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Michigan State
University

This is to certify that the
dissertation entitled

ANATOMY AND FUNCTION OF OREXlN-CONTAINING
NEURONS IN DAY- AND NIGHT-ACTIVE ANIMALS

presented by
JOSHUA PATRICK NIXON

has been accepted towards fulfillment
of the requirements for the

PhD. degree in Department of Zoology and
Ecology. Evolutionary Biology

 

 

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ANATOMY AND FUNCTION OF OREXIN-CONTAINING NEURONS IN DAY-
AND NIGHT-ACTIVE ANIMALS

By

Joshua Patrick Nixon

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Zoology and Ecology, Evolutionary Biology, and Behavior
Program

2005

ABSTRACT

ANATOMY AND FUNCTION OF OREXIN-CONTAINING NEURONS IN DAY-
AND NIGHT-ACTIVE ANIMALS

By
Joshua Patrick Nixon

The orexins are a family of hypothalamic peptides implicated in the
modulation of feeding, arousal state, and somatomotor functions. The first part of
this dissertation examines the distribution of cell bodies and fibers expressing
immunoreactivity for orexin A (OXA) or orexin B (OXB) in two nocturnal rodents,
the Long-Evans rat (Rattus norvegicus) and Syrian hamster (Mesocricetus
auratus), and two diurnal rodents, the Nile grass rat (Arvicanthis niloticus) and
the degu (Octodon degus). I show that although the overall distribution of orexin
is very similar in these species, there are significant species differences in the
distribution of orexin cell bodies as well as in the density of orexin-IR fibers in
some regions. Specifically, I show species differences in the organization of the
main body of orexin neurons in the lateral hypothalamus, and provide evidence
for a previously undescribed population of OXA neurons in the magnocellular
neurosecretory nuclei of the hypothalamus. With respect to orexin fiber density,
the differences observed were few, but dramatic. The majority of the differences
were observed in the degu, suggesting that this species is an outlier.

The second series of experiments investigated the functional and
anatomical relationships between the orexins and patterns of activity in the grass
rat. Some individuals of this species switch to a more nocturnal pattern of activity

when given access to a running wheel, while others continue to be most active

during the day. In both day- and night-active grass rats, the percentages of
orexin A (OXA) and orexin B (OXB) cells expressing Fos were highest when
animals were actively running in wheels. In night-active animals, removal of the
running wheel significantly decreased Fos expression in OXA and OXB cells.
Additionally, in night-active animals, clear regional differences were apparent,
such that the presence of a wheel induced Fos in a higher percentage of orexin
cells in medial regions of the lateral hypothalamus (LH) than in lateral regions.
No regional differences were observed in day-active animals.

I also characterize the relationship between the orexins and neuropeptide-
Y cells in the intergeniculate leaflet (IGL). These cells are known to modulate
effects of arousal on the mammalian circadian system. However, the route
through which this information reaches the IGL has not been established. I
present evidence that OXA and OXB cells have projections to the IGL that
appear to make contact with NPY cells, and discuss the possibility that these
fibers may be involved in relaying feedback regarding the activity state of the
animal to the circadian system through these projections. Next, I demonstrate
that many NPY cells in the grass rat IGL exhibiting OXA fiber appositions are
significantly more likely to express Fos during nocturnal wheel running than are
NPY cells without such contacts (p<0.001). Finally, using the retrograde tract-
tracer cholera toxin B, I show that 20 percent of all OXA neurons project to the
IGL, and that these OXA neurons projecting to the IGL do not form a

topographically isolated cluster of cells within the lateral hypothalamus.

Copyright by
JOSHUA PATRICK NIXON

2005

For my mother, Jude, who encouraged me to dream; and my wife, Amy, for

reminding me to work towards those dreams.

Acknowledgements

I owe a debt of gratitude to a number of people who have helped me to
complete this dissertation. First and foremost I would like to thank my advisor, Dr.
Laura Smale, for taking me on as a graduate student. Despite my tendency to
procrastinate, Laura has been incredibly patient and generous with her time, and
I greatly appreciate her advice and friendship.

I would also like to thank the members of my committee, Drs. Kay
Holekamp, Antonio Nunez, and Lynwood Clemens for their input and feedback. I
would like to specifically thank Kay for cutting me off when I got too wordy, Tony
for correcting me the many times I was completely wrong, and Lyn for never
losing his sense of humor every time I had to re-schedule a committee meeting.

I wish to extend thanks to past and present members of the Smale and
Nufiez labs. I would like to thank Dr. Megan Mahoney, Michael Schwartz, Dr.
Chidambaram Ramanathan, Dr. Gladys Martinez, Jessica St. John, and
Alexandra Castillo-Ruiz for their friendship, feedback, and willingness to put up
with my awful sense of humor. Special thanks to Megan and Mike for helping me
to develop as a graduate student - their friendship, humor, and willingness to go
for coffee at any time helped make this whole thing fun. I would like to thank Dr.
Colleen Novak for getting me interested in orexin in the first place. I was also
fortunate to have the assistance of a number of superb undergraduates and
technicians working in the Smale Lab, including Heather Ross, Betty Gubik, Joel

Breen, Janaina Gamez, Anna Baumgras, and Nicole Timm.

vi

Thanks also to the many people in the Department who helped me
develop as a student as well as an instructor, especially Dr. Susan Hill, Dr. Will
Kopachik, and numerous fellow graduate students who have served as teaching
assistants in the Biological Science, Developmental Biology and Comparative
Anatomy courses over the last six years.

The work described in this thesis could not have been completed without
the assistance of a number of additional people. I thank Drs. Theresa Lee, Cheryl
Sisk, and Lyn Clemens for donating specimens used in Chapter 2. Dr. Sharleen
Sakai provided invaluable advice on technical procedures for Chapters 4 and 5,
and Dr. John I. Johnson graciously offered his assistance in the organization and
analysis of data for Chapter 2. l also thank David McFarlane for the many hours
he spent helping to set up and troubleshoot the data recording systems and
computers used in our lab.

Financial support for this research came from NIMH RO1-MH53433
awarded to Laura Smale, a grant from the Sigma Xi Foundation, and funding
from the Graduate School, the Department of Zoology, and the Ecology,
Evolutionary Biology and Behavior Program at Michigan State University.

Finally, I would like to thank my friends and family for their support and
encouragement. Thanks to my fellow Spartan alumni from 8 South Hubbard for
your friendship from our undergrad days to the present. Special thanks to the
Pavelka family for your amazing generosity and friendship over the years. Last
but not least, to my wife, my parents, my siblings, and my extended family, thank

you all for believing in me.

vii

TABLE OF CONTENTS

LIST OF TABLES ............................................................................... x
LIST OF FIGURES ............................................................................ xi
KEY TO ABBREVIATIONS .............................................................. xv
CHAPTER 1
Introduction ....................................................................................... 1
General Introduction .......................................................................................... 1
Overview of orexin ............................................................................................. 1
Orexin discovery ............................................................................................ 1
Orexin and feeding ......................................................................................... 4
Orexin and arousal ......................................................................................... 8
Other actions of orexin ................................................................................... 9
Introduction to the grass rat ............................................................................. 11
Overview of chapters ....................................................................................... 13
CHAPTER 2
A comparative analysis of the distribution of orexin in the brains
of nocturnal and diurnal rodents ................................................... 17
Introduction ...................................................................................................... 17
Methods and materials .................................................................................... 23
Animal handling ........................................................................................... 23
Tissue collection and processing ................................................................. 23
Cell and fiber counts and analysis ............................................................... 25
Results ............................................................................................................ 26
Orexin cell bodies ........................................................................................ 26
Orexin fiber distribution ................................................................................ 49
Discussion ....................................................................................................... 74
General observations ................................................................................... 74
Species differences in OXA and OXB cell distribution ................................. 77
Species differences in OXA and OXB fiber distribution ................................ 82
Summary ...................................................................................................... 85
CHAPTER 3

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) ........................... 88
Introduction ...................................................................................................... 88
Methods ........................................................................................................... 91

Animal housing and determination of activity patterns ................................. 91
Tissue processing and analysis ................................................................... 92

viii

Patterns of Fos Expression in Orexin Cells .................................................. 97

IGL Anatomy .............................................................................................. 100
Results .......................................................................................................... 101
Patterns of Fos Expression in Orexin Cells ................................................ 101
IGL Anatomy .............................................................................................. 105
Discussion ..................................................................................................... 108
IGL Anatomy .............................................................................................. 108
Fos expression in orexin cells: general patterns ........................................ 110
Fos expression in orexin cells: heterogeneity of orexin cell function .......... 111
General conclusions .................................................................................. 113
CHAPTER 4

Orexin fibers form appositions with Fos expressing
neuropeptide-Y cells in the grass rat intergeniculate Ieaflet....117

Introduction .................................................................................................... 1 17
Materials and Methods .................................................................................. 120
Results .......................................................................................................... 123
Discussion ..................................................................................................... 125
CHAPTER 5
Retrograde labeling of orexin neurons projecting to the grass rat
intergeniculate leaflet ................................................................... 130
Introduction .................................................................................................... 130
Methods ......................................................................................................... 133
Animal handling ......................................................................................... 133
Surgical procedures ................................................................................... 134
Tissue collection and processing ............................................................... 135
Cell counts and analysis ............................................................................ 136
Results .......................................................................................................... 137
Discussion ..................................................................................................... 139
References ..................................................................................... 150

LIST OF TABLES

Table 2.1. Distribution of orexin A and B cell bodies in the Long-Evans rat, grass
rat, Syrian hamster, and degu. The relative densities of OXA- or OXB-containing
cells are indicated in each column as very dense (++++), dense (+++),
moderately dense (++), sparse (+), or absent (-). ............................................... 33

Table 2.2. Pairwise correlation matrix of orexin A (top) and orexin B (bottom) fiber
distribution in the Long-Evans rat, grass rat, Syrian hamster, and degu.
Correlation values were obtained from a principal component factor analysis of
orexin A and orexin B fiber density patterns in all four species. Values listed at
intersections between rows and columns represent the correlation in fiber density
between species indicated by the corresponding row and column labels. All
values represent significant correlations (p < 0.001 ). .......................................... 51

Table 2.3. Distribution of orexin A and B fibers in the Long-Evans rat, grass rat,
Syrian hamster, and degu. The relative densities of OXA- or OXB-containing
fibers are indicated in each column as very dense (indicated by ++++), dense
(+++), moderately dense (++), sparse (+), or absent (-). Unmarked rows indicate
areas for which no data are available .................................................................. 52

LIST OF FIGURES

Images in this dissertation are presented in color.

Figure 1.1. Actogram, double-plotted, depicting wheel running patterns typical of
day-active (top) and night-active (bottom) grass rats maintained in a 12:12 light-
dark cycle. Each horizontal line in the actogram represents 48 hours; vertical
marks indicate wheel running activity. Lighting conditions are indicated by the bar
at the bottom. Note that both day- and night-active animals exhibit a bout of
activity just prior to lights-on. ............................................................................... 14

Figure 2.1. Photomicrographs of orexin A and orexin B cell bodies in the Long-
Evans rat, grass rat, Syrian hamster, and degu. Scale bar = 100 um. ................ 27

Figure 2.2. Photomicrographs of orexin A cell bodies in the paraventricular
nucleus (Pa) of the Long-Evans rat (A), grass rat (B), and Syrian hamster (C).
Note lack of similar cell bodies in the degu (D). 3V: third ventricle. Scale bar =
200 pm. ............................................................................................................... 30

Figure 2.3. Photomicrographs of orexin A cell bodies in the supraoptic nucleus
($0) of the Long-Evans rat (A), grass rat (B), and Syrian hamster (C). Note lack
of similar cell bodies in the degu (D). OC: optic chiasm. Scale bar = 200 ,um. ....31

Figure 2.4. Photomicrographs of paraventricular nucleus (Pa; column 1) and
supraoptic nucleus (SO; column 2) of the Long-Evans rat, grass rat, and Syrian
hamster after preabsorption with orexin A blocking peptide. Note that
preabsorption with blocking peptide eliminates cell bodies seen in Figures 2.6
and 2.7. 3V: third ventricle; OC: optic chiasm. Scale bar = 200 pm. ................... 32

Figure 2.5. Line drawing of every 6th section through the region of the Long-
Evans rat hypothalamus that contains orexin cells. Sections are ordered from
rostral (A) to caudal (N). Filled circles indicate locations where both orexin A and
orexin B neurons are found, while open circles indicate orexin A neurons only.
3V: third ventricle; SCN: suprachiasmatic nucleus; OC: optic chiasm; f: fornix; mt:
mammillothalamic tract. Scale bar = 500 ,um. ................................................. 34

Figure 2.6. Line drawing of every 6th section through the region of the grass rat
hypothalamus that contains orexin cells. Sections are ordered from rostral (A) to
caudal (L). Filled circles indicate locations where both orexin A and orexin B
neurons are found, while open circles indicate orexin A neurons only. 3V: third
ventricle; SCN: suprachiasmatic nucleus; OC: optic chiasm; f: fornix; mt:
mammillothalamic tract. Scale bar = 500 ,um. ..................................................... 38

Figure 2.7. Line drawing of every 6th section through the region of the Syrian
hamster hypothalamus that contains orexin cells. Sections are ordered from
rostral (A) to caudal (J). Filled circles indicate locations where both orexin A and

xi

orexin B neurons are found, while open circles indicate orexin A neurons only.
3V: third ventricle; SCN: suprachiasmatic nucleus; OC: optic chiasm; f: fornix; mt:
mammillothalamic tract. Scale bar = 500 pm. ..................................................... 41

Figure 2.8. Line drawing of every 6th section through the region of the degu
hypothalamus that contains orexin cells. Sections are ordered from rostral (A) to
caudal (N). Filled circles indicate locations where both orexin A and orexin B
neurons are found. 3V: third ventricle; OC: optic chiasm; f: fornix; mt:
mammillothalamic tract; aq: cerebral aqueduct. Scale bar = 500 pm. ................. 44

Figure 2.9. Photomicrographs of orexin A fibers around the suprachiasmatic
nucleus (SCN) of the Long-Evans rat (A), grass rat (B), Syrian hamster (C), and
degu (D). 3V: third ventricle; OC: optic chiasm. Scale bar = 200 pm. ................. 64

Figure 2.10. Photomicrographs of orexin A fibers in the ventromedial
hypothalamic nucleus (VMH) of the Long-Evans rat (A), grass rat (B), Syrian
hamster (C), and degu (D). Note markedly higher density of orexin fibers in the
degu VMH than in the other three species. 3V: third ventricle; Arc: arcuate
nucleus. Scale bar = 300 pm ............................................................................... 65

Figure 2.11. Photomicrographs of orexin A fibers in the lateral mammillary
nucleus (LM) of the Long-Evans rat (A), grass rat (B), Syrian hamster (C), and
degu (D). Note higher density of orexin fibers in the hamster LM than in the other
three species. MM: medial mammillary nucleus. Scale bar = 200 pm ................. 66

Figure 2.12. Photomicrographs of orexin A fibers in the intergeniculate leaflet
(IGL) of the Long-Evans rat (A), grass rat (B), Syrian hamster (C), and degu (D).
DLG: dorsolateral geniculate nucleus; VLG: ventrolateral geniculate nucleus.
Scale bar = 200 pm. ............................................................................................ 68

Figure 2.13. Photomicrographs of orexin A fibers in the centrolateral nucleus (CL)
of the Long-Evans rat (A), grass rat (B), Syrian hamster (C), and degu (D). Note
higher density of orexin fibers in the degu CL than in the other three species. LV:
lateral ventricle; PVT: paraventricular thalamic nucleus, MHb: medial habenular
nucleus; LHb: lateral habenular nucleus. Scale bar = 300 pm. ........................... 69

Figure 2.14. Photomicrographs of orexin A fibers in the xiphoid nucleus (Xi) of
the Long-Evans rat (A), grass rat (B), Syrian hamster (C), and degu (D). Note
relative lack of orexin fibers in the degu Xi in comparison with the other three
species. 3V: third ventricle. Scale bar = 200 pm. ................................................ 70

Figure 2.15. Photomicrographs of orexin A fibers in the dorsal raphé nucleus
(DR) of the Long-Evans rat (A), grass rat (B), Syrian hamster (C), and degu (D).
4: trochlear nucleus; Aq: cerebral aqueduct. Scale bar = 300 pm. ...................... 72

Figure 2.16. Photomicrographs of orexin A fibers in the locus coeruleus (LC) of
the Long-Evans rat (A), grass rat (B), Syrian hamster (C), and degu (D). 4V:
fourth ventricle. Scale bar = 200 pm. .................................................................. 73

xii

Figure 2.17. Photomicrographs of orexin A fibers in the flocculus (Fl) of the Long-
Evans rat (A), grass rat (B), Syrian hamster (C), and degu (D). Note higher
density of orexin fibers in the degu Fl than in the other three species. PFI:
paraflocculus; VC: ventral cochlear nucleus. Scale bar = 200 pm. ..................... 75

Figure 3.1. (A) Actogram illustrating wheel running activity of an individual grass
rat. Although initially day-active, this individual exhibited a spontaneous switch to
a night-active pattern, illustrating both the two basic patterns and the lack of
transients during a switch. This spontaneous switch is atypical; most animals in
our colony establish stable day- or night-active rhythms shortly after introduction
of a wheel. (B) Line graph illustrating average hourly wheel revolutions over a 5
day monitoring period of all day-active (n = 18, open circles) and night-active (n =
18, closed circles) grass rats used in this study. Arrows at ZT 4 and ZT 16
indicate sampling times for animals used in this study. Bar at bottom indicates
light cycle. ........................................................................................................... 93

Figure 3.2. Drawing of representative sections through (A) rostral, (B) middle and
(C) caudal sections of the grass rat LH. Boxes representing sampling area are
1200 x 700 pm, divided from left to right into medial, central, and lateral divisions.
Dots represent cells stained for OXA. Distribution of OXB cells were similar (not
shown). 3V = 3rd ventricle; f= fornix. ................................................................... 99

Figure 3.3. Photomicrograph (40x) of orexin A and B cells in the grass rat LH.
Cells marked * are expressing Fos-IR only; Open arrows indicate cells
expressing only OXA (Panel A) or OXB (Panel B); Solid arrows point to double-
labeled cells. Scale bar = 50 um. ...................................................................... 102

Figure 3.4. Temporal patterns of mean (:t SEM) levels of Fos expression in OXA
(top) and OXB (bottom) cells in day- and night-active animals sacrificed at 2T 4
and ZT 16. Light bars represent day-active animals; dark bars represent night-
active grass rats. Significant differences (p < 0.05) noted by different letters. ..103

Figure 3.5. Fos-IR in orexin cells in grass rats housed with (+) and without (-)
running wheels. Day-active grass rats (top) were sacrificed at ZT 4, night-active
animals (bottom) at ZT 16. Dark bars = OXA; light bars = OXB. Significant
differences (p < 0.05) noted by different letters. Removal of running wheel
caused significant reductions n Fos-IR only in night-active animals. Effects of
wheels were not significant in day-active animals, although trends were similar (p
= 0.06 and p = 0.08 for OXA and OXB, respectively). ....................................... 104

Figure 3.6. Regional patterns of orexin Fos-IR in night-active grass rats housed
with (top) or without (bottom) running wheels at ZT 16 demonstrating both the
medial to lateral gradient in orexin cell activity, and the differences and
similarities between OXA- and OXB-labeled cells. Significant differences (p <
0.05) noted by different letters. Dark bars = OXA; light bars = OXB. ................ 106

xiii

Figure 3.7. Photomicrographs of OXA (Panels A and B) and OXB (Panels C and
D) fibers and NPY cells in the grass rat IGL. Arrows indicate where contacts
between NPY cells and orexin fibers appear to be present. Panels (A) and (C)
are 40x; scale bar is 50 pm. Panels (B) and (D) are 100x close-ups of regions
marked with * in panels (A) and (C), respectively; scale bar is 20 pm. ............. 107

Figure 4.1. Photomicrographs (100x) of orexin A (OXA) fiber appositions with
neuropeptide-Y (NPY) cells (Panels A, B, C) or NPY cells expressing Fos
(Panels D, E, F) in the grass rat IGL. Arrows indicate appositions between OXA
fibers and NPY cells; Scale bar is 50 pm. ......................................................... 124

Figure 4.2. Percentages (iSEM) of Fos expression in neuropeptide-Y (NPY) cells
that do (NPY-OXA) or do not (NPY-Only) exhibit appositions with orexin A (OXA)
fibers in the IGL of day- and night-active grass rats. Asterisk indicates significant
difference between columns (p < 0.0001 ). Dark bars = NPY+OXA cells; light bars
= NPY-Only cells. .............................................................................................. 126

Figure 5.1. Line drawing of the lateral hypothalamic area at low magnification
(5x) depicting the 1600 pm x 700 pm sampling area used in this study. The
sampling box is aligned to the third ventricle and ventral border of the fornix, and
divided from left to right into 40 pm divisions labeled Medial (M), Central (C),
Lateral 1 (L1), and Lateral 2 (L2), respectively. Circles represent cells containing
orexin A. Scale bar = 500 pm; f = fornix, mt = mammalothalamic tract, 3V = third
ventricle. ............................................................................................................ 138

Figure 5.2. Line drawings depicting injection sites for animals described in this
study. Darkly shaded area indicates placement of needle tip; lighter shaded area
indicates spread of cholera toxin tracer from injection site. All drawings were
traced under low magnification (10x). Drawings are arranged rostrally to caudally
through the intergeniculate leaflet. Scale bar = 300 pm. APT: anterior pretectal
nucleus; ar: acoustic radiation; DLG: dorsolateral geniculate nucleus; IGL:
intergeniculate leaflet; LP: lateral posterior thalamic nucleus; ml: medial
lemniscus; opt: optic tract; Po: posterior thalamic nuclear group; SubG:
subgeniculate nucleus; VLG: ventrolateral geniculate nucleus; VPM: ventral
posteromedial thalamic nucleus; ZID: zona inserta, dorsal; ZIV: zona incerta,
ventral. .............................................................................................................. 140

Figure 5.3. Photomicrographs depicting cells in the lateral hypothalamus after
staining with Texas Red labeled orexin (red fluorescence, panels A, D, and G)
and injection of FITC-conjugated cholera toxin B (green fluorescence, panels C,
F, and I). Panels B, E, and H are composite images showing both labels.
Neurons exhibiting immunoreactivity for both orexin and cholera toxin B are
marked with an asterisk (*). Arrows indicate cells labeled with cholera toxin B
only. Scale bar = 25 ,um. ................................................................................... 142

xiv

KEY TO ABBREVIATIONS

 

Abbreviation Definition

AB complex ............................................ avidin-biotin complex

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

Arc .......................................................... arcuate nucleus

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

BNST ..................................................... bed nucleus of the stria terminalis
CL ....... - ................................................... c entrolateral thalamic nucleus
CRF ........................................................ corticotrophin releasing factor
CSF ........................................................ cerebrospinal fluid

CTB ........................................................ cholera toxin subunit B

CTB-FITC ............................................... CTB conjugated with AlexaFluor 488
DAB ........................................................ diaminobenzidine

DH .......................................................... dorsal hypothalamic area

DMH ....................................................... dorsomedial hypothalamic nucleus
GFAP ..................................................... glial fibrillary acidic protein

GHT ....................................................... geniculohypothalamic tract

GnRH ..................................................... gonadotropin releasing hormone
HPA ........................................................ hypothalamic-pituitary-adrenal axis
HPG ....................................................... hypothalamic-pituitary-gonadal axis
IGL ......................................................... intergeniculate leaflet

IR ........................................................... immunoreactive, immunoreactivity
LC .......................................................... locus coeruleus

LD .......................................................... light-dark

LE ........................................................... Long-Evans

LH .......................................................... lateral hypothalamus

LHA ........................................................ lateral hypothalamic area

LM .......................................................... lateral mammillary nucleus

MCH ....................................................... melanin concentrating hormone
NDS ....................................................... normal donkey serum

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

NPY ........................................................ neuropeptide-Y

OT .......................................................... oxytocin

OX1R ...................................................... orexin receptor 1

OX2R ...................................................... orexin receptor 2

OXA ....................................................... orexin A

OXB ....................................................... orexin B

Pa ........................................................... paraventricular hypothalamic nucleus
PBS ........................................................ phosphate buffered saline

PBS-TX .................................................. PBS with 0.3% Triton-X 100

PC .......................................................... paracentral thalamic nucleus

PCA ........................................................ principal components factor analysis
PeF ........................................................ perifornical region

PH .......................................................... posterior hypothalamic area

XV

Abbreviation Definition

 

 

PPO ....................................................... preproorexin

PV .......................................................... paraventricular thalamic nucleus
RCh ........................................................ retrochiasmatic area

REM ....................................................... rapid eye movement

RHT ........................................................ retinohypothalamic tract

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

SD .......................................................... Sprague-Dawley

SO .......................................................... supraoptic nucleus

Sol .......................................................... nucleus of the solitary tract

SOR ....................................................... supraoptic retrochiasmatic nucleus
Subl ........................................................ subincertal thalamic nucleus

SuM ........................................................ supramammillary nucleus

TC .......................................................... tuberum cinerum

VMH ....................................................... ventromedial hypothalamic nucleus
Xi ............................................................ xiphoid nucleus

ZT ........................................................... Zeitgeber time

xvi

-.n. . .. -._ . - _ . _. .—.- q- .. ——--—- -.-— -..-..._.7. ._._._.

CHAPTER 1

Introduction

General Introduction

The research in this dissertation focuses upon two major goals. One is to
describe species differences in the distribution of the orexins, peptides implicated
in the regulation of ingestive behavior, sleep-wake and general arousal, and the
modulation of somatomotor functions. The second is to examine the functional
and anatomical relationships between the orexins and activity state in the diurnal
grass rat, Arvicanthis niloticus. In this introductory chapter I will provide (1) an
overview of the orexins and their function, (2) a short description of the grass rat,
the primary animal model used in these studies, and (3) a summary of the

questions addressed in each chapter of this thesis.

Overview of orexin

Orexin discovery

When the discovery of a novel peptide apparently limited to cell bodies in
the hypothalamus was announced in 1998 (de Lecea et al. 1998; Sakurai et al.
1998), interest was high due to the possibility of its involvement with feeding. The
peptide, dubbed orexin by Sakurai et. al. (1998) and hypocretin by de Lecea et.
al. (1998), was independently discovered in two laboratories using very different
methods. One group isolated the long form of orexin, orexin A (OXA), by
searching for ligands for “orphaned” G-protein coupled receptors (Sakurai et al.

1998). The second group first isolated the precursor protein, preproorexin, in

1996 using a subtractive PCR technique to recover hypothalamus-specific
proteins (Gautvik et al. 1996) but did not publish a detailed investigation of the
precursor or its derivatives until early in 1998 (de Lecea et al. 1998).

The initial reports of these discoveries showed that the orexins are a
family containing two peptides, the 33 amino acid OXA (hypocretin-1) and the
shorter 28 amino acid orexin B (OXB, hypocretin-2), both derived from the
precursor protein, preproorexin (PPO), through proteolytic processing (de Lecea
et al. 1998; Sakurai et al. 1998). The PPO gene, which is highly conserved
across species, has some similarities with the secretin/incretin family of peptides
(de Lecea et al. 1998), and appears to have arisen early during chordate
evolution through a circular mutation of an incretin gene (Alvarez and Sutcliffe
2002). Orexin has been identified in all major vertebrate taxa, including fish
(Kaslin et al. 2004; Huesa et al. 2005), amphibians (Shibahara et al. 1999;
Yamamoto et al. 2004; Singletary et al. 2005), reptiles (Farrell et al. 2003), birds
(Ohkubo et al. 2002), and mammals (Sakurai et al. 1999). Preproorexin mRNA
was initially reported to be limited to cell bodies in the lateral hypothalamus (LH)
(Gautvik et al. 1996), although more recent reports have provided evidence for
orexin neurons in other brain regions, including the amygdala, median eminence,
and ependyma (Chen et al. 1999; Kummer et al. 2001; Ciriello et al. 2003c).
Because the orexin cells outside of the lateral hypothalamus have not been
extensively studied, references to orexin neurons in this dissertation will refer
only to neurons in the main population of orexin cells located near the fornix,

unless otherwise specified. To avoid confusion when discussing the distribution

of orexin-containing neurons, references to the lateral hypothalamus as a whole
in this dissertation will be designated using the abbreviation LH, while the lateral
hypothalamic area, a specifically defined subregion of the LH, will be referred to
as LHA.

The orexins bind to two G-protein coupled receptors; OXA binds equally to
either orexin receptor 1 (OX1R) or orexin receptor 2 (OX2R); OXB binds to both
receptors but displays moderate selectivity for OX2R (Sakurai et al. 1998; Smart
et al. 1999). Orexin A and B have been shown to act directly upon axon terminals
to increase the release of both GABA and glutamate (van den Pol et al. 1998).
The orexins may also affect the presynaptic effect of CaZ*-dependent transmitters
by increasing calcium levels, both through mobilization of internal Ca2+ stores
and through secondary influx of external calcium (Smart et al. 1999).

Although the total number of orexin neurons is fairly small, axonal
projections from these cells extend from the LH to many regions of the rat brain
and spinal cord (Peyron et al. 1998; Chen et al. 1999; Cutler et al. 1999; Date et
al. 1999; Nambu et al. 1999). The overall distribution of orexin fibers in the rat
brain and spinal cord suggested that, in addition to feeding actions, the orexins
might be involved in the sleep-wake cycle, emotional responses,
thermoregulation and nocioception (Peyron et al. 1998; Cutler et al. 1999;
Nambu et al. 1999).

There is also strong evidence for an important role for orexin outside of
the central nervous system. Both orexin and orexin receptors are present in

peripheral tissues. Both PPO and orexin receptor mRNA are present in the gut in

several species, including rats, guinea pigs, sheep, and humans (Kirchgessner
and Liu 1999; Ehrstrom et al. 2005). Additionally, PPO mRNA has been identified
in the heart and testicular tissue of rats (Johren et al. 2001 ), and orexin receptors
have been found in rat lung, as well as the adrenal glands and gonads of both

rats and sheep (Johren et al. 2001; Zhang et al. 2005a).

Orexin and feeding

The hypothalamic distribution of cell bodies containing the precursor
protein suggested that orexins are involved in feeding behavior. Prior to the
discovery of orexin, the only other peptide known to be found in cell bodies
limited to the LH was melanin concentrating hormone (MCH), a peptide known to
be involved the regulation of feeding (Du et al. 1996). Early experiments showed
that 48 hours of fasting resulted in a 2.4-fold increase in rat preproorexin mRNA
(Sakurai et al. 1998), and that injections of OXA and OXB could elicit ingestion in
rats, although the effects of OXA appeared to be stronger than those of OXB,
perhaps due to its more stable structure (Sakurai et al. 1998). This difference in
the orexigenic effects of OXB in comparison to OXA has been replicated several
times, with most studies suggesting that OXB is less effective than OXA in
eliciting feeding or drinking behavior (Sakurai et al. 1998; Edwards et al. 1999;
Kunii et al. 1999). In some cases OXB has been ineffective in eliciting any
ingestive behavior whatsoever (Lubkin and Stricker—Krongrad 1998; Sweet et al.
1999)

Orexin effects on ingestive behavior appear to depend upon interactions

with other food-related signaling systems, such as neuropeptide-Y (NPY), Ieptin,

MCH, grehlin, galanin, and agouti-related protein (Broberger et al. 1998; Horvath
et al. 1999a; Rauch et al. 2000; Schwartz et al. 2000; Sakurai 2003; Ehrstrom et
al. 2005; Takenoya et al. 2005). For example, the ingestive behavior stimulated
by orexin is attenuated or blocked by Ieptin, a potent inhibitor of food intake
(reviewed in Schwartz et al. 2000). In one study, pretreatment with Ieptin blocked
orexin-induced activity in nearly half of the orexin responsive neurons identified in
the arcuate nucleus (Rauch et al. 2000). In addition, Ieptin injections in the rat are
capable of blocking both OXA-induced feeding behavior and NPY-induced Fos
immunoreactivity in OXA cells (Niimi et al. 2001). Leptin may block the effects of
orexins directly or indirectly, as some OXB cells have been shown to express
Ieptin receptors, and NPY cells in the rat and monkey Arc receiving orexin fiber
contacts also expressed Ieptin receptors (Horvath et al. 1999a). Orexin appears
to have a reciprocal blocking effect on Ieptin, as OXA administered intravenously
reduces plasma leptin concentrations in humans (Ehrstrom et al. 2005).

There are several lines of evidence that suggest interactions between
orexin and blood glucose levels. Insulin-induced hypoglycemia results in a rapid
rise in nuclear c-Fos expression in OXA cells of the rat (Moriguchi et al. 1999).
Orexin-containing pancreatic islet cells also contain insulin in humans, and some
of these cells express orexin receptors (Ehrstrom et al. 2005). Intravenous orexin
administration raises insulin levels in the blood, presumably by stimulating
pancreatic cells expressing orexin receptors (Ehrstrom et al. 2005). In addition,
there are some indications that defects in the orexin system may affect the

regulation of glucose in humans. For example, in humans with narcolepsy, a

condition associated with low or nonexistent levels of orexins (Nishino et al.
2000; Nishino et al. 2001), there appears to be a higher risk of non-insulin
dependent diabetes (Honda et al. 1986).

Despite the documented relationship between the orexins and feeding and
satiety systems, there is some controversy over the actual effect orexins have on
feeding behavior. The administration of orexins into the central nervous system
has not always reliably increased feeding behavior (reviewed in Sutcliffe and de
Lecea 2000). While it is generally agreed that the orexins do not represent as
potent a stimulator of feeding as NPY, for example, the relative strength or
weakness of the orexins as compared to other peptides such as MCH has not
been clearly established (Lubkin and Stricker-Krongrad 1998; Edwards et al.
1999). Indeed, in studies performed in various laboratories, the orexins elicited
an ingestive response ranging between very robust (Sakurai et al. 1998;
Yamanaka et al. 2000), moderate (Edwards et al. 1999; Sweet et al. 1999), and
weak (Lubkin and Stricker—Krongrad 1998).

The variation in feeding behavior elicited in individual studies may be
explained by several factors. First, orexins have been shown to increase both
GABA and glutamate release in the rat in vitro (van den Pol et al. 1998). These
peptides thus appear to have the ability to affect the fast synaptic excitatory or
inhibitory activity of many parts of the hypothalamus. Therefore, the reported
effects of centrally administered orexins may not be physiologically relevant, as
spillover into other brain regions might activate or inhibit systems not normally

involved in the feeding effects of orexin. The actual discrete local effects of

naturally released peptides are presumably much more finely controlled by the
nervous system than even the most carefully placed injection. Indirect actions or
spillover effects of injected orexins have been proposed as explanations for
differences seen among several studies (Lubkin and Stricker-Krongrad 1998;
Edwards et al. 1999). Secondly, the relative degree of feeding behavior observed
after introduction of orexin may be related to stress, as at least some orexin-
induced ingestive responses rely upon interactions between orexin, NPY, and
corticosterone levels (Horvath et al. 1999a; Ida et al. 20003; Jaszberényi et al.
2000; Yamanaka et al. 2000). Finally, orexin-induced feeding might be time-
dependent. Circadian responsiveness of the feeding effect of OXA in the rat has
been observed in at least one study, with an increase in food intake following
OXA injection only occurring during the light phase of the daily light-dark cycle
(Kotz et al. 2002).

Although the exact role of the orexins in feeding has yet to be established,
it is possible that the orexins are involved in the coordination of locomotor activity
and arousal in response to stress and variation in food availability. During short-
term food deprivation, orexin receptor mRNA is up-regulated (Lu et al. 2000).
Orexins promote wakefulness, occasionally leading to increased searching and
exploratory behavior (Ida et al. 1999; Jones et al. 2001). A decrease in food
availability may thus increase arousal at times when the animal is normally
quiescent, leading to increased locomotion and searching behaviors. By
modifying the timing of arousal, the orexin system might increase the probability

of the animal encountering a food source that is not available at other times of

the day. The orexin system is clearly uniquely situated for involvement in the
coordination of an interrelated suite of behaviors related to food intake and

arousal.

Orexin and arousal

The overall distribution of orexin fibers in the brain has suggested that the
orexins play a role in a number of behaviors, including the maintenance of
arousal (Hagan et al. 1999; Horvath et al. 1999b). Orexin fibers have been
shown to project to various brain nuclei implicated in the control of sleep state
(Peyron et al. 1998; Date et al. 1999; Nambu et al. 1999; Mintz et al. 2001;
Moore et al. 2001). Application of OXA in the locus coeruleus (Hagan et al. 1999;
Bourgin et al. 2000) and lateral preoptic area (Methippara et al. 2000) of the rat
have been shown to increase wakefulness, primarily through a decrease in rapid
eye movement (REM) sleep (Bourgin et al. 2000). Activity in locus coeruleus
neurons increases following application of OXA (Hagan et al. 1999; Horvath et al.
1999b; Bourgin et al. 2000). In contrast to OXA, OXB does not seem to affect
wakefulness (Bourgin et al. 2000).

The orexin cells also receive input from brain circuits involved in regulation
of sleep-wakefulness. In mammals, circadian organization of activity including
sleep-wake behavior is regulated by an endogenous clock located in the
suprachiasmatic nucleus (SCN) (reviewed in Weaver 1998). Orexin cell bodies in
the lateral hypothalamus receive both limited direct contact from the SCN
(Abrahamson et al. 2001), as well as substantial indirect contact from the SCN

via the medial preoptic area (Deurveilher and Semba 2005). Introduction of

chemicals known to increase arousal in rats, such as methamphetamines or the
anti-narcoleptic drug Modafanil, increase nuclear Fos expression in orexin cell
bodies (Weaver 1998; Chemelli et al. 1999; Estabrooke et al. 2001).
Furthermore, increasing the behavioral arousal of rats by sleep deprivation
induced by handling also increases the expression of nuclear Fos in OXA cells
(Estabrooke et al. 2001). Orexin cells thus appear to be capable of both receiving
information relating to the arousal state of the animal, and relaying arousal
information to other nuclei known to promote wakefulness. The recent finding
that a defect in the orexin system is associated with the sleep disorder
narcolepsy (Chemelli et al. 1999; Lin et al. 1999; Here et al. 2001; Hungs et al.

2001) has strengthened the association between the orexins and arousal.

Other actions of orexin

In addition to the feeding and arousal-related actions of the orexins, there
is evidence for the involvement of orexins in the modulation of general activity.
For example, in rats, injections of OXA and, to a lesser extent OXB have been
shown to increase locomotor activity (Ida et al. 2000b; Nakamura et al. 2000;
Jones et al. 2001; Yoshimichi et al. 2001; Kotz et al. 2002) and burrowing
behavior (Ida et al. 1999). Orexin B does not appear to have as strong an effect
on general locomotor activity in lab rats as does OXA, but has been shown to be
more effective than OXA in eliciting searching behavior (Ida et al. 1999; Jones et
al. 2001) or exploration of novel environments (Jones et al. 2001). The increase
in locomotor activity observed in rats after application of OXA does not appear to

be related to the concurrent increase in feeding often observed following orexin

injections (Kotz et al. 2002). Face washing and grooming behavior also increase
in frequency following injections of OXA but not OXB (Ida et al. 1999; Ida et al.
2000b; Nakamura et al. 2000; Duxon et al. 2001; Jones et al. 2001). The
increase in grooming following injection of OXA in the rat is blocked by prior
application of a CRF antagonist, suggesting that the behavior may be linked to
stress (Ida et al. 2000b). Both the grooming and locomotor effects of orexins may
also involve interactions with dopaminergic (Nakamura et al. 2000) and
serotonergic (Nakamura et al. 2000; Duxon et al. 2001) systems.

Orexin may also be involved in the regulation of autonomic functions.
There are extensive projections from orexin neurons to hindbrain nuclei that
regulate cardiovascular and sympathetic processes (Date et al. 1999; Zheng et
al. 2005). Several studies have shown that application of OXA increases heart
rate, blood pressure, and respiration rate in rats and mice (Shirasaka et al. 2002;
Ciriello et al. 2003a; de Oliveira and Ciriello 2003; Zhang et al. 2005b; Zheng et
al. 2005). Body temperature, which generally rises during active periods and
decreases when animals are quiescent, increases following injection of OXA
(Yoshimichi et al. 2001), but not after injection of OXB (Jones et al. 2001). The
increase in body temperature following application of OXA does not appear to be
a result of increased locomotor activity (Yoshimichi et al. 2001).

Finally, orexins have been implicated in modulation of the hypothalamic-
pituitary-gonadal (HPG) axis at several levels. First, within the hypothalamus,
orexin has been shown to stimulate the release of gonadotropin releasing

hormone (GnRH) (Russell et al. 2001). Cells containing GnRH receive direct

10

contact from orexin fibers in rats and sheep (Iqbal et al. 2001; Campbell et al.
2003), and in rats GnRH neurons have also been shown to express orexin
receptors (Campbell et al. 2003). In addition, orexin projections to the
hypothalamic magnocellular nuclei that in turn project to the pituitary also appear
to be important in HPG regulation. Magnocellular neurons in the Pa express
orexin receptors, and these receptors are selectively upregulated during the
estrous cycle and early lactation in rats (Wang et al. 2003).

At the level of the pituitary, more evidence for orexin involvement in HPG
regulation has been found. Specifically, both rat and human pituitary express
orexin receptors (Blanco et al. 2001; Johren et al. 2001), and OXA acting on
these receptors appears to directly block GnRH-mediated release of luteinizing
hormone in proestrous female rats (Russell et al. 2001). With respect to the
gonads, testicular tissue in rats expresses orexin, and testicular tissue in both
rats and sheep expresses orexin receptor mRNA (Johren et al. 2001; Zhang et
al. 20053). Although orexin mRNA appears to be absent in rat ovary, orexin
receptors are present (Johren et al. 2001). Although the specific actions of orexin
on gonadal tissue are currently unknown, the presence of orexin and orexin
receptors in the gonads suggests the possibility that orexins may affect the HPG

axis at this level as well as at the hypothalamic and pituitary levels.

Introduction to the grass rat

The unstriped Nile grass rat (Arvicanthis niloticus, hereafter referred to as
the grass rat) is a diurnal murid rodent indigenous to large portions of sub-

Saharan Africa. Our laboratory maintains a breeding colony of grass rats, derived

11

from wild animals captured in 1993 in the Masai Mara National Reserve in Kenya
(Katona and Smale 1997). Although there has been some confusion over the
taxonomic grouping of the genus Arvicanthis (reviewed in McElhinny 1996), a
recent karyological analysis performed at the Museum National d’Histoire
Naturelle (Paris) confirms that the grass rats in our colony are Arvicanthis
niloticus Desmarest 1822 (Challet et al. 2002). The grass rat exhibits strong
diurnal rhythms of daily activity in the wild (Blanchong and Smale 2000). In the
laboratory, the grass rat is diurnal with respect to body temperature, general
activity, and reproductive behavior (McElhinny et al. 1997). The grass rat is a
murid rodent, and as such is closely related to the standard laboratory rat (Rattus
norvegicus). The grass rat has proven to be a useful diurnal animal model for
studies of sleep (Novak et al. 1999, 2000b), reproduction (McElhinny et al. 1997;
Mahoney et al. 2004; Mahoney and Smale 2005b, 20053), and the circadian
regulation of activity (Katona et al. 1998; Nunez et al. 1999; Mahoney et al. 2000;
Novak et al. 20003; Smale et al. 2001a; Schwartz et al. 2004).

Although the grass rat appears to be a strictly diurnal animal, there is
some degree of plasticity in their behavior. Specifically, when given continuous
access to a running wheel, some individual grass rats exhibit what appears to be
an unusual form of masking, switching from a day-active to a night-active pattern
of activity (Blanchong et al. 1999). In our colony, this shift from day- to night-
active patterns occurs in approximately 30% of animals given free access to a
wheel (Mahoney et al. 2001). Unlike circadian phase-shifting, which involves a

gradual transition to a new activity pattern due to re-entrainment of the circadian

12

clock, this switch to a night-active pattern appears to happen quickly, and is not
associated with the transients typical of a phase shift (Nixon and Smale 2004;
Redlin and Mrosovsky 2004), and appears to reflect a masking effect of the
wheel rather than a shift in the internal clock (Nixon and Smale 2004). Day-active
grass rats in a 12:12 light-dark (LD) cycle begin to run in the wheel about one
hour prior to lights-on, exhibit moderate to high levels of wheel running
throughout the day, and cease running in the wheel within 1 to 2 hours after
lights-out (Katona and Smale 1997) (Figure 1.1). The night-active grass rats are
like the day-active ones in that they show a rise in activity just prior to lights-on.
However, in the night-active grass rats, this bout ends when the lights come on,
and there is virtually no activity during the 12 hours of light. They begin to run in
the wheel just before lights-out, and this activity continues for five or more hours

into the dark period (Blanchong et al. 1999).

Overview of chapters

I present here 3 series of investigations intended to (1) evaluate species
differences and similarities in the distribution of orexin cell bodies and fibers; and
(2) shed light on the functional relationship between orexin cells and activity in
the grass rat. Two of the four chapters that are a part of this dissertation (Chapter
3 and Chapter 4) have been published with a co-author; for the sake of
consistency l have therefore elected to use the term “we” in all chapters that
present data. I address the first issue in Chapter 2, where I examine the
distribution of OXA and OXB-containing cell bodies and fibers in the brains of two

nocturnal and two diurnal species. This study has two primary goals: first, to

13

Figure 1.1. Actogram, double-plotted, depicting wheel running patterns typical of
day-active (top) and night-active (bottom) grass rats maintained in a 12:12 light-
dark cycle. Each horizontal line in the actogram represents 48 hours; vertical
marks indicate wheel running activity. Lighting conditions are indicated by the bar
at the bottom. Note that both day- and night-active animals exhibit a bout of
activity just prior to lights-on.

14

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time (hours)

Figure 1.1. Actogram, double-plotted, depicting wheel running patterns typical of
day-active (top) and night-active (bottom) grass rats. Each horizontal line in the
actogram represents 48 hours; vertical marks indicate wheel running activity.
Lighting conditions are indicated by the bar at the bottom. Note that both day-
and night-active animals exhibit a bout of activity just prior to lights-on.

15

identify potential species differences in the overall distribution of orexin in these
animals, and second, to determine whether differences in orexin distribution
might be related to behavioral or anatomical differences in the species examined.

In the remaining chapters, I address the second theme of this dissertation,
related to the relationship between orexin and activity patterns in the grass rat.
We have previously shown that a difference in Fos activation in NPY cells of the
intergeniculate leaflet (IGL) is associated with the differences in wheel running
patterns expressed by the individual grass rats (Smale et al. 2001b). The
existence of a projection from orexin cells to the IGL led us to hypothesize that
orexin plays a role in the modulation of these NPY cells by activity. In Chapter 3,
I examine orexin neurons in wheel running grass rats to determine whether
patterns of Fos activation in these cells are consistent with a role for orexin in the
activation of NPY neurons in the IGL. I then determine whether orexin cells are in
a position to modulate NPY cells within the IGL through direct projections to
them. In Chapter 4, I continue my investigation of orexin-NPY interactions by
determining whether NPY cells that receive input from orexin cells express Fos,
and whether this varies as a function of the activity sate of the animal. Finally, in
Chapter 5, I use a retrograde tract-tracer to determine the distribution of orexin

cells that project to the grass rat IGL.

16

CHAPTER 2
A comparative analysis of the distribution of orexin in the brains

of nocturnal and diurnal rodents

Introduction

The orexins (hypocretins) are a recently described family of peptides
originating in cells of the lateral hypothalamus (Sakurai et al. 1998; de Lecea and
Sutcliffe 1999). Orexins are thought to be primarily involved in the regulation of
arousal and sleep-wake behavior, general activity, body temperature, drinking,
and feeding (Lubkin and Stricker-Krongrad 1998; Edwards et al. 1999; Hagan et
al. 1999; Kunii et al. 1999; Mondal et al. 1999b; Piper et al. 2000; Estabrooke et
al. 2001; Hungs et al. 2001; Yoshimichi et al. 2001; Kotz et al. 2002; Diano et al.
2003; Yamanaka et al. 2003; Kiwaki et al. 2004; Berthoud et al. 2005; Kodama et
al. 2005; Thorpe and Kotz 2005). Anatomical studies of orexin fiber distribution in
the rat brain show that the densest projections extend to the locus coeruleus,
raphé nuclei, periaqueductal central gray, paraventricular hypothalamic nucleus,
arcuate nucleus, and the lining of the third ventricle (Peyron et al. 1998; Chen et
al. 1999; Cutler et al. 1999; Date et al. 1999; Mondal et al. 1999a; Nambu et al.
1999). Orexin cell bodies in the rat are primarily limited to the perifornical nucleus
and lateral hypothalamic area, with more sparse distributions in the dorsal
hypothalamic area, posterior hypothalamic area and the dorsomedial
hypothalamic nuclei (Peyron et al. 1998; Chen et al. 1999; Cutler et al. 1999;
Nambu et al. 1999). Published descriptions of orexin cell and fiber distributions

are similar to those in the rat for the Syrian and Djungarian hamster

17

(McGranaghan and Piggins 2001; Mintz et al. 2001; Khorooshi and Klingenspor
2005) as well as for humans (Moore et al. 1998; Arihara et al. 2000; Moore et al.
2001)

The orexins consist of two peptides, orexin A (OXA, hypocretin-1) and
orexin B (OXB, hypocretin-2), derived from the same precursor protein,
preproorexin (de Lecea et al. 1998; Sakurai et al. 1998). The orexins bind to two
G-coupled protein receptors, orexin receptors 1 (OX1R, HCRTR-1) and 2 (OX2R,
HCRTR—Z) (Sakurai et al. 1998). Although OXA and OXB appear to be equally
effective in activation of OX1R, OXA is 30- to 100-fold more effective than OXB in
activating OX1R (Sakurai et al. 1998; Smart et al. 1999). The two orexin
receptors exhibit distinctly different distribution patterns in the rat brain (reviewed
in Kilduff and de Lecea 2001). For example, while the raphé nuclei, thalamus,
and layer 6 of the cortex express OX1R and OX2R equally, only OX1R is present
in cortical layer 5, hippocampal field CA1, and locus coeruleus (LC), whereas
cortical layer 2, hippocampal field CA3, septal nuclei, and tuberomammillary
nuclei express only OX2R (Lu et al. 2000; Hervieu et al. 2001; Marcus et al.
2001). The differential distribution and potential selectivity of the two orexin
receptors raises the possibility that there may be some differences in the
functional roles played by OXA and OXB within the central nervous system.

Several other lines of evidence have also suggested that that OXA and
OXB may be differentially involved in particular functional systems. First,
repeated studies have shown that OXA is more effective than OXB in promoting

ingestive behavior (Sakurai et al. 1998; Edwards et al. 1999; Kunii et al. 1999).

18

This conclusion is supported by data from orexin receptor studies. Orexin-
induced ingestive behavior is attenuated by OX1R antagonists (Rodgers et al.
2000; Thorpe and Kotz 2005), and food deprivation selectively up-regulates
OX1R mRNA in the amygdala without affecting OX2R mRNA in this structure (Lu
et al. 2000). Second, although OXB is generally ineffective in eliciting feeding or
drinking behavior, there is evidence that OXB may be important in the promotion
of arousal. Several studies have shown that the effects of orexins on arousal in
thalamic midline and raphé nuclei depend primarily upon OX2R (Bayer et al.
2002; Brown et al. 2002), and disruption of OX2R has been linked to the sleep
disorder narcolepsy in dogs (Lin et al. 1999). In at least one study, OXB has
been shown to be more effective than OXA in activation of wakefulness-
promoting thalamic nuclei in the rat (Bayer et al. 2002), although this is not likely
to be the case for all structures involved in the regulation of sleep and
wakefulness, (Marcus et al. 2001). While both orexins are clearly involved in a
multitude of homeostatic and regulatory processes, most published reports on
their functions have not fully addressed potential differences between OXA and
OXB.

Most published data on the distribution of orexin cells has focused on
OXA, and relatively little is known about OXB. The distribution of OXB is
generally described as being identical to that of OXA but is often presented
incompletely (Cutler et al. 1999; Date et al. 1999; Nambu et al. 1999) or not at all
(Peyron et al. 1998; Chen et al. 1999). Because the two orexins are produced by

the same precursor protein, there may have been a tendency in the early

19

literature on the orexins to assume that the peptides are produced equally in
each orexin cell, and at least one study has provided some support for this
assumption (Zhang et al. 2002). However, there is evidence that the up-
regulation of one orexin peptide over the other is very possible, and may serve
some physiologically relevant function (Mondal et al. 19993; Cai et al. 2001).

A second issue that has received little attention involves the tendency to
assume that distributions of orexin-containing cells and fibers seen in one strain
or species of research animal are representative of all strains and species.
Although partial descriptions of orexin distribution in within the central nervous
system have been published for many species, including mice (Broberger et al.
1998; van den Pol 1999; Tritos et al. 2001), cats (Zhang et al. 2002, 2004), and
humans (Moore et al. 1998), the majority of studies examining orexin distribution
in detail throughout the brain have been in rats (Rattus norvegicus). In addition,
most descriptions of the orexins in the rat were performed using the highly inbred
Wistar strain (Peyron et al. 1998; Cutler et al. 1999; Nambu et al. 1999). The
occasional difference found in orexin distribution between rat strains tends to be
largely ignored or overlooked, even for differences as fundamental as the
distribution of orexin-immunoreactive (IR) cell bodies. For example, OXA-IR cells
have been described in the median eminence (Chen et al. 1999) in the Sprague-
Dawley (SD) rat, a less inbred strain, but not in the Wistar rat. Although previous
studies of the Wistar rat showed orexin-IR cell bodies only in the lateral
hypothalamus, at least one study has shown OXB-IR, but not OXA-IR cells in the

amygdala of both Wistar and SD rats (Ciriello et al. 2003c). These studies

20

highlight both the importance of examining orexin distribution in more than one
specific animal type, and the previously mentioned importance of examining both
forms of orexin.

While the orexins have been studied in some depth in nocturnal laboratory
rodents, little attention has been paid to them in diurnal animal models. Detailed
descriptions of orexin cell or fiber distributions in diurnal animals are limited, and
currently there are no descriptions of the distribution of both peptides in any
diurnal species that are as complete as those available for the Wistar rat. Studies
in humans (Moore et al. 1998) and sheep (Archer et al. 2002) describe OXA in
limited portions of the brain, and the distribution of OXA-IR cell bodies has also
been described in the Korean chipmunk (Tamias sibiricus barben) (Kodama et al.
2005) and the African green (vervet) monkey (Cercopithecus aethiops) (Diano et
al. 2003). The most complete study to date in a diurnal animal describes OXB,
but not OXA in the Nile grass rat (Arvicanthis niloticus) (Novak and Albers 2002).
Given the reported relationship between the orexins and regulation of sleep and
arousal (reviewed in Hungs and Mignot 2001), it is especially important to
investigate potential differences in orexin distribution in animals with completely
different sleep patterns, such as those seen in nocturnal and diurnal animals.
This is especially important because the loss of orexin or the dysfunction of its
receptors have been linked to several disorders in humans (Nishino et al. 2000;
Thannickal et al. 2000; Nevsimalova et al. 2005; Petersen et al. 2005), a diurnal

species.

21

One potential diurnal animal model with which to investigate this issue is
the Nile grass rat, a rodent native to sub-Saharan Africa. The grass rat exhibits
strong diurnal patterns of activity both in the laboratory and in the field
(McElhinny et al. 1997; Blanchong and Smale 2000). The grass rat is a murid
rodent, and as such is closely related to the standard laboratory rat (hereafter
referred to as the lab rat). The grass rat is an excellent model for investigation of
various physiological differences between nocturnal and diurnal animals,
including those associated with sleep (Novak et al. 1999, 2000b), reproduction
(McElhinny et al. 1997; Mahoney et al. 2004; Mahoney and Smale 2005b,
20053), and the circadian regulation of activity (Katona et al. 1998; Nunez et al.
1999; Mahoney et al. 2000; Novak et al. 20003; Smale et al. 2001a; Schwartz et
al. 2004). We have recently demonstrated that the grass rat exhibits a diurnal
rhythm in Fos-immunoreactivity in OXB cells (Martinez et al. 2002).

A second diurnal animal model being used in laboratory study of circadian
biology is the degu (Octodon degus), a highly social South American
hystricomorph rodent (Nowak 1999). The degu is relatively long-lived and
matures slowly, unlike the precocial rats and mice commonly used in laboratory
research (Reviewed in Lee 2004). Like hamsters, rhythms in degus are strongly
influenced by social and olfactory cues (Governale and Lee 2001; Jechura and
Lee 2004), and there is some evidence for seasonal (photoperiodic) changes in
reproductive structures (Reviewed in Lee 2004). The degu is a suitable model for

research on sleep and the circadian regulation of behavior (Krajnak et al. 1997;

22

Goel et al. 1999; Jiao et al. 1999; K35 and Edgar 1999; Jechura et al. 2000;
Mohawk et al. 2005; Mohawk and Lee 2005).

In this study we characterized the distribution of OXA and OXB cell bodies
and fibers and systematically compared them in two diurnal species (the grass
rat and the degu) and two nocturnal species, the lab rat and golden hamster
(Mesocricetus auratus). As previous studies have already described orexin
distributions in the highly inbred Wistar rat, we chose to use a less inbred strain,

the Long-Evans (LE) rat, in the current study.

Methods and materials

Animal handling

Adult male grass rats (n = 4) and degus (n = 4) were obtained from
captive breeding colonies at Michigan State University and the University of
Michigan, respectively. Long Evans rats (n = 3) and hamsters (n = 4) were
obtained from a commercial breeder (Charles River Laboratories, Raleigh, North
Carolina). All animals were housed under standard light-dark (LD) cycles (LE rat,
grass rat, and degu, 12:12 LD; hamster, 14:10 LD) with food and water provided
ad Iibitum prior to perfusion. All animal handling procedures in this study followed
National Institutes for Health guidelines and were approved by the Michigan

State University All-University Committee for Animal Use and Care.

Tissue collection and processing

At the time of sacrifice, all animals were anesthetized with sodium

pentobarbital (Nembutal; Abbot Laboratories, North Chicago, IN) and perfused

23

transcardially with 0.01 M phosphate-buffered saline (PBS; pH 7.4, 150-300
mllanimal), followed by 150 to 300 ml of fixative (4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4). Brains were post-fixed for 4 to 8 hours in 4%
paraformaldehyde before being transferred to 20% sucrose in 0.1 M phosphate
buffer. After 24 h in sucrose, brains were sectioned in three series at either 30
pm (grass rat and hamster) or 40 um (LE rat and degu) using a freezing
microtome. Coronal sections from the olfactory bulb through the brain stem of
four grass rats, three LE rats, four hamsters and three degus were used to
determine orexin A and B fiber distribution. A third series of sections from one
grass rat and one degu was stained with Cresyl violet, mounted on gelatin-
coated slides and coverslipped to aid in delineation and identification of different
structures. Additional sections through the preoptic area and lateral
hypothalamus from two LE rats, three grass rats and one hamster were used in
OXA blocking experiments.

Tissue was processed for OXA or OXB immunoreactivity in the following
manner. Unless otherwise specified, all steps were carried out at room
temperature. Free-floating tissue was incubated in 5% normal donkey serum
(NDS; Jackson Laboratories), in PBS with 0.3% Triton-X 100 (Research Products
International, Mount Prospect, IL; PBS-TX) for 1 h. Tissue was then incubated in
primary antibody for 42 h at 4°C (goat anti-orexin A 1210,000, or goat anti-orexin
B 1210,000, Santa Cruz Biotechnology; in PBS-TX and 3% N08), and then in
biotinylated secondary antibody for 1 h (donkey anti-goat 1:500, Santa Cruz; in

PBS—TX and 3% NDS), followed by 1 h in avidin-biotin complex (0.9% each

24

avidin and biotin solutions, Vector Laboratories, Burlingame, CA; in PBS-TX).
Tissue was rinsed and reacted in diaminobenzidine (DAB, 0.5 mg/ml, Sigma) in a
tris-hydrochloride buffer (Trizma, Sigma; pH 7.2) with hydrogen peroxide (0.35 pl
30% hydrogen peroxide/ml buffer). Control sections were incubated in the PBS-
TX/NDS solution, with the primary antibody omitted. Tissue used in blocking
experiments was processed as described above, but prior to adding tissue to the
primary antibody solution, the primary antibody was preabsorbed with one or
both orexin blocking peptides for 48 h at 4°C (1 :50 OXA, 1:50 OXB, or 1:50 each
OXA and OXB blocking peptide, Santa Cruz; in PBS with 0.3% Triton-X 100 and
3% NDS). All tissue was mounted on gelatin-coated slides, dehydrated, and

coverslipped.

Cell and fiber counts and analysis

For analysis of orexin A or B fiber distribution, all sections were examined
under a light microscope (Leitz, Laborlux S, Wetzlar, Germany), and the
presence or absence of labeled fibers, as well as their density, was recorded for
brain regions from the olfactory bulbs through the brainstem. The distribution of
all OXA or OXB-IR cell bodies was also mapped for each species. High-
resolution digital photographs of representative sections were taken using a
digital camera (Carl Zeiss, AxioCam MRc; thtingen, Germany) attached to a
Zeiss light microscope (Axioskop 2 Plus). Image contrast and color balance were
optimized using Zeiss AxioVision software (Carl Zeiss Vision). Final figures were
prepared using Adobe Illustrator and Adobe Photoshop (Adobe Systems, San

Jose, CA).

25

To systematically record the distribution and relative density of OXA and
OXB fibers within each species, fiber densities were divided into the following five
categories: very dense (++++), dense (+++), moderately dense (++), low density
(+), and absent (-). Terminology and abbreviations for neural structures in this
study follow Paxinos and Watson (1998), except for divisions of the hamster bed
nucleus of the stria terminalis (BNST) which follows Morin and Wood (2001).
Comparisons of BNST divisions between species follow the comparison of
terminologies between rat and hamster outlined in Alheid et al. (1995).
Stereotaxic atlases for the rat (Paxinos and Watson 1998) and Syrian hamster
(Morin and Wood 2001) were used to aid in identification of specific structures in
these species. In the degu and grass rat, NissI-stained tissue was used in
concert with the rat atlas (Paxinos and Watson 1998) for identification of nuclei.
Functional and anatomical grouping of brain structures for analysis followed
Paxinos (1995), as modified by John I. Johnson (personal communication).
Because of the resulting size and complexity of the orexin fiber data set, fiber
densities in the regions examined in this study were subjected to a principal
components factor analysis (PCA; Statistica, StatSoft, Tulsa, OK) to identify

patterns in the data.

Results

Orexin cell bodies

General
In all animals examined, OXA- and OXB-IR cell bodies were present in the

lateral hypothalamus (Figure 2.1). Although the distribution of orexin neurons

26

 

Figure 2.1. Photomicrographs of orexin A and orexin B cell bodies in the Long-
Evans rat, grass rat, Syrian hamster, and degu. Scale bar = 100 pm.

27

OmfinA

Orexin B

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28

differed among species, the majority of the orexin cells in all species were
observed in the perifornical region (PeF) and lateral hypothalamic area (LHA). In
the LE rat, grass rat and hamster, cell bodies immunoreactive for OXA were also
found in the paraventricular hypothalamic nucleus (Pa), supraoptic nucleus (SO),
and the supraoptic retrochiasmatic nucleus (SOR) (Figure 2.2, Figure 2.3).
Orexin A-IR neurons in the Pa and 80 were generally smaller and less intensely
stained for OXA than were cells in the PeF. For all three species exhibiting
orexin-IR neurons in the Pa and SO, the OXA-IR nuclei did not appear to
represent nonspecific binding of the primary antibody, as preabsorption of the
primary antibody with OXA blocking peptide supplied by the manufacturer
reduced or eliminated OXA immunoreactivity in these nuclei (Figure 2.4). No
OXB-IR neurons were observed in Pa, SO, or SOR of any species. Orexin A and
OXB cell body distribution in the LE rat, grass rat, degu and hamster are
summarized in Table 2.1 and are depicted in Figures 2.5, 2.6, 2.7, and 2.8,

respectively.

Orexin cell bodies in the LE Rat

In the LE rat, OXA- and OXB-IR cells were observed at high density in the
PeF, LHA, and dorsomedial hypothalamic nucleus (DMH) (Figure 2.5). Moderate
densities of labeled cells were present in the posterior hypothalamic area (PH)
and tuberum cinerum (TC). Sparsely scattered cells were also observed in the
dorsal hypothalamic area (DH), retrochiasmatic area (RCh), and the subincertal

thalamic nucleus (Subl). Orexin A-IR cell bodies in SO and magnocellular Pa

29

Paraventricular Nucleus

 

Figure 2.2. Photomicrographs of orexin A cell bodies in the paraventricular
nucleus (P3) of the Long-Evans rat (A), grass rat (8), and Syrian hamster (C).
Note lack of similar cell bodies in the degu (D). 3V: third ventricle. Scale bar =
200 um.

30

Supraoptic Nucleus

 

Figure 2.3. Photomicrographs of orexin A cell bodies in the supraoptic nucleus
(80) of the Long-Evans rat (A), grass rat (B), and Syrian hamster (C). Note lack
of similar cell bodies in the degu (D). OC: optic chiasm. Scale bar = 200 um.

31

Paraventricular Supraoptic

Pa - 3V 00
SO

Grass rat LE rat

Hamster

Figure 2.4. Photomicrographs of paraventricular nucleus (Pa; column 1) and
supraoptic nucleus (SO; column 2) of the Long-Evans rat, grass rat, and Syrian
hamster after preabsorption with orexin A blocking peptide. Note that
preabsorption with blocking peptide eliminates cell bodies seen in Figures 2.2
and 2.3. 3V: third ventricle; OC: optic chiasm. Scale bar = 200 pm.

32

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33

Figure 2.5. Line drawing of every 6th section through the region of the Long-
Evans rat hypothalamus that contains orexin cells. Sections are ordered from
rostral (A) to caudal (N). Filled circles indicate locations where both orexin A and
orexin B neurons are found, while open circles indicate orexin A neurons only.
3V: third ventricle; SCN: suprachiasmatic nucleus; OC: optic chiasm; f: fornix; mt:
mammillothalamic tract. Scale bar = 500 pm.

34

Long-Evans rat

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

35

Long-Evans rat (continued)

 

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9

 

 

 

 

 

or... ..., 2. L

e. . ' :. ' e e.
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Figure 2.5 (continued)

36

Long-Evans rat (continued)

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t I
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Figure 2.5 (continued)

37

Figure 2.6. Line drawing of every 6th section through the region of the grass rat
hypothalamus that contains orexin cells. Sections are ordered from rostral (A) to
caudal (L). Filled circles indicate locations where both orexin A and orexin B
neurons are found, while open circles indicate orexin A neurons only. 3V: third
ventricle; SCN: suprachiasmatic nucleus; OC: optic chiasm; f: fornix; mt:

mammillothalamic tract. Scale bar = 500 pm.

38

Grass rat

 

'__--'-

 

 

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mu

0

 

 

 

 

s

 

 

   

    
 

 

 

Grass rat (continued)

 

G . H

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2.6 (continued)

40

Figure 2.7. Line drawing of every 6th section through the region of the Syrian
hamster hypothalamus that contains orexin cells. Sections are ordered from
rostral (A) to caudal (J). Filled circles indicate locations where both orexin A and
orexin B neurons are found, while open circles indicate orexin A neurons only.
3V: third ventricle; SCN: suprachiasmatic nucleus; OC: optic chiasm; f: fornix; mt:
mammillothalamic tract. Scale bar = 500 pm.

41

Syrian hamster

 

 

 

A

 

 

 

 

 

 

 

 

 

 

 

42

 

Syrian hamster (continued)

 

 

 

 

 

 

 

 

 

Figure 2.7 (continued)

43

 

 

- -. C
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u
s
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s l
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4
\

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2.8. Line drawing of every 6th section through the region of the degu
hypothalamus that contains orexin cells. Sections are ordered from rostral (A) to
caudal (N). Filled circles indicate locations where both orexin A and orexin B
neurons are found. 3V: third ventricle; OC: optic chiasm; f: fornix; mt:
mammillothalamic tract; aq: cerebral aqueduct. Scale bar = 500 pm.

44

Degu(confinued)

 

 

 

 

 

 

 

 

 

 

Figure 2.8 (continued)

45

Degu(confinued)

 

 

 

 

 

 

 

 

 

 

Figure 2.8 (continued)

46

 

Degu(confinued)

()m M O N

 

 

 

 

 

 

 

Figure 2.8 (continued)

47

were very dense (Figure 2.2), while cell bodies in SOR were only moderately

dense (Figure 2.3). No OXB-IR neurons were seen in the P3 or SO.

Orexin cell bodies in. the grass rat

The distribution of OXA- and OXB-IR neurons in the grass rat was similar
to the pattern seen in the LE rat. Cell bodies expressing OXA and OXB were
observed at very high density in PeF, TC, LHA, and PH (Figure 2.6). Unlike the
LE rat, OXA- and OXB-IR neurons in the PeF were often observed inside the
grass rat fornix. Moderate densities of OXA- and OXB-IR cell bodies were visible
in DMH and Subl, with sparsely scattered cells also present in RCh and DA. As
in the LE rat, cell bodies expressing strong OXA immunoreactivity were present
in PA, SO, and SOR (Figure 2.2, Figure 2.3). No OXB-IR neurons were present
in these nuclei. Orexin A-IR cell bodies were very densely distributed in the
magnocellular Pa and SO. Moderately dense OXA-IR cell bodies were present in

SOR.

Orexin cell bodies in the hamster

Unlike the LE rat and grass rat, OXA- and OXB-IR neurons in the hamster
were conspicuously absent in PeF; the majority of orexin cells were observed in
LHA and DMH, dorsal or dorsomedial to PeF, with sparsely scattered cells also
present in TC and Subl (Figure 2.7). Although OXA-IR neurons were present in
the hamster magnocellular PA, SO, and SOR (Figure 2.2, Figure 2.3), the overall

density of OXA-IR cell bodies in these structures was far lower than that in the

48

LE rat or grass rat. As in the LE rat and grass rat, no OXB-IR neurons were

present in these nuclei.

Orexin cell bodies in the degu
The distribution of OXA- and OXB-IR cell bodies was very different in the

degu than in the LE rat, grass rat and hamster. The regions of highest density of
OXA and OXB cells formed two distinct clusters, in the DMH and in the LHA
ventrolateral to the fornix (Figure 2.8). Orexin A and OXB neurons were less
densely distributed in the PeF, DA, and PH. In the PeF, orexin-IR neurons were
clustered tightly against the boundaries of the fornix, but did not intrude into this
structure as observed in the grass rat. Cells labeled for OXA and OXB were
sparsely distributed throughout the RCh and Subl. Unlike the hamster, grass rat
and LE rat, no orexin-IR neurons were visible in the Pa, SO, or SOR. Orexin A-
and B-IR cells in the degu were also different from those seen in the other three
species in that orexin neurons were scattered through the TC caudal to the
ventromedial hypothalamic nucleus (VMH), with small numbers of cells observed

near the midline in sections through the supramammillary nucleus (SuM).

Orexin fiber distribution

General
Orexin A- and OXB-IR fibers were visible in many regions throughout the
forebrain, midbrain, and hindbrain in all species examined. Numerous varicosities

were noted on these fibers, especially in the most dense projections extending

49

rostrally from the lateral hypothalamus to the preoptic area and amygdala, and
posteriorly through the periaqueductal gray to the raphé nuclei and brainstem.
The average density of OXA and OXB fiber innervation in over two hundred
individual brain regions was examined for each species. A PCA analysis of these
fiber densities revealed only one significant eigenvector (eigenvalue = 5.740,
cumulative variance explained = 71.748%); no other factors with eigenvalues
greater than 1 were revealed. Factor coordinates for all species along this
eigenvector were similar (LE Rat OXA = -0.8753, OXB = -0.8669; Grass rat OXA
= -0.8275, OXB = -0.8178; Hamster OXA = -0.8657, OXB = -0.8446; Degu OXA
= -0.8270, OXB = -0.8498), suggesting a strong overall similarity between OXA
and OXB fiber densities across species. Overall, the PCA analysis revealed that
correlations in OXA measurements between pairs of species ranged from 59.2%
to 72.2% similarity, while these measurements for OXB ranged from 57.1% to
68.6%. Within species, correlations between OXA and OXB fiber densities were
quite high (LE rat: 85.8%; grass rat: 85.2%; hamster: 92.4%; degu: 98.5%).
Significance levels for all correlations were < 0.001. The PCA correlation matrix
summarizing between-species correlations for OXA and OXB is presented in
Table 2.2. The overall details of the distribution and relative density of OXA and
OXB fibers for each species are summarized in Table 2.3. Below we provide a
general description of the patterns of fiber distribution and highlight the

differences between species.

50

OmfinA

LE rat Grass rat Hamster Degu

LE rat 1.0000
Grass rat 0.7220 1.0000
Hamster 0.6982 0.6086 1 .0000
Degu 0.6412 0.5706 0.6458 1 .0000

 

 

 

 

 

 

 

 

OmflnB

LE rat Grass rat Hamster Degu

LE rat 1.0000
Grass rat 0.6989 1.0000
Hamster 0.6862 0.6054 1 .0000
Degu 0.6321 0.5709 0.6401 1 .0000

 

 

 

 

 

 

 

 

Table 2.2. Pairwise correlation matrix of orexin A (top) and orexin B (bottom)
fiber distribution in the Long-Evans rat, grass rat, Syrian hamster, and degu.
Correlation values were obtained from a principal component factor analysis of
orexin A and orexin B fiber density patterns in all four species. Values listed at
intersections between rows and columns represent the correlation in fiber density
between species indicated by the corresponding row and column labels. All
values represent significant correlations (p < 0.001).

51

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62

Hygothalamus

In all species examined, OXA- and OXB-IR fibers extended from the
lateral hypothalamus throughout much of the brain. These fibers were
moderately to densely distributed throughout much of the hypothalamus, with the
exception of the zona incerta, where fiber density was low, and the
suprachiasmatic (Figure 2.9) and medial mammilary nuclei, which exhibited little
to no orexin fibers in any species. Several species differences were observed.
First, differences in fiber density were seen in the main regions in which we saw
substantial differences in the distribution of labeled cells. In the hamster PeF,
fiber density was reduced in comparison with the other species, and in the LE rat
and grass rat, many OXA fibers were also present in the magnocellular Pa and
SO. OXB fibers in all species and OXA fibers in the hamster and degu were
moderate to low in the Pa and SO. Second, in the degu, a very dense network of
OXA and OXB fibers was present in the VMH (Figure 2.10), but in the other
species fiber densities in this region were moderate to low. The OXA and OXB
fiber complex in the degu VMH was the single most densely distributed network
of orexin fibers in this animal. Third, in the hamster, orexin-IR fibers in the lateral
mammillary nucleus (LM) were fairly dense, while in the other three species
fibers in the LM were sparse (Figure 2.11).

Thalamus

In contrast to the dense networks of OXA and OXB fibers in the
hypothalamus, orexin fibers were sparse or absent throughout much of the

thalamus. The majority of orexin projections to the thalamus targeted midline

63

Suprachiasmatic Nucleus

Ag" _- B

SCN

0C

Figure 2.9. Photomicrographs of orexin A fibers around the suprachiasmatic
nucleus (SCN) of the Long-Evans rat (A), grass rat (8), Syrian hamster (C), and
degu (D). 3V: third ventricle; OC: optic chiasm. Scale bar = 200 pm.

64

Ventromedial Hypothalamic Nucleus

A_‘-“B

VMH

 

Figure 2.10. Photomicrographs of orexin A fibers in the ventromedial
hypothalamic nucleus (VMH) of the Long-Evans rat (A), grass rat (B), Syrian
hamster (C), and degu (D). Note markedly higher density of orexin fibers in the
degu VMH than in the other three species. 3V: third ventricle; Arc: arcuate
nucleus. Scale bar = 300 um.

65

Lateral Mammillary Nucleus

‘\\ , MM
' LM

 

Figure 2.11. Photomicrographs of orexin A fibers in the lateral mammillary
nucleus (LM) of the Long-Evans rat (A), grass rat (B), Syrian hamster (C), and
degu (D). Note higher density of orexin fibers in the hamster LM than in the other
three species. MM: medial mammillary nucleus. Scale bar = 200 pm.

66

structures, most notably the paraventricular thalamic nucleus (PV), which
received dense or very dense orexin projections in all four species. In all species
examined, projections of OXA or OXB fibers to lateral regions of the thalamus
were most dense in the intergeniculate leaflet (IGL) (Figure 2.12); other lateral or
ventral thalamic nuclei received little to no orexin input. Although the pattern of .
orexin innervation in the thalamus was fairly consistent, the degu differed from
most or all of the other species examined in several regions. First, dense orexin
fibers were present in the degu centrolateral (CL) and paracentral nuclei (PC). In
the other three species, orexin fibers in these nuclei were mostly sparse or
absent, although the LE rat did exhibit moderate innervation in the PC (Figure
2.13). In contrast, the opposite pattern was observed in the xiphoid nucleus (Xi),
which contained sparse fibers in the degu and dense orexin innervation in the
other three species (Figure 2.14).

Telencephalon

Outside of the diencephalon, the distribution of orexin-IR fibers was more
heterogeneous. Orexin-A and OXB fibers were scattered throughout all areas of
the cortex; these fibers were more concentrated in inner and outer cortical layers,
and slightly reduced in Layers 3 and 4 in all animals. The olfactory bulbs
exhibited no fibers in the degu and hamster (no data were available here for LE
rat and grass rat), but sparsely scattered fibers were present in the olfactory
cortex and tubercle in all species. Orexin fiber density was uniformly low to
moderate in the septal nuclei, claustrum, and amygdala, with the exception of the

hamster medial amygdaloid nucleus, where orexin fibers were more dense than

67

Intergeniculate Leaflet

A DLG B

IGL
VLG

 

Figure 2.12. Photomicrographs of orexin A fibers in the intergeniculate leaflet
(IGL) of the Long-Evans rat (A), grass rat (B), Syrian hamster (C), and degu (D).
DLG: dorsolateral geniculate nucleus; VLG: ventrolateral geniculate nucleus.
Scale bar = 200 pm.

68

Centrolateral Thalamic Nucleus

ALV-.‘B

MHb LHb

CL

PVT

O
, D
e‘

 

;, f at!
,/~,-* . 3'
- 3351!?»-

Figure 2.13. Photomicrographs of orexin A fibers in the centrolateral nucleus
(CL) of the Long-Evans rat (A), grass rat (B), Syrian hamster (C), and degu (D).
Note higher density of orexin fibers in the degu CL than in the other three
species. LV: lateral ventricle; PVT: paraventricular thalamic nucleus, MHb:
medial habenular nucleus; LHb: lateral habenular nucleus. Scale bar = 300 pm.

69

Xiphoid Nucleus

A _, B

'\
_ ‘~ ‘- V‘r. (
* 7",
‘
XI
'1 " ‘ ‘. H a ‘ I b L
. " _ ‘f‘r... "- - ..., VN J
. , l .‘ - , f . . ' Y A‘- " "’- ::‘1$'$§«.'~?‘ 1'“: :3 -,:" .I ‘5’."
’ ' ‘1‘ V " I 1' 1'" " (‘3Vi . . , I "me“. .

Figure 2.14. Photomicrographs of orexin A fibers in the xiphoid nucleus (Xi) of
the Long-Evans rat (A), grass rat (B), Syrian hamster (C), and degu (D). Note
relative lack of orexin fibers in the degu Xi in comparison with the other three
species. 3V: third ventricle. Scale bar = 200 pm.

70

in the grass rat and degu, but not significantly more so than in the LE rat. The
density of both OXA and OXB fibers was very low in the hippocampus and basal
ganglia in all animals. The bed nucleus of the anterior commissure exhibited
moderate to dense orexin fiber innervation in all species.

Midbrain

In general, OXA and OXB innervation of midbrain structures was
moderate to low, with the exception of the dense fiber tracts in the periaqueductal
gray and the very dense plexus of OXA and OXB fibers projecting to the raphé
nuclei observed in all species (Figure 2.15).

Hindbrain

In the hindbrain, OXA and OXB-IR fiber densities were moderate to low in
the ascending brainstem tracts, motor and precerebellar nuclei, and were
sparsely distributed or absent in the cochlear, vestibular, and olivary nuclei in all
species. Visceral sensory nuclei, such as the nucleus of the solitary tract (Sol)
and the Kdlliker-Fuse nucleus, exhibited moderate to dense innervation by OXA
and OXB fibers in all species, but the nucleus with the most extensive network of
orexin fibers observed in the hindbrain was the LC (Figure 2.16). In the LE rat,
grass rat, and hamster, the LC was the most densely innervated structure
observed in the brain. In the degu, the network of OXA and OXB fibers present in
the LC was quite dense, but was slightly less so than that observed in the VMH.

Cerebellum

In the cerebellum, very few orexin fibers were observed in the LE rat,

grass rat or hamster, but the degu was markedly different. In the degu

71

Dorsal Raphé Nucleus

A “ B
' \. 1' you , _
, '2
' ) . “K“; .1 I',t“ .
..' DR‘ .‘ J"
> m H , .. \K'
2""
I.
4
—'
C . D _
\
E - J v t,
r, I‘. . '
\_ L'-
.1»: {3‘ j- :-:)V;
1 _; :5;
, -:‘~,cr-,,¢l,,
c ‘3 4 .
",9!
93'".

Figure 2.15. Photomicrographs of orexin A fibers in the dorsal raphé nucleus
(DR) of the Long-Evans rat (A), grass rat (B), Syrian hamster (C), and degu (D).
4: trochlear nucleus; Aq: cerebral aqueduct. Scale bar = 300 um.

72

Locus Coeruleus

   

   

‘1.
.: ~
.v; , ‘
_'~_’ I
. . r
‘x‘;q"_
. I. (I.
w '
u' i I
X
_ . .
, ..
. ., _‘ .
j- -- 4 ,‘ .
' “‘7‘ . ' ‘1 ‘ ..
‘x'r ..‘l'u, - I ,
, .’ Cl ,, ‘
.‘ ‘.’* I ‘
r . I) 7“ 't - ".1:
e. ., y. 2“ '_ \ "5 ...,
l, 'I I a ' " . s
,Ll--':t"~ -~.'.w .\
"- ‘. J ! ,s—r , 7 '~'
I _ ‘A‘V‘. c. P
..f; . 3. .
.x a“, y
r‘ ./ ~ -
4 oil.
, .J .‘
' I v-r
4 .

Figure 2.16. Photomicrographs of orexin A fibers in the locus coeruleus (LC) of
the Long-Evans rat (A), grass rat (B), Syrian hamster (C), and degu (D). 4V:
fourth ventricle. Scale bar = 200 pm.

73

cerebellum, moderate OXA and OXB fiber densities were observed in some deep
cerebellar nuclei, as well as the flocculus (Figure 2.17).

Circumventricular regions and ependyma

In Circumventricular organs, OXA innervation in all animals was moderate
to low, and there were even fewer OXB fibers. In the LE rat and grass rat, the
median eminence exhibited greater densities of OXA-IR fibers relative to the
other two species, although this difference is likely explained by axonal

projections from the OXA-IR cells in the Pa and SO in these two species.

Discussion

General observations

We present here a thorough examination of the distribution of OXA— and
OXB-IR cell bodies and fibers in four species. Prior to this study, no published
data were available describing orexin A or B in the LE rat strain or in the degu,
and no data were available for OXA in the grass rat or OXB in the Syrian
hamster. The data presented here correspond well with previously published
examinations of OXA in the Syrian hamster (McGranaghan and Piggins 2001;
Mintz et al. 2001) and of OXB in the grass rat (Novak and Albers 2002).

At the most general level, the three most significant observations in this
study were: (1) There is a high degree of correspondence within species
between OXA and OXB; (2) The overall pattern of orexin cell and fiber
distribution is very similar in these four species and to those described previously

for other strains and species of mammal, including Wistar and Sprague-Dawley

74

F locculus

A . B ‘ PFI

Fl

VC

_ '
. . , '3‘
. ° ‘ , I.
' ' ., . J;
- \
I ‘l
\’ . ' ‘
. .: ‘
.
I

4"."
C

Figure 2.17. Photomicrographs of orexin A fibers in the flocculus (Fl) of the
Long-Evans rat (A), grass rat (B), Syrian hamster (C), and degu (D). Note higher
density of orexin fibers in the degu Fl than in the other three species. PFI:
paraflocculus; VC: ventral cochlear nucleus. Scale bar = 200 um.

75

rats (Peyron et al. 1998; Chen et al. 1999; Cutler et al. 1999; Date et al. 1999;
Nambu et al. 1999), Djungarian hamsters (McGranaghan and Piggins 2001;
Khorooshi and Klingenspor 2005) and primates (Horvath et al. 1999a; Arihara et
al. 2000; Moore et al. 2001); and (3) Despite the overall similarity between
species, there are several striking differences, both with respect to the
distribution of orexin-IR cells, as well as in the relative density of orexin-IR fibers
in some regions.

The implications of the observation that there are high levels of
correspondence between OXA and OXB distribution within species are twofold.
First, although at least some OXA-only cell types were identified in the current
study (see below), our data support the general assumption that the majority of
cells and fibers expressing OXA immunoreactivity also express OXB, consistent
with previous reports (Zhang et al. 2002). Second, the overlap between OXA-
and OXB-IR fibers in all species studied suggests that the individual actions of
OXA and OXB in regulation of physiology and behavior are more likely due to
differences in the distribution of the orexin receptor subtypes within the brain than
to differential distribution of the two forms of the peptide. The two orexin receptor
subtypes do exhibit marked differences in distribution in rats (Lu et al. 2000;
Hervieu et al. 2001; Marcus et al. 2001), and both rat and human orexin
receptors express differential responsiveness to OXA or OXB (Sakurai et al.
1998; Smart et al. 1999). We did not map the distribution of orexin receptor
subtypes in the species examined in the present study and are therefore unable

to directly address this issue in the four rodents studied here.

76

The second major pattern seen in this study, that there are overall
similarities in the distribution of orexins between species, suggests that orexin
networks are strongly conserved among species. This implies that the functions
of orexin are likely to be similar in the LE rat, grass rat, Syrian hamster, and
degu, as well as in other species. The orexins are strongly conserved peptides
(Sakurai et al. 1999; Shibahara et al. 1999; Ohkubo et al. 2002). The orexin gene
arose early in chordate evolution (Alvarez and Sutcliffe 2002), and has been
retained in all major vertebrate classes, including fish (Kaslin et al. 2004; Huesa
et al. 2005), amphibians (Shibahara et al. 1999; Yamamoto et al. 2004;
Singletary et al. 2005), reptiles (Farrell et al. 2003), birds (Ohkubo et al. 2002),
and mammals (Sakurai et al. 1999). This pattern is evidence for an essential
functional role for the orexins in mediation of behavioral and physiological
processes common to all vertebrates, and further implies strong evolutionary
constraints against modification of projections from orexin cells.

Lastly, the differences observed between species in such a strongly
conserved peptide family may reflect subtle functional differences related to the
life history, behavior, or physiological organization of different animals. We focus
the rest of our discussion on the species differences in the distribution of orexin-

IR cells and fibers.

Species differences in OXA and OXB cell distribution

Though few in number, the three differences observed between species in

the overall distribution of orexin-IR cell bodies were striking. First, orexin cells

77

were conspicuously absent in the hamster PeF. Second, the distribution of the
main body of orexin-IR perikarya within the caudal diencephalon in the degu was
markedly different from that seen in the other species examined in this study.
Third, the presence of OXA-IR cell bodies in the Pa and SO was noted in three of
the four species examined.

In the hamster, previous reports stated that orexin-IR neurons were
primarily located in LHA and PeF (McGranaghan and Piggins 2001; Mintz et al.
2001). In the present study, orexin-IR cells were largely absent in the PeF, and
were primarily found in the LHA dorsal to the perifornical region (Figure 2.7). It is
possible that the difference observed between the present data and those
described in previous reports reflect differences in delineation of specific
hypothalamic areas. The present study relied on a hamster-specific atlas to
identify brain regions (Morin and Wood 2001); as noted by the authors of this
atlas, the hamster brain is in many respects organized quite differently from that
of the rat (Morin and Wood 2001). At least one previous study (McGranaghan
and Piggins 2001) used a rat brain atlas (Paxinos and Watson 1998); the second
report (Mintz et al. 2001) did not indicate the method used for identification of
different regions. In comparison with the rat brain atlas, the region identified as
PeF in the hamster atlas is much smaller and situated closer to the fornix
(Paxinos and Watson 1998; Morin and Wood 2001). However, even if an
expanded rat-like definition of the PeF is applied to the hamster, it is clear that
the number of orexin neurons observed near the fornix is lower in the hamster

than in the other three species examined in this study (see Figures 2.5 - 2.8).

78

In the degu, two main differences were noted in the distribution of orexin-
lR perikarya within the caudal diencephalon in comparison with the other three
species. First, the degu exhibited orexin neurons through a much larger extent of
the rostral-caudal axis of the hypothalamus, with cells expressing OXA or OXB
extending from the RCh to the SuM (Figure 2.8). Second, the overall organization
of the main body or orexin neurons differed in the degu relative to the other
species in that two distinct clusters of orexin-IR cells were present, one
dorsomedial to the fornix, and a second group ventrolateral to the fornix. In the
caudal portion of their distributions, these groups merged into a single scattered
group of orexin-IR cells near the midline, below the third ventricle. The
differences in the overall distribution of orexin neurons in the degu relative to the
other three species raise the possibility that orexin cells in the ventrolateral LHA,
TC, and SuM of the degu receive different inputs or project to different targets
than orexin cells in the dorsal LHA and PeF of the rat, grass rat, or hamster.
Projections from these neurons might contribute to the overall differences in
orexin fiber distribution in the degu relative to the other three species (see
below).

Although the main body of orexin neurons in the LE rat, grass rat and
hamster LHA were fairly consistent with those described previously, the presence
of OXA-IR perikarya in the magnocellular nuclei of the hypothalamus in these
three species has not been described previously. The presence of orexin-IR
neurons outside of the lateral hypothalamus is not without precedent. Earlier

reports suggested the presence of OXA-IR neurons in the median eminence

79

(Chen et al. 1999), and OXB-IR neurons in limbic structures (Ciriello et al.
2003c). In general, the OXA-IR perikarya in the Pa, SO and SOR were not as
darkly stained as were OXA-IR neurons in the LHA and PeF, and the number of
OXA-IR cells in these nuclei varied between species (Figure 2.2, Figure 2.3).
Orexin A-IR cells in the Pa and SO were fairly dense in the grass rat, with fewer
OXA-IR cells visible in the LE rat Pa and SO. In the hamster, immunoreactivity
for OXA was generally negligible in cells of the Pa, but a small number of well-
defined OXA-IR neurons were visible in the SO.

The magnocellular neurosecretory cells of the Pa and SO are primarily
known for the production of arginine vasopressin (AVP) and oxytocin (OT),
peptide hormones delivered to the pituitary via projections to the median
eminence (reviewed in Armstrong 1995). These cells have been studied
extensively, and the normal functions of these nuclei are well-understood. The
presence of a previously unreported peptide in these cells is therefore quite
surprising. Several lines of evidence suggest that the OXA immunoreactivity in
these nuclei reflects the presence of the peptide rather than non-specific
immunoreactivity. First, tissue that has been blocked by preabsorption of the
OXA antibody with OXA blocking peptide reduces or eliminates the staining of
cell bodies in the Pa, 80, and LHA of all species examined (Figure 2.4). Second,
as immunohistochemical procedures for both OXA and OXB were identical
except for the primary antibody used, nonspecific binding of the secondary
antibody is unlikely (see Methods). Third, the OXA immunoreactivity seen in the

Pa, SO, and SOR does not appear to be the result of the primary antibody

80

binding to an orexin-like peptide that is normally found in these magnocellular
nuclei, as tissue reacted with the same antibodies does not show any evidence
of orexin-IR perikarya in the Pa or SO of the degu (present study), Sprague-
Dawley rat (personal observation), or the golden-mantled ground squirrel
(Sperrnophilus Iateralis) (personal observation). Finally, the OXA-IR neurons in
the SO and Pa are not the result of the antibody binding to AVP, as other regions
known to produce AVP, such as the SCN, do not exhibit similar immunoreactivity
(see Figure 2.9)

The Pa and SO are involved in a number of systems in the brain. These
nuclei project to the pituitary via the median eminence, where they release
vasopressin and oxytocin, peptides involved in reproduction, regulation of blood
pressure, and control of the hypothalamic-pituitary-adrenal (HPA) axis (reviewed
in Hatton 1990). The presence of OXA in these magnocellular nuclei in the LE
rat, but not in the inbred Wistar rat, might be related to the stress response,
which is higher in the LE rat than in the Wistar rat (Tohei et al. 2003). Orexin A
actions in the Pa appear to potentiate the stress response, in part by modulating
the release of corticotrophin releasing factor (CRF) (Ida et al. 2000b; Jaszberényi
et al. 2000; Samson et al. 2002). In contrast, OXB appears to have a suppressive
rather than a stimulatory effect on CRF release (Malendowicz et al. 1999), raising
the possibility that differences in OXA distribution might contribute to differences
in stress-related behavior and physiology between different strains of rats.

In addition to a potential role in the stress response, the presence of OXA-

IR neurons in the magnocellular hypothalamic nuclei could potentially be involved

81

in strain and species differences in some reproductive behavior, cardiovascular
response, or even feeding. With respect to reproductive functions, OXA has been
shown to both induce the release of gonadotropin releasing hormone (GnRH) in
the hypothalamus, and paradoxically to directly inhibit GnRH actions in the
pituitary (Russell et al. 2001). The influence of OXA on the release of both GnRH
and CRF seems to be mediated in part by neuropeptide-Y (NPY) (lda et al.
2000a; lda et al. 2000b; Jaszberényi et al. 2000; Russell et al. 2001), a peptide
known both to interact with orexin in the control of ingestive behavior (Yamanaka
et al. 2000) and to stimulate the release of OT and AVP from the Pa and SO
(Kapoor and Sladek 2001). Furthermore, the magnocellular Pa and SO receive
inputs from brain stem nuclei (such as the Sol) involved in regulation of
cardiovascular and visceral responses (Armstrong 1995). Many of these nuclei
are also heavily innervated by orexin fibers, suggesting a possibility for not only
direct actions of orexins within the Pa and SO, but indirect actions through orexin

efferents from the main body of orexin neurons within the LHA.

Species differences in OXA and OXB fiber distribution

There were several specific regions in which one or two species tended to
strongly diverge from the rest in terms of orexin-IR fiber density. In two of these
regions -the magnocellular neurosecretory nuclei of the hypothalamus in the LE
rat and grass rat, and the PeF in the hamster — differences in OXA and OXB fiber
density are directly related to the presence OXA cell bodies or the absence of

any orexin-IR neurons, respectively. A third area, the median eminence in the LE

82

rat and grass rat, is also likely different with respect to OXA fiber density than in
the hamster and degu because of projections originating from OXA-IR cells in the
LE rat and grass rat Pa and SO (although it is worth mentioning that the hamster,
which also exhibited OXA neurons in the SO, did not differ from the degu with
respect to OXA fiber density in this region). The species differences in relative
fiber density in other brain regions raise interesting questions. We will focus the
remainder of this discussion on the brain regions within each species that
showed the most unique patterns of innervation — specifically, the LM in the
hamster, and the VMH, CL, Xi, and cerebellum in the degu.

Orexin fiber density in the lateral mammillary nucleus was much higher in
the hamster than in the other three species, in which orexin fibers were sparse or
absent in this region (Figure 2.11). The mammillary nuclei in general receive
dense input from the hippocampus (reviewed in Simerly 1995), and lesion
studies suggest a role for the LM in spatial learning and memory in rats (Vann
2005). However, it is unclear at this time why the hamster LM should exhibit
increased orexin fiber input relative to the other species.

The most striking differences in orexin fiber distribution in this study were
seen in the degu. In this species, orexin fiber density was lower in the xiphoid
nucleus (Figure 2.14) and much higher in the centrolateral nucleus of the
thalamus (Figure 2.13) in comparison to the other three species. The degu also
exhibited an extremely dense network of orexin-IR fibers in the VMH,
predominantly in the ventromedial portion of this nucleus (Figure 2.10). In the

other species, orexin fiber density in the VMH was moderate to low, while this

83

nucleus represented the region of greatest fiber density observed in the degu.
With respect to the cerebellum, the degu exhibited moderately dense orexin-IR
innervation of the cerebellar nuclei and flocculus, while in the other three species
these regions were sparsely innervated at best (Figure 2.17). The grass rat, LE
rat, and Syrian hamster are relatively closely related to each other (all are
members of the Suborder Sciurognathi, Family Muridae), and the degu
(Suborder Hystricognathi, Family Octodontidae) is relatively distantly related to
them (Nowak 1999). It is therefore possible that the differences in fiber
distribution observed between the degu and the other three species are a
reflection of phylogenetic history and constrains associated with it, rather than
functional differences among these rodents. However, it is tempting to speculate
on the function of orexin projections to these regions in the degu.

Although the implication of the lack of orexin input to the xiphoid nucleus is
unclear, the networks of orexin-IR fibers in the degu CL, VMH and cerebellum
could all play a role in anti-predator behavior during periods of heightened
arousal, such as sentinel activity. When feeding, degus alternate sentinel duties
and produce alarm calls to warn conspecifics of danger (reviewed in Lee 2004).
Sentinel duty in the degu requires that an animal both detect potential threats,
and respond properly to threats once identified. The orexin-IR fibers in the
flocculus might aid in the visual detection of threats. Predator avoidance behavior
in the degu appears to be based primarily upon visual scans for aerial or ground-
level threats (Ebensperger and Hurtado 2005). Pursuit of a visually acquired

target requires smooth, coordinated eye movements to maintain visual contact

84

with a constantly moving stimulus (Lisberger et al. 1987). The flocculus has been
shown to be important in maintaining such predictive eye movements during
visual tracking (Robinson and Fuchs 2001). The CL and the VMH may be
involved in appropriate response to predators. Amygdalar projections from the
CL and other intralaminar thalamic nuclei are thought to engage fear responses
in rats (reviewed in Price 1995), and the CL specifically appears to be important
in cognitive awareness and executive decision making (Van der Werf et al.
2002). The VMH is part of the cerebral-hypothalamic “behavior control column”
mediating somatomotor aspects of complex motivated behavior (Swanson 2000).
The dorsomedial region of this nucleus is specifically implicated in predator-
induced defensive responses (Canteras 2002). As the three brain regions in the
degu receiving heavier than average orexin innervation may all be involved in
anti-predator behaviors, coordination of predator detection, tracking, and
appropriate fear and avoidance responses during periods of heightened arousal

may be an important function of orexins in the degu.

Summary

In summary, with respect to the overall distribution of orexin, this study
confirms the high degree of similarity among species but also reveals some
significant differences. The distribution of orexin-IR cell bodies and fibers is quite
similar among the Long-Evans rat, grass rat, hamster and degu, and is largely
consistent with previously published reports (Peyron et al. 1998; Chen et al.
1999; Cutler et al. 1999; Date et al. 1999; Nambu et al. 1999; McGranaghan and

Piggins 2001; Mintz et al. 2001; Novak and Albers 2002). With respect to orexin

85

fibers, the high levels of congruence between species suggest a strongly
conserved common role for OXA and OXB in the maintenance of arousal state,
modulation of somatomotor activity, and control of ingestive behavior. On the
other hand, there are some significant species differences in the distribution of
orexin cell bodies as well as the density of orexin-IR fibers in some regions. With
respect to cell bodies, the present study describes specific species differences in
the organization of the main body of OXA- and OXB-IR neurons in the lateral
hypothalamus, and also provides evidence for a previously undescribed
population of OXA-IR neurons in the magnocellular neurosecretory nuclei of the
hypothalamus. These differences in orexin cell distribution raise the possibility
that some subpopulations of orexin-IR neurons might differ with respect to
afferent inputs, suggesting potential differences in activation of orexin neurons
among species. In addition, the sometimes considerable differences in the
relative density of orexin-IR fiber input to different regions might reflect

differences in the importance or function of acute orexin actions in specific nuclei.

86

Chapter 3

Nixon, J. P. and L. Smale (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.

87

CHAPTER 3
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)

Introduction

In mammals, circadian rhythms in physiology and behavior are generated
by the suprachiasmatic nucleus (SCN) and entrained by the light-dark cycle
through the retinohypothalamic tract (reviewed in Weaver 1998). In addition to
photic cues, circadