
DLG………………………………………………………………………………55
OPN………………………………………………………………………………56
cFOS expression in Arousal/Sleep-Related Regions ………….………….....57
VLPO……………………………………………………………………………57
viii

LH, DR, LC……………………………………………………………………..58
Summary and Conclusions………………………………………………59

CHAPTER 3 Ventral subparaventricular zone lesions and light-induced masking
behavior in the diurnal rodent, Arvicanthis niloticus………………….………61
ABSTRACT………………………………………………………….…….……...…61
INTRODUCTION………………………………………………………….………..63
METHODS…………………………………………………………………….….....65
Animals………………………………………………………………………....…65
Experimental Procedures..………………………………………………….…..66
Surgical Procedures……………………………………………………….….…67
Assessment of the Lesions……………………………………………….…….68
Data Analysis…………………………………………………………………….69
RESULTS…………………………………………………………………………..70
Histology………………………………………………………………………….70
Masking…………………………………………………………………………..71
Circadian Rhythms……………………………………………………………...78
DISCUSSION………………………………………………………….…………..85
Masking…………………………………………………………………………..85
Circadian Rhythms……………………………………………………………..103
SCN Damage……………………………………………………………………104
Summary and Conclusion………………………………………….………….104
CHAPTER 4 The pineal gland does not influence the daily patterning of behavior
of diurnal Arvicanthis niloticus……..………………………………..…………..106
ABSTRACT………………………………………………………….…………….106
INTRODUCTION………………………………………………………………….108
METHODS…………………………………………………………………………110
Animals…………………………………………………………………………..111
Experimental Procedures………………………………………………………111
Experiment 1: Effects of exogenous melatonin on activity and body
temperature…………………………………………………………….…..….111
Experiment 2: Effects of pinealectomy on activity and sleep behavior…112
Measurement of Plasma Melatonin Levels…………………………………..114
Data Analysis………..…..………………………………………………………114
Experiment 1: Effects of exogenous melatonin on activity and body
temperature…………………………………………………………….…..….115
Experiment 2: Effects of pinealectomy on activity and sleep behavior…115
RESULTS.…………………………………………………………………….…...117
Experiment 1: Effects of exogenous melatonin on activity and body
temperature…………………………………………………………………..….117
Experiment 2: Effects of pinealectomy on activity and sleep behavior…...117
LD Conditions…………………………………………………………………117
Masking………………………………………………………………………..120
Period and Phase Shifts……………………………………………………..124
Melatonin Levels……………………………………………………………...124
ix

DISCUSSION…………………………………………………………………..…124
LD Conditions………………………………………………………………..…127
Masking………………………………………………………………………….128
Period and Phase Shifts……………………………………………………….129
Melatonin Level…………………………………………………………………129
Summary and Conclusions…………………………………………………....130
CHAPTER 5 Summary and Conclusions………………………………………132
Temporal niche specific response to light…………………………………..…132
Neural Mechanisms of Masking………………………………………………...133
Implications and recommendations…………………………………………….136
REFERENCES…………………………………………………………………..…139

x

LIST OF TABLES
Table 2.1. Literature review of cFOS activation in response to light pulses in nocturnal
mice, rats, and hamsters * Indicates that the study did not statistically analyze their
data.
…………………………………………………………………………….…………….17

Table 2.2. Literature review of masking in12:12LD condition for animals with
suprachiasmatic nucleus lesions(†) indicates studies that observed extreme damage to
the optic chiasm. (*) indicates studies that use lack of rhythms in LD as a criteria for
good SCN lesion.
……...……………………………………………………………………….………….49

Table 4.1. Analysis of light pulse data masking data in PINX and SHAM animals
………………………..………………………………………………………………..121

xi

LIST OF FIGURES
Figure 2.1. Line drawings of retinal projections in Mus musculus and Arvicanthis
niloticus. Animals were injected bilaterally with the tract tracer CTβ and their brains were
processed using immunocytochemistry. Sections 1-8 depict the rostral to caudal
progression of projections in the grass rat (A) and mice (B). A magnification of sections
1-5 (C), illustrates that mice had fewer fibers within the vSPZ than grass rats, section 3.
For interpretation of the references to color in this and all other figures, the reader is
referred to the electronic version of this dissertation.
………………………………………………………………………………………….26

Figure 2.2. Behavioral masking response to 1 hr of light exposure. Grass increased
activity as observed in a representative actogram (A) and group data (B). The opposite
behavior was observed in mice as both a representative actogram (C) and group data
(D) had a decrease in activity. An asterisk (*) indicates significance p < .05.
………………………………………………………………………………………….31

Figure 2.3. Photomicrographs of cFOS in retino-recipient regions. Grass rats on the
right, have controls with relatively low cFOS activation while the light pulse (LP) have
higher levels with the exception of the VMH that remained unchanged. The mice on the
left, had an increase in cFOS activation after the LP (SCN), no change between control
and LP (vSPZ, LHb, IGL, and VMH), or a decrease in cFOS activation after LP (DGN
and OPN).
………………………………………………………………………………………….33

Figure 2.4. Patterns in cFOS expression in retino-recipient regions. Panel A shows a
significant increase in activation within the SCN. Three regions, the vSPZ (B), LHb (C)
and IGL (E) respond with a significant increase in grass rats but not in the mice. Two
regions, the DLG (D) and OPN (F) have an opposite response to light exposure with
mice experiencing a decrease and grass rats an increase in activation. Panel G shows
no response within the VMH of either species. An asterisk (*) indicates significance p <
.05.
………………………………………………………………………………………….35

Figure 2.5. Photomicrographs of the dorsal lateral geniculate nucleus for all mouse
subjects. On the left, 3 control mice have very high levels of cFOS (MM06, MM09 &
MM10) while the remaining two have moderate levels of expression (MM07 & MM08).
On the right, all LP animals have low levels of cFOS expression.
………………………………………………………………………………………….38

xii

Figure 2.6. Photomicrographs of the olivary pretectal area for all mouse subjects. On
the left, 4 control mice have high levels of cFOS (MM06, MM07, MM09 & MM10) while
all but MM4 of the LP animals (Right) have low levels of cFOS expression.
………………………………………………………………………………………….39

Figure 2.7. Photomicrographs of cFOS in arousal/sleep-related regions. Grass rats on
the right, have controls with relatively low cFOS activation while the light pulse (LP)
animals have higher levels in all areas examined (VLPO, LH, DR, LC). The mice on the
left, had no change between control and LP for all these areas.
………………………………………………………………………………………….40

Figure 2.8. Patterns in cFOS expression in arousal/sleep-related regions. Panel A-D
show the same pattern of response for within each species, respectively. In the grass
rat cFOS activation is observed in the VLPO (A), LH (B), DR (C), and LC (D). No
response was observed in any of the arousal/sleep-related regions in the mouse tissue.
An asterisk (*) indicates significance p < .05.
………………………………………………………………………………………….42

Figure 2.9. Photomicrographs of the suprachiasmatic nucleus and ventral
subparaventricualr zone for all mouse subjects. On the left, all control mice have low
levels of cFOS in the SCN while on the right LP animals have high levels of cFOS
expression. An examination if the vSPZ, reveals that control animals vary greatly as
three animals have high levels of expression (MM07, MM08, & MM10) and two have
lower levels (MM06 & MM09).
………………………………………………………………………………………….46

Figure 2.10. Photomicrographs of the intergeniculate leaflet for all mouse subjects.
Both control (Left) and LP (Right) animals have high levels of cFOS expression.
………………………………………………………………………………………….52

Figure 3.1. Photomicrographs of Nissl-stained tissue in all SHAM lesioned animals.
Sections were selected to represent one section before the vSPZ, three spanning the
rostral-caudal extent of the regions and one section after. No damage is observed in the
tissue with the exception of needle marks.
………………………………………………………………………………………….72

Figure 3.2. Photomicrographs of Nissl-stained tissue in 2 representative Miss animals
and all unilaterally lesioned animals. Sections were selected to represent one section
before the vSPZ, three spanning the rostral-caudal extent of the regions and one
section after. Miss animals ( MASPZ-13 & MASPZ-23) had no damage observed in the
xiii

vSPZ or SCN, while unilaterally lesioned animals (MASPZ-05, MASPZ-08, & MASPZ22) had one side of the vSPZ completely ablated. Areas damaged by the toxin are
outlined in red.
………………………………………………………………………………………….74

Figure 3.3. Photomicrographs of Nissl-stainedtissue in all bilaterally lesioned animals.
Sections were selected to represent one section before the vSPZ, three spanning the
rostral-caudal extent of the regions and one section after. All lesioned animals had
extensive damage observed in the vSPZ, 2 of these animals also had damage within
the SCN (MASPZ-03 & MASPZ-04). Areas damaged by the toxin are outlined in red.
………………………………………………………………………………………….76

Figure 3.4. Patterns of behavioral response of activity to light exposure in Miss and
Unilateral animals. On the x-axis, 4 different time points, ZT6, ZT14, ZT18 and ZT22,
were examined. The black bars represent acivity during 1 hr of a control day while grey
bars are the same hour during the light exposure. All animals respond to light at most
time points (16/20), however, two animals had a time point where there was no
response (MASPZ-13 & MASPZ-22) and one animal had two time points were activity
was decreased (MASPZ-08).
………………………………………………………………………………………….79

Figure 3.5. Patterns of behavioral response of activity to light exposure in SHAM
animals. On the x-axis, 4 different time points, ZT6, ZT14, ZT18 and ZT22, were
examined. The black bars represent acivity during 1 hr of a control day while grey bars
are the same hour during the light exposure. Two animals had strong positive masking
behavior, with an increase for all time points (MASPZ-14 & MASPZ-21). One animal did
not display a constant masking response, with both increases and decreases in activity
(MASPZ-07). The two final animals had very low activity level readings during this
portion of the experiment making it hard to interpret their masking behavior (MASPZ-15
& MASPZ-18).
………………………………………………………………………………………….81

Figure 3.6. Patterns of behavioral response of activity to light exposure in Miss and
Unilateral animals. On the x-axis, 4 different time points, ZT6, ZT14, ZT18 and ZT22,
were examined. The black bars represent acivity during 1 hr of a control day while grey
bars are the same hour during the light exposure. All animals respond to light at most
time points (12/16), however, two animals had a time point where there was no
response (MASPZ-03 & MASPZ-10) and one animal had two time points were activity
was decreased (MASPZ-19).
………………………………………………………………………………………….83

xiv

Figure 3.7. 3.5:3.5 LD Ultradian masking protocol in Miss and Unilateral animals. The
far left panel shows the 28hr actograms of animals during the ultradian cycle, grey bars
indicate the dark phases of the cycle. The middle panel shows the Chi-Squared
periodogram of animals during the last 7 days of the protocol. All animals have a
significant 24 hr rhythm and only one animal has a significant 7 hr cycle (MASPZ-13).
On the far right panel, total activity during the light and dark period were summed and
turned into a percentage of activity. All animals have the expected higher percentage of
activity during the light phases with the exception of MASPZ-08, who had more activity
during the dark phases.
………………………………………………………………………………………….86

Figure 3.8. 3.5:3.5 LD Ultradian masking protocol in SHAM animals. The far left panel
shows the 28hr actograms of animals during the ultradian cycle, grey bars indicate the
dark phases of the cycle. The middle panel shows the Chi-Squared periodogram of
animals during the last 7 days of the protocol. All animals have a significant 24 hr
rhythm and only one animal has a significant 7 hr cycle (MASPZ-14). On the far right
panel, total activity during the light and dark period were summed and turned into a
percentage of activity. Only 2 animals (MASPZ-14 & MASPZ-21) have the expected
higher percentage of activity during the light phases, the other 3 animals (MASPZ-7,
MASPZ-15 & MASPZ-18) did not respond with equal amounts of activity during the light
and dark phases.
…………………………………………………………………………………………89

Figure 3.9. 3.5:3.5 LD Ultradian masking protocol in SHAM animals. The far left panel
shows the 28hr actograms of animals during the ultradian cycle, grey bars indicate the
dark phases of the cycle. The middle panel shows the Chi-Squared periodogram of
animals during the last 7 days of the protocol. Three animals have a significant 24 hr
rhythm and one animal is arrhythmic (MASPZ-04). Only one animal had a significant 7
hr cycle (MASPZ-03). On the far right panel, total activity during the light and dark
period were summed and turned into a percentage of activity. Three animals (MASPZ03, MASPZ-10 & MASPZ-19) had the expected higher percentage of activity during the
light phases, the other animals (MASPZ-04) did not respond and had higher activity
during the dark phases.
……………………………………………………………………………………...….92

Figure 3.10. Circadian rhythms analysis in varying lighting conditions in SHAM. On the
Far left, actograms of all animals across three different lighting conditions, 12:12LD,
constant light, and constant darkness. Chi-squared analysis was conducted to detect
the presence of significant rhythms were run for all lighting conditions. All animals had
rhythms at the three lighting conditions except MASPZ-15 in LL.
………………………………………………………………………………………….95

xv

Figure 3.11. Circadian rhythms analysis in varying lighting conditions in Miss and
Unilateral groups. On the Far left, actograms of all animals across three different lighting
conditions, 12:12LD, constant light, and constant darkness. Chi-squared analysis was
conducted to detect the presence of significant rhythms were run for all lighting
conditions. All animals had significant rhythms at the three lighting conditions.
…………………………………………………………………………………….……98

Figure 3.12. Circadian rhythms analysis in varying lighting conditions in Bilateral lesion
animals. On the Far left, actograms of all animals across three different lighting
conditions, 12:12LD, constant light, and constant darkness. Chi-squared analysis was
conducted to detect the presence of significant rhythms were run for all lighting
conditions. One animal had no rhythms at any light conditions (MASPZ-04). In Constant
darkness MASPZ-10 became arrhythmic and in constant light MASPZ-03 was no longer
rhythmic.
……………………………………………………………………………………….101

Figure 4.1. The effects of exogenous melatonin on activity and body temperature (BT).
Group data of the hour after administration of melatonin does display alteration of
activity (A) nor BT (C) at any concentration level, ranging from 0.00-5.00 mg/kg. When
Individuals are plotted across 24 hr none display an alteration in activity (B) or BT (D).
The dotted red line at ZT11 indicates the administration of melatonin.
………………………………………………………………………………………118

Figure 4.2. 24 hr activity profile of PINX and SHAM animals. Grey bar indicates lights
off at ZT12.
………………………………………………………………………………………119

Figure 4.3. Sleep patterns in pinealectomized animals. Panel A demonstrates the
behavior we classified as “sleep-like,” the animals adopted a distinct posture, lying down
in a slumped position with the head on the floor of the cage. The total number of sleep
bouts (B) and the average sleep bout duration (C) were not different between PINX and
SHAM animals. Additionally, the spread of total sleep amount across the night was
identical between the two groups.
……………………………………………………………………………………….122

Figure 4.4. Patterns of the masking responses of activity to light. Both SHAM (Black)
and PINX (Grey) animals experienced increases in activity at all time points, ZT14,
ZT18 and ZT22. An asterisk (*) indicates significance p < .05.
………………………………………………………………………………………123

xvi

Figure 4.5. Circadian rhythms of activity in 12:12 LD cycle, constant light (LL), and
constant darkness (DD). Panel A shows two representative actograms of PINX (right)
and SHAM (left) animals. There was no significant difference between the two groups
for measurement of alpha (B) or tau (C).
………………………………………………………………………………………125

Figure 4.6. Phase shifting ability of pinealectomized animals. Panel A and B are
representative of PINX and SHAM animals in response to phase advances and delays.
No significant differences were observed between the two groups (C).
………………………………………………………………………………………126

xvii

KEY TO ABBREVIATIONS

Abbreviation

Definition

ABC……………………………..Avidin-Biotin Peroxidase Complex
AHA……………………………..Anterior Hypothalamic Area
ANOVA…………………………Analysis of Variance
BT……………………………….Body Temperature
CT……………………………….Circadian Time
CTβ……………………………..Cholera Toxin Beta-subunit
DAB…………………………….3,3’-diaminobenzidine
DD………………………………Constant Dark Conditions
DLG…………………………….Dorsal Lateral Geniculate
DMH……………………………Dorsomedial Hypothalamic Nucleus
DP………………………………Dark Pulse
DR………………………………Dorsal Raphe
GA ……………………….…….General Activity
GHT……………………….……Geniculohypothalamic Tract
HR ……………………………..Heart Rate
IACUC………………………….Institutional Animal Care and Use Committee
ICC……………………………..Immunocytochemistry
IGL …………………………….Intergeniculate Leaflet
IML …………………………….Intermediolateral Nucleus

xviii

ipRGC ………………………...melanopsin-expressing retinal ganglion cells
IRs……………………………..Infrared Motion Detectors
LC ……………………………..Locus Coeruleus
LD ……………………………..Light:Dark Cycle
LH ……………………………..Lateral Hypothalamus
LHb ……………………………Lateral Habenula
LP ……………………………..Light Pulse
LL ……………………………..Constant Light Conditions
NMA…………………………...N-Methyl-DL-aspartic acid
NMDA…………………………N-Methyl-D-aspartic acid
NPY.…………………………..Neuropeptide Y
PBS………………………...…Phosphate Buffered Saline
PINX…………………………..Pinealectomy
PVN …………………………..Paraventricular Nucleus of the Hypothalamus
PVT……………………………Paraventricuar thalamus
OC .…………………………...Optic Chiasm
OD…………………………….Optical Density
OPN …………………………..Olivary Pretectal Nucleus
opt……………………………..Optic Tract
RCh……………………………Retrochiasmatic Area
REM …………………………..Rapid Eye Movement Sleep
RT-PCR……………………….Real Time-Polymerase Chain Reaction
SCG …………………………..Superior Cervical Ganglion

xix

SCN …………………………..Suprachiasmatic Nucleus of the Hypothalamus
SCNx …………………………Suprachiasmatic Nucleus Lesions
VLPO ………………………...Ventrolateral Preoptic Nucleus
VMH…………………………..Ventromedial Hypothalamic Nucleus
vSPZ …………………………Ventral Subparaventricular Zone
SPZx………………………… Ventral Subparaventricular Zone Lesion
ZT.…………………………....Zeitgeber Time

xx

CHAPTER 1
Introduction
General Introduction
The cyclic pattern of the earth’s rotation has created systematic and predictable
daily rhythms in a range of environmental variables. Changes in the amount of ambient
light and in temperature across the day produce unique patterns of resources available
at specific phases of the day-night cycle; this variation had led organisms to become
specialized for specific temporal niches (Kronfeld-Schor & Dayan, 2008). At the two
extremes of this adaptive spectrum are (1) animals that are active primarily during the
day, (diurnal animals), and (2) animals active at night, (nocturnal animals; Refinetti,
2008). Species that occupy these two temporal niches have developed adaptations that
optimize functioning in their respective environments. Most notably, in vertebrates the
visual system differs greatly between diurnal and nocturnal species (Peichl, 2005). The
relative number of their photoreceptors that are cones, and the variety of types of
cones, are greater in diurnal than nocturnal animals, which allows them to use in the
daylight for color vision (Ahnelt & Kolb, 2000). Additionally, regions of the brain devoted
to vision are larger in animals that are active during the day than those most active at
night (Campi & Krubitzer, 2010; Gaillard, Karten, & Sauve, 2013). Both of these
adaptations presumably improve survival and reproduction by allowing animals to more
effectively locate food and identify potential dangers (Daan, 1981). The evolutionary
transitions between nocturnal and diurnal species have, therefore, required many
changes within the body, including the brain.

1

The expression of diurnal and nocturnal activity patterns is regulated by two
distinct mechanisms, masking and circadian timekeeping systems. It has been
hypothesized that these systems first evolved because single-cell organisms were
reliant on the sun for the production of energy through photosynthesis, and cells that
were able to restrict this process to the day, wasted less energy and had an adaptive
advantage. Simons (2009) suggests that before circadian systems evolved, organisms
developed the ability to respond directly and immediately to the presence of the sun, a
masking response. Eventually, such organisms acquired the cellular machinery needed
to maintain an endogenous circadian timekeeping mechanism that enabled them to
anticipate and prepare for daily changes in the photic environment. These systems are
also present in multicellular organisms but have become more complex, such that, for
example, in animals there are neural mechanisms that regulate behavior in a time-ofday dependant manner. Species that switch their temporal niche undergo changes in
both circadian systems and masking mechanisms to properly adapt to a new temporal
environment.
When mammals evolved from reptiles they moved from a diurnal to a nocturnal
niche, but there have been multiple, parallel evolutionary pathways back to diurnality
(Smale, Lee, & Nunez, 2003). Many studies have examined the differences between the
circadian systems of nocturnal and diurnal rodents, and these have found numerous
similarities in the central circadian pacemaker of mammals, the suprachiasmatic
nucleus (SCN), and many differences in rhythms in brain regions beyond the SCN
(Smale, Nunez, & Schwartz, 2008). Little, however, is known about the neural changes
that occurred at these transitions to alter masking behavior.

2

In this introductory chapter, I will first provide an overview of masking, with a
focus on the differences observed in nocturnal and diurnal species. I will define the
terms associated with masking and provide examples of niche specific masking
responses. I will provide an overview of current research into the neural mechanisms of
masking, which has focused almost exclusively on nocturnal rodents. This work has
been aimed at identification of the retinal cells that are components of the input system
pivotal to the expression of masking, as well as brain regions that are innervated by
these cells. Here, I will also review papers that describe studies of effects of lesions of
particular brain regions on masking. Finally, I will outline the research questions that are
addressed in chapters two, three and four.

Masking Definitions
Aschoff (1960) first described masking in terms of how it affects circadian
rhythms. He deemed environmental factors that obscure the observation of the
endogenous rhythm as masking factors, as they directly influenced the variable being
measured and masked the influence of the pacemaker (Aschoff, Daan, & Honma,
1982). The masking response can, therefore, be thought of as an immediate behavioral
or physiological response to the presentation of an environmental stimulus, and the
mechanism that produce it can be broken down into three components: (1) the input
system that signals the presentation of the masking stimulus, (2) neural mechanisms
that process the signal, and (3) the behavioral or physiological endpoint. More recently,
researchers have proposed that the masking response helps animals specialize for their
temporal niche (Mrosovsky, Foster, & Salmon, 1999). Masking enhances or represses

3

the expression of the endogenous rhythm and allows animals to adjust their behavior in
response to small but important changes in the environment, providing animals more
flexibility in the expression of their rhythms (Marques & Waterhouse, 1994).
Many different environmental stimuli can function as masking agents, such as
temperature (Hoffman, 1969), social cues (Aschoff & Vongoetz, 1988) and, most
importantly, light (Aschoff & Vongoetz, 1989). I will focus the remainder of the review
below on light-induced masking. There are also both behavioral and physiological
variables that respond in an immediate manner to changes in the photic environment.
Light, for example, immediately suppresses the secretion of endogenous melatonin at
night in all animals with functioning pineal glands (Challet, 2007). Light also impacts the
rest-activity cycle of animals but the direction of the response is dependent on the
temporal niche an animal occupies (Redlin, 2001). Among day-active organisms, such
as diurnal birds (Aschoff & Vongoetz, 1989), rodents (Shuboni, Cramm, Yan, Nunez, &
Smale, 2012), and primates (Gander & Mooreede, 1983), light increases the level of
activity, a process referred to as positive masking. Among nocturnal rodents (Butler &
Silver, 2011; Mrosovsky et al., 1999; Pendergast & Yamazaki, 2011; Redlin &
Mrosovsky, 1999b) and primates (Erkert, Gburek, & Scheideler, 2006), light decreases
activity, a processes referred to as negative masking. Additionally, exposure to light
increases sleep in nocturnal species, such as mice (Morin & Studholme, 2009;
Studholme, Gompf, & Morin, 2013) and decreases it in diurnal ones, such as humans
(Cajochen, 2007; Lockley et al., 2006; Teixeira et al., 2013). These responses to light
help restrict animals to the temporal niche to which they are optimally adapted, and

4

masking responses become a major barrier standing in the way of evolutionary
transitions between nocturnality and diurnality (Kronfeld-Schor & Dayan, 2008).
In the wild, different selective pressures, including the introduction of predators
(Fenn & Macdonald, 1995), competition (Harrington et al., 2009; Shkolnik, 1971) and
anthropogenic disturbances (Rasmussen & Macdonald, 2012), induce temporal niche
switches in species. One nice illustration of how masking is altered by the presence of
competition comes from studies of two species of spiny mice whose ranges overlap in
the vicinity of the Dead Sea and that have partitioned their resources by occupying
different temporal niches (Shkolnik, 1971). Masking behavior of the diurnal golden spiny
mouse is different from that of the nocturnal common spiny mouse but it is not simply
reversed. Rather, the golden spiny mouse has a positive masking response to daytime
dark exposure and does not respond to nighttime light (Cohen, Smale, & KronfeldSchor, 2010; Rotics, Dayan, Levy, & Kronfeld-Schor, 2011). However, when the golden
spiny mouse is removed from the competitive environment in the field and placed in a
semi-natural enclosure it displays nocturnal activity patterns (Gutman & Dayan, 2005),
something that also occurs within the laboratory (Cohen, Smale, & Kronfeld-Schor,
2009). This suggests that the golden spiny mouse may represent a transitional state in
which it maintains some features of an originally nocturnal ancestor but has undergone
some changes in masking, such that it can be described as neither nocturnal nor
diurnal.
In the laboratory, some other diurnal rodents can experience inversions in their
behavioral patterns with the introduction of a running wheel. Plasticity in this artificial
context has been observed in the Octodon degus (referred to below as a degus; Kas &

5

Edgar, 1999; Otalora, Vivanco, Madariaga, Madrid, & Rol, 2010), Mongolian gerbil
(Weinert, Weinandy, & Gattermann, 2007), and the Nile grass rat (referred to below
simply as a grass rat; Blanchong, McElhinny, Mahoney, & Smale, 1999; Blanchong &
Smale, 2000). It should be noted that not all degus and grass rats are capable of this
nocturnal activity pattern, and that the literature on daily rhythms and niche classification
of Mongolian gerbils is conflicting (Weinert et al., 2007). However, when members of
these species are night-active in the lab, they all have patterns of masking responses to
light and darkness typically seen in nocturnal species (degus: Vivanco, Rol, & Madrid,
2010; grass rats: Redlin & Mrosovsky, 2004; Mongolian gerbils: Weinert et al., 2007).
Although this work demonstrates the remarkable plasticity found in diurnal rodents, the
context is highly artificial and it is difficult to know if it can provide insight into more
fundamental mechanisms.

Neural Mechanisms of Masking
The first attempts to understand the neural mechanisms of masking in mammals
focused on the identification of the input systems through which light might drive the
behavioral response. Mrosovsky et al. (1999) initially noticed the impact of retinal
degeneration on the masking response when they saw that rd/rd mice, which have a
mutation that induced the loss of rods and cones as the animal aged, no longer showed
positive masking in response to darkness but maintained the ability to respond to light.
This indicated positive and negative masking responses are mediated by two different
types of photoreceptive cells at the level of the eye. Further evidence for this
segregation came from examination of the spectral sensitivities of the two masking

6

responses. Specifically, the negative masking seen in nocturnal mice is more sensitive
to short-wavelengths while positive masking in these animals is induced by dimming
medium-wavelength light in the range associated with the activation of rods and cones
(Thompson, Foster, Stone, Sheffield, & Mrosovsky, 2008). Little was known, however,
about the type of photosensitive retinal cell that mediates the negative masking seen in
these animals, until the mid-2000s.
The search for the photoreceptors responsible for negative masking in nocturnal
species involved many investigators because these cells were also believed to mediate
photic influences on the circadian pacemaker and to induce the papillary light reflex
(Gooley, Lu, Fischer, & Saper, 2003). The short-wavelength photoreceptive cells,
referred to now as intrinsically photo-responsive retinal ganglion cells (ipRGCs), contain
a newly discovered photopigment, melanopsin, and these cells were found to be the
ones that project to the SCN (Gooley, Lu, Chou, Scammell, & Saper, 2001). In mice that
have been genetically engineered to no longer express (knocked-out) the gene
-/-

encoding of the protein melanopsin (Opn4 ), photic influences on circadian rhythms
(Panda et al., 2002), the pupilary reflex (Lucas et al., 2003), and masking (Mrosovsky &
Hattar, 2003) were all diminished. However, when these ipRGCs are completely
eliminated in mice by knocking-in a toxin that kills the cells, mice no longer exhibit
evidence of either positive nor negative masking (Guler et al., 2008). This work
established that melanopsin-containing ipRGCs are the conduit that relays photic
information into the brain to produce both positive and negative masking. Since the
discovery of these cells, all examinations into the neural mechanisms of masking have
focused on brain regions that receive input from them.
7

The melanopsin-containing ipRGCs project to many areas of the mouse brain
(Hattar et al., 2006), including the suprachiasmatic nucleus (SCN), intergeniculate
leaflet (IGL), dorsal lateral geniculate (DLG), and olivary pretectal nucleus (OPN). All of
these areas have been investigated in nocturnal animals, to determine if they play a role
in the mediation of masking responses to light. Such studies have led Mrosovsky and
colleagues propose that multiple areas mediate masking of activity in response to light
but that no single one of these areas is necessary for its maintenance (for review see
Redlin, 2001). Based on their early work with rd/rd mice, Mrosovsky and colleagues
targeted areas that were associated with vision. Specifically, these investigators have
ablated the IGL (Redlin, Vrang, & Mrosovsky, 1999), DLG (Edelstein & Mrosovsky,
2001), and visual cortex (Redlin, Cooper, & Mrosovsky, 2003) and observed an actual
increase in the masking response to light, with greater suppression of activity in
lesioned animals. Recently, the effects of IGL lesions on masking have been examined
in diurnal grass rats (Gall, Smale, Yan, & Nunez, 2013). Large lesions that
encompassed both the IGL and part, or all, of the DLG produce a startling inversion of
masking behavior, such that animals no longer positively masked to light but instead
had a negative masking response. Therefore, here too there is no overall destruction of
the masking behavior but an intriguing reversal in its direction, suggesting that the IGLregion is an important structure for maintenance of diurnal activity in grass rats.
Redlin and Mrosovsky (1999a) also destroyed the SCN of hamsters and saw no
effect on masking in the context of an ultradian cycle, but they did not rule out the
possibility that the SCN contributes to masking behavior. However, another study in
which the same protocol was used, found that lesions directed at the SCN did block

8

masking in hamsters (Li, Gilbert, & Davis, 2005). One explanation of the discrepant
findings proposed by Li et al. (2005) is that damage to areas of the hypothalamus that
are innervated by retinal fibers that pass through the SCN (e.g. ventral
subparaventicular zone, vSPZ) might be responsible for the effects they observed. The
question of whether and how the SCN might play a role in masking in nocturnal
mammals has thus not been settled, and nothing at all is known about this issue in
diurnal species.
Finally, in his review of the neural mechanisms of masking, Redlin (2001) also
proposes that the OPN might play an important role in masking. This region of the optic
tectum lies downstream of the SCN and has reciprocal connections with the IGL. While
no study has directly examined its role in regulation of general activity, one study has
investigated the effects of OPN lesions on the ability of albino rats to experience a
strain-specific form of masking, dark-induced REM sleep (Miller, Miller, Obermeyer,
Behan, & Benca, 1999). Removal of the OPN greatly reduced the ability of animals to
experience the induction of REM sleep by darkness, however, total sleep was not
affected and activity was not monitored in that study.
Neither the SCN nor the OPN have been investigated in relation to their potential
roles in masking in a diurnal species. Additionally, other brain regions such as the vSPZ
and the pineal gland should be considered in the regulation of masking but have yet to
be directly examined for their role in masking in either diurnal or nocturnal animals.

Overview of the chapters

9

In this dissertation, I present experiments that were designed to examine (1)
what neural mechanisms drive the light-induced masking behaviors of a diurnal species
and (2) how they may differ from those of their nocturnal counterparts. To address this
question, I have used the diurnal grass rat and the nocturnal mouse to contrast
responses of different brain regions to light at a time-of-day at which light induces
activity in the nocturnal species and increases it in a diurnal one. Differences in the
patterns of neural activation can provide a first step in the identification of brain regions
that might be responsible for the behavioral differences between nocturnal and diurnal
rodents (Chapter 2). Direct examination into the functions of one brain region in the
grass rat, the vSPZ, will then be explored by ablating it with chemotoxic lesions
(Chapter 3). In addition, I have removed the pineal gland in order to test the hypothesis
that it may contribute to masking, as well as to diurnality more generally (Chapter 4).
The immediate response of activity to light exposure, masking, is important in promoting
the most advantageous activity pattern for an animal and identifying brain regions
involved in the process of light-induced masking behavior will provide insight into the
changes in the nervous system that have occurred during evolutionary transitions from
one temporal niche to another. Therefore, these experiments examine a new avenue in
the understanding of the evolution of diurnality.

10

CHAPTER 2
Acute effects of light on the brain and behavior of Arvicanthis niloticus and Mus
Musculus
ABSTRACT
Photic cues influence daily patterns of activity via two complementary
mechanisms: (1) entraining the internal circadian clock and (2) directly increasing or
decreasing activity levels, a phenomenon referred to as “masking”. The direction of this
masking response is dependent on the temporal niche an organism occupies; nocturnal
animals typically decrease activity when exposed to light, while diurnal animals
generally increase activity. Little is known about the neural mechanisms underlying
these differences. Here, we directly compared the masking effects of light on behavior,
and the activation of several brain regions by that light, in diurnal Arvicanthis niloticus
and nocturnal Mus musculus. Each species displayed the temporal niche appropriate
behavioral response to light, with the diurnal grass rats increasing their activity and the
nocturnal mice decreasing their activity when presented with a 1 hr pulse of light two
hours after lights off (ZT14, where ZT0 is lights on). The patterns of cFOS activation
varied between the two species. Grass rats showed an increase in activation in all areas
that receive retinal input while mice showed an increase in one of these regions (the
suprachiasmatic nucleus), no change in others (the ventral subparaventricular zone,
intergeniculate leaflet and lateral habenula) and a decrease in two (the olivary pretectal
nucleus and dorsal lateral geniculate). In addition, cFOS was increased by light in three
arousal-related brain regions (the lateral hypothalamus, dorsal raphe, and locus
coeruleus) and one sleep-promoting region (the ventrolateral preoptic area) in grass

11

rats but not mice. Taken together, these results suggest the possibility that the retinorecipient and sleep/arousal-related brain regions that exhibit different patterns of
responsiveness to light in the two species may contribute to differences in their behavior
responses to photic stimuli.

12

INTRODUCTION
Light is a powerful environmental cue that can have a major impact on the daily
behaviors and physiology of an organism (Aschoff, 1999). Specifically, light can have
acute effects on behavior and physiology (masking effects) and it can impact the
endogenous pacemaker (circadian rhythms effect; Mrosovsky, 1999; Redfern, Minors, &
Waterhouse, 1994). Disentangling the influences of these two processes can be difficult
in natural conditions because in environments with rhythmic alteration of light and
darkness masking and circadian rhythms complement each other to coordinate the daily
patterning of behavior and physiology (Redlin, 2001). Early circadian biologists devised
experimental protocols to disentangle the influences of these two systems in the
laboratory. However, in doing so they created a certain negative stigma associated with
masking (Minors & Waterhouse, 1989a). This is evident in the original definition of the
term, “…certain (sometimes overlooked) experimental conditions can obscure the real
Zeitgeber-mechanism. We may call them masking conditions” (Aschoff, 1960). Masking,
however, can reflect adaptive mechanisms that contribute to regulation of the daily
patterning of activity, rather than processes that simply obscure the influences of
circadian mechanisms.
Diurnal and nocturnal species differ with respect to the direction of the acute
response of their locomotor activity to photic stimuli. Light produces an increase in
activity (positive masking) in diurnal animals, whereas in a nocturnal species it typically
decreases activity (negative masking; Mrosovsky, 1999). Many experiments have
documented the suppression of activity by light in nocturnal mice (Butler & Silver, 2011;
Morin, Lituma, & Studholme, 2010; Pendergast & Yamazaki, 2011), rats (Scheer, Ter

13

Horst, Van der Vliet, Buij, 2001), and hamsters (Redlin & Mrosovsky, 1999b). Masking
effects of light on activity have been less well-studied in diurnal rodents, though they
have recently been described in Nile grass rats (Redlin & Mrosovsky, 2004; Shuboni et
al., 2012), degus (Vivanco et al., 2010), and Mongolian gerbils (Weinert et al., 2007).
Several approaches have been taken to investigate the neural basis of masking.
One approach has been to examine effects of lesions of retino-recipient regions on
acute behavioral responsiveness to photic stimuli. The first area to be examined was
the suprachiasmatic nucleus (SCN), site of the primary circadian oscillator in mammals.
Redlin and Mrosovsky (1999a) saw no effect of SCN lesions on masking in hamsters
but they did not rule out the SCN as a possible contributor to masking. Another study
found that lesions directed at the SCN did actually block masking (Li et al., 2005); these
investigators propose that either the SCN or other hypothalamic areas that are
innervated by retinal fibers that pass through it (e.g. ventral subparaventricular zone,
vSPZ) play a major role in masking (Li et al., 2005). Effects of lesions of the
intergeniculate leaflet (IGL) on masking have also been examined in two studies. Redlin
et al. (1999) reported that IGL lesions actually increased negative masking in hamsters,
while Gall et al. (2013) found that the lesions caused a change in the direction of the
masking response of diurnal grass rats. Control grass rats showed positive masking of
activity in response to a light pulse, while lesioned animals showed a negative masking
response to light. In nocturnal rodents, lesions of the dorsal lateral geniculate nucleus
(DLG; Edelstein & Mrosovsky, 2001) and visual cortex (Redlin et al., 2003) have also
led to an increase in light-induced suppression of activity. Lesion studies have also
implicated the retino-recipient olivary pretectal nucleus (OPN) in mediation of direct

14

effects of light on masking in nocturnal laboratory rats; specifically, light-induced REM
sleep was attenuated by ablation of the OPN in these animals (Miller et al., 1999).
Consideration of the effects of lesions of different retino-recipient areas of the brain led
Mrosovsky and colleagues to propose that multiple areas mediate negative masking of
activity by light in nocturnal species (Redlin, 2001).
A second approach to exploration of neural substrates of masking has focused
on the recently discovered melanopsin-containing intrinsically photo-responsive retinal
ganglion cells (ipRGCs) and the brain regions to which they project. When these cells
are completely eliminated in mice, the animals no longer exhibit evidence of positive
and negative masking (Guler et al., 2008). ipRGCs project to many areas of the brain
(Hattar et al., 2006), one or more of which is likely to be functionally linked to masking.
Finally, several studies of nocturnal rodents have used the immediate early gene
cFOS to characterize responsiveness to light of cells in regions that receive input from
the retina, especially regions to which the ipRGCs project. Results from these studies
have revealed region, species, and strain differences, summarized in Table 2.1. There
are very few studies that have examined light-induced cFOS activation in diurnal
species and most of these have focused exclusively on the SCN (Abe, Honma,
Shinohara, & Honma, 1995; Krajnak, Dickenson, & Lee, 1997; M. M. Mahoney, Smale,
& Lee, 2009; Schumann, Cooper, Hofmeyr, & Bennett, 2006). Only two study have
examined light-induced cFOS beyond the SCN in a diurnal species, and this revealed
an increase in cFOS in the peri-SCN region of Nile grass rats (Mahoney, Bult, & Smale,
2001) and the IGL of degus (Krajnak et al., 1997). Nothing is known about patterns of

15

responsiveness to photic stimuli in other brain region that receive input from ipRGCs in
a diurnal species.
Here we present data collected with the aim of better understanding pathways
that might contribute to masking, and where along these pathways differences between
nocturnal and diurnal species might emerge. We first examined and directly compared
the distribution of retinal projections in the grass rat and mouse to determine the extent
to which regions known to receive input from melanopsin cells in the mouse also
receive input from the retina in grass rats. We then examined the responsiveness of
several of these regions to light administered at a time of day at which it suppresses
activity in mice and increases it in grass rats. We also characterized the effects of this
light on cFOS in sleep/arousal-related areas to determine how they may differ between
the two species.

METHODS
Animals
Adult female grass rats were obtained from the breeding colony at Michigan State
University and adult male CD1 mice were obtained from Charles River Laboratory
(Wilmington, MA, USA). These animals were maintained on a 12:12 light-dark (LD)
cycle with 300 lux of white light during the light phase and <1lux of red light during the
dark phase. For the duration of the experimental procedures all animals were singly
housed in Plexiglas cages (34x28x17 cm) equipped with an enrichment device (PVC,
length: 8 cm, diameter: 6 cm); food (PMI Nutrition Prolab RMH 2000, Brentwood, MO)
and water were available ad libitum. The Institutional Animal Care and Use Committee
(IACUC) of Michigan State University approved these experimental procedures.
16

Table 2.1. Literature review of cFOS activation in response to light pulses in mice, rats, and hamsters. *
Indicates that the study did not statistically analyze their data.

17

Table 2.1. (cont’d)

18

Table 2.1. (cont’d)

19

Experimental Procedures
Experiment 1: Mapping of retinal projections.
We used Cholera Toxin Beta-subunit (CTβ) injections into the eye to map all
retinal projections in a grass rat (n=1) and mouse (n=1). Low salt CTβ (List Biological,
Campbell, CA, USA) was diluted to a concentration of 3 mg/mL in a mixture of 1.8%
DMSO and saline. The grass rat or mouse was deeply anesthetized using isoflorane
(2.5-5%, Abbott Laboratories, IL, USA), the eye was then protruded from the socket and
5 µL of CTβ was injected into the posterior chamber using a 10 µL Hamilton syringe
(Hamilton, Reno, Nevada, USA). The injection was given over a thirty-second period
and the needle was left in the eye for an additional minute to ensure the diffusion of the
substance into the vitreous humor. To obtain maximal labeling, both eyes were injected
with the tracer. One week after the eye injections, the animals were deeply anesthetized
using an i.p. injection of sodium pentobarbital (400 mg/kg). The animals were then
transcardially perfused with 0.1M phosphate buffered saline (PBS, pH 7.2) and then a
4% paraformaldehyde solution in PBS, also containing 0.075M lysine and 0.01M
sodium periodate (PLP, Sigma-Aldrich, St. Louis, MO, USA). The brains were post-fixed
for an additional 4 hrs in the PLP solution; they were then transferred into sucrose for a
minimum of 24 hrs, after which they were cut along the coronal plane at 30 µm using a
freezing-microtome. These sections were divided into three series and stored in
cyroprotectant at -20° until further processing.
C

Experiment 2: Behavioral and cFOS responses to nighttime light

20

Eight grass rats and ten mice were used in this experiment. Activity levels were
monitored via infrared motion detectors (IRs, Visonic Tel Aviv, Israel) that were attached
to each cage lid near the food and water. These monitors function by using infrared
beams that, when broken, trigger a count; all of the counts were recorded with the
VitalView Program (Minimitter, Bend, OR, USA) on a personal computer in an adjacent
room. After two weeks of baseline data were collected, the grass rats and mice were
both randomly assigned to either a control group (grass rat: n=4, mice: n=5) or a group
that was exposed to a light pulse (LP; grass rats: n=4, mice: n=5). All of these animals
were perfused (see above) at zeitgeber time (ZT) 15. Animals in the LP group were
exposed to a pulse of light for an hour prior to sacrifice (ZT14-15). All grass rats in this
experiment were also injected bilaterally with cholera toxin the week before sacrifice,
but their brains were not processed for visualization of the tracer.

Immunocytochemistry (ICC)
Experiment 1: Mapping of retinal projections
One series of sections was reacted for the CTβ tracer. Unless otherwise
mentioned, free-floating sections were incubated at room temperature and washed
three times between incubations (10 minutes/wash) with 0.01M PBS (pH 7.2). The initial
incubation was preceded by six 10-minute rinses in PBS to remove excess
cryoprotectant. Sections were then blocked with 5% normal horse serum (Vector
Laboratory, Burlingame, CA, USA) in PBS and 0.3% Triton X-100. Tissue was then
incubated with goat anti- CTβ (1:100,000, List Biological) at 4° on a shaker f or 24 hrs.
C
The tissue was then incubated with a biotinylated horse anti-goat secondary (1:100,

21

Vector Laboratory, Burlingame, CA, USA) and then avidin-biotin peroxidase complex
(ABC) with the ABC Vectastain Kit (Vector Laboratory, Burlingame, CA, USA). The final
reaction of the tissue used 3,3’-diaminobenzidine (DAB, 0.5 mg/mL, Sigma-Aldrich) and
30% hydrogen peroxide in a Trizma buffer (Sigma-Aldrich) to visualized the retinal fibers
as a dark brown stain. After two minutes, the reaction was stopped with three rinses of
0.01M PBS, 10min/rinse. The sections were then mounted onto gelatin-coated slides,
dehydrated, and coverslipped with Permount (Fisher Scientific, Fair Lawn, NJ, USA).
Experiment 2: Behavioral and cFOS responses to nighttime light
One series of sections from each animal (i.e. every 3

rd

section) was processed

with immunohistochemistry to visualize the distribution of the protein cFOS. The
protocol for grass rats followed the same steps outlined for the CTβ reaction. In brief,
sections were incubated in (i) 5% normal donkey serum (Jackson ImmunoResearch,
West Grow, PA, USA), (ii) the primary rabbit anti-cFOS antibody (1:50,000, Santa Cruz
Biochemistry, Santa Cruz, CA, USA), (iii) biotinylated donkey anti-rabbit antibody
(1:200, Jackson ImmunoResearch) and (iv) the ABC complex (Vector Laboratory). From
this point the procedure differs from that used for the CTβ. Sections were rinsed in a
0.14M acetate buffer (pH 7.2) three times, 10 min/wash. To visualize the protein, a
mixture of DAB (0.5mg/mL) and nickel sulfate in 0.14M acetate buffer (pH 7.2) was
reacted with 3% hydrogen peroxide to produce a bluish-purple stain. Once the reaction
was complete (6 minutes), the tissue was washed three times in PBS, 10 min/wash.
Sections were mounted, dehydrated, and coverslipped with dibutyl phthalate xylene
(Sigma-Aldrich).

22

The procedure for processing tissue from the mice followed similar steps except
that: (1) the tissue was rinsed in 0.1% PBT (PBS with .01% Triton X-100) rather than
PBS, (2) the concentration of the primary antibody, rabbit anti-cFOS, was higher
(1:20,000), and (3) the tissue was incubated over-night in the ABC solution. Finally, the
DAB reaction was carried out in a Trizma buffer (as in the CTβ protocol described
above) and lasted for only 3 minutes.
A second series of brain sections from each animal was stained with cresyl violet
and used to delineate regions of interest for analysis.

Data Analysis
Experiment 1: Mapping of retinal projections
Detailed maps of all sections containing retinal fibers were drawn and
corresponding photographs were taken. The drawings were constructed using a light
microscope (Leitz, Wetzlar, Germany) equipped with a drawing tube; drawings were
made using a low power objective but fibers were confirmed using a higher power
objective. From the complete map, areas of interest were identified and photographed
using a CCD camera (CX900, MBF Biosciences, Williston, VT, USA) attached to a light
microscope (Ziess, Gottingen, Germany).

Experiment 2: Behavioral and cFOS responses to nighttime light
To analyze the behavioral data, Vital View files were converted into actograms
via ClockLab (Actimetrics, Wilmette, IL, USA) and raw data were transferred into
Microsoft Excel. The actograms provided visual confirmation of masking behavior during

23

the light pulse, while Excel allowed for quantitative assessment of the data. To
statistically compare activity between groups, the raw data were corrected for
differences in motion detector sensitivity by (1) converting the data into percentages
(activity during the hour before sacrifice/24 hr of baseline activity) and (2) subjecting
them to an arcsine transformation. Effects of light on behavior were examined in each
species by comparing these values from light pulsed and control animals with
independent-sample t-tests using SPSS.
For the analysis of cFOS we focused primarily on areas that receive retinal input
and/or are associated with sleep/arousal. Observers that were blind to which
experimental group the animal had been in did counts of numbers of cFOS+ cells in all
brain regions. A light microscope equipped with a drawing tube was used to produce
bilateral maps of labeled cells in all areas, with the exception of the dorsal raphe (DR),
which had one centrally located counting box, from at least one section. Counts were
done on two sections for the SCN, vSPZ, IGL, DLG, the ventrolateral preoptic nucleus
(VLPO), the lateral hypothalamus (LH), locus coeruleus (LC) and DR. Counting boxes
described in earlier reports were used to delineate regions sampled in the VLPO
(Novak, Smale, & Nunez, 1999), vSPZ (Schwartz, Nunez, & Smale, 2004), LH
(Martinez, Smale, & Nunez, 2002; Nixon & Smale, 2004), LC (Castillo-Ruiz & Nunez,
2011), and DR (Castillo-Ruiz & Nunez, 2011). For the remaining regions whole areas
were identified and counted. The boundaries of the SCN and OPN were easily
visualized, while the VMH, IGL and DLG were delineated with the aid of the nissl
stained tissue. Independent sample t-tests were used to determine if numbers of FOS+

24

cells differed between light-exposed and control groups; counts from each region, and
from each species, were analyzed separately.

RESULTS
Experiment 1: Mapping of retinal projections
Drawings of retinal fibers in a sequence of sections, and photomicrographs of
such sections, are shown for grass rats and mice in Figure 2.1. Here, we focus on
retinal projections outside the primary optic tract. The first fibers observed were in the
VLPO; both species had fibers in this region but they were sparse (3 to 6 per section)
(Figure 1A-B, section 1). Grass rats, unlike mice, also had staining medial to the VLPO,
in the ventromedial preoptic nucleus. In subsequent sections (Figure 2.1A-B, sections 24) dense retinal fibers could be seen across the full rostral-caudal extent of the SCN.
These findings are similar to those previously reported for the grass rat (Smale &
Boverhof, 1999) and mouse (Hattar et al., 2006; Mikkelsen, 1992). In the most rostral
section containing the SCN, fibers also became visible within the supraoptic nucleus
(SON, Figure 2.1A-B, section 2) in both species; innervation of the SON increased as
sections progressed caudally. Fibers along this pathway extended beyond the SON into
the amygdaloid complex and the lateral hypothalamus (LH, Figure 2.1A-B, section 5).
The mice appeared to have a few more fibers extending toward the amygdaloid
complex but were otherwise identical to the grass rats. In the caudal sections of the
SCN (Figure 2.1A-B, section 3-4), a group of fibers radiated dorsally from the nuclei,
extending into the vSPZ; this was the case in both species, however, in grass rats these
fibers extended further beyond the SCN than they did in mice. Many fibers were also

25

Figure 2.1. Line drawings of retinal projections in Mus musculus and Arvicanthis
niloticus. Animals were injected bilaterally with the tract tracer CTβ and their brains were
processed using immunocytochemistry. Sections 1-8 depict the rostral to caudal
progression of projections in the grass rat (A) and mice (B). A magnification of sections
1-5 (C), illustrates that mice had fewer fibers within the vSPZ than grass rats, section 3.
For interpretation of the references to color in this and all other figures, the reader is
referred to the electronic version of this dissertation.

26

Figure 2.1. (cont’d)

27

Figure 2.1. (cont’d)

29

observed within the retrochiasmatic area (RCh, Figure 2.1A-B, section 5), and
extending dorsally into the anterior hypothalamic area (AHA). As sections progressed
caudally, hypothalamic innervations became restricted to the area immediately dorsal to
the optic tract (opt). In grass rats, fibers were observed in the dorsomedial hypothalamic
nucleus (DMH) but not in the ventromedial hypothalamic nucleus (VMH). The
distribution of fibers in the DMH was similar in the two species, but in mice these fibers
extended into the anterior sections of the VMH. Dense retinal fibers were seen in all
regions of the geniculate complex of both species (Figure 2.1A-B, section 6-7); (i.e. the
dorsal lateral geniculate nucleus (DLG), intergeniculate leaflet (IGL) and the ventral
lateral geniculate (VLG). The DLG appeared first; at this level, fibers also began to
extend toward the midline. Progressing caudally, we saw one fiber in the lateral
habenula (LHb) of each species, and in the paraventricuar thalamus (PVT), 2-3 fibers
were visible in the grass rat and 1-2 fibers in the mouse. Fibers were also observed
medial to the DLG where they became denser in both species as the OPN appeared
(Figure 2.1A-B, section 7). These nuclei migrated laterally as the superior colliculus
emerged with very dense staining of the tracer (Figure 2.1A-B, section 8). Overall, the
distribution of the retinal fibers was very similar in the grass rat and mouse; the only
regions that differed substantially were the vSPZ, where there were more labeled fibers
in the grass rat, and VMH, where there were more fibers in the mouse than grass rat.

Experiment 2: Behavioral and cFOS responses to nighttime light
Behavior

30

Figure 2.2. Behavioral masking response to 1 hr of light exposure. Grass increased
activity as observed in a representative actogram (A) and group data (B). The opposite
behavior was observed in mice as both a representative actogram (C) and group data
(D) had a decrease in activity. An asterisk (*) indicates significance p < .05.

31

Exposure of grass rats to a 1 hr light pulse at ZT14 induced a marked increase in
general activity (Figure 2.2A). Specifically, among control animals, 4.69±0.67% of daily
activity occurred during the hour beginning at ZT14, whereas among animals exposed
to 1 hr of light at ZT14 this figure was significantly higher, at 15.72±4.59% (t(5)=2.74,
p=0.041). The opposite response was observed in the mice. In this case, the
percentages of daily activity occurring during the hour beginning at ZT14 were
17.66±3.52% among control mice but only 4.06±1.53% among light-exposed animals
(t(8)=3.75, p=0.006; Figure 2.2B).

cFOS expression in Retino-Recipent Regions
In our examination of cFOS responses to light in retinorecipient regions of mice
and grass rats we found four basic patterns: (1) light induced an increase in cFOS in
both species, (2) neither species responded to the light, (3) the responses of the two
species were in opposite directions, and (4) a cFOS response was seen in grass rats
but not mice. The first pattern was evident in the SCN, where the number of cFOS+
cells increased in response to the 1 hr light pulse in both species (Grass Rat: t(5)=4.04,
p=0.01 and Mouse: t(8)=4.77, p=0.001; Figure 2.3). The second pattern occurred in the
VMH, where neither species responded to the light; (Figure 2.3; Grass rat: (t(5)=1.10,
p=0.32; Mouse: t(8)=0.67, p=0.59). The VMH was visually striking in grass rats exposed
to light, as it was relatively void of cFOS+ cells compared to the highly reactive
surrounding regions (the arcuate nucleus and DMH, Figure 2.3). The two species
exhibited opposite patterns of response to light in the DLG and OPN (Figure 2.3).
Specifically, light induced an increase in cFOS in both areas in grass rats

32

Figure 2.3. Photomicrographs of cFOS in retino-recipient regions. Grass rats on the right, have controls with relatively
low cFOS activation while the light pulse (LP) have higher levels with the exception of the VMH that remained
unchanged. The mice on the left, had an increase in cFOS activation after the LP (SCN), no change between control
and LP (vSPZ, LHb, IGL, and VMH), or a decrease in cFOS activation after LP (DLG and OPN).

33

Figure 2.3. (cont’d)

34

Figure 2.4. Patterns in cFOS expression in retino-recipient regions. Panel A shows a significant increase in activation
within the SCN. Three regions, the vSPZ (B), LHb (C) and IGL (E) respond with a significant increase in grass rats but not
in the mice. Two regions, the DLG (D) and OPN (F) have an opposite response to light exposure with mice experiencing a
decrease and grass rats an increase in activation. Panel G shows no response within the VMH of either species. An
asterisk (*) indicates significance p < .05.

35

Figure 2.4. (cont’d)

36

(DLG: t(5)=3.61, p=0.012; OPN: t(5)=3.61, p=0.012), and a decrease in these regions in
mice (DGL: t(8)=4.308, p=0.003 and OPN: t(8)=3.486, p=0.008). Closer inspection
revealed that several of the control mice had very high concentrations of FOS+ cells in
these two regions; this was the case for the DLG in three of five mice (Figure 2.5) and
the OPN in four of five mice (Figure 2.6).
In all other retinorecipient areas that we examined light induced an increase in
cFOS in grass rats and had no significant effect in mice. The grass rats responded to
light with an increase in activation within the vSPZ (t(5)=7.57, p<0.01; Figure 2.3), IGL
(t(5)=31.22, p<0.001; Figure 2.3), and LHb (t(5)=5.67, p<0.01; Figure 2.3). In mice, light
had no effect on cFOS in these regions (vSPZ: t(8)=0.12, p=0.908; IGL: t(8)=0.22,
p=0.828; LHb: t(8)=1.33, p=0.220).

cFOS expression in Arousal/Sleep-Related Regions
As light-induced masking has been shown to inhibit sleep in diurnal species and
induce it in nocturnal ones, we examined regions that are associated with sleep and
arousal. In the grass rat, all arousal-inducing areas examined showed an increase in
cFOS+ cells after the light pulse; this included the DR (t(5)=7.67, p<0.01; Figure 2.7)
and the LC (t(5)=7.11, p<0.01; Figure 2.5). Among mice, there was no response to a
light pulse in the DR (t(8)=0.774, p=0.461) or the LC (t(8)=1.753, p=0.118). In the LH
(Figure 2.7), where orexin neurons are found, control grass rats had significantly lower
numbers of cFOS+ cells than grass rats exposed to the light pulse (t(5)=11.50,
p<0.001). Among mice cFOS did not differ in the two groups (t(8)=0.66, p=0.527).

37

Figure 2.5. Photomicrographs of the dorsal lateral geniculate nucleus for all mouse
subjects. On the left, 3 control mice have very high levels of cFOS (MM06, MM09 &
MM10) while the remaining two have moderate levels of expression (MM07 & MM08).
On the right, all LP animals have low levels of cFOS expression.

38

Figure 2.6. Photomicrographs of the olivary pretectal area for all mouse subjects. On
the left, 4 control mice have high levels of cFOS (MM06, MM07, MM09 & MM10) while
all but MM4 of the LP animals (Right) have low levels of cFOS expression.

39

Figure 2.7. Photomicrographs of cFOS in arousal/sleep-related regions. Grass rats on the right, have controls with
relatively low cFOS activation while the light pulse (LP) animals have higher levels in all areas examined (VLPO, LH,
DR, LC). The mice on the left, had no change between control and LP for all these areas.

40

Figure 2.7. (cont’d)

41

Figure 2.8. Patterns in cFOS expression in arousal/sleep-related regions. Panel A-D show the same pattern of response
for within each species, respectively. In the grass rat cFOS activation is observed in the VLPO (A), LH (B), DR (C), and
LC (D). No response was observed in any of the arousal/sleep-related regions in the mouse tissue. An asterisk (*)
indicates significance p < .05.

42

Finally, in the VLPO (Figure 2.7), a sleep-promoting of the hypothalamus, light induced
an increase in cFOS in the grass rats (t(5)=5.87, p<0.01) but had no effect in mice
(t(8)=0.74, p=0.481).

DISCUSSION
In the current study we found that behavioral responses to light were clearly
inverted in diurnal grass rats compared to nocturnal mice. While 1 hr of exposure to a
300 lux photic stimulus increased general activity in diurnal grass rats, it decreased
activity of nocturnal mice. This result was not surprising, as it replicates our earlier
findings in the same species (Shuboni et al., 2012), and the suppression of activity by
light has been seen in many studies in nocturnal rodents (Butler & Silver, 2011; Morin et
al., 2010; Pendergast & Yamazaki, 2011; Redlin & Mrosovsky, 1999b). It should be
noted that the increase in activity observed in the grass rats was not a fear-induced
startle response, as this behavior is only produced with high levels of light ( >1000 lux,
data not shown). However, relationships between masking and photic induction of cFOS
in areas that are retino-recipient, or related to sleep/arousal, were more complicated.

cFOS expression in Retino-Recipent Regions
Retinal projections have been well described by others in both mice (Hattar et al.,
2006; Mikkelsen, 1992) and grass rats (Smale & Boverhof, 1999), but our tract tracing
results enabled us to confirm that retinal projections extend to the regions discussed
below and that these projections do not differ substantially in grass rats and CD1 mice
(Figure 2.1 A-B). When considering the cFOS responses of different retino-recipient

43

areas in grass rats and mice, four general patterns become apparent: (1) In one area,
the SCN, grass rats and mice responded to light in the same manner. This, result is
consistent with earlier data from both species and provides assurance that the
fundamental parameters of the experimental manipulation (e.g. light intensities) and
methods used to assess cFOS were sound. (2) In a second area, the VMH, there was
no response in either species, providing assurance that light-induced changes in cFOS
observed here did not reflect a simple widespread reaction to the stimulus, but can
provide insight into more focused functional responses to photic stimuli that induce
masking. (3) In three retinorecipient areas, the vSPZ, IGL and LHb, light induced an
increase in cFOS in grass rats but had no effect in mice. Thus, there is a relationship
between the increase of activity by light and photic induction of cFOS in these regions in
grass, whereas these responses are dissociable in mice, suggesting that masking of
behavior by light does not depend on induction of cFOS in these areas in mice, though
it might in grass rats. (4) Finally, in two brain regions, the DLG and OPN, the responses
seen in mice and grass rats were in opposite directions. The implications of these
species differences are discussed below, but first it should be noted that in some
regions the patterns of cFOS in our mice were different from those described in earlier
published reports; these are highlighted in Table 2.1. Many potentially important
experimental parameters vary among these studies, including light intensity, time of day
at which animals were exposed to light, the duration of the light pulse and, perhaps
most importantly, the strain of mouse used in these studies. To the author’s knowledge,
there are no reports of cFOS activation in CD1 mice after exposure to a nighttime light
pulse. We chose this strain because, unlike many others, melatonin is elevated at night

44

and directly suppressed by light (i.e. masked), as it is in other mammals. Neural
pathways associated with the masking response of the pineal gland to light may also
play a role in direct responses of some brain regions to photic stimuli. It is also possible
that a light-induced decrease in melatonin could cause a change in the nature of the
cFOS response to light in these regions (though the results described in Chapter 4
suggest that this is unlikely).

SCN
In the SCN, we found that both species showed a clear increase in cFOS after
the light pulse (Figure 2.3, 2.4 & 2.9). This pattern of activation has been demonstrated
before in both grass rats and mice pulsed with light during the dark phase of a 24 hr LD
cycle as in the current study (Grass rat: Katona, Rose, & Smale, 1998; Schwartz et al.,
2004; Mouse: Colwell & Foster, 1992; Masana, Benloucif, & Dubocovich, 1996), as well
as when they are kept in DD and exposed to a pulse of light during the subjective night
(Grass rats: Mahoney et al., 2001; Mouse: Geusz et al., 1997; Miller et al., 1999). The
focus of research on induction of cFOS in the SCN by light has been on its role in phase
shifting of the endogenous circadian oscillator, and there is good evidence that it does
play such a role in nocturnal rodents (Honrado et al., 1996; Wollnik et al., 1995). This is
likely to be the case in grass rats as well, as effects of light on the circadian system in
these animals are very similar to those seen in nocturnal species (Smale et al., 2003).
The possibility that light-induced cFOS in the SCN plays a role in masking has
not been examined in any species, though the question of whether the SCN itself is

45

Figure 2.9. Photomicrographs of the suprachiasmatic nucleus and ventral
subparaventricualr zone for all mouse subjects. On the left, all control mice have low
levels of cFOS in the SCN while on the right LP animals have high levels of cFOS
expression. An examination if the vSPZ, reveals that control animals vary greatly as
three animals have high levels of expression (MM07, MM08, & MM10) and two have
lower levels (MM06 & MM09).

46

important for masking has been addressed in numerous studies of SCN lesioned
nocturnal rodents. As noted above, one form of masking that is the same in nocturnal
and diurnal species is the suppression of melatonin secretion by light at night, and this
appears to depend on the SCN (Perreau-Lenz et al., 2003; Perreau-Lenz, Kalsbeek,
Pevet, & Buijs, 2004; Studholme et al., 2013). The role of the SCN in masking of
behavior has been directly tested in only two studies of nocturnal animals (hamsters),
and these produced conflicting results (Li et al., 2005; Redlin & Mrosovsky, 1999a). The
presence of behavioral rhythms in SCN-lesioned animals maintained in a 24 hr LD cycle
can also indicate the maintenance of masking, but here too, the results are mixed
(Table 2.2); they are also complicated by the fact that SCN lesions may cause varying
degrees of damage to the optic chiasm (Mistlberger, 1992). The role of the SCN in
masking thus remains unclear in nocturnal species, and there are no data on this issue
in diurnal species (however, see Table 2.2 and Chapter 3).

vSPZ
The vSPZ is one of the three retino-recipient regions in which light produced a
robust increase in cFOS expression in grass rats but had no effect in mice (Figure
2.10). The induction of cFOS in this region by light has been seen previously in grass
rats (Mahoney et al., 2001), as well as nocturnal rodents, including hamsters, rats, and
mice (Brooks et al., 2011; LeGates et al., 2012; Todd, Gall, Weiner, & Blumberg, 2012;
Y. Zhang et al., 1993). Thus, the lack of a response in our mice is somewhat surprising.
It is not likely to be due to technical difficulties, as in these same animals we were able
to see clear light-induced masking in their SCN-lesioned hamsters might be attributable

47

to the destruction of retinal input to that area. In rats, lesions of the vSPZ that leave the
SCN intact eliminate increases and decreases in cFOS in other retino-recipient brain
regions (Figure 2.9). As noted above, the differences among studies might be
attributable to the fact that our strain of mice was not the same as those used in the two
earlier studies (Brooks et al., 2011; LeGates et al., 2012). In any case, the current
results suggest that in these mice, the masking of activity by a photic stimulus can occur
in the absence of a cFOS response of vSPZ neurons to that stimulus.
The question of whether the presence of a cFOS response of cells in the vSPZ
reflects a role that this region may play in masking is more complex. It is an interesting
candidate region because it receives input from both the retina (Figure 2.1A-B; (Hattar
et al., 2006; Johnson, Morin, & Moore, 1988; Smale & Boverhof, 1999), and the SCN
(Kriegsfeld, Leak, Yackulic, LeSauter, & Silver, 2004; Morin, Goodless-Sanchez, Smale,
& Moore, 1994; Schwartz, Urbanski, Nunez, & Smale, 2011), and it projects to many of
the same areas as the SCN (Canteras, Ribeiro-Barbosa, Goto, Cipolla-Neto, &
Swanson, 2011; Schwartz et al., 2011; Watts, Swanson, & Sanchez-Watts, 1987). It is
thus well positioned to integrate direct photic signals from the retina with circadian
signals, and to modulate the same functions (e.g. activity) that are regulated by the
SCN. There is some evidence that the vSPZ may contribute to diurnality of the grass rat
circadian system, as it exhibits rhythms in cFOS that are very different in from those
seen in nocturnal lab rats (Nunez, Bult, McElhinny, & Smale, 1999) and that persist in
DD (Schwartz et al., 2004). Lesions in this area also lead to a reduction in the ratio of
activity during subjective day relative to night in these animals and altered

48

Table 2.2. Literature review of masking in12:12 LD condition for animals with suprachiasmatic nucleus lesions. (†)
indicates studies that observed extreme damage to the optic chiasm. (*) indicates studies that use lack of rhythms in
LD as a criteria for good SCN lesion.

49

Table 2.2. (cont’d)

50

reentrainment rates (Schwartz, Nunez, & Smale, 2009). The role of the vSPZ in
masking has not been directly tested, though Li et al. (2005) suggested that the loss of
endogenous rhythms in activity, suggesting that this region it is an essential output
pathway for the circadian regulation of activity. However, these lesioned animals do
exhibit rhythms in LD conditions (Abrahamson & Moore, 2006; Cipolla-Neto et al., 1995;
Lu et al., 2001; Moore & Danchenko, 2002), suggesting that the vSPZ is not required for
the masking response of activity to light in these animals. Finally in Chapter 3, I
describe evidence that direct masking to light pulses is maintained in grass rats with
ablated vSPZ, suggesting that although cells in this region respond in an acute manner
to light (Figure 2.3) they are not necessary for masking in grass rats.

IGL
The second of the three retino-recipient regions in which light produced an
increase in cFOS in grass rats but had no effect in mice is the IGL (Figure 2.10). This
result suggests that mechanisms associated with light responsiveness of the IGL of
grass rats are similar to those of another diurnal species, the Octodon degus, as, when
those animals are held in DD 1 hr of light at CT16 can stimulate an increase in cFOS in
the IGL (Krajnak et al., 1997). There are far more studies of this issue in nocturnal
rodents, and here the story are somewhat more complicated. Although there is good
evidence that light induces cFOS in the hamster IGL, the published data on rats and
mice are inconsistent (Table 2.2). Several studies have shown that light can induce
cFOS in the rat IGL but most of these have looked at least 2 hrs after exposure to the

51

Figure 2.10. Photomicrographs of the intergeniculate leaflet for all mouse subjects.
Both control (Left) and LP (Right) animals have high levels of cFOS expression.

52

light began (Aronin et al., 1990; Janik & Mrosovsky, 1992; Peters et al., 1996; Rusak et
al., 1990; Todd et al., 2012), and in one study in which rats were sacrificed just 30 and
90 minutes after lights were turned on there was no increase in cFOS (Park et al.,
1993). In their review of data on photic induction of cFOS, (Kornhauser, Mayo, &
Takahashi, 1996) suggest that in rats an increase in cFOS in the IGL can only be
detected two or more hours after the light stimulus begins. Interestingly, Juhl et al.
(2007) found that while 90 minutes of light at ZT14 or ZT19 did not induce a change in
overall cFOS in the rat IGL, it did elevate cFOS within the subset of cells that contain
enkephlin; no change was seen in neuropeptide Y-containing calls. One important
question is whether light induces cFOS in the same subpopulations of IGL cells in
diurnal grass rats as in nocturnal lab rats. Perhaps, for example, cFOS is induced in
projection neurons in lab rats and in inhibitory interneurons in grass rats. It should be
noted that the experiments described above were conducted on Sprague-Dawley and
Wistar rats, and that a more recent study using Fischer 344 rats did find a significant
rise in cFOS in the IGL after only 1 hr of exposure to light at midday and midnight
(Prichard et al., 2002). In the case of mice, there are two studies that show an increase
in cFOS in the IGL (Delogu et al., 2012; Lupi et al., 2012) and one in which the animals
did not respond to a light pulse (Lupi et al., 1999). Again, the strain of the mice might
account for the differing results, as C57/BL/6J and C3H/He mice responded differently
even when kept in the same laboratory and exposed to the same irradiance levels at the
same times of day (Lupi et al., 1999; Lupi et al., 2012). Taken together, the published
data on light-induced cFOS in the IGL of rats and mice suggest that the absence of

53

light-induced cFOS in our CD1 mice might reflect a strain difference, and/or that
induction of cFOS takes longer than 1 hr in these animals.
The patterns of responsiveness of grass rats and mice seen in the current study
raise the question of whether the IGL might contribute to differences in their behavioral
responses to that light (i.e. masking). Several kinds of data suggest that the IGL might
play a role in masking. As is the case for the SCN and vSPZ, the retinal input to the IGL
comes from ipRGCs in mice (Hattar et al., 2006), and these cells play an important role
in masking (Mrosovsky & Hattar, 2003). Lesion studies have also produced interesting
results relating the IGL to masking, in both nocturnal and diurnal animals. In nocturnal
rodents, masking is retained under LD conditions after the IGL is lesioned (Cipolla-Neto
et al., 1995; Edelstein & Amir, 1999; Redlin et al., 1999) and in hamsters the masking
response of wheel running to light is actually increased after the IGL has been
destroyed (Edelstein & Amir, 1999). The most striking effect of IGL lesions on masking,
however, is that seen in diurnal grass rats, as Gall et al. (2013) showed that destruction
of the IGL actually reversed the direction of the masking response, causing animals to
respond to light with a decrease, rather than an increase, in activity. Results from lesion
studies, as well as the cFOS response shown here, suggest that the IGL plays an
important role in the ability of grass rats to sustain a masking response typical of diurnal
animals.

LHb
The third retino-recipient brain region in which light increased cFOS in the grass
rat but had no effect in mice is the LHb. Only one other study that we are aware of has

54

directly assessed effects of light on cFOS in this region in nocturnal mice, and that study
found a large stimulatory effect (LeGates et al., 2012). On the other hand, in an LD
cycle, cFOS in this region is higher during the night than during the day in nocturnal
rodents, including mice, rats and hamsters (Tavakoli-Nezhad & Schwartz, 2006). It
should also be noted that cFOS patterns suggest that the habenula is activated in
response to activity (Engber et al., 1998; Paul, Indic, & Schwartz, 2011; TavakoliNezhad & Schwartz, 2006), and stress (Chastrette, Pfaff, & Gibbs, 1991; Lehner et al.,
2004). Perhaps differences in these parameters could account for the differences
between our results with mice and those described by LeGates et al. (2012).
We were particularly interested in the habenula because its direct access to light
information from the retina (Hattar et al., 2006) and its projections to arousal-inducing
areas of the brain (Aizawa, Kobayashi, Tanaka, Fukai, & Okamoto, 2012; Andres, von
During, & Veh, 1999; Herkenham & Nauta, 1979) putting it in a good position to mediate
masking. In vivo electrophysiological studies in rats have shown that neurons in the
habenula, particularly its lateral subregion, respond to acute presentation of light, and in
vitro recordings reveal a circadian rhythm in its firing rate (Zhao & Rusak, 2005).
Though it is not yet clear whether the LHb contributes to masking, or to species
differences in its manifestation, its responsiveness to light in grass rats suggest that it is
a region that should be explored further in efforts to better understand these issues.

DLG
The dorsal lateral geniculate (DLG) of the thalamus is one of the two brain
regions in which light induced an increase in cFOS in the grass rat and a decrease in

55

mice (Figure 2.3). Although a light-induced decrease in cFOS in the habenula has not
been reported before, it should be noted that (Prichard et al., 2002) saw no effect of a 2
hr pulse of light at midnight on cFOS in the DLG of rats. In contrast to these nocturnal
rodents, grass rats exhibited a robust increase in response to light, as has been
reported in two other diurnal species, treeshrews (Poveda & Kretz, 2009) and
Mongolian gerbils (Fite, Wu, & Bellemer, 2005). The DLG is an important part of the
primary visual system (Hendrickson, Wilson, & Ogren, 1978; Hoffmann, Stone, &
Sherman, 1972), but it also plays a role in the masking behavior of mice, as lesions in
this area enhance the inhibitory effect of light on activity in these animals (Edelstein &
Mrosovsky, 2001). The possibility that the DLG plays a role in masking in grass rats and
other diurnal species has not yet been examined, but the data here suggest the
possibility of interesting and important differences in the role that it might play in
nocturnal and diurnal species.

OPN
The second region in which light increased cFOS in the grass rat and decreased
it in mice was the OPN (Figure 2.3). In this case, the control mice appeared to have
very high levels of cFOS, and light led to a decrease of more than 50% (Figure 2.6).
This was surprising to us because an earlier study, by Prichard et al. (2002) had found
that a 2 hr pulse of light at midnight can induce a significant increase in cFOS within the
OPN of nocturnal lab rats. However, when light exposure occurred during the day it did
not have an effect. Differences in the time of sampling in these studies may, therefore,

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account for the apparent inconsistency, although it is possible that it reflect differences
between lab rats and mice.
The focus of research on the OPN has been on the role that it plays in the
papillary light reflex (Allen, Brown, & Lucas, 2011) but the same characteristics that
enable it to do that are ones that would enable it to contribute to masking. It receives
substantial input from melanopsin-containing cells in the retina (Hattar et al., 2006),
contains neurons that are capable of coding illumination levels (Szkudlarek, Orlowska,
& Lewandowski, 2012) and it projects to at least one region that appears to play a role
in masking, the IGL (Morin & Blanchard, 1995). Additionally, removal of the OPN
interferes with masking of REM sleep by darkness in albino rats (Miller et al., 1999).
However, the role of this region in masking of general activity by photic stimuli has not
been examined in either nocturnal or diurnal species.

cFOS expression in Arousal/Sleep-Related Regions
When considering the cFOS responses to light in the 4 arousal/sleep-related
areas examined here, only one general pattern becomes apparent: in all of them light
induced an increase in cFOS in grass rats but had no effect in mice. The implications of
these species differences are discussed below, but first it should be noted that in this
case there are few published data with which to compare our results.

VLPO
In the VLPO, a region of the hypothalamus known to promote sleep in some
species (Lu et al., 2001; Sherin, Shiromani, McCarley, & Saper, 1996), we found that

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the same light that stimulated activity actually increased cFOS in grass rats (Figure 2.7).
The reasons for this paradoxical response are not clear, but one possibility involves the
internal circuitry of the VLPO, where the subset of cells that actually stimulate sleep in
nocturnal rodents are known to contain galanin, and cFOS is elevated in them during
sleep (Gaus, Strecker, Tate, Parker, & Saper, 2002). Thus, it may be that in grass rats,
but not mice, light activates inhibitory interneurons that suppress galanin-containing
cells in the VLPO, leading to a reduction in sleep.
In mice, we found that while light decreased activity it did not affect cFOS in the
VLPO. Previous experiments examining light-induced cFOS in this area in mice have
produced results that are somewhat contradictory. Lupi et al. (2008), using RT-PCR,
saw a significant light-induced increase in fos mRNA when mice were pulsed for 1 hr
with light. However, Tsai et al. (2009) saw no effect of light on overall levels of cFOS in
this region but they did when they focused specifically on the sleep-promoting galaninpositive cells there. It should be noted that the VLPO is innervated by fibers originating
in the retina in grass rats and mice (Figure 2.1), though there is some evidence that the
density of that retinal input is higher in nocturnal lab rats than grass rats (Nunez,
unpublished results); this does not appear to be the case in mice. Many of these fibers
are known in mice to come from melanopsin-containing retinal ganglion cells (Gooley et
al., 2003). Several investigators have suggested that this pathway may play a role in
masking (Lupi, Oster, Thompson, and Foster, 2008; Redlin, 2001) but no on has directly
examined the region’s role in the masking response.

LH, DR, LC

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The LH, DR and LC are of interest in understanding the neural mechanisms of
masking as each plays an important role in induction and maintenance of arousal
(Mistlberger, Antle, Kilduff, & Jones, 2003; Sara & Bouret, 2012; Tsujino & Sakurai,
2013). In all three of these brain regions, grass rats were responsive to light, while mice
were not. The absence of an effect of light on cFOS in arousal-inducing structures of
mice has been seen by others (Mendoza, Clesse, Pevet, & Challet, 2010), and is likely
not a simple reflection of the rate of decay in the protein. Light-induced cFOS has been
seen previously in the LH and DR of grass rats (Adidharma, Leach, & Yan, 2012), as
well as the DR of another diurnal murid rodent, the Mongolian gerbil (Fite et al., 2005).
Interestingly, the retina projects directly to the LH of grass rats (Figure 2.1), and to the
DR of Mongolian gerbils (Fite, Birkett, Smith, Janusonis, & McLaughlin, 2003). It is
tempting to speculate that these pathways play a role in the induction of cFOS and
masking in these diurnal species.

Summary and Conclusions
The behavioral response of nocturnal and diurnal rodents to light exposure are
polar opposites, with nocturnal species decreasing activity and diurnal species
increasing activity after a light pulse. Most of the brain regions we examined showed a
very different response to light in mice and rats. In some cases, the response of the
mice was very different than those observed in other nocturnal rodents. Above we
explored several possible explanations for the discrepancies, including age, duration of
exposure, other FOS-inducing stimuli, and most importantly strain differences. Although
those regions varied, we did observe a clear and similar uni-direction response in the

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SCN in grass rats and mice, indicating that we replicated a well-established pattern in
our animals. Therefore, we believe that these findings demonstrate a interesting
differences between nocturnal mice and diurnal grass rats in the responsiveness of
other regions to light that will provide the framework for targeting regions in future
attempts to better understand the masking response to light and how the system varies
between species that occupy different temporal niches.

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CHAPTER 3
Ventral subparaventricular zone lesions and light-induced masking behavior in
the diurnal rodent, Arvicanthis niloticus
ABSTRACT
The ventral subparaventricular zone (vSPZ) is a region of the brain that plays a major
role in regulation of circadian rhythms in sleep and activity. Animals most active during
the day (diurnal animals), display different patterns of cellular activation and firing rates
in the vSPZ when compared to animals active during the night, (nocturnal animals).
Masking behavior, an acute behavioral response to an external stimulus, also differs
between diurnal and nocturnal animals, with light inhibiting activity in nocturnal animals
and stimulating it in diurnal ones. Little is known about the neural structures that
mediate masking, but the vSPZ is a promising candidate as it receives direct retinal
input and it responds to light with a rapid increase in cFOS. Here we tested the effects
of chemical ablation of the vSPZ on the ability of animals to exhibit acute responses to
the presentation of light and to maintain circadian rhythmicity under different lighting
conditions. The vSPZ was successfully abolished in 4 animals; in 2 of these, the SCN
was also virtually destroyed. Two of the 4 animals retained the ability to respond to light
pulses with an immediate increase in activity; 3 of these 4 animals with no vSPZ
showed masking in the context of a 3.5:3.5 ultradian light:dark (LD) cycle. In a 12:12 LD
cycle, the same three animals had clear daily rhythms, but when placed into constant
darkness and then constant light, only one of these animals displayed a significant
circadian rhythm. These data establish that the vSPZ is not necessary for the masking
response to light and suggest that the grass rat vSPZ may contribute to circadian

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regulation. Additionally, in the grass rat the NMA is capable of destroying the SCN, a
structure generally believed to be resistant to this chemotoxin.

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INTRODUCTION
Light is a powerful daily signal, indicating the progression of time across the 24 hr
day. In mammals, light influences the daily pattern of activity through two important
mechanisms, entrainment of circadian rhythms and masking, that function in a
synergistic manner to promote the appropriate behavior at the correct time-of-day
(Mrosovsky & Thompson, 2008). The immediate change in behavior, such as general
activity, after the acute presentation of light is called the masking response (Aschoff,
1960). The direction of this response is dependent on the temporal niche an animal
occupies. Among diurnal species light triggers an increase in activity while for nocturnal
species the opposite is observed (Mrosovsky, 1999). This difference has long been
established (Aschoff & Vongoetz, 1989; Borbely & Huston, 1974), however, until
recently the importance of masking in the evolution of diurnality has received little
attention (Chiesa, Aguzzi, Garcia, Sarda, & de la Iglesia, 2010; Cohen et al., 2010;
Mrosovsky & Hattar, 2005; Rotics et al., 2011). Understanding where and how the brain
responds to a masking pulse of light would provide greater insight into the changes that
must occur for an organism to transition from nocturnal to diurnal pattern of behavior.
However, almost nothing is known about the neural mechanisms of masking in diurnal
species.
In nocturnal species, investigations into the brain structures important for the
masking response to light have established two facts. First, masking is dependent on
the signal generated by cells within the retina that contain melanopsin (ipRGCs) after
they are exposed to light (Hatori et al., 2008; Mrosovsky & Hattar, 2003; Panda et al.,
2003). This suggests that areas innervated by ipRGCs could play an important role in

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the regulation of masking. ipRGC projections extend to many regions that receive retinal
input outside of the primary visual pathway, including the suprachiasmatic nucleus
(SCN), olivary pretectal nucleus (OPN), intergeniculate leaflet (IGL), and the ventral
subparaventricular zone (vSPZ; Hattar et al., 2006). Second, lesions of no one region
have thus far clearly eliminated masking (Redlin, 2001). The destruction of the IGL
enhances the inhibitory effects of light on activity in hamsters (Redlin & Mrosovsky,
1999b), while removal of the OPN attenuates the masking of REM by darkness in albino
rats (Miller et al., 1999). Lesions of the SCN have produced mixed results, with both
positive (Li et al., 2005) and negative (Redlin & Mrosovsky, 1999a) findings. One
explanation posed by Li et al. (2005) for the discrepancy, is that extensive damage to
the retinal fibers innervating the vSPZ caused the loss of the masking response in their
animals. The vSPZ could, therefore, play an important role in masking.
The vSPZ is a region of the brain found dorsal to the SCN that receives major
input from the SCN, and that projects to many of the same areas as the SCN, in both
nocturnal (Kriegsfeld et al., 2004; Morin et al., 1994) and diurnal (Schwartz et al., 2011)
species. The vSPZ is of interest in the understanding of diurnality for a variety of
reasons. Diurnal animal have different daily patterns of cellular activation (Nunez et al.,
1999; Schwartz et al., 2004) and firing rates (Sato & Kawamura, 1984) in the vSPZ
when compared to nocturnal animals. The area is known for playing a major role in
regulation of circadian rhythms of sleep and activity. Chemotoxic and electrolytic lesions
of the vSPZ abolish circadian activity rhythms of nocturnal rodents when they are kept
in constant darkness (Abrahamson & Moore, 2006; Cipolla-Neto et al., 1995; Lu et al.,
2001; Moore & Danchenko, 2002). Partial lesions of the vSPZ of grass rats do not have

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the same dramatic effect, but there is a correlation between the size of the lesion and
rhythm strength (Schwartz et al., 2009). Beyond the region’s role in circadian rhythms,
the vSPZ may also impact the expression of daily activity by influencing masking. As
mentioned above, the vSPZ receives input from melanopsin cells in mice, and data
described in Chapter 2 revealed that the region has a niche dependent cFOS response
to light exposure. Additionally, grass rats with partial lesions in the area have bouts of
arrhythmia in a 12:12 light:dark (LD) cycle (Schwartz et al., 2009), which suggests a
diminished masking response to photic stimuli. Taken together, the data suggest that
the vSPZ is an ideal target area for the investigation of the neural mechanisms of
masking.
Here we used the diurnal rodent, Arvicanthis niloticus (Blanchong et al., 1999), to
test the hypothesis that the vSPZ is a part of the neural circuitry that mediates masking
and promotes the differences in the masking response associated with an animal’s
temporal niche. We did this by determining whether lesions of the region affect the
masking of general activity by acute presentations of light and in the context of an
ultradian LD cycle. We also asked how circadian activity rhythms of vSPZ-lesioned
animals are affected by different light conditions.

METHODS
Animals
Thirty-five adult female Arvicanthis niloticus (grass rats) were obtained from the
breeding colony at Michigan State University. All animals were singly housed in
Plexiglas cages (34x28x17cm) and given food (PMI Nutrition Prolab RMH 2000,

65

Brentwood, MO) and water ad libitum for the duration of the study. The facilities were
maintained at a temperature ranging between 20-25°C with a 12:12 LD schedule. All
experimental procedures were approved by the Institutional Animal Care and Use
Committee (IACUC) of Michigan State University and were in accordance with the
guidelines of the National Institutes of Health on the care and use of animals.

Experimental Procedures
At the onset of the experiment, animals were transferred into a room with uniform
fluorescent lighting, which exposed all animals to 300 lux. Each animal was housed in
an individual cage equipped with infrared detectors (IRs, Visonic Tel Aviv, Israel)
attached to the cage top. Activity was recorded in 5-minute bins using the VitalView 4.0
Software (Minimiter, Bend, OR) on a computer located in an adjacent room. Animals
were monitored for at least 10 days in 12:12 LD conditions prior to the surgery. Animals
then were all assigned to either a vSPZ lesioned group (SPZx) or a sham control group.
After the surgeries (described below) animals were allowed to recover for a minimum of
two weeks.
Animals were first tested for their masking response to light using a procedure
outlined in Shuboni et al. (2012). In brief, animals were exposed to 1 hr light pulses
(LPs) at three different circadian times (CTs, CT0 = activity onset) during the subjective
night (CT14, CT18, CT22) and once during the subjective day (CT6). This was
accomplished by releasing animals into constant darkness for two days, once every four
days; animals were exposed 1 h pulses of light on the second of these two days; the
order of the pulses was randomly selected. Additionally, animals were exposed to a 7 h

66

ultradian light:dark cycle (3.5:3.5 LD) for nine days, during which time they exhibited
free-running circadian rhythms; analysis of the final seven days in these lighting
conditions reveals effects of masking independent of the circadian influence on activity.
Finally, some features of the circadian regulation of activity were examined by placing
animals into 12:12 LD for two weeks, constant darkness (DD) for two weeks, and
constant light (LL) for three weeks. At the completion of the study, animals were
sacrificed and brains collected for histological assessment of the lesions.

Surgical Procedures
Thirty animals underwent the operation to bilaterally lesion the vSPZs with NMA
and five experienced the same procedure but were only injected with the control vehicle
(SHAM). In brief, grass rats were first anesthetized using isoflorane (2.5-5%, Abbott
Laboratories, IL, USA) and then injected s.c. with Ketoprofen (5 mg/kg of body weight).
Prior to the surgery, the surface of the head was shaved and sterilized with betadine.
Animals were then secured in a steretaxic apparatus (Stoelting Co.; Wood Dale, IL) with
the tooth bar set to -4.5 mm and then injected s.c. on the top of the head with lidocaine
(0.03 mL). A 2 cm incision was made on the scalp to expose the surface of the skull
and, using a surgical drill, two small 2 mm holes were made at the appropriate
coordinates. Injection coordinates were +0.16 mm anterior and ±0.05 mm lateral to
bregma and 6 mm ventral to the dura. Injections were made using a 0.5 µL Hamilton
syringe (Hamilton, Reno, Nevada, USA) filled with either 0.3M NMA (Sigma, St. Louis,
Missouri, USA) in 0.2M saline, or the vehicle control. All animals were injected with 180
nL of solution per side using a manual stereotaxic microinjector (Stoelting), injections

67

were given over 5 a minute period (36 nL/min) and the needle was left for 1 minute
before being withdrawn. At the conclusion of the surgery, the incision was closed with
autoclips and animals were injected with sterile saline (2 cc, 0.9% NaCl, s.c.). For the
next 2 days, all animals received the analgesic Melaxicam (0.1 mg/kg of body weight)
orally in apple every 24hrs.

Assessment of the Lesions
At the completion of the experimental procedures, all animals were transcardially
perfused at ZT2 as described in Schwartz et al. (2009). In brief, animals were deeply
anesthetized using an i.p. injection of sodium penabarital (400mg/kg). The animals were
then transcardially perfused with 0.1M phosphate buffer saline (PBS, pH 7.2) and then a
4% paraformaldehyde solution in PBS, also containing 0.075M lysine and 0.01M
sodium periodate (PLP, Sigma-Aldrich, St. Louis, MO, USA). The brains were post-fixed
for an additional 4 hr in the PLP solution; they were then transferred into sucrose for a
minimum of 24 hr, after which the brains were cut at 30 µm along a coronal plane using
a freezing-microtome. These sections were divided into three series and stored in
cyroprotectant at -20° until further processing. One series was mounted onto gelatinC
coated slides, stained for Nissl using thionin, and coverslipped with Permount (Fisher
Scientific, Fair Lawn, NJ, USA). To delineate the extent of the lesions, two
experimenters independently evaluated the tissue. They examined 5 sections using a
light microscope (Leitz, Wetzlar, Germany) equipped with a drawing tube at 5x
magnification. Both experimenters drew the lesioned area for one section rostral to the
SCN, 3 sections that included the SCN (rostral, middle, and caudal representatives),

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and another section caudal to the SCN. The drawings were then compared between the
experimenters and each lesion was classified as bilateral hit, unilateral hit, or miss.

Data Analysis
To analyze masking, we converted the VitalView data into Microsoft excel
spreadsheets. The degree of masking during the light pulses was examined by
comparing the total activity of the animal during the hour of the pulse to the same hour
on the previous day. A three-way mixed ANOVA was used to assess within-subjects
effects of lighting condition (control and LP) and time (CT6, CT14, CT18, CT22), and
the between-subjects effects of surgical condition (sham, bilateral hit, unilateral hit, and
miss). Masking behavior in the context of the ultradian LD cycle was examined by
comparing the total amount of activity during all the light periods to the sum of activity
during all the dark periods. A two-way mixed ANOVA was used to assess withinsubjects effects of lighting condition (light and dark) and the between-subjects effects of
surgical condition (sham, bilateral hit, unilateral hit, and miss). Additionally, Chi-squared
periodograms for all the animals were constructed for the last seven days of the
ultradian LD cycle using ClockLab (Actimetrics, Wilmette, IL, USA). This analysis
distinguishes significant rhythms from random fluctuations in the data (Sokolove &
Bushell, 1978).
To analyze the circadian rhythms data, we converted our Vitalview files into
actograms using ClockLab (Actimetrics, Wilmette, IL, USA). We also analyzed the last
week in LD, DD, and LL using Chi-squared periodograms to identify circadian rhythms
of the animals. Then determined onset and offset of activity and the period of the

69

rhythm, tau (τ) with the program, ClockLab identifies these points independently,
however, the experimenter also visually inspected these points prior to analysis, to
identify and manually corrected any errors (Schrader, Walaszczyk, & Smale, 2009).
From the onsets and offsets we also calculated the duration of the active period, alpha
(α), and inactive period (ρ), and an alpha/rho ratio was determined from those values. A
two-way mixed ANOVA was used to assess effects of lighting condition (LL and DD,
within subjects) and surgical condition (sham, bilateral hit, unilateral hit, and miss,
between-subjects) on τ and the alpha/rho ratio. All analyses were further examined if
significant interaction or main effects were present using one-way ANOVAs and t-test.
Significant differences were ascribed when p < 0.05.

RESULTS
Histology
Though needle tracts were observed in the brains of sham animals (Figure 3.1;
n=5), their SCN and vSPZ were intact (Figure 3.1). In 5 animals the toxin failed to
damage the SCN or vSPZ on either side of the brain (“miss”, Figure 3.2), and in 3
animals, damage to these structures was unilateral (“unilateral hit”). In these animals,
the lesioned areas showed a dramatic reduction in the number of neurons (Figure 3.2).
Finally, we had four successful bilateral lesions that destroyed the entire vSPZ (Figure
3.3). In all of these animals (referred to as “lesion”) the retrochiasmatic area was also
completely destroyed, one also sustained unilateral damage to the supraoptic nucleus
and in another the caudal PVN was partially destroyed. Finally, two animals in the

70

bilateral hit group had extensive damage to the SCN with only small fragments of the
nuclei remaining.

Masking
The mixed three-way ANOVA used to assess effects of masking pulses of light
on activity violated the assumption of sphericity. We therefore analyzed each of the 4
groups individually (i.e. sham, miss, unilaterally hit, and bilateral hit), using withinsubjects two-way ANOVAs. Unilaterally lesioned and sham groups continued to violate
assumptions of sphericity. In the miss group there were no main effects of either lighting
(F[1, 5] = 2.747, p = 0.158) or time (F[3, 15] = 1.249, p = 0.327), but there was an
interaction (F[3, 15] = 8.644, p < 0.014). Among animals in the bilateral hit group there
was a near-significant main effect of lighting condition, with activity levels tending to be
higher during the light (F[1, 3] = 9.282, p < 0.056). To better understand the presence of
masking in our animals, we looked at each individual’s masking response at all timepoints. Miss animals all had higher levels of activity when pulsed with light (Figure 3.4).
However, this was not the case in unilateral hit (Figure 3.4) or sham animals (Figure
3.5), as one animal in each group showed no response to the light. Examination of
activity of bilaterally lesioned animas also reveals a great deal of variation (Figure 3.6).
Although all animals appeared to respond to light overall, this was not the case for each
animal at one or more of the time points examined; the times at which activity was not
higher during the light pulse than the control pulse varied from animal to animal.
However, 12 of the 16 total cases had increased in activity during the light periods and
only for 3 cases was there no change or a decrease in activity.

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Figure 3.1. Photomicrographs of Nissl-stained tissue in all SHAM lesioned animals. Sections were selected to
represent one section before the vSPZ, three spanning the rostral-caudal extent of the regions and one section after.
No damage is observed in the tissue with the exception of needle marks.

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Figure 3.1. (cont’d)

73

Figure 3.2. Photomicrographs of Nissl-stained tissue in 2 representative Miss animals and all unilaterally lesioned
animals. Sections were selected to represent one section before the vSPZ, three spanning the rostral-caudal extent of
the regions and one section after. Miss animals (MASPZ-13 & MASPZ-23) had no damage observed in the vSPZ or
SCN, while unilaterally lesioned animals (MASPZ-05, MASPZ-08, & MASPZ-22) had one side of the vSPZ completely
ablated. Areas damaged by the toxin are outlined in red.

74

Figure 3.2. (cont’d)

75

Figure 3.3. Photomicrographs of Nissl-stained tissue in all bilaterally lesioned animals. Sections were selected to
represent one section before the vSPZ, three spanning the rostral-caudal extent of the regions and one section after.
All lesioned animals had extensive damage observed in the vSPZ, 2 of these animals also had damage within the
SCN (MASPZ-03 & MASPZ-04). Areas damaged by the toxin are outlined in red.

76

Figure 3.3. (cont’d)

77

The analysis of the variation between groups for the ultradian cycle also revealed that it
violated the assumption of sphericity. Only in the miss group was there a significant
difference in activity between the lights on and lights off conditions (t(6)=3.067,
p=0.028). Within this group all individuals exhibited higher activity levels during the light
period and displayed 24 hr rhythms, and in one individual a 7 hr rhythm was evident in
the chi-squared periodogram (Figure 3.7). Among unilateral (Figure 3.7) and sham
groups (Figure 3.8) there was no difference in activity between dark and light periods. In
the sham animals, 3 grass rats had little or no difference between activity in the light
and dark periods (MASPZ-07, MASPZ-15, and MASPZ-18; Figure 3.8) while the other
two were more active during the light (MASPZ-14 and MASPZ-21). Among bilaterally
lesioned animals 3 of 4 individuals were more active during the light than dark periods
(Figure 3.9). Only one grass rat in this group showed a significant 7 hr rhythm; this
animal also showed the greatest masking response to the 1 hr light pulses (MASPZ-03).
The other 3 animals with bilateral lesions had no 7 hr cycling, two had 24 hr rhythms
and one was arrhythmic. Among animals in this group that had a significant 24 hr
rhythm, activity was higher in the light than dark (MASPZ-03, MASPZ-10 and MASPZ19), whereas among the arrhythmic animal the activity was higher during the dark than
the light (MASPZ-04; Figure 3.9).

Circadian Rhythms
Analysis of the variance in τ in constant darkness and in constant light again
showed that it violated the assumption of sphericity. Paired-sample t-tests of the

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Figure 3.4. Patterns of behavioral response of activity to light exposure in Miss and
Unilateral animals. On the x-axis, 4 different time points, ZT6, ZT14, ZT18 and ZT22,
were examined. The black bars represent acivity during 1 hr of a control day while grey
bars are the same hour during the light exposure. All animals respond to light at most
time points (16/20), however, two animals had a time-point where there was no
response (MASPZ-13 & MASPZ-22) and one animal had two time points were activity
was decreased (MASPZ-08).

79

Figure 3.4. (cont’d)

80

Figure 3.5. Patterns of behavioral response of activity to light exposure in SHAM
animals. On the x-axis, 4 different time points, ZT6, ZT14, ZT18 and ZT22, were
examined. The black bars represent acivity during 1 hr of a control day while grey
bars are the same hour during the light exposure. Two animals had strong
positive masking behavior, with an increase for all time points (MASPZ-14 &
MASPZ-21). One animal did not display a constant masking response, with both
increases and decreases in activity (MASPZ-07). The two final animals had very
low activity level readings during this portion of the experiment making it hard to
interpret their masking behavior (MASPZ-15 & MASPZ-18).

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Figure 3.5 (cont’d)

82

Figure 3.6. Patterns of behavioral response of activity to light exposure in Miss and
Unilateral animals. On the x-axis, 4 different time points, ZT6, ZT14, ZT18 and ZT22,
were examined. The black bars represent acivity during 1 hr of a control day while grey
bars are the same hour during the light exposure. All animals respond to light at most
time points (12/16), however, two animals had a time point where there was no
response (MASPZ-03 & MASPZ-10) and one animal had two time points were activity
was decreased (MASPZ-19).

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Figure 3.6 (cont’d)

84

remaining groups showed a significantly longer τ in LL when compared to DD for the
miss group (t(4)=4.453, p=0.011) and a trend toward significance for the sham group
(t(4)=2.482, p=0.068). The Chi-squared analysis showed the presence of circadian
rhythms in all individuals in sham (Figure 3.10), unilateral lesion, and miss groups
(Figure 3.11) but not in all bilaterally lesioned animals (Figure 3.12). Three of the 4
animals in this group had rhythms in LD conditions, however, this decreased to 2 in DD
and 1 in LL. Therefore, there was greater variance in the bilateral lesion group, which
led to the violation of the assumption of sphericity. This was also the case for the
analysis of the alpha/rho ratio in this group. Separate one-way ANOVAs of the data
from the miss (F[2, 16] = 63.382, p < 0.001) and sham (F[2, 8] = 9.056, p = 0.012)
animals both showed a significantly higher alpha/rho ratio in LL compared to DD and
LD.

DISCUSSION
In this experiment we lesioned the vSPZ, a structure found deep the
hypothalamus, in diurnal grass rats, and assessed masking behavior as well as
circadian rhythms in these animals. Previous attempts to ablate the vSPZ in grass rats
yielded no complete lesions but demonstrated a relationship between lesion size and
rhythm dysfunction. We were able to successfully destroy the vSPZ but these lesions
had no effect on the masking response to light.

Masking

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Figure 3.7. 3.5:3.5 LD Ultradian masking protocol in Miss and Unilateral animals. The far left panel shows the 28hr
actograms of animals during the ultradian cycle, grey bars indicate the dark phases of the cycle. The middle panel
shows the Chi-Squared periodogram of animals during the last 7 days of the protocol. All animals have a significant 24
Squared
hr rhythm and only one animal has a significant 7 hr cycle (MASPZ 13). On the far right panel, total activity during the
(MASPZ-13).
light and dark period were summed and turned into a percentage of activity. All animals have the expected higher
e
percentage of activity during the light phases with the exception of MASPZ 08, who had more activity during the dark
MASPZ-08,
phases.

86

Figure 3.7. (cont’d)

87

Figure 3.7. (cont’d)

88

Figure 3.8. 3.5:3.5 LD Ultradian masking protocol in SHAM animals. The far left panel shows the 28hr actograms of
animals during the ultradian cycle, grey bars indicate the dark phases of the cycle. The middle panel shows the ChiSquared periodogram of animals during the last 7 days of the protocol. All animals have a significant 24 hr rhythm and
only one animal has a significant 7 hr cycle (MASPZ-14). On the far right panel, total activity during the light and dark
period were summed and turned into a percentage of activity. Only 2 animals (MASPZ-14 & MASPZ-21) have the
expected higher percentage of activity during the light phases, the other 3 animals (MASPZ-7, MASPZ-15 & MASPZ-18)
did not respond with equal amounts of activity during the light and dark phases.

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Figure 3.8. (cont’d)

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Figure 3.8. (cont’d)

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Figure 3.9. 3.5:3.5 LD Ultradian masking protocol in SHAM animals. The far left panel shows the 28hr actograms of
animals during the ultradian cycle, grey bars indicate the dark phases of the cycle. The middle panel shows the ChiSquared periodogram of animals during the last 7 days of the protocol. Three animals have a significant 24 hr rhythm and
one animal is arrhythmic (MASPZ-04). Only one animal had a significant 7 hr cycle (MASPZ-03). On the far right panel,
total activity during the light and dark period were summed and turned into a percentage of activity. Three animals
(MASPZ-03, MASPZ-10 & MASPZ-19) had the expected higher percentage of activity during the light phases, the other
animals (MASPZ-04) did not respond and had higher activity during the dark phases.

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Figure 3.9. (cont’d)

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Results from both approaches that we took for the assessment of masking
revealed that some animals in which the vSPV was completely destroyed were able to
exhibit masking. First, in animals with bilateral lesions of the vSPZ there was a near
significant increase in activity in response to a 1 hr pulse of light (p = 0.056). When we
consider the 4 light pulses in all of the 4 animals with complete vSPZ lesions, we find
that in only 1 of 16 cases was activity higher during the control period than during the
light pulse, whereas in 13 of the 16 cases the reverse was true; on two occasions there
was no difference. Secondly, a significant 7 hr rhythm in the ultradian LD cycle was
observed in one individual (MASPZ-03; Figure 3.9) that sustained complete bilateral
damage of the vSPZ; in this animal more activity occurred during the light than dark
phase of the cycle. Furthermore, 3 of these 4 animals showed more activity in the light
than dark phase of the ultradian cycle. Together, these data have established that the
vSPZ is not necessary for masking.
There are no published reports of effects of vSPZ lesions on masking. However,
it is impossible to destroy the SCN without damaging some of the vSPZ, and there are
several studies that have examined effects of SCN lesions on masking. In one report of
a study in which SCN lesions impaired masking in hamsters, in the context of a 7 h
ultradian LD cycle, the authors noted that the effect could have been due to destruction
of retinal fibers projecting to the vSPZ (Li et al., 2005). Additionally, in circadian rhythms
studies in which the vSPZ was eliminated in rats (Abrahamson & Moore, 2006; CipollaNeto et al., 1995; Lu et al., 2001; Moore & Danchenko, 2002) animals continued to have
nighttime activity in LD conditions after ablation of the region. Demonstrating that for
nocturnal animals, which have a disrupted expression of activity rhythms in constant

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Figure 3.10. Circadian rhythms analysis in varying lighting conditions in SHAM. On the Far left, actograms of all
animals across three different lighting conditions, 12:12 LD, constant light, and constant darkness. Chi-squared
analysis was conducted to detect the presence of significant rhythms were run for all lighting conditions. All animals
had rhythms at the three lighting conditions except MASPZ-15 in LL.

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Figure 3.10. (cont’d)

96

Figure 3.10. (cont’d)

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Figure 3.11. Circadian rhythms analysis in varying lighting conditions in Miss and Unilateral groups. On the Far left,
actograms of all animals across three different lighting conditions, 12:12 LD, constant light, and constant darkness. Chisquared analysis was conducted to detect the presence of significant rhythms were run for all lighting conditions. All
animals had significant rhythms at the three lighting conditions.

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Figure 3.11. (cont’d)

99

Figure 3.11. (cont’d)

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Figure 3.12. Circadian rhythms analysis in varying lighting conditions in Bilateral lesion animals. On the Far left,
actograms of all animals across three different lighting conditions, 12:12 LD, constant light, and constant darkness. Chisquared analysis was conducted to detect the presence of significant rhythms were run for all lighting conditions. One
animal had no rhythms at any light conditions (MASPZ-04). In Constant darkness MASPZ-10 became arrhythmic and in
constant light MASPZ-03 was no longer rhythmic.

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Figure 3.12. (cont’d)

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conditions after vSPZ lesion, are still able to mask to a LD cycle. Here too we observed
that a majority (3 of 4 animals) of our completely lesioned animals displayed diurnal
activity patterns in LD conditions with the exception of the one animal with the greatest
SCN damage (MASPZ-04). Altogether, our current data supports the idea that the vSPZ
is not crucial for the masking response of grass rats.

Circadian Rhythms
Complete ablation of the vSPZ in our animals greatly reduced the expression of
the rest-activity cycles in constant conditions. Previous attempts to lesion this area in
the grass rat did not succeed in destroying the entire vSPZ, however, the size of the
partial lesions was negatively correlated with both the strength of the rhythms and
reentrainment rate. In nocturnal animals, the destruction of the vSPZ eliminates
circadian rhythms in activity (Abrahamson & Moore, 2006; Lu et al., 2001) and sleep (Lu
et al., 2001) but does not impact temperature rhythms. Here we observed the same
diminished expression of circadian rhythms in activity for the grass rats. Most animals
(3/4) with complete bilateral lesions of the vSPZ no longer had significant circadian
rhythms as detected by chi-squared analysis in constant light. However, one of these
arrhythmic animals (MASPZ-04) had the most damaged SCN of all animals in the study.
We, therefore, cannot draw conclusions about the effects of vSPZ lesions on circadian
rhythms in the grass rat because of our small sample size. More investigation into the
role of the vSPZ in the circadian regulation of activity is needed to determine if it plays a
similar role in circadian regulation in nocturnal and diurnal animals.

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SCN Damage
Two of our 4 animals in which the vSPZ was completely lesioned also had
extensive damage to the SCN (Figure 3.3). It thus appears that in the grass rat NMA is
an effective agent at high doses for inducing the destruction of the SCN. An earlier
study by Schwartz et al. (2009) found that the SCN of grass rats appeared to be intact
while the vSPZ was damaged by this chemotoxin, but in the current study we used a
substantially higher concentration and volume of NMA. Previous work in nocturnal
rodents has suggested that the SCN is resistant to direct injections of chemotoxins,
such as NMDA (Hastings, Roberts, & Herbert, 1985) and kainic acid (Schwarcz &
Kohler, 1983).The concentration of toxin used by Hastings et al. (1985) was half that
used in our experiment, but the volume was almost three times higher; the total amount
administered in that study was therefore slightly higher than in this one. The current
finding that the SCN of grass rats is vulnerable to chemical lesions may thus reflect the
high concentration of the toxin that we used, but it also raises the possibility of a very
interesting species difference in SCN function.

Summary and Conclusions
The data described here must be described as preliminary; however they do lead
to two clear conclusions: (1) The vSPZ, which had been a prime candidate for the
mediation of masking, is not necessary for it in grass rats, and (2) the SCN of these
animals is susceptible to chemotoxic effects of NMDA, challenging prevailing views
about the resistance of the SCN to such agents.

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The hypothesis that the vSPZ plays a role in mediation of differences in masking
responses of diurnal and nocturnal species had emerged for several reasons. The
region has input from ipRGCs (Hattar et al., 2006), and cells in it undergo an increase in
cFOS after light exposure in grass rats, which is not the case in nocturnal mice (Chapter
2). Furthermore, although there are no other experiments that have directly examined
the role of the vSPZ in masking, lesions that have damaged both the SCN and the
vSPZ have abolished masking in hamsters (Li et al., 2005). Data from the current
study, suggest that this is not the case in diurnal grass rats. Although the number of
animals with complete lesions was small (n=4), we saw a difference between activity
levels in control and LP conditions in these animals, and one whose vSPZ was
completely destroyed showed clear evidence of masking in a 7 hr LD cycle. It should
also be noted that investigations into the neural mechanisms of masking in nocturnal
species has suggested that masking is mediated by several redundant retino-recipient
regions (Redlin, 2001). These may operate within a neural network that balances
positive and negative influences of light on activity in ways that lead to species patterns
of masking. Further tests using gradients of light intensity (Redlin et al., 1999) should
therefore be used to determine if the vSPZ plays a supportive in the masking response
under other conditions. Although the neural mechanisms of masking remain elusive, the
current data have established that the vSPZ of diurnal grass rats is not crucial for its
maintenance.

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CHAPTER 4
The pineal gland does not influence the daily patterning of behavior of diurnal
Arvicanthis niloticus
ABSTRACT
Melatonin is a hormone rhythmically secreted by the pineal gland, with peak levels
occurring during the night. The hormone in rats and hamsters improves the
reentrainment rate of the circadian activity rhythms and alters the masking response to
a reversed light:dark (LD) cycle. In diurnal animals, melatonin is present during the
inactive phase of the rest/active cycle and in primates, it directly influences sleep and
body temperature. Pinealectomized humans also have a higher incidence of sleep
disruption after surgery. Few studies have used a non-primate, diurnal mammalian
species to examine the role of the pineal in promotion of the patterning of activity and
sleep at night, masking responses to light, or basic parameters of the circadian
timekeeping system, such as period and phase of the rhythms. Here, the authors
directly examined the hypothesis that melatonin contributes to diurnal patterns of
behavior and physiology of a diurnal rodent, Arvicanthis niloticus, first by administering
the hormone to intact animals, and then by removing the pineal gland, the primary
producer of the circulating melatonin. Exogenous melatonin ranging from physiological
levels to high pharmacological doses did not suppress body temperature or reduce
activity when administered at ZT11, a very different response than observed in
primates. Removing the pineal gland had no effect on the hourly distribution of activity
across a 12:12 h LD cycle, or on the patterns of sleep across the dark phase of that
cycle. The direct, masking, effects of light at night on activity were also the same in

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pinealectomized and control grass rats, as 1 hr pulses of light across the dark phase of
a 24 h LD cycle stimulated increases in activity of control and pinealectomized animals
to a similar extent. In addition, the circadian regulation of activity, as indicated by the
responses of rhythm period and phase to changes in lighting conditions, was unaffected
by the surgical condition of the animals. Taken together, therefore, these data suggest
that the pineal gland does not contribute to the expression of diurnality in grass rats, as
it does in primates. These data are discussed in light of what they may tell us about the
evolution of diurnality in mammals.

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INTRODUCTION
Melatonin is a hormone rhythmically produced by the pineal gland that is
elevated in both nocturnal and diurnal species during the dark phase of the daily
light:dark (LD) cycle and during the subjective night when animals are held in constant
conditions (Reiter, 1993). The nighttime rise in melatonin is driven by circadian
mechanisms, but its secretion can be directly suppressed by light (Lewy, Wehr,
Goodwin, Newsome, & Markey, 1980; Nelson & Takahashi, 1991). Receptors for the
hormone are found in many regions of the brain and in peripheral tissues (Ishii, Tanaka,
Kobayashi, Kato, & Sakuma, 2009; Slominski, Reiter, Schlabritz-Loutsevitch, Ostrom, &
Slominski, 2012; Stankov, Fraschini, & Reiter, 1991). The functions of melatonin are
wide ranging, and it appears to play a role in immune function (Carrillo-Vico et al., 2006;
Haldar, Rai, & Singh, 2004), neuroprotection (Katona et al., 1998; Mayo, Sainz, Antolin,
& Rodriguez, 1999; Mayo et al., 1998), reproduction (Reiter et al., 2009) and rates of
disease progression (Baydas, Koz, Tuzcu, & Nedzvetsky, 2008; Otalora, Madrid,
Alvarez, Vicente, & Rol, 2008; Xia et al., 2008). Melatonin can also influence the period
and phase of internally generated circadian rhythms of many species, both nocturnal
and diurnal (Pevet, 2003). In addition, there is evidence that melatonin can influence
sleep, activity, and body temperature in a direct, circadian-independent, manner in
some species (Badia, Myers, Boecker, Culpepper, & Harsh, 1991; Cajochen, 2007;
Zhdanova, 2005).
Activity-suppressing effects of melatonin have been noted in diurnal fish (LopezOlmeda, Madrid, & Sanchez-Vazquez, 2006; Zhdanova, Wang, Leclair, & Danilova,
2001), birds (Murakami, Kawano, Nakahara, Nasu, & Shiota, 2001), old world monkeys

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(Hao & Rivkees, 2000; Inui & Hazeki, 2010; Zhdanova et al., 2002), and in humans
(reviewed in Lavie, 1997). Two studies have directly compared diurnal and nocturnal
species. In nocturnal owls and trench, activity is not altered by melatonin exposures,
whereas in diurnal sparrows, quail and goldfish activity is decreased (Lopez-Olmeda et
al., 2006; Murakami et al., 2001). Many studies have examined the sleep-promoting
effects of exogenously administered melatonin in humans (e.g. Dollins et al., 1993;
Dollins, Zhdanova, Wurtman, Lynch, & Deng, 1994; Nave, Herer, Haimov, Shlitner, &
Lavie, 1996; Zhdanova et al., 1995; Zhdanova, Wurtman, Morabito, Piotrovska, &
Lynch, 1996). In his analysis of the published data, (Lavie, 1997) concluded that if
melatonin is administered at times of day during which it is not already elevated via
endogenous mechanisms, melatonin has a sleep promoting effect, and suggested that
its endogenous role is to inhibit wakefulness-generating mechanisms during the night.
Further data on the role of the pineal gland in the regulation of daily sleep patterns in
humans has come from studies of individuals that experience injuries that interfere with
signals to the pineal (Biering-Sorensen & Biering-Sorensen, 2001; Scheer et al., 2006)
or have had the organ surgically removed (Kocher, Brun, Borson-Chazot, Gonnaud, &
Claustrat, 2006; Petterborg, Thalen, Kjellman, & Wetterberg, 1991). These studies have
revealed that such individuals have an increase in sleep dysfunction, though it is
impossible to rule out the possibility that other features of their conditions are the source
of the problem. However, in some such cases, administration of melatonin improves
sleep-related symptoms (Etzioni et al., 1996; Jan, Tai, Hahn, & Rothstein, 2001;
Lehmann, Cockerell, & Rudge, 1996). Little is known about direct effects that the pineal
may have on sleep and activity in diurnal mammals other than primates.

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Another way in which the pineal gland could promote sleep is via an influence on
the masking response of animals to light. Masking refers to a process whereby light
directly affects behavior, such as the induction of sleep in nocturnal species and
wakefulness in diurnal ones. Two studies have revealed elevated masking responses to
light in pinealectomized vs. control nocturnal rats (Quay, 1970; Vilaplana, Cambras, &
Dieznoguera, 1994). Comparable studies with diurnal species have not been reported,
however, treatment with melatonin has been found to alter the masking response of
body temperature and sleep to light at night in humans. Young men given an infusion of
melatonin and then exposed to bright lights showed a reduction in the light-induced
increase of body temperature (BT; Strassman, Qualls, Lisansky, & Peake, 1991) and
had shortened latencies to sleep onset (Burgess, Sletten, Savic, Gilbert, & Dawson,
2001) when compared to the controls.
Virtually nothing is known about the role of the pineal gland in regulation of
behavior of non-primate diurnal mammals. Here, our primary goal was to examine the
hypothesis that the pineal gland contributes to the decrease in activity and increase in
sleep that occurs at night in the Nile grass rat, a diurnal murid rodent from East Africa.
We did this through examination of the possibilities that it has direct effects on these
behaviors and that it might mediate masking responses to light. In addition, we tested
whether it modulates the circadian regulation of activity. We did this first by
administering melatonin to these animals and monitoring their activity and Tb, and then
by determining whether removal of the pineal gland would affect these basic processes.

METHODS

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Animals
Adult male and female Arvicanthis niloticus (grass rats) from the breeding colony
at Michigan State University were singly housed in Plexiglas cages (34x28x17 cm) for
the duration of these experiments. The facilities were maintained at a temperature
ranging between 20-25°C with a 12:12 light:dark (LD) schedule. Animals were also
given food (PMI Nutrition Prolab RMH 2000, Brentwood, MO) and water ad libitum. All
experimental procedures were approved by the Institutional Animal Care and Use
Committee (IACUC) of Michigan State University and were treated in accordance with
the guidelines of the National Institutes of Health on the care and use of animals.

Experimental Procedures
Experiment 1: Effects of exogenous melatonin on activity and body temperature
Six adult male grass rats were surgically implanted with E-mitters (PDT ER-4000
G e-mitters, Minimitter, Bend, OR) to monitor body temperature and general activity,
following the protocol outlined by (Schrader et al., 2009). In brief, grass rats were first
anesthetized using isoflorane (2.5-5%, Abbott Laboratories, IL, USA) and then injected
s.c. with buprenorphine hydrochloride (Buprenex, 0.06 mg/kg of body weight) prior to
the surgery. Along the midline of the peritoneal cavity we made a 1 cm incision and the
transmitter was placed into the cavity in a position underneath the liver. The abdominal
muscle was then closed with nylon suture and the skin was secured with dissolvable
chromic gut sutures, which were then reinforced with autoclips. At the conclusion of the
surgery, animals were injected with sterile saline (2 cc, 0.9% NaCl) and Ketoprofen

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(5mg/kg of body weight). Animals received the analgesic Melaxicam (0.1 mg/kg of body
weight) orally in apple every 24 hrs for the next 2 days.
After a recovery period of at least two weeks, animals were administered varying
doses of melatonin (0.33 mg/kg, 1.0 mg/kg, 4 mg/kg, 5 mg/kg of body weight) or a
vehicle control (ethanol) in a piece of apple, using a protocol similar to that described in
Hiebert et al. (2006). Melatonin (Sigma Chemical, St. Louis, MO) was first dissolved in
100% ethanol to an appropriate concentration that allowed for 10-25 µL of solution to be
administered, based on the weight of the animal. Solutions were made on the day of
exposure and stored at 4°C in a 2 mL microcentrifuge tube shielded from direct light by
3

foil until use. A 5 mm piece of apple was injected with the solutions using a Hamilton
syringe and then the ethanol was allowed to evaporate for at least 30 minutes before
being fed to the animals. Grass rats were habituated to receiving apple prior to the
experiment; animals always took samples voluntarily and consumed them immediately.
Each animal was exposed to the control and then the experimental conditions from the
lowest-to-highest dose in a within-subjects design; each exposure was administered at
least 4 days after the previous one.

Experiment 2: Effects of pinealectomy on activity and sleep behavior
Twenty adult female grass rats were set up with infrared motion detectors (IRs,
Visonic Tel Aviv, Israel) to monitor general activity. Animals were randomly assigned to
either the pinealectomy (n=10, PINX) or control (n=8, SHAM) group. All animals were
anesthetized using isoflorane and then the surface of the head were shaved and
sterilized with betadine. Animals were secured in a steretaxic apparatus (Stoelting Co.;
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Wood Dale, IL) and then injected s.c. with Buprenex and lidocaine (0.03 mL) prior to the
surgery. A 2 cm incision was made on the scalp to expose the surface of the skull.
Above Lambda, a circular hole was made with a 5 mm trephine and, using fine forceps,
the pineal gland was quickly extracted. Two researchers examined the extracted gland
to confirm that it had the distinct texture and shape of the pineal; samples were also
stored in PBS and later examined by D.D.S. under a dissecting scope to further confirm
that the pineal gland had been removed. The hole in the skull was packed with gel foam
and the incision was closed with autoclips. Post-operative care was the same as
described above. SHAM animals followed the same surgical protocol without the
removal of the pineal gland.
After a recovery period of at least two weeks, animals went through multiple tests
to examine patterns of activity and sleep as well as responses to changes in lighting
conditions. First, grass rats were monitored in 12:12 LD cycle for 1 week. The animals
were then placed into constant darkness (DD) for 2 weeks and then transferred into
constant light (LL) for 2 weeks (Figure 4.5A). Animals were then placed in a 12:12 LD
cycle until they all displayed stable entrainment. At this point, over an eight-day period,
each animal was videotaped across the dark phase of the LD cycle with two low-light
lens CCTV cameras connected to a time-lapse video recorder that condensed a 12 hr
period onto a 2 hr tape. These tapes were then converted into DVDs that could be seen
on a computer screen and the behavior was scored with a custom-made data
acquisition program (S & K Computer Products, Toronto, Ontario, Canada; Lonstein &
Stern, 1997). The details of the scoring are described below. Animals were then
exposed to a masking protocol in a 12:12 LD cycle (Shuboni et al., 2012). Specifically,

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animals went through a series of three day cycles during which they were exposed to a
1 hr light pulse (LP) or dark pulse (DP) on the third day; DPs were administered first (at
ZT2, 10, and 6) and then LPs (at ZT14, 22, and 18). Finally, grass rats were phase
shifted to determine the rate of reentrainment after a 6 hr phase delay and then a 6 hr
phase advance. At the end of the experiments blood samples were collected and
analyzed to determine melatonin concentrations (described below).

Measurement of Plasma Melatonin Levels
Animals were anesthetized using intraperitoneal injections of sodium penabarital
(400mg/kg), and blood samples were collected with heparinized 5cc syringes via
cardiac puncture. Each sample was alliquoted into a 2 mL microcentrifuge tubes and
spun down in a centrifuge at 1,000 g at 4° for 20 minutes. Plasma was transferred into
C
new microcentrifuge tubes and stored at -20° Plas ma and quality controlled samples
C.
were analyzed for melatonin concentrations in duplicate using a commercial ELISA kit
(GenWay Biotech, San Diego, CA) according to the manufacturer’s protocol utilizing the
competition principle and microtiter plate separation. Six standards and 2 controls
included in the kit were used for comparison. A standard curve was obtained by plotting
the optical densities (ODs) of the standards (y-axis, linear) against their provided
melatonin concentrations (x-axis, logarithmic) in a regression equation using a Hillslope
plot with the program, SigmaPlot (Systat, San Jose, CA). The concentration of each
unknown sample was then calculated using the equation generated by the standards.

Data Analysis

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Experiment 1: Effects of exogenous melatonin on activity and body temperature
Signals from the transmitters interfaced with receiver (ER-4000, Minimiter, Bend,
OR) on which each animal’s cage was placed. Each base was connected to a computer
in an adjacent room, where the data were collected in one-minute bins using Vitalview
4.0 Software (Minimiter, Bend, OR). General activity was recorded by summing counts
(movements) across each one-minute period, while body temperature was taken with a
single recording at the end of the sampling interval. Data were imported into Microsoft
Excel, were all counts were summed into 1 hr samples for activity or averaged for each
1 hr interval for temperature. Two within-subjects one-way ANOVAs were conducted
separately for both general activity and body temperature to compare the reaction of
animals at ZT11 to varying concentrations of melatonin.

Experiment 2: Effects of pinealectomy on activity and sleep behavior
The IR detectors were attached to the lids of the cages, near the food and water.
Each monitor sent information about the motion of an animal (infrared beams breaks) to
a personal computer in an adjacent room equipped with the VitalView Program
(Minimitter, Bend, OR, USA). To analyze different parameters of the circadian rhythms
files were converted into actograms via ClockLab (Actimetrics, Wilmette, IL, USA). The
program automatically determined onset and offset of activity, and calculated the period
of the rhythm, tau (τ). The experimenter visually inspected these points prior to analysis,
as the program occasionally improperly designated onset/offset, and manually corrected
errors (as in Schrader et al., 2009). From the onsets and offsets we also calculated the
duration of the active period of the daily/circadian rhythms, alpha (α). Using Microsoft

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Excel we also examined the raw activity data and determined the total activity per cycle,
based on the circadian period in DD, LL, and LD. Additionally, for 5 days in LD we
calculated the activity profile in 1 hr-bins across 24 hrs. Finally, two experimenters
independently examined the actograms to determine when rhythms had reentrained
after phase shifts of the LD cycle; this point was defined as the first day of a period of at
least three days during which the time of activity onsets were consistent (e.g. Jechura,
Mahoney, Stimpson, & Lee, 2006). Results were all analyzed with mixed two-way
ANOVAs. Specifically, for analyses of alpha and total activity the within-subjects
variable was lighting condition (DD, LL, and LD) and the between-subjects variable was
surgical condition (PINX and SHAM). For tau, we also did a two-way ANOVA; here the
within subjects variable was lighting conditions (DD and LL) and the between subjects
variable was again surgical condition (PINX and SHAM). Finally, we used independentsample t-tests to determine the differences between PINX and SHAM animals for both
phase delays and phase advances.
“Sleep-like behavior” (referred to here as “sleep”) was scored from videotapes
when the animal adopted a distinct posture, lying down in a slumped position with the
head on the floor of the cage (Figure 4.3A). An observer blind to the condition of the
animal recorded onsets and offsets of each sleep bout across the night (using the
custom-made data scoring program described above). From these data we calculated
the total amount of sleep, the number of sleep bouts, and the duration of those bouts,
during the 12 hr period of recording. For analysis of the total sleep bout number and
average bout duration, we used independent sample t-tests to compare PINX and

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SHAM groups. To analyze the effect of surgery on the amount of sleep per hour across
the 12 hr period of darkness we used a mixed two-way ANOVA (Time X Surgery).
To analyze the masking data, we used Microsoft Excel to sum the total amount of
activity during the 1 hr of each of the 6 pulses, and the same h on the corresponding
control day. For each pulse, we used a mixed two-way ANOVA to compare the pulse
condition (dark/light pulse and control) and the surgical conditions (PINX and SHAM).
Finally, we compared the absorbance level of the melatonin assay between the PINX
and SHAM group using an independent samples t-test.

RESULTS
Experiment 1: Effects of exogenous melatonin on activity and body temperature
General activity was not affected by melatonin during the hour of its
administration (Figure 4.1A ; F[4, 16] = 0.605, p = 0.623). Additionally, we did not
observe any alteration in profile of activity when we examined the behavior of the
animals across the day after the exposures (Figure 4.1B). There was a near-significant
effect of melatonin on body temperature (Figure 4.1C; F[4, 16] = 4.982, p = 0.060).
However, when we examined the 24 hr profile of activity we observed a lower
temperature rhythm during one of the exposures (Figure 4.1D). When we corrected for
average body temperature across the day, there was no longer a trend toward
significance (F[4, 16] = 1.649, p = 0.265).

Experiment 2: Effects of pinealectomy on activity and sleep behavior
LD Conditions

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Figure 4.1. The effects of exogenous melatonin on activity and body temperature (BT). Group data of the hour after
administration of melatonin does display alteration of activity (A) nor BT (C) at any concentration level, ranging from 0.005.00 mg/kg. When Individuals are plotted across 24 hr none display an alteration in activity (B) or BT (D). The dotted red
line at ZT11 indicates the administration of melatonin.

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Figure 4.2. 24 hr activity profile of PINX and SHAM animals. Grey bar indicates lights off at ZT12.

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In LD conditions, there was a main effect of time on hourly rates of activity
(F[23, 345] = 17.954, p < 0.001), but there was no significant effect of surgical condition
(F[1, 15] = 0.194, p = 0.666), and there was no interaction between these two variables
(F[23, 345] = 2.068, p = 0.085). In both groups of animals the highest levels of activity
occurred during the 12 hr light period and at the transitions between light and dark
phases of the LD cycle (Figure 4.2B). Under these same lighting conditions sleep bout
numbers (Figure 4.3B) and durations (Figure 4.3C) also did not differ in PINX and
SHAM groups (t(12)=1.416, p=0.182 and t(12)=0.364, p=0.727, respectively). Hourly
rates of sleep across the night were (Figure 4.3D) affected by time (F[11, 132] = 8.401,
p < 0.001) but not by surgical condition (F[1, 12] = 0.007, p = 0.934), nor were they
affected by an interaction between these two variables (F[11, 132] = 0.361, p = 0.862).

Masking
The masking responses to light did not differ significantly as a function of surgical
condition; this was the case for both dark pulses and light pulses (Table 4.1). Among
both PINX and control grass rats activity increased in response to light at all three times
sampled during the dark period; there was no significant effect of dark pulses during the
day (Figure 4.4). The duration of the active period in different lighting periods can also
be an indicator of masking, and this too was unaffected by removal of the pineal gland.
Specifically, alpha was affected by lighting condition (F[2, 26] = 30.318, p < 0.001) but
not by surgery (F[1, 13] = 0.000, p = 0.993), or by an interaction between lighting and
surgery (F[2, 26] = 0.379, p = 0.636). Alpha was significantly longer in LL than both LD
and DD (Figure 4.5B).

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Table 4.1. Analysis of light pulse data masking data in PINX and SHAM animals

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Figure 4.3. Sleep patterns in pinealectomized animals. Panel A demonstrates the
behavior we classified as “sleep-like,” the animals adopted a distinct posture, lying down
in a slumped position with the head on the floor of the cage. The total number of sleep
bouts (B) and the average sleep bout duration (C) were not different between PINX and
SHAM animals. Additionally, the spread of total sleep amount across the night was
identical between the two groups.

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Figure 4.4. Patterns of the masking responses of activity to light. Both SHAM (Black)
and PINX (Grey) animals experienced increases in activity at all time points, ZT14,
ZT18 and ZT22. An asterisk (*) indicates significance p < .05.

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Period and Phase Shifts
Tau did not differ in PINX and SHAM groups (F[1, 14] = 1.808, p = 0.200), and
there was no interaction between lighting condition and surgery (F[1, 14] =
0.114, p = 0.740). There was a main effect of lighting condition (F[1, 14] = 90.789, p <
0.001) such that the period of the rhythm was significantly longer in LL than in DD
(Figure 4.5C). Finally, animals reentrained from phase delays significantly faster than
phase advances (Figure 4.6C; F[1, 11] = 5.542, p = 0.038), but, again there was no
effect of surgery (F[1, 11] = 0.188, p = 0.673) nor was there an interaction (F[1, 11] =
0.009, p = 0.927).

Melatonin Levels
At the completion of the behavioral experiments, plasma melatonin levels were
compared between PINX and SHAM animals. The standard curve created from the set
standards provided by the company had an R squared value of 0.9954. One animal was
removed from the analysis because of a large difference between the duplicate
samples, with a Confidence Variance percentage greater than 25%. Animals without
pineal glands had a lower mean level of plasma melatonin, 19.299±2.252 pg/mL, than
the SHAMS, 37.565±9.382 pg/mL. When we compared the absorbance levels of the
two groups, PINX animals had significantly lower levels than SHAM (t(8)=2.423,
p=0.042).

DISCUSSION

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Figure 4.5. Circadian rhythms of activity in 12:12 LD cycle, constant light (LL), and
constant darkness (DD). Panel A shows two representative actograms of PINX (right)
and SHAM (left) animals. There was no significant difference between the two groups
for measurement of alpha (B) or tau (C).

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Figure 4.6. Phase shifting ability of pinealectomized animals. Panel A and B are
representative of PINX and SHAM animals in response to phase advances and delays.
No significant differences were observed between the two groups (C).

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The collection of conditions in which we compared intact and pinealectomized
grass rats with respect to many features of behavioral rhythms under a variety of
lighting conditions, strongly suggest that melatonin does not contribute to diurnality in
these animals as it does in primates. Whereas there is good evidence that melatonin
can help reduce activity and consolidate sleep at night in primates, this does not appear
to be the case in the grass rat. This conclusion stems from data, discussed below, that
were obtained in the context of a 24 h LD cycle, a masking paradigm, shifts in a light
dark cycle as well as constant lighting conditions.

LD Conditions
Temporal patterns of general activity and sleep in an LD cycle did not differ
between pinealectomized and control groups. This was the case for the average overall
amount of activity across the day as well as the waveform of the activity pattern across
the 24 h LD cycle (Figure 4.2). Furthermore, there was no indication that removal of the
pineal gland affected sleep. Video analysis revealed no difference in the overall
temporal distribution of sleep across the dark phase or the duration or number of sleep
bouts during that time. It should be noted that studies of sleep in pinealectomized
nocturnal rodents have yielded contradictory results (Fisher & Sugden, 2010;
Mendelson & Bergmann, 2001; Mouret, Coindet, & Chouvet, 1974; Wang et al., 2003).
However, there is some evidence that humans may be different in this regard. There are
data suggesting that people without a functional pineal gland have sleep that is less
consolidated and that extends into the day considerably more than it does in people
with normal patterns of melatonin secretion (Macchi & Bruce, 2004; Slawik et al., 2012).

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Though most of this information comes from case studies (Macchi & Bruce, 2004), it
does suggest that the pineal contributes to consolidation of human sleep at night,
presumably through secretion of its hormone melatonin, which has clear soporific
effects in other diurnal primates, birds and fish (Hao & Rivkees, 2000; Hughes & Badia,
1997; Inui & Hazeki, 2010; Lopez-Olmeda et al., 2006; Murakami et al., 2001;
Zhdanova, 2005; Zhdanova et al., 2002; Zhdanova et al., 2001). Although the pineal
may contribute to diurnality in those animals, the current data suggest that this is not the
case in grass rats.

Masking
In intact diurnal mammals, light pulses at night directly suppress melatonin and
stimulate an increase in activity (Redlin, 2001), raising the possibility that the light
induced decrease in melatonin could contribute to positive masking. If that is the case,
then pinealectomy should decrease the masking responses to photic stimuli. However,
this did not appear to be the case, as we found 1 hr light pulses during the D phase of a
12:12 LD cycle increased activity in both groups and that the magnitude of the response
did not differ between the two groups. The stimulatory effect of light at night on activity
is thus unlikely to be facilitated by a decrease in melatonin, providing further evidence
that melatonin does not influence activity/rest state in these animals.
Data from grass rats maintained in constant conditions also suggests that
masking was unaffected by pinealectomy. Specifically, the duration of the active period
increased when conditions changed from DD to LL (Figure 4.5B), and the increase was
identical in intact and PINX animals. The effect of light intensity on alpha is likely to

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reflect masking, with light expanding the active period and darkness reducing it in a
diurnal mammal.

Period and Phase Shifts
Though our primary purpose has been to determine whether the pineal gland has
any direct influence on activity/rest state, we also examined some features of circadian
regulation. In some mammals, pineal melatonin has a small influence on tau and on
how it responds to changes in lighting conditions, but in most it does not (AguilarRoblero & Vega-Gonzalez, 1993; Aschoff et al., 1982; Cassone, 1992; Cheung &
McCormack, 1982; Morin, 1993; Morin & Cummings, 1981; Yanovski, Rosenwasser,
Levine, & Adler, 1990). The evidence that we obtained by removing the pineal gland
suggests that this is also the case in grass rats. We saw no significant difference
between PINX and SHAM animals in period, or in its lengthening upon transfer from DD
to LL (Figure 4.5C). In another diurnal rodent, the golden mantled ground squirrel, tau
changes in response to photic stimuli were similarly unaffected by light
intensity(Martinet & Zucker, 1985). Rates of reentrainment were also unaffected by
PINX in grass rats (Figure 4.6), which was also the case in ground squirrels (Martinet &
Zucker, 1985), but they are in some nocturnal rodents under some conditions
(Finkelstein, Baum, & Campbell, 1978; Quay, 1970, 1971).

Melatonin Levels
Circulating melatonin was higher in control than PINX animals, but the magnitude
of the difference was not as great as we expected. The fact that some melatonin was

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present in PINX grass rats was not surprising, as surgical removal of the pineal does
not completely eliminate circulating melatonin in birds (Jin et al., 2011) or rats (Agez et
al., 2009). The low levels of melatonin in our intact animals were likely due to the time at
which we collected the samples. In a sister species (Arvicanthis ansorgei) the decline in
production of melatonin occurs between ZT23 and ZT0 (Garidou et al., 2002).

Summary and Conclusions
As noted above, there are numerous reports of soporific effects of melatonin in
diurnal vertebrates (for review, Zhdanova & Wurtman, 1997), such as fish (LopezOlmeda et al., 2006; Zhdanova et al., 2001) and birds (Murakami et al., 2001). The
pineal gland also has sleep-promoting effects in diurnal primates (Hao & Rivkees, 2000;
Hughes & Badia, 1997; Inui & Hazeki, 2010; Zhdanova, 2005; Zhdanova et al., 2002),
including humans (Matsumoto, 1999; Tzischinsky & Lavie, 1994). There are no data
that we are aware of addressing the question of whether the pineal gland might play a
direct role in the regulation of activity or sleep in non-primate mammals that have
independently evolved a diurnal pattern of adaptation to the day-night cycle. Katz (2011)
has argued at a very general level that when common behaviors evolve independently it
is likely to occur via similar changes in neural organization, because the potential
mechanisms that could produce a given behavioral patterns are limited. In the current
study we examined whether this might be the case when it comes to potential changes
in the role played by the pineal gland at two independent evolutionary transitions from
nocturnality to diurnality, one that occurred among primates and the other within a murid
clade. The data here suggests that this is not the case. That is, whereas the pineal

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contributes to decreases in activity and increases in sleep at night in diurnal primates,
the current data suggest that this is not the case in diurnal grass rats. Further
investigation into the distribution of melatonin receptors within diurnal grass rats and
humans may provide insight into why melatonin has such a different role. Other, basic
mechanisms that promote sleep at night and activity during the day may be the same,
but the role that the pineal gland in primates appears to be absent in diurnal grass rats.

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CHAPTER 5
Summary and Conclusions
Temporal niche specific responses to light
In the first, introductory chapter of this dissertation, I outline the importance of the
masking system for the structuring of daily behavioral rhythms associated with temporal
niches. I proposed that during evolutionary transitions between temporal niches,
changes must occur in the neural mechanisms that regulate masking of behavior. The
results I presented in Chapter 2 indicated that the neural response to light of nocturnal
mice and diurnal grass rats is very different, with more areas within the diurnal brain
responding to exposure. These differences could be a result of alterations within the
input systems that transmit the signal or in how the brain responds to the stimuli.
Previous work in nocturnal rodents has established the importance of
melanopsin-containing retinal ganglion cells (ipRGCs) in production of signals for
negative masking (Mrosovsky, Salmon, Foster, & McCall, 2000) and as conduits for
relaying rod and cone information to induce positive masking (Guler et al., 2008). Some
researchers suggest that alterations within this input system are sufficient for inverting
rhythms and play a role in temporal niche switches (Mrosovsky & Hattar, 2005).
However, these non-visual photoreceptors are also responsible for other functions
including the suppression of melatonin and the entrainment of the rhythms within the
suprachiasmatic nucleus (SCN), the circadian pacemaker in mammal. Both the pineal
and the SCN remain similar after the transition between diurnal and nocturnal species
(Challet, 2007). Melatonin levels peak at night in both nocturnal and diurnal species and
glutamate rhythms of the SCN are also similar. These similarities also include the peak

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in rhythmic expression of clock genes, the transcription-translation feedback loop that is
important for generating endogenous rhythms, within both regions (Pineal: Ackermann,
Dehghani, Bux, Kauert, & Stehle, 2007; Karolczak, Burbach, Sties, Korf, & Stehle, 2004;
SCN: Ramanathan, Nunez, & Smale, 2008). When mice are mutated to alter the input
system and display diurnal activity patterns (Doyle, Castrucci, McCall, Provencio, &
Menaker, 2006), the pineal and SCN are both altered (Doyle, Yoshikawa, Hillson, &
Menaker, 2008). Specifically, mutant animals have later peaks in their rhythms in the
clock gene Per1 within SCN cells and peripheral tissue, like the pineal. Alterations in the
input systems needed to induce diurnality in a nocturnal rodent, therefore, have broad
repercussions that produce animals that are not similar to those found in naturally
occurring populations.
However, nothing is definitively known about the input system mediating masking
in diurnal species as no direct examination of photoreceptor function has been
conducted in a diurnal model. Although, for a human experiencing masking to light, lowspectrum blue light is associated with arousal, i.e. positive masking (Iskra-Golec,
Wazna, & Smith, 2012). These intensities are within the activation range of the ipRGCs
that produce negative masking in nocturnal animals. The signal carried by these
photoreceptors may therefore remain the same, light activating ipRGCs and darkness
activating traditional photoreceptors. The difference between nocturnal and diurnal
species with respect to their responses to the stimuli probably lies downstream of the
ipRGCs, in the neural mechanisms that dictate the masking response.

Neural Mechanisms of Masking

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Differences in masking behavior of nocturnal and diurnal species are likely the
product of alterations within the neural mechanisms of masking. Literature on nocturnal
rodents suggests that there is no singular region that controls the masking response to
light (Redlin, 2001); however, several areas have been targeted for investigation into
their functional role in the masking response. While the role of the SCN is controversial
(Li et al., 2005; Redlin & Mrosovsky, 1999a), lesions of the intergeniculate leaflet (IGL;
Redlin et al., 1999) and olivary pretectal nucleus (OPN; Miller et al., 1999) have been
shown to alter but not ablate masking of activity and REM induction, respectively. The
work in Chapter 2 spurred Gall et al. (2013) to ablate the IGL in diurnal grass rats; here
too they observed an alteration and not an ablation in masking after lesion. In the work
described in Chapter 3, I further investigated the functional role of one brain area, the
ventral subparaventricular zone (vSPZ), which responded differently to light in mice and
grass rats, by using directed chemical lesions in the diurnal grass rat. Earlier work in the
laboratory suggested that vSPZ lesioned grass rats might be less sensitive to the 12:12
light/dark (LD) cycle, with random bouts of arrhythmia occurring in animals with the
largest lesions (Schwartz et al., 2009). We, however, did not observe an alteration in
how a small cohort of vSPZ lesioned grass rats responded to light in two different
masking protocols. These findings do not eliminate the vSPZ as a possible component
of the masking system but they do demonstrate that the region is not necessary for
light-induced masking.
Chapter 3 also describes an interesting discovery about the responsiveness of
the grass rat SCN to chemotoxins. The SCN was believed to have endogenous
protection from toxic agents (Bottum, Poon, Haley, Karmarkar, & Tischkau, 2010).

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Unlike other regions, such as the hippocampus, the SCN cells of nocturnal rodents were
not damaged by exposure to N-methyl-D- aspartate (NMDA) in vivo (Hastings et al.,
1985), in vitro (Colwell & Levine, 1996) or in cell culture (Bottum et al., 2010). This
difference poses the question of why the grass rat SCN responded differently to the
toxin when compared to the SCN of nocturnal rodents. It is believed that the SCN of
nocturnal and diurnal rodents is very similar (Smale et al., 2003). However, (Novak &
Albers, 2002) suggest that the SCN of grass rat is less sensitive to the phase shifting
ability of NMDA when compared to nocturnal rodents.
An alternative way to influence the neural mechanisms of masking could be
through hormones. The hormone melatonin is produced at night by both nocturnal and
diurnal species (Challet, 2007). However, in primates melatonin has a very specific
soporific effect not observed in nocturnal animals (Zhdanova & Wurtman, 1997).
Melatonin has also been shown to prevent the light-induced increases in body
temperature (Strassman et al., 1991) and arousal (Burgess et al., 2001) in humans.
Additionally, pinealectomy in nocturnal rodents alters the response of the SCN to light
(Gonzalez & Dyball, 2006) and increase negative masking behavior (Quay, 1970;
Vilaplana et al., 1994). Pineal derived melatonin may, therefore, play different roles in
nocturnal and diurnal animals. In the research described in chapter 4, we removed the
pineal gland, effectively eliminating circulating melatonin, and then tested the masking
behavior of our diurnal grass rats. Exogenous melatonin presentation did not induce
sleep in our grass rats nor did the loss of melatonin after pineal removal alter sleeping
patterns. This finding is different from those obtained in research on humans and could
be reflective of differences in the pathway for the evolution of diurnality in these two

135

species. It should also be noted that humans and grass rats have different patterns of
melatonin secretion. The onset of melatonin during the night is much earlier for humans
than it is for grass rats (Garidou et al., 2002; Wehr, 1991). Masking to light exposure
was also not influenced by pineal removal in the grass rat; nocturnal rats are different in
this regard. Effects of melatonin on the grass rat are clearly different from those seen in
both nocturnal rats and diurnal primates, demonstrating that the hormone is not
universally involved in masking behavior.

Implications and Recommendations
Our current data has demonstrated a clear activity pattern dependent
responsiveness of the brain to light exposure. Diurnal grass rats had more areas
respond to a 1 hr pulse of light when compared to nocturnal mice. This was not due to
differences in staining of tissue as both species responded with a strong increase in
cFOS activation within the SCN. We propose that the differences between our nocturnal
and diurnal rodents are likely the product of alterations within the neural mechanisms of
masking and not the input system within the retina. We also established that the vSPZ
and pineal gland are not necessary for the masking response to light in grass rats. This
research has added to the growing list of regions examined to determine the neural
mechanisms of masking.
Understanding the masking system is important because it plays an integral role
in how organisms regulate their activity patterns. Beyond the investigation into the
evolution of diurnality, masking behavior is also an important contributor to the success
of our modern society. As masking has allowed us to overcome the endogenous

136

circadian clock and to continue to function beyond the natural 24 hr constraints
established by the sun. For humans, light stimulates positive masking behaviors by
increasing arousal and decreasing sleepiness (Minors & Waterhouse, 1989b). With the
invention of high intensity artificial light, people have gained the ability to regulate light
exposure and have extended their activity further into the night. This ability to expose
oneself to light during the natural rest-phase of the activity cycle has caused problems
within our bodies. Specifically, the ability to remain awake through light-induced
masking also phase shifts the circadian pacemaker. Chronic inconsistent schedules of
light exposures can lead to many different health consequences, including cancer,
immune problems and gastrointestinal illness (Erren & Reiter, 2013). In mice, these
phase shifts in the circadian system can, over time, lead to the death of older animals
(Davidson et al., 2006). Masking, therefore, for humans can lead to detrimental effects
because society pushes the system past where it has short-term beneficial effects.
Additionally, human light pollution has altered the light landscape for populations of
organisms, possibly altering masking behaviors especially within urban communities.
In summary, this dissertation demonstrates that there are clear differences in the
masking behavior of nocturnal and diurnal rodents and that these differences are
evident within the brain. The neural substrates that coordinate the behaviors are
complex and may vary among animals that have taken different evolutionary paths to
diurnality, as is demonstrated by the effectiveness of melatonin in humans and not
grass rats. However, understanding these systems is important for both humans and
wildlife as the environmental landscape continues to change naturally and at the hands

137

of man. Therefore, further more detailed analysis of the neural mechanisms of masking
is merited.

138

REFERENCES

139

REFERENCES

Abe, H., Honma, S., Shinohara, K., & Honma, K. I. (1995). Circadian modulation in
photic induction of Fos-like immunoreactivity in the suprachiasmatic nucleus cells
of diurnal chipmunk, Eutamias asiaticus. J Comp Physiol A, 176(2), 159-167.
Abrahamson, E. E., & Moore, R. Y. (2006). Lesions of suprachiasmatic nucleus
efferents selectively affect rest-activity rhythm. Mol Cell Endocrinol, 252(1-2), 4656. doi: 10.1016/j.mce.2006.03.036
Ackermann, K., Dehghani, F., Bux, R., Kauert, G., & Stehle, J. H. (2007). Day-night
expression patterns of clock genes in the human pineal gland. J Pineal Res,
43(2), 185-194. doi: 10.1111/j.1600-079X.2007.00461.x
Adidharma, W., Leach, G., & Yan, L. (2012). OREXINERGIC SIGNALING MEDIATES
LIGHT-INDUCED NEURONAL ACTIVATION IN THE DORSAL RAPHE
NUCLEUS. Neuroscience, 220, 201-207. doi:
10.1016/j.neuroscience.2012.06.020
Agez, L., Laurent, V., Guerrero, H. Y., Pevet, P., Masson-Pevet, M., & Gauer, F. (2009).
Endogenous melatonin provides an effective circadian message to both the
suprachiasmatic nuclei and the pars tuberalis of the rat. J Pineal Res, 46(1), 95105. doi: 10.1111/j.1600-079X.2008.00636.x
Aguilar-Roblero, R., Garciahernandez, F., Aguilar, R., Arankowskysandoval, G., &
Druckercolin, R. (1986). SUPRACHIASMATIC NUCLEUS TRANSPLANTS
FUNCTION AS AN ENDOGENOUS OSCILLATOR ONLY IN CONSTANT
DARKNESS. Neurosci Lett, 69(1), 47-52. doi: 10.1016/0304-3940(86)90412-x
Aguilar-Roblero, R., & Vega-Gonzalez, A. (1993). Splitting of locomotor circadian
rhythmicity in hamsters is facilitated by pinealectomy. Brain Res, 605(2), 229236.
Ahnelt, P. K., & Kolb, H. (2000). The mammalian photoreceptor mosaic-adaptive
design. Progress in Retinal and Eye Research, 19(6), 711-777. doi:
10.1016/s1350-9462(00)00012-4

140

Aizawa, H., Kobayashi, M., Tanaka, S., Fukai, T., & Okamoto, H. (2012). Molecular
characterization of the subnuclei in rat habenula. J Comp Neurol, 520(18), 40514066. doi: 10.1002/cne.23167
Allen, A. E., Brown, T. M., & Lucas, R. J. (2011). A distinct contribution of shortwavelength-sensitive cones to light-evoked activity in the mouse pretectal olivary
nucleus. J Neurosci, 31(46), 16833-16843. doi: 10.1523/jneurosci.2505-11.2011
Amir, S., Lamont, E. W., Robinson, B., & Stewart, J. (2004). A circadian rhythm in the
expression of PERIOD2 protein reveals a novel SCN-controlled oscillator in the
oval nucleus of the bed nucleus of the stria terminalis. Journal of Neuroscience,
24(4), 781-790. doi: 10.1523/jneurosci.4488-03.2004
Andres, K. H., von During, M., & Veh, R. W. (1999). Subnuclear organization of the rat
habenular complexes. J Comp Neurol, 407(1), 130-150.
Angeles-Castellanos, M., Salgado-Delgado, R., Rodriguez, K., Buijs, R. M., & Escobar,
C. (2010). THE SUPRACHIASMATIC NUCLEUS PARTICIPATES IN FOOD
ENTRAINMENT: A LESION STUDY. Neuroscience, 165(4), 1115-1126. doi:
10.1016/j.neuroscience.2009.11.061
Aronin, N., Sagar, S. M., Sharp, F. R., & Schwartz, W. J. (1990). Light regulates
expression of a Fos-related protein in rat suprachiasmatic nuclei. Proc Natl Acad
Sci U S A, 87(15), 5959-5962.
Aschoff, J. (1960). EXOGENOUS AND ENDOGENOUS COMPONENTS IN
CIRCADIAN RHYTHMS. Cold Spring Harbor Symposia on Quantitative Biology,
25, 11-28.
Aschoff, J. (1999). Masking and parametric effects of high-frequency light-dark cycles.
Jpn J Physiol, 49(1), 11-18.
Aschoff, J., Daan, S., & Honma, K. I. (1982). Zeitgebers, Entrainment, and Masking:
some Unsettled Questions. Berlin: Springer-Verlag.
Aschoff, J., & Vongoetz, C. (1988). MASKING OF CIRCADIAN ACTIVITY RHYTHMS
IN MALE GOLDEN-HAMSTERS BY THE PRESENCE OF FEMALES. Behavioral
Ecology and Sociobiology, 22(6), 409-412. doi: 10.1007/bf00294978

141

Aschoff, J., & Vongoetz, C. (1989). MASKING OF CIRCADIAN ACTIVITY RHYTHMS
IN CANARIES BY LIGHT AND DARK. Journal of Biological Rhythms, 4(1), 2938. doi: 10.1177/074873048900400102
Badia, P., Myers, B., Boecker, M., Culpepper, J., & Harsh, J. R. (1991). BRIGHT LIGHT
EFFECTS ON BODY-TEMPERATURE, ALERTNESS, EEG AND BEHAVIOR.
Physiology & Behavior, 50(3), 583-588. doi: 10.1016/0031-9384(91)90549-4
Baydas, G., Koz, S. T., Tuzcu, M., & Nedzvetsky, V. S. (2008). Melatonin prevents
gestational hyperhomocysteinemia-associated alterations in neurobehavioral
developments in rats. J Pineal Res, 44(2), 181-188. doi: 10.1111/j.1600079X.2007.00506.x
Biering-Sorensen, F., & Biering-Sorensen, M. (2001). Sleep disturbances in the spinal
cord injured: an epidemiological questionnaire investigation, including a normal
population. Spinal Cord, 39(10), 505-513. doi: 10.1038/sj.sc.3101197
Blanchong, J. A., McElhinny, T. L., Mahoney, M. M., & Smale, L. (1999). Nocturnal and
diurnal rhythms in the unstriped Nile rat, Arvicanthis niloticus. Journal of
Biological Rhythms, 14(5), 364-377. doi: 10.1177/074873099129000777
Blanchong, J. A., & Smale, L. (2000). Temporal patterns of activity of the unstriped Nile
rat, Arvicanthis niloticus. Journal of Mammalogy, 81(2), 595-599. doi:
10.1644/1545-1542(2000)081<0595:tpoaot>2.0.co;2
Borbely, A. A., & Huston, J. P. (1974). Effects of two-hour light-dark cycles on feeding,
drinking and motor activity of the rat. Physiol Behav, 13(6), 795-802.
Bottum, K., Poon, E., Haley, B., Karmarkar, S., & Tischkau, S. A. (2010).
Suprachiasmatic nucleus neurons display endogenous resistance to
excitotoxicity. Exp Biol Med (Maywood), 235(2), 237-246. doi:
10.1258/ebm.2009.009244
Brooks, E., Waters, E., Farrington, L., & Canal, M. M. (2011). Differential hypothalamic
tyrosine hydroxylase distribution and activation by light in adult mice reared
under different light conditions during the suckling period. Brain Struct Funct,
216(4), 357-370. doi: 10.1007/s00429-011-0318-9

142

Burgess, H. J., Sletten, T., Savic, N., Gilbert, S. S., & Dawson, D. (2001). Effects of
bright light and melatonin on sleep propensity, temperature, and cardiac activity
at night. Journal of Applied Physiology, 91(3), 1214-1222.
Butler, M. P., & Silver, R. (2011). Divergent photic thresholds in the non-image-forming
visual system: entrainment, masking and pupillary light reflex. Proceedings of the
Royal Society B-Biological Sciences, 278(1706), 745-750. doi:
10.1098/rspb.2010.1509
Cajochen, C. (2007). Alerting effects of light. Sleep Medicine Reviews, 11(6), 453-464.
doi: 10.1016/j.smrv.2007.07.009
Campi, K. L., & Krubitzer, L. (2010). Comparative Studies of Diurnal and Nocturnal
Rodents: Differences in Lifestyle Result in Alterations in Cortical Field Size and
Number. Journal of Comparative Neurology, 518(22), 4491-4512. doi:
10.1002/cne.22466
Canteras, N. S., Ribeiro-Barbosa, E. R., Goto, M., Cipolla-Neto, J., & Swanson, L. W.
(2011). The retinohypothalamic tract: comparison of axonal projection patterns
from four major targets. Brain Res Rev, 65(2), 150-183. doi:
10.1016/j.brainresrev.2010.09.006
Carrillo-Vico, A., Reiter, R. J., Lardone, P. J., Herrera, J. L., Fernandez-Montesinos, R.,
Guerrero, J. M., & Pozo, D. (2006). The modulatory role of melatonin on immune
responsiveness. Curr Opin Investig Drugs, 7(5), 423-431.
Cassone, V. M. (1992). The pineal gland influences rat circadian activity rhythms in
constant light. J Biol Rhythms, 7(1), 27-40.
Castillo-Ruiz, A., & Nunez, A. A. (2011). Fos expression in arousal and reward areas of
the brain in grass rats following induced wakefulness. Physiol Behav, 103(3-4),
384-392. doi: 10.1016/j.physbeh.2011.03.011
Cha, Y. J., Lee, J. H., Baik, T. K., & Park, J. S. (2011). c-Fos immunoreactivity in the
neurons of the lateral geniculate nucleus in albino rats by light exposure after
dark rearing. Korean J Ophthalmol, 25(6), 434-439. doi:
10.3341/kjo.2011.25.6.434

143

Challet, E. (2007). Minireview: Entrainment of the Suprachiasmatic clockwork in diurnal
and nocturnal mammals. Endocrinology, 148(12), 5648-5655. doi:
10.1210/en.2007-0804
Chastrette, N., Pfaff, D. W., & Gibbs, R. B. (1991). Effects of daytime and nighttime
stress on Fos-like immunoreactivity in the paraventricular nucleus of the
hypothalamus, the habenula, and the posterior paraventricular nucleus of the
thalamus. Brain Res, 563(1-2), 339-344.
Cheung, P. W., & McCormack, C. E. (1982). Failure of pinealectomy or melatonin to
alter circadian activity rhythm of the rat. Am J Physiol, 242(3), R261-264.
Chiesa, J. J., Aguzzi, J., Garcia, J. A., Sarda, F., & de la Iglesia, H. O. (2010). Light
intensity determines temporal niche switching of behavioral activity in deep-water
Nephrops norvegicus (Crustacea: Decapoda). J Biol Rhythms, 25(4), 277-287.
doi: 10.1177/0748730410376159
Cipolla-Neto, J., Bartol, I., Seraphim, P. M., Afeche, S. C., Scialfa, J. H., & Peracoli, A.
M. (1995). The effects of lesions of the thalamic intergeniculate leaflet on the
pineal metabolism. Brain Res, 691(1-2), 133-141.
Cohen, R., Smale, L., & Kronfeld-Schor, N. (2009). Plasticity of Circadian Activity and
Body Temperature Rhythms in Golden Spiny Mice. Chronobiology International,
26(3), 430-446. doi: 10.1080/07420520902820939
Cohen, R., Smale, L., & Kronfeld-Schor, N. (2010). Masking and Temporal Niche
Switches in Spiny Mice. Journal of Biological Rhythms, 25(1), 47-52. doi:
10.1177/0748730409351672
Coindet, J., Chouvet, G., & Mouret, J. (1975). EFFECTS OF LESIONS OF
SUPRACHIASMATIC NUCLEI ON PARADOXICAL SLEEP AND SLOW-WAVE
SLEEP CIRCADIAN-RHYTHMS IN RAT. Neurosci Lett, 1(4), 243-247. doi:
10.1016/0304-3940(75)90068-3
Colwell, C. S., & Foster, R. G. (1992). Photic regulation of Fos-like immunoreactivity in
the suprachiasmatic nucleus of the mouse. J Comp Neurol, 324(2), 135-142. doi:
10.1002/cne.903240202
Colwell, C. S., & Levine, M. S. (1996). Glutamate receptor-induced toxicity in neostriatal
cells. Brain Res, 724(2), 205-212.
144

Daan, S. (1981). Adaptive daily strategies in behavior. In J. Aschoff (Ed.), Handbook of
neurobiology and behavior, Biological Clocks (Vol. 4, pp. 275-298). New York:
Plenum Press.
Davidson, A. J., Sellix, M. T., Daniel, J., Yamazaki, S., Menaker, M., & Block, G. D.
(2006). Chronic jet-lag increases mortality in aged mice. Curr Biol, 16(21), R914916. doi: 10.1016/j.cub.2006.09.058
DeCoursey, P. J., Krulas, J. R., Mele, G., & Holley, D. C. (1997). Circadian performance
of suprachiasmatic nuclei (SCN)-lesioned antelope ground squirrels in a desert
enclosure. Physiology & Behavior, 62(5), 1099-1108. doi: 10.1016/s00319384(97)00263-1
Delogu, A., Sellers, K., Zagoraiou, L., Bocianowska-Zbrog, A., Mandal, S., Guimera, J.,
. . . Lumsden, A. (2012). Subcortical visual shell nuclei targeted by ipRGCs
develop from a Sox14+-GABAergic progenitor and require Sox14 to regulate
daily activity rhythms. Neuron, 75(4), 648-662. doi: 10.1016/j.neuron.2012.06.013
Dollins, A. B., Lynch, H. J., Wurtman, R. J., Deng, M. H., Kischka, K. U., Gleason, R. E.,
& Lieberman, H. R. (1993). EFFECT OF PHARMACOLOGICAL DAYTIME
DOSES OF MELATONIN ON HUMAN MOOD AND PERFORMANCE.
Psychopharmacology, 112(4), 490-496. doi: 10.1007/bf02244899
Dollins, A. B., Zhdanova, I. V., Wurtman, R. J., Lynch, H. J., & Deng, M. H. (1994).
EFFECT OF INDUCING NOCTURNAL SERUM MELATONIN
CONCENTRATIONS IN DAYTIME ON SLEEP, MOOD, BODY-TEMPERATURE,
AND PERFORMANCE. Proc Natl Acad Sci U S A, 91(5), 1824-1828. doi:
10.1073/pnas.91.5.1824
Doyle, S. E., Castrucci, A. M., McCall, M., Provencio, I., & Menaker, M. (2006).
Nonvisual light responses in the Rpe65 knockout mouse: rod loss restores
sensitivity to the melanopsin system. Proc Natl Acad Sci U S A, 103(27), 1043210437. doi: 10.1073/pnas.0600934103
Doyle, S. E., Yoshikawa, T., Hillson, H., & Menaker, M. (2008). Retinal pathways
influence temporal niche. Proc Natl Acad Sci U S A, 105(35), 13133-13138. doi:
10.1073/pnas.0801728105
Easton, A., Meerlo, P., Bergmann, B., & Turek, F. W. (2004). The suprachiasmatic
nucleus regulates sleep timing and amount in mice. Sleep, 27(7), 1307-1318.
145

Edelstein, K., & Amir, S. (1999). The role of the intergeniculate leaflet in entrainment of
circadian rhythms to a skeleton photoperiod. J Neurosci, 19(1), 372-380.
Edelstein, K., & Mrosovsky, N. (2001). Behavioral responses to light in mice with dorsal
lateral geniculate lesions. Brain Res, 918(1-2), 107-112. doi: 10.1016/s00068993(01)02966-3
Engber, T. M., Koury, E. J., Dennis, S. A., Miller, M. S., Contreras, P. C., & Bhat, R. V.
(1998). Differential patterns of regional c-Fos induction in the rat brain by
amphetamine and the novel wakefulness-promoting agent modafinil. Neurosci
Lett, 241(2-3), 95-98.
Erkert, H. G., Gburek, V., & Scheideler, A. (2006). Photic entrainment and masking of
prosimian circadian rhythms (Otolemur garnettii, Primates). Physiol Behav, 88(12), 39-46. doi: 10.1016/j.physbeh.2006.03.003
Erren, T. C., & Reiter, R. J. (2013). Revisiting chronodisruption: when the physiological
nexus between internal and external times splits in humans.
Naturwissenschaften, 100(4), 291-298. doi: 10.1007/s00114-013-1026-5
Etzioni, A., Luboshitzky, R., Tiosano, D., BenHarush, M., Goldsher, D., & Lavie, P.
(1996). Melatonin replacement corrects sleep disturbances in a child with pineal
tumor. Neurology, 46(1), 261-263.
Fenn, M. G. P., & Macdonald, D. W. (1995). USE OF MIDDENS BY RED FOXES RISK REVERSES RHYTHMS OF RATS. Journal of Mammalogy, 76(1), 130-136.
doi: 10.2307/1382321
Finkelstein, J. S., Baum, F. R., & Campbell, C. S. (1978). Entrainment of the female
hamster to reversed photoperiod: role of the pineal. Physiol Behav, 21(1), 105111.
Fisher, S. P., & Sugden, D. (2010). Endogenous Melatonin is Not Obligatory for the
Regulation of the Rat Sleep-Wake Cycle. Sleep, 33(6), 833-840.
Fite, K. V., Birkett, M. A., Smith, A., Janusonis, S. F., & McLaughlin, S. (2003). Retinal
ganglion cells projecting to the dorsal raphe and lateral geniculate complex in
Mongolian gerbils. Brain Res, 973(1), 146-150. doi: 10.1016/s00068993(03)02549-6
146

Fite, K. V., Wu, P. S., & Bellemer, A. (2005). Photostimulation alters c-Fos expression in
the dorsal raphe nucleus. Brain Res, 1031(2), 245-252. doi:
10.1016/j.brainres.2004.10.054
Fuller, C. A., Lydic, R., Sulzman, F. M., Albers, H. E., Tepper, B., & Mooreede, M. C.
(1981). CIRCADIAN-RHYTHM OF BODY-TEMPERATURE PERSISTS AFTER
SUPRACHIASMATIC LESIONS IN THE SQUIRREL-MONKEY. American
Journal of Physiology, 241(5), R385-R391.
Gaillard, F., Karten, H. J., & Sauve, Y. (2013). Retinorecipient areas in the diurnal
murine rodent Arvicanthis niloticus: A disproportionally large superior colliculus.
Journal of Comparative Neurology, 521(8), 1699-1726. doi: 10.1002/cne.23303
Gall, A. J., Smale, L., Yan, L., & Nunez, A. A. (2013). Lesions of the Intergeniculate
Leaflet Lead to a Reorganization in Circadian Regulation and a Reversal in
Masking Responses to Photic Stimuli in the Nile Grass Rat. PLoS One, 8(6),
e67387. doi: 10.1371/journal.pone.0067387
Gander, P. H., & Mooreede, M. C. (1983). LIGHT-DARK MASKING OF CIRCADIAN
TEMPERATURE AND ACTIVITY RHYTHMS IN SQUIRREL-MONKEYS.
American Journal of Physiology, 245(6), R927-R934.
Garidou, M. L., Gauer, F., Vivien-Roels, B., Sicard, B., Pevet, P., & Simonneaux, V.
(2002). Pineal arylalkylamine N-acetyltransferase gene expression is highly
stimulated at night in the diurnal rodent, Arvicanthis ansorgei. Eur J Neurosci,
15(10), 1632-1640.
Gaus, S. E., Strecker, R. E., Tate, B. A., Parker, R. A., & Saper, C. B. (2002).
Ventrolateral preoptic nucleus contains sleep-active, galaninergic neurons in
multiple mammalian species. Neuroscience, 115(1), 285-294.
Geusz, M. E., Fletcher, C., Block, G. D., Straume, M., Copeland, N. G., Jenkins, N. A., .
. . Day, R. N. (1997). Long-term monitoring of circadian rhythms in c-fos gene
expression from suprachiasmatic nucleus cultures. Curr Biol, 7(10), 758-766.
Gonzalez, J. A., & Dyball, R. E. (2006). Pinealectomy reduces optic nerve but not
intergeniculate leaflet input to the suprachiasmatic nucleus at night. J
Neuroendocrinol, 18(2), 146-153. doi: 10.1111/j.1365-2826.2005.01395.x

147

Gooley, J. J., Lu, J., Chou, T. C., Scammell, T. E., & Saper, C. B. (2001). Melanopsin in
cells of origin of the retinohypothalamic tract. Nat Neurosci, 4(12), 1165. doi:
10.1038/nn768
Gooley, J. J., Lu, J., Fischer, D., & Saper, C. B. (2003). Broad role for melanopsin in
nonvisual photoreception. Journal of Neuroscience, 23(18), 7093-7106.
Guler, A. D., Ecker, J. L., Lall, G. S., Haq, S., Altimus, C. M., Liao, H. W., . . . Hattar, S.
(2008). Melanopsin cells are the principal conduits for rod-cone input to nonimage-forming vision. Nature, 453(7191), 102-+. doi: 10.1038/nature06829
Gutman, R., & Dayan, T. (2005). Temporal partitioning: An experiment with two species
of spiny mice. Ecology, 86(1), 164-173. doi: 10.1890/03-0369
Haldar, C., Rai, S., & Singh, R. (2004). Melatonin blocks dexamethasone-induced
immunosuppression in a seasonally breeding rodent Indian palm squirrel,
Funambulus pennanti. Steroids, 69(6), 367-377. doi:
10.1016/j.steroids.2004.03.006
Hao, H. P., & Rivkees, S. (2000). Melatonin does not shift circadian phase in baboons.
Journal of Clinical Endocrinology & Metabolism, 85(10), 3618-3622. doi:
10.1210/jc.85.10.3618
Harrington, L. A., Harrington, A. L., Yamaguchi, N., Thom, M. D., Ferreras, P.,
Windham, T. R., & Macdonald, D. W. (2009). The impact of native competitors on
an alien invasive: temporal niche shifts to avoid interspecific aggression?
Ecology, 90(5), 1207-1216. doi: 10.1890/08-0302.1
Hastings, M. H., Roberts, A. C., & Herbert, J. (1985). Neurotoxic lesions of the anterior
hypothalamus disrupt the photoperiodic but not the circadian system of the
Syrian hamster. Neuroendocrinology, 40(4), 316-324.
Hatori, M., Le, H., Vollmers, C., Keding, S. R., Tanaka, N., Buch, T., . . . Panda, S.
(2008). Inducible ablation of melanopsin-expressing retinal ganglion cells reveals
their central role in non-image forming visual responses. PLoS One, 3(6), e2451.
doi: 10.1371/journal.pone.0002451
Hattar, S., Kumar, M., Park, A., Tong, P., Tung, J., Yau, K. W., & Berson, D. M. (2006).
Central projections of melanopsin-expressing retinal ganglion cells in the mouse.
Journal of Comparative Neurology, 497(3), 326-349. doi: 10.1002/cne.20970
148

Hendrickson, A. E., Wilson, J. R., & Ogren, M. P. (1978). The neuroanatomical
organization of pathways between the dorsal lateral geniculate nucleus and
visual cortex in Old World and New World primates. J Comp Neurol, 182(1), 123136. doi: 10.1002/cne.901820108
Herkenham, M., & Nauta, W. J. (1979). Efferent connections of the habenular nuclei in
the rat. J Comp Neurol, 187(1), 19-47. doi: 10.1002/cne.901870103
Hoffmann, K. P., Stone, J., & Sherman, S. M. (1972). Relay of receptive-field properties
in dorsal lateral geniculate nucleus of the cat. J Neurophysiol, 35(4), 518-531.
Honrado, G. I., Johnson, R. S., Golombek, D. A., Spiegelman, B. M., Papaioannou, V.
E., & Ralph, M. R. (1996). The circadian system of c-fos deficient mice. J Comp
Physiol A, 178(4), 563-570.
Hu, K., Scheer, F., Ivanov, P. C., Buijs, R. M., & Shea, S. A. (2007). The
suprachiasmatic nucleus functions beyond circadian rhythm generation.
Neuroscience, 149(3), 508-517. doi: 10.1016/j.neuroscience.2007.03.058
Huerta, J. J., Llamosas, M. M., Cernuda-Cernuda, R., & Garcia-Fernandez, J. M.
(1999). Spatio-temporal analysis of light-induced Fos expression in the retina of
rd mutant mice. Brain Res, 834(1-2), 122-127.
Hughes, R. J., & Badia, P. (1997). Melatonin and sleep - Sleep-promoting and
hypothermic effects of daytime melatonin administration in humans. Sleep, 20(2),
124-131.
Ibuka, N., Inouye, S. I. T., & Kawamura, H. (1977). ANALYSIS OF SLEEPWAKEFULNESS RHYTHMS IN MALE RATS AFTER SUPRACHIASMATIC
NUCLEUS LESIONS AND OCULAR ENUCLEATION. Brain Res, 122(1), 33-47.
doi: 10.1016/0006-8993(77)90660-6
Ibuka, N., & Kawamura, H. (1975). LOSS OF CIRCADIAN-RHYTHM IN SLEEPWAKEFULNESS CYCLE IN RAT BY SUPRACHIASMATIC NUCLEUS
LESIONS. Brain Res, 96(1), 76-81. doi: 10.1016/0006-8993(75)90574-0
Inui, Y., & Hazeki, O. (2010). Acute effects of melatonin and its time of administration on
core body temperature and heart rate in cynomolgus monkeys. Journal of
Toxicological Sciences, 35(3), 383-391.
149

Ishii, H., Tanaka, N., Kobayashi, M., Kato, M., & Sakuma, Y. (2009). Gene structures,
biochemical characterization and distribution of rat melatonin receptors. J Physiol
Sci, 59(1), 37-47. doi: 10.1007/s12576-008-0003-9
Iskra-Golec, I. M., Wazna, A., & Smith, L. (2012). Effects of blue-enriched light on the
daily course of mood, sleepiness and light perception: A field experiment.
Lighting Research & Technology, 44(4), 506-513. doi:
10.1177/1477153512447528
Jan, J. E., Tai, J., Hahn, G., & Rothstein, R. R. (2001). Melatonin replacement therapy
in a child with a pineal tumor. Journal of Child Neurology, 16(2), 139-140. doi:
10.1177/088307380101600215
Janik, D., Mikkelsen, J. D., & Mrosovsky, N. (1995). Cellular colocalization of Fos and
neuropeptide Y in the intergeniculate leaflet after nonphotic phase-shifting
events. Brain Res, 698(1-2), 137-145.
Janik, D., & Mrosovsky, N. (1992). Gene expression in the geniculate induced by a
nonphotic circadian phase shifting stimulus. Neuroreport, 3(7), 575-578.
Jechura, T. J., Mahoney, M. M., Stimpson, C. D., & Lee, T. M. (2006). Odor-specific
effects on reentrainment following phase advances in the diurnal rodent, Octodon
degus. American Journal of Physiology-Regulatory Integrative and Comparative
Physiology, 291(6), R1808-R1816. doi: 10.1152/ajpregu.00005.2006
Jin, E., Jia, L., Li, J., Yang, G., Wang, Z., Cao, J., & Chen, Y. (2011). Effect of
monochromatic light on melatonin secretion and arylalkylamine Nacetyltransferase mRNA expression in the retina and pineal gland of broilers.
Anat Rec (Hoboken), 294(7), 1233-1241. doi: 10.1002/ar.21408
Johnson, R. F., Morin, L. P., & Moore, R. Y. (1988). Retinohypothalamic projections in
the hamster and rat demonstrated using cholera toxin. Brain Res, 462(2), 301312.
Juhl, F., Hannibal, J., & Fahrenkrug, J. (2007). Photic induction of c-Fos in enkephalin
neurons of the rat intergeniculate leaflet innervated by retinal PACAP fibres. Cell
Tissue Res, 329(3), 491-502. doi: 10.1007/s00441-007-0422-6

150

Karolczak, M., Burbach, G. J., Sties, G., Korf, H. W., & Stehle, J. H. (2004). Clock gene
mRNA and protein rhythms in the pineal gland of mice. Eur J Neurosci, 19(12),
3382-3388. doi: 10.1111/j.0953-816X.2004.03444.x
Kas, M. J. H., & Edgar, D. M. (1999). A nonphotic stimulus inverts the diurnal-nocturnal
phase preference in Octodon degus. Journal of Neuroscience, 19(1), 328-333.
Katona, C., Rose, S., & Smale, L. (1998). The expression of Fos within the
suprachiasmatic nucleus of the diurnal rodent Arvicanthis niloticus. Brain Res,
791(1-2), 27-34.
Katz, P. S. (2011). Neural mechanisms underlying the evolvability of behaviour. Philos
Trans R Soc Lond B Biol Sci, 366(1574), 2086-2099. doi:
10.1098/rstb.2010.0336
Kocher, L., Brun, J., Borson-Chazot, F., Gonnaud, P.-M., & Claustrat, B. (2006).
Increased REM sleep associated with melatonin deficiency after pinealectomy: A
case study. Chronobiology International, 23(4), 889-901. doi:
10.1080/07420520600827095
Kornhauser, J. M., Mayo, K. E., & Takahashi, J. S. (1996). Light, immediate-early
genes, and circadian rhythms. Behav Genet, 26(3), 221-240.
Krajnak, K., Dickenson, L., & Lee, T. M. (1997). The induction of Fos-like proteins in the
suprachiasmatic nuclei and intergeniculate leaflet by light pulses in degus
(Octodon degus) and rats. J Biol Rhythms, 12(5), 401-412.
Kriegsfeld, L. J., Leak, R. K., Yackulic, C. B., LeSauter, J., & Silver, R. (2004).
Organization of suprachiasmatic nucleus projections in Syrian hamsters
(Mesocricetus auratus): an anterograde and retrograde analysis. J Comp Neurol,
468(3), 361-379. doi: 10.1002/cne.10995
Kronfeld-Schor, N., & Dayan, T. (2008). Activity patterns of rodents: the physiological
ecology of biological rhythms. Biol Rhythm Res, 39(3), 193-211. doi:
10.1080/09291010701683268
Lavie, P. (1997). Melatonin: Role in gating nocturnal rise in sleep propensity. Journal of
Biological Rhythms, 12(6), 657-665. doi: 10.1177/074873049701200622

151

LeGates, T. A., Altimus, C. M., Wang, H., Lee, H. K., Yang, S., Zhao, H., . . . Hattar, S.
(2012). Aberrant light directly impairs mood and learning through melanopsinexpressing neurons. Nature, 491(7425), 594-598. doi: 10.1038/nature11673
Lehmann, E. D., Cockerell, O. C., & Rudge, P. (1996). Somnolence associated with
melatonin deficiency after pinealectomy. Lancet, 347(8997), 323-323. doi:
10.1016/s0140-6736(96)90496-1
Lehner, M., Taracha, E., Skorzewska, A., Wislowska, A., Zienowicz, M., Maciejak, P., . .
. Plaznik, A. (2004). Sensitivity to pain and c-Fos expression in brain structures in
rats. Neurosci Lett, 370(1), 74-79. doi: 10.1016/j.neulet.2004.07.089
Lewy, A. J., Wehr, T. A., Goodwin, F. K., Newsome, D. A., & Markey, S. P. (1980). Light
suppresses melatonin secretion in humans. Science, 210(4475), 1267-1269.
Li, X. D., Gilbert, J., & Davis, F. C. (2005). Disruption of masking by hypothalamic
lesions in Syrian hamsters. Journal of Comparative Physiology a-Neuroethology
Sensory Neural and Behavioral Physiology, 191(1), 23-30. doi: 10.1007/s00359004-0569-5
Lima, A. G., Britto, L. R., Hossokawa, N. M., & Hamassaki-Britto, D. E. (2003). Static,
but not optokinetic visual stimuli induce Fos expression in the retina and brain of
retinal degeneration mice. Neurosci Lett, 342(1-2), 9-12.
Liu, X. G., Zhang, B. J., Xu, X. H., Huang, Z. L., & Qu, W. M. (2012). Lesions of
suprachiasmatic nucleus modify sleep structure but do not alter the total amount
of daily sleep in rats. Sleep and Biological Rhythms, 10(4), 293-301. doi:
10.1111/j.1479-8425.2012.00572.x
Lockley, S. W., Evans, E. E., Scheer, F., Brainard, G. C., Czeisler, C. A., & Aeschbach,
D. (2006). Short-wavelength sensitivity for the direct effects of light on alertness,
vigilance, and the waking electroencephalogram in humans. Sleep, 29(2), 161168.
Lonstein, J. S., & Stern, J. M. (1997). Role of the midbrain periaqueductal gray in
maternal nurturance and aggression: c-fos and electrolytic lesion studies in
lactating rats. Journal of Neuroscience, 17(9), 3364-3378.
Lopez-Olmeda, J. F., Madrid, J. A., & Sanchez-Vazquez, F. J. (2006). Melatonin effects
on food intake and activity rhythms in two fish species with different activity
152

patterns: Diurnal (goldfish) and nocturnal (tench). Comparative Biochemistry and
Physiology a-Molecular & Integrative Physiology, 144(2), 180-187. doi:
10.1016/j.cbpa.2006.02.031
Lu, J., Zhang, Y. H., Chou, T. C., Gaus, S. E., Elmquist, J. K., Shiromani, P., & Saper,
C. B. (2001). Contrasting effects of ibotenate lesions of the paraventricular
nucleus and subparaventricular zone on sleep-wake cycle and temperature
regulation. J Neurosci, 21(13), 4864-4874.
Lucas, R. J., Hattar, S., Takao, M., Berson, D. M., Foster, R. G., & Yau, K. W. (2003).
Diminished pupillary light reflex at high irradiances in melanopsin-knockout mice.
Science, 299(5604), 245-247. doi: 10.1126/science.1077293
Lupi, D., Cooper, H. M., Froehlich, A., Standford, L., McCall, M. A., & Foster, R. G.
(1999). Transgenic ablation of rod photoreceptors alters the circadian phenotype
of mice. Neuroscience, 89(2), 363-374.
Lupi, D., Oster, H., Thompson, S., & Foster, R. G. (2008). The acute light-induction of
sleep is mediated by OPN4-based photoreception. Nat Neurosci, 11(9), 10681073. doi: 10.1038/nn.2179
Lupi, D., Semo, M., & Foster, R. G. (2012). Impact of age and retinal degeneration on
the light input to circadian brain structures. Neurobiol Aging, 33(2), 383-392. doi:
10.1016/j.neurobiolaging.2010.03.006
Macchi, M. M., & Bruce, J. N. (2004). Human pineal physiology and functional
significance of melatonin. Front Neuroendocrinol, 25(3-4), 177-195. doi:
10.1016/j.yfrne.2004.08.001
Mahoney, M., Bult, A., & Smale, L. (2001). Phase response curve and light-induced fos
expression in the suprachiasmatic nucleus and adjacent hypothalamus of
Arvicanthis niloticus. J Biol Rhythms, 16(2), 149-162.
Mahoney, M. M., Smale, L., & Lee, T. M. (2009). Daily immediate early gene expression
in the suprachiasmatic nucleus of male and female Octodon degus. Chronobiol
Int, 26(5), 821-837. doi: 10.1080/07420520903044265
Marchant, E. G., & Morin, L. P. (1999). The hamster circadian rhythm system includes
nuclei of the subcortical visual shell. J Neurosci, 19(23), 10482-10493.
153

Marques, M. D., & Waterhouse, J. M. (1994). MASKING AND THE EVOLUTION OF
CIRCADIAN RHYTHMICITY. Chronobiology International, 11(3), 146-155. doi:
10.3109/07420529409057234
Martinet, L., & Zucker, I. (1985). Role of the pineal gland in circadian organization of
diurnal ground squirrels. Physiol Behav, 34(5), 799-803.
Martinez, G. S., Smale, L., & Nunez, A. A. (2002). Diurnal and nocturnal rodents show
rhythms in orexinergic neurons. Brain Res, 955(1-2), 1-7.
Masana, M. I., Benloucif, S., & Dubocovich, M. L. (1996). Light-induced c-fos mRNA
expression in the suprachiasmatic nucleus and the retina of C3H/HeN mice.
Brain Res Mol Brain Res, 42(2), 193-201.
Matsumoto, M. (1999). The hypnotic effects of melatonin treatment on diurnal sleep in
humans. Psychiatry Clin Neurosci, 53(2), 243-245. doi: 10.1046/j.14401819.1999.00480.x
Mayo, J. C., Sainz, R. M., Antolin, I., & Rodriguez, C. (1999). Ultrastructural
confirmation of neuronal protection by melatonin against the neurotoxin 6hydroxydopamine cell damage. Brain Res, 818(2), 221-227.
Mayo, J. C., Sainz, R. M., Uria, H., Antolin, I., Esteban, M. M., & Rodriguez, C. (1998).
Melatonin prevents apoptosis induced by 6-hydroxydopamine in neuronal cells:
implications for Parkinson's disease. J Pineal Res, 24(3), 179-192.
Mendelson, W. B., & Bergmann, B. M. (2001). Effects of pinealectomy on baseline
sleep and response to sleep deprivation. Sleep, 24(4), 369-373.
Mendoza, J., Clesse, D., Pevet, P., & Challet, E. (2010). Food-reward signalling in the
suprachiasmatic clock. Journal of Neurochemistry, 112(6), 1489-1499. doi:
10.1111/j.1471-4159.2010.06570.x
Mikkelsen, J. D. (1992). Visualization of efferent retinal projections by
immunohistochemical identification of cholera toxin subunit B. Brain Res Bull,
28(4), 619-623.

154

Miller, A. M., Miller, R. B., Obermeyer, W. H., Behan, M., & Benca, R. M. (1999). The
pretectum mediates rapid eye movement sleep regulation by light. Behav
Neurosci, 113(4), 755-765.
Minors, D. S., & Waterhouse, J. M. (1989a). MASKING IN HUMANS - THE PROBLEM
AND SOME ATTEMPTS TO SOLVE IT. Chronobiology International, 6(1), 29-53.
doi: 10.3109/07420528909059140
Minors, D. S., & Waterhouse, J. M. (1989b). Masking in humans: the problem and some
attempts to solve it. Chronobiol Int, 6(1), 29-53.
Mistlberger, R. E. (1992). Nonphotic entrainment of circadian activity rhythms in
suprachiasmatic nuclei-ablated hamsters. Behav Neurosci, 106(1), 192-202.
Mistlberger, R. E., Antle, M. C., Kilduff, T. S., & Jones, M. (2003). Food- and lightentrained circadian rhythms in rats with hypocretin-2-saporin ablations of the
lateral hypothalamus. Brain Res, 980(2), 161-168.
Mistlberger, R. E., Bergmann, B. M., Waldenar, W., & Rechtschaffen, A. (1983).
Recovery sleep following sleep deprivation in intact and suprachiasmatic nucleilesioned rats. Sleep, 6(3), 217-233.
Moore, R. Y., & Danchenko, R. L. (2002). Paraventricular-subparaventricular
hypothalamic lesions selectively affect circadian function. Chronobiol Int, 19(2),
345-360.
Morin, L. P. (1993). AGE, BUT NOT PINEAL STATUS, MODULATES CIRCADIAN
PERIODICITY OF GOLDEN-HAMSTERS. Journal of Biological Rhythms, 8(3),
189-197. doi: 10.1177/074873049300800302
Morin, L. P., & Blanchard, J. (1995). Organization of the hamster intergeniculate leaflet:
NPY and ENK projections to the suprachiasmatic nucleus, intergeniculate leaflet
and posterior limitans nucleus. Vis Neurosci, 12(1), 57-67.
Morin, L. P., & Cummings, L. A. (1981). EFFECT OF SURGICAL OR
PHOTOPERIODIC CASTRATION, TESTOSTERONE REPLACEMENT OR
PINEALECTOMY ON MALE HAMSTER RUNNING RHYTHMICITY. Physiology
& Behavior, 26(5), 825-838. doi: 10.1016/0031-9384(81)90106-2

155

Morin, L. P., Goodless-Sanchez, N., Smale, L., & Moore, R. Y. (1994). Projections of
the suprachiasmatic nuclei, subparaventricular zone and retrochiasmatic area in
the golden hamster. Neuroscience, 61(2), 391-410.
Morin, L. P., Lituma, P. J., & Studholme, K. M. (2010). Two Components of Nocturnal
Locomotor Suppression by Light. Journal of Biological Rhythms, 25(3), 197-207.
doi: 10.1177/0748730410369890
Morin, L. P., & Studholme, K. M. (2009). Millisecond Light Pulses Make Mice Stop
Running, then Display Prolonged Sleep-Like Behavior in the Absence of Light.
Journal of Biological Rhythms, 24(6), 497-508. doi: 10.1177/0748730409349059
Mouret, J., Coindet, J., & Chouvet, G. (1974). EFFECT OF PINEALECTOMY ON
SLEEP STAGES AND RHYTHMS OF MALE RAT. Brain Res, 81(1), 97-105. doi:
10.1016/0006-8993(74)90480-6
Mrosovsky, N. (1999). Masking: history, definitions, and measurement. Chronobiol Int,
16(4), 415-429.
Mrosovsky, N., Foster, R. G., & Salmon, P. A. (1999). Thresholds for masking
responses to light in three strains of retinally degenerate mice. Journal of
Comparative Physiology a-Sensory Neural and Behavioral Physiology, 184(4),
423-428. doi: 10.1007/s003590050341
Mrosovsky, N., & Hattar, S. (2003). Impaired masking responses to light in melanopsinknockout mice. Chronobiology International, 20(6), 989-999. doi: 10.1081/cbi120026043
Mrosovsky, N., & Hattar, S. (2005). Diurnal mice (Mus musculus) and other examples of
temporal niche switching. J Comp Physiol A Neuroethol Sens Neural Behav
Physiol, 191(11), 1011-1024. doi: 10.1007/s00359-005-0017-1
Mrosovsky, N., Salmon, P. A., Foster, R. G., & McCall, M. A. (2000). Responses to light
after retinal degeneration. Vision Res, 40(6), 575-578.
Mrosovsky, N., & Thompson, S. (2008). Negative and positive masking responses to
light in retinal degenerate slow (rds/rds) mice during aging. Vision Res, 48(10),
1270-1273. doi: 10.1016/j.visres.2008.02.016

156

Murakami, N., Kawano, T., Nakahara, K., Nasu, T., & Shiota, K. (2001). Effect of
melatonin on circadian rhythm, locomotor activity and body temperature in the
intact house sparrow, Japanese quail and owl. Brain Res, 889(1-2), 220-224. doi:
10.1016/s0006-8993(00)03205-4
Muscat, L., & Morin, L. P. (2006). Intergeniculate leaflet: contributions to photic and nonphotic responsiveness of the hamster circadian system. Neuroscience, 140(1),
305-320. doi: 10.1016/j.neuroscience.2006.01.050
Nave, R., Herer, P., Haimov, I., Shlitner, A., & Lavie, P. (1996). Hypnotic and
hypothermic effects of melatonin on daytime sleep in humans: Lack of
antagonism by flumazenil. Neurosci Lett, 214(2-3), 123-126. doi: 10.1016/03043940(96)12899-8
Nelson, D. E., & Takahashi, J. S. (1991). Comparison of visual sensitivity for
suppression of pineal melatonin and circadian phase-shifting in the golden
hamster. Brain Res, 554(1-2), 272-277.
Nixon, J. P., & Smale, L. (2004). Individual differences in wheel-running rhythms are
related to temporal and spatial patterns of activation of orexin A and B cells in a
diurnal rodent (Arvicanthis niloticus). Neuroscience, 127(1), 25-34. doi:
10.1016/j.neuroscience.2004.04.052
Novak, C. M., & Albers, H. E. (2002). N-Methyl-D-aspartate microinjected into the
suprachiasmatic nucleus mimics the phase-shifting effects of light in the diurnal
Nile grass rat (Arvicanthis niloticus). Brain Res, 951(2), 255-263.
Novak, C. M., Smale, L., & Nunez, A. A. (1999). Fos expression in the sleep-active cell
group of the ventrolateral preoptic area in the diurnal murid rodent, Arvicanthis
niloticus. Brain Res, 818(2), 375-382.
Nunez, A. A., Bult, A., McElhinny, T. L., & Smale, L. (1999). Daily rhythms of Fos
expression in hypothalamic targets of the suprachiasmatic nucleus in diurnal and
nocturnal rodents. J Biol Rhythms, 14(4), 300-306.
Otalora, B. B., Madrid, J. A., Alvarez, N., Vicente, V., & Rol, M. A. (2008). Effects of
exogenous melatonin and circadian synchronization on tumor progression in
melanoma-bearing C57BL6 mice. J Pineal Res, 44(3), 307-315. doi:
10.1111/j.1600-079X.2007.00531.x

157

Otalora, B. B., Vivanco, P., Madariaga, A. M., Madrid, J. A., & Rol, M. A. (2010).
INTERNAL TEMPORAL ORDER IN THE CIRCADIAN SYSTEM OF A DUALPHASING RODENT, THE OCTODON DEGUS. Chronobiology International,
27(8), 1564-1579. doi: 10.3109/07420528.2010.503294
Panda, S., Provencio, I., Tu, D. C., Pires, S. S., Rollag, M. D., Castrucci, A. M., . . .
Hogenesch, J. B. (2003). Melanopsin is required for non-image-forming photic
responses in blind mice. Science, 301(5632), 525-527. doi:
10.1126/science.1086179
Panda, S., Sato, T. K., Castrucci, A. M., Rollag, M. D., DeGrip, W. J., Hogenesch, J. B.,
. . . Kay, S. A. (2002). Melanopsin (Opn4) requirement for normal light-induced
circadian phase shifting. Science, 298(5601), 2213-2216. doi:
10.1126/science.1076848
Park, H. T., Baek, S. Y., Kim, B. S., Kim, J. B., & Kim, J. J. (1993). Profile of Fos-like
immunoreactivity induction by light stimuli in the intergeniculate leaflet is different
from that of the suprachiasmatic nucleus. Brain Res, 610(2), 334-339.
Paul, M. J., Indic, P., & Schwartz, W. J. (2011). A role for the habenula in the regulation
of locomotor activity cycles. Eur J Neurosci, 34(3), 478-488. doi: 10.1111/j.14609568.2011.07762.x
Peichl, L. (2005). Diversity of mammalian photoreceptor properties: Adaptations to
habitat and lifestyle? Anatomical Record Part a-Discoveries in Molecular Cellular
and Evolutionary Biology, 287A(1), 1001-1012. doi: 10.1002/ar.a.20262
Pendergast, J. S., & Yamazaki, S. (2011). Masking Responses to Light in Period Mutant
Mice. Chronobiology International, 28(8), 657-663. doi:
10.3109/07420528.2011.596296
Perreau-Lenz, S., Kalsbeek, A., Garidou, M. L., Wortel, J., van der Vliet, J., van
Heijningen, C., . . . Buijs, R. M. (2003). Suprachiasmatic control of melatonin
synthesis in rats: inhibitory and stimulatory mechanisms. Eur J Neurosci, 17(2),
221-228.
Perreau-Lenz, S., Kalsbeek, A., Pevet, P., & Buijs, R. M. (2004). Glutamatergic clock
output stimulates melatonin synthesis at night. Eur J Neurosci, 19(2), 318-324.

158

Peters, R. V., Aronin, N., & Schwartz, W. J. (1996). c-Fos expression in the rat
intergeniculate leaflet: photic regulation, co-localization with Fos-B, and cellular
identification. Brain Res, 728(2), 231-241.
Petterborg, L. J., Thalen, B. E., Kjellman, B. F., & Wetterberg, L. (1991). EFFECT OF
MELATONIN REPLACEMENT ON SERUM HORMONE RHYTHMS IN A
PATIENT LACKING ENDOGENOUS MELATONIN. Brain Research Bulletin,
27(2), 181-185. doi: 10.1016/0361-9230(91)90065-r
Pevet, P. (2003). Melatonin: from seasonal to circadian signal. J Neuroendocrinol,
15(4), 422-426.
Poveda, A., & Kretz, R. (2009). c-Fos expression in the visual system of the tree shrew
(Tupaia belangeri). J Chem Neuroanat, 37(4), 214-228. doi:
10.1016/j.jchemneu.2009.03.006
Prichard, J. R., Stoffel, R. T., Quimby, D. L., Obermeyer, W. H., Benca, R. M., & Behan,
M. (2002). Fos immunoreactivity in rat subcortical visual shell in response to
illuminance changes. Neuroscience, 114(3), 781-793.
Quay, W. B. (1970). PRECOCIOUS ENTRAINMENT AND ASSOCIATED
CHARACTERISTICS OF ACTIVITY PATTERNS FOLLOWING PINEALECTOMY
AND REVERSAL OF PHOTOPERIOD. Physiology & Behavior, 5(11), 1281-&.
doi: 10.1016/0031-9384(70)90041-7
Quay, W. B. (1971). Dissimilar functional effects of pineal stalk and cerebral meningeal
interruptions on phase shifts of circadian activity rhythms. Physiol Behav, 7(4),
557-567.
Ramanathan, C., Nunez, A. A., & Smale, L. (2008). Daily rhythms in PER1 within and
beyond the suprachiasmatic nucleus of female grass rats (Arvicanthis niloticus).
Neuroscience, 156(1), 48-58. doi: 10.1016/j.neuroscience.2008.07.020
Rasmussen, G. S. A., & Macdonald, D. W. (2012). Masking of the zeitgeber: African
wild dogs mitigate persecution by balancing time. Journal of Zoology, 286(3),
232-242. doi: 10.1111/j.1469-7998.2011.00874.x
Redfern, P., Minors, D., & Waterhouse, J. (1994). CIRCADIAN-RHYTHMS, JET-LAG,
AND CHRONOBIOTICS - AN OVERVIEW. Chronobiology International, 11(4),
253-265. doi: 10.3109/07420529409067793
159

Redlin, U. (2001). Neural basis and biological function of masking by light in mammals:
Suppression of melatonin and locomotor activity. Chronobiology International,
18(5), 737-758. doi: 10.1081/cbi-100107511
Redlin, U., Cooper, H. M., & Mrosovsky, N. (2003). Increased masking response to light
after ablation of the visual cortex in mice. Brain Res, 965(1-2), 1-8. doi:
10.1016/s0006-8993(02)03844-1
Redlin, U., & Mrosovsky, N. (1999a). Masking by light in hamsters with SCN lesions.
Journal of Comparative Physiology a-Sensory Neural and Behavioral Physiology,
184(4), 439-448. doi: 10.1007/s003590050343
Redlin, U., & Mrosovsky, N. (1999b). Masking of locomotor activity in hamsters. Journal
of Comparative Physiology a-Sensory Neural and Behavioral Physiology, 184(4),
429-437. doi: 10.1007/s003590050342
Redlin, U., & Mrosovsky, N. (2004). Nocturnal activity in a diurnal rodent (Arvicanthis
niloticus): The importance of masking. Journal of Biological Rhythms, 19(1), 5867. doi: 10.1177/0748730403260371
Redlin, U., Vrang, N., & Mrosovsky, N. (1999). Enhanced masking response to light in
hamsters with IGL lesions. Journal of Comparative Physiology a-Sensory Neural
and Behavioral Physiology, 184(4), 449-456. doi: 10.1007/s003590050344
Refinetti, R. (2008). The diversity of temporal niches in mammals. Biol Rhythm Res,
39(3), 173-192. doi: 10.1080/09291010701682690
Reiter, R. J. (1993). The melatonin rhythm: both a clock and a calendar. Experientia,
49(8), 654-664.
Reiter, R. J., Tan, D. X., Manchester, L. C., Paredes, S. D., Mayo, J. C., & Sainz, R. M.
(2009). Melatonin and reproduction revisited. Biol Reprod, 81(3), 445-456. doi:
10.1095/biolreprod.108.075655
Rotics, S., Dayan, T., Levy, O., & Kronfeld-Schor, N. (2011). Light Masking in the Field:
An Experiment with Nocturnal and Diurnal Spiny Mice Under Semi-natural Field
Conditions. Chronobiology International, 28(1), 70-75. doi:
10.3109/07420528.2010.525674

160

Rusak, B., Robertson, H. A., Wisden, W., & Hunt, S. P. (1990). Light pulses that shift
rhythms induce gene expression in the suprachiasmatic nucleus. Science,
248(4960), 1237-1240.
Sara, S. J., & Bouret, S. (2012). Orienting and reorienting: the locus coeruleus mediates
cognition through arousal. Neuron, 76(1), 130-141. doi:
10.1016/j.neuron.2012.09.011
Sato, T., & Kawamura, H. (1984). Circadian rhythms in multiple unit activity inside and
outside the suprachiasmatic nucleus in the diurnal chipmunk (Eutamias sibiricus).
Neurosci Res, 1(1), 45-52.
Scheer, F., Ter Horst, G. J., Van der Vliet, J., & Buijs, R. M. (2001). Physiological and
anatomic evidence for regulation of the heart by suprachiasmatic nucleus in rats.
American Journal of Physiology-Heart and Circulatory Physiology, 280(3),
H1391-H1399.
Scheer, F., Zeitzer, J. M., Ayas, N. T., Brown, R., Czeisler, C. A., & Shea, S. A. (2006).
Reduced sleep efficiency in cervical spinal cord injury; association with abolished
night time melatonin secretion. Spinal Cord, 44(2), 78-81. doi:
10.1038/sj.sc.3101784
Scheer, F. A., Ter Horst, G. J., van Der Vliet, J., & Buijs, R. M. (2001). Physiological and
anatomic evidence for regulation of the heart by suprachiasmatic nucleus in rats.
Am J Physiol Heart Circ Physiol, 280(3), H1391-1399.
Schrader, J. A., Walaszczyk, E. J., & Smale, L. (2009). Changing patterns of daily
rhythmicity across reproductive states in diurnal female Nile grass rats
(Arvicanthis niloticus). Physiology & Behavior, 98(5), 547-556. doi:
10.1016/j.physbeh.2009.08.012
Schumann, D. M., Cooper, H. M., Hofmeyr, M. D., & Bennett, N. C. (2006). Lightinduced Fos expression in the suprachiasmatic nucleus of the four-striped field
mouse, Rhabdomys pumilio: A southern African diurnal rodent. Brain Res Bull,
70(4-6), 270-277. doi: 10.1016/j.brainresbull.2006.04.009
Schwarcz, R., & Kohler, C. (1983). Differential vulnerability of central neurons of the rat
to quinolinic acid. Neurosci Lett, 38(1), 85-90.

161

Schwartz, M. D., Nunez, A. A., & Smale, L. (2004). Differences in the suprachiasmatic
nucleus and lower subparaventricular zone of diurnal and nocturnal rodents.
Neuroscience, 127(1), 13-23. doi: 10.1016/j.neuroscience.2004.04.049
Schwartz, M. D., Nunez, A. A., & Smale, L. (2009). Rhythmic cFos expression in the
ventral subparaventricular zone influences general activity rhythms in the Nile
grass rat, Arvicanthis niloticus. Chronobiol Int, 26(7), 1290-1306. doi:
10.3109/07420520903415742
Schwartz, M. D., Urbanski, H. F., Nunez, A. A., & Smale, L. (2011). Projections of the
suprachiasmatic nucleus and ventral subparaventricular zone in the Nile grass
rat (Arvicanthis niloticus). Brain Res, 1367, 146-161. doi:
10.1016/j.brainres.2010.10.058
Sherin, J. E., Shiromani, P. J., McCarley, R. W., & Saper, C. B. (1996). Activation of
ventrolateral preoptic neurons during sleep. Science, 271(5246), 216-219.
Shkolnik, A. (1971). Diurnal activity in a small desert rodent. Int J Biometeorol, 15(2),
115-120.
Shuboni, D. D., Cramm, S., Yan, L., Nunez, A. A., & Smale, L. (2012). Acute Behavioral
Responses to Light and Darkness in Nocturnal Mus musculus and Diurnal
Arvicanthis niloticus. Journal of Biological Rhythms, 27(4), 299-307. doi:
10.1177/0748730412449723
Simons, M. J. P. (2009). The Evolution of the Cyanobacterial Posttranslational Clock
from a Primitive "Phoscillator". Journal of Biological Rhythms, 24(3), 175-182.
doi: 10.1177/0748730409333953
Slawik, H. C., Stoffel, M., Vesely, Z., Lehmberg, J., Pohl, C., Meyer, B., . . . Krieg, S. M.
(2012). Salivary melatonin concentration and sleep patterns before and after
pinealectomy in patients with pineocytoma WHO degrees I. Journal of Sleep
Research, 21, 210-210.
Slominski, R. M., Reiter, R. J., Schlabritz-Loutsevitch, N., Ostrom, R. S., & Slominski, A.
T. (2012). Melatonin membrane receptors in peripheral tissues: distribution and
functions. Mol Cell Endocrinol, 351(2), 152-166. doi: 10.1016/j.mce.2012.01.004

162

Smale, L., & Boverhof, J. (1999). The suprachiasmatic nucleus and intergeniculate
leaflet of Arvicanthis niloticus, a diurnal murid rodent from East Africa. J Comp
Neurol, 403(2), 190-208.
Smale, L., Lee, T., & Nunez, A. A. (2003). Mammalian diurnality: Some facts and gaps.
Journal of Biological Rhythms, 18(5), 356-366. doi: 10.1177/0748730403256651
Smale, L., Nunez, A. A., & Schwartz, M. D. (2008). Rhythms in a diurnal brain. Biol
Rhythm Res, 39(3), 305-318. doi: 10.1080/09291010701682666
Stankov, B., Fraschini, F., & Reiter, R. J. (1991). Melatonin binding sites in the central
nervous system. Brain Res Brain Res Rev, 16(3), 245-256.
Stephan, F. K., & Zucker, I. (1972). Circadian rhythms in drinking behavior and
locomotor activity of rats are eliminated by hypothalamic lesions. Proc Natl Acad
Sci U S A, 69(6), 1583-1586.
Strassman, R. J., Qualls, C. R., Lisansky, E. J., & Peake, G. T. (1991). ELEVATED
RECTAL TEMPERATURE PRODUCED BY ALL-NIGHT BRIGHT LIGHT IS
REVERSED BY MELATONIN INFUSION IN MEN. Journal of Applied
Physiology, 71(6), 2178-2182.
Studholme, K. M., Gompf, H. S., & Morin, L. P. (2013). Brief light stimulation during the
mouse nocturnal activity phase simultaneously induces a decline in core
temperature and locomotor activity followed by EEG-determined sleep. American
Journal of Physiology-Regulatory Integrative and Comparative Physiology,
304(6), R459-R471. doi: 10.1152/ajpregu.00460.2012
Szkudlarek, H. J., Orlowska, P., & Lewandowski, M. H. (2012). Light-induced responses
of slow oscillatory neurons of the rat olivary pretectal nucleus. PLoS One, 7(3),
e33083. doi: 10.1371/journal.pone.0033083
Tavakoli-Nezhad, M., & Schwartz, W. J. (2006). Hamsters running on time: is the lateral
habenula a part of the clock? Chronobiol Int, 23(1-2), 217-224. doi:
10.1080/07420520500521947
Teixeira, L., Lowden, A., da Luz, A. A., Turte, S. L., Moreno, C. R., Valente, D., . . .
Fischer, F. M. (2013). Exposure to bright light during evening class hours
increases alertness among working college students. Sleep Medicine, 14(1), 9197. doi: 10.1016/j.sleep.2012.08.017
163

Thompson, S., Foster, R. G., Stone, E. M., Sheffield, V. C., & Mrosovsky, N. (2008).
Classical and melanopsin photoreception in irradiance detection: negative
masking of locomotor activity by light. European Journal of Neuroscience, 27(8),
1973-1979. doi: 10.1111/j.1460-9568.2008.06168.x
Todd, W. D., Gall, A. J., Weiner, J. A., & Blumberg, M. S. (2012). Distinct
retinohypothalamic innervation patterns predict the developmental emergence of
species-typical circadian phase preference in nocturnal Norway rats and diurnal
nile grass rats. J Comp Neurol, 520(14), 3277-3292. doi: 10.1002/cne.23098
Tong, M., Watanabe, E., Yamamoto, N., Nagahata-Ishiguro, M., Maemura, K., Takeda,
N., . . . Ozaki, Y. (2013). Circadian expressions of cardiac ion channel genes in
mouse might be associated with the central clock in the SCN but not the
peripheral clock in the heart. Biol Rhythm Res, 44(4), 519-530. doi:
10.1080/09291016.2012.704801
Tsai, J. W., Hannibal, J., Hagiwara, G., Colas, D., Ruppert, E., Ruby, N. F., . . . Bourgin,
P. (2009). Melanopsin as a sleep modulator: circadian gating of the direct effects
of light on sleep and altered sleep homeostasis in Opn4(-/-) mice. PLoS Biol,
7(6), e1000125. doi: 10.1371/journal.pbio.1000125
Tsujino, N., & Sakurai, T. (2013). Role of orexin in modulating arousal, feeding, and
motivation. Front Behav Neurosci, 7, 28. doi: 10.3389/fnbeh.2013.00028
Tzischinsky, O., & Lavie, P. (1994). Melatonin possesses time-dependent hypnotic
effects. Sleep, 17(7), 638-645.
Vilaplana, J., Cambras, T., & Dieznoguera, A. (1994). EFFECTS OF SHORT LIGHTDARK CYCLES ON THE MOTOR-ACTIVITY RHYTHM OF PINEALECTOMIZED
RATS. Biol Rhythm Res, 25(2), 198-201. doi: 10.1080/09291019409360289
Vivanco, P., Rol, M. A., & Madrid, J. A. (2010). PACEMAKER PHASE CONTROL
VERSUS MASKING BY LIGHT: SETTING THE CIRCADIAN CHRONOTYPE IN
DUAL OCTODON DEGUS. Chronobiology International, 27(7), 1365-1379. doi:
10.3109/07420528.2010.502984
Wachulec, M., Li, H., Tanaka, H., Peloso, E., & Satinoff, E. (1997). Suprachiasmatic
nuclei lesions do not eliminate homeostatic thermoregulatory responses in rats.
Journal of Biological Rhythms, 12(3), 226-234. doi:
10.1177/074873049701200304
164

Wang, F., Li, J. C., Wu, C. F., Yang, J. Y., Zhang, R. M., & Chai, H. F. (2003).
Influences of a light-dark profile and the pineal gland on the hypnotic activity of
melatonin in mice and rats. Journal of Pharmacy and Pharmacology, 55(9),
1307-1312. doi: 10.1211/0022357021729
Warren, W. S., Champney, T. H., & Cassone, V. M. (1994). THE SUPRACHIASMATIC
NUCLEUS CONTROLS THE CIRCADIAN-RHYTHM OF HEART-RATE VIA THE
SYMPATHETIC NERVOUS-SYSTEM. Physiology & Behavior, 55(6), 1091-1099.
doi: 10.1016/0031-9384(94)90392-1
Watts, A. G., Swanson, L. W., & Sanchez-Watts, G. (1987). Efferent projections of the
suprachiasmatic nucleus: I. Studies using anterograde transport of Phaseolus
vulgaris leucoagglutinin in the rat. J Comp Neurol, 258(2), 204-229. doi:
10.1002/cne.902580204
Wehr, T. A. (1991). The durations of human melatonin secretion and sleep respond to
changes in daylength (photoperiod). J Clin Endocrinol Metab, 73(6), 1276-1280.
Weinert, D., Weinandy, R., & Gattermann, R. (2007). Photic and non-photic effects on
the daily activity pattern of Mongolian gerbils. Physiology & Behavior, 90(2-3),
325-333. doi: 10.1016/j.physbeh.2006.09.019
Wollnik, F., Brysch, W., Uhlmann, E., Gillardon, F., Bravo, R., Zimmermann, M., . . .
Herdegen, T. (1995). Block of c-Fos and JunB expression by antisense
oligonucleotides inhibits light-induced phase shifts of the mammalian circadian
clock. Eur J Neurosci, 7(3), 388-393.
Xia, C.-M., Shao, C.-H., Xin, L., Wang, Y.-R., Ding, C.-N., Wang, J., . . . Zhu, D.-N.
(2008). Effects of melatonin on blood pressure in stress-induced hypertension in
rats. Clinical and Experimental Pharmacology and Physiology, 35(10), 12581264. doi: 10.1111/j.1440-1681.2008.05000.x
Yanovski, J. A., Rosenwasser, A. M., Levine, J. D., & Adler, N. T. (1990). The circadian
activity rhythms of rats with mid- and parasagittal 'split-SCN' knife cuts and
pinealectomy. Brain Res, 537(1-2), 216-226.
Zhang, S. W., Zeitzer, J. M., Yoshida, Y., Wisor, J. P., Nishino, S., Edgar, D. M., &
Mignot, E. (2004). Lesions of the suprachiasmatic nucleus eliminate the daily
rhythm of hypocretin-1 release. Sleep, 27(4), 619-627.

165

Zhang, Y., Kornhauser, J. M., Zee, P. C., Mayo, K. E., Takahashi, J. S., & Turek, F. W.
(1996). Effects of aging on light-induced phase-shifting of circadian behavioral
rhythms, fos expression and CREB phosphorylation in the hamster
suprachiasmatic nucleus. Neuroscience, 70(4), 951-961.
Zhang, Y., Van Reeth, O., Zee, P. C., Takahashi, J. S., & Turek, F. W. (1993). Fos
protein expression in the circadian clock is not associated with phase shifts
induced by a nonphotic stimulus, triazolam. Neurosci Lett, 164(1-2), 203-208.
Zhao, H., & Rusak, B. (2005). Circadian firing-rate rhythms and light responses of rat
habenular nucleus neurons in vivo and in vitro. Neuroscience, 132(2), 519-528.
doi: 10.1016/j.neuroscience.2005.01.012
Zhdanova, I. V. (2005). Melatonin as a hypnotic: Pro. Sleep Medicine Reviews, 9(1), 5165. doi: 10.1016/j.smrc.2004.04.003
Zhdanova, I. V., Geiger, D. A., Schwagerl, A. L., Leclair, O. U., Killiany, R., Taylor, J. A.,
. . . Madras, B. K. (2002). Melatonin promotes sleep in three species of diurnal
nonhuman primates. Physiology & Behavior, 75(4), 523-529. doi: 10.1016/s00319384(02)00654-6
Zhdanova, I. V., Wang, S. Y., Leclair, O. U., & Danilova, N. P. (2001). Melatonin
promotes sleep-like state in zebrafish. Brain Res, 903(1-2), 263-268. doi:
10.1016/s0006-8993(01)02444-1
Zhdanova, I. V., & Wurtman, R. J. (1997). Efficacy of melatonin as a sleep-promoting
agent. J Biol Rhythms, 12(6), 644-650.
Zhdanova, I. V., Wurtman, R. J., Lynch, H. J., Ives, J. R., Dollins, A. B., Morabito, C., . .
. Schomer, D. L. (1995). SLEEP-INDUCING EFFECTS OF LOW-DOSES OF
MELATONIN INGESTED IN THE EVENING. Clinical Pharmacology &
Therapeutics, 57(5), 552-558. doi: 10.1016/0009-9236(95)90040-3
Zhdanova, I. V., Wurtman, R. J., Morabito, C., Piotrovska, V. R., & Lynch, H. J. (1996).
Effects of low oral doses of melatonin, given 2-4 hours before habitual bedtime,
on sleep in normal young humans. Sleep, 19(5), 423-431.

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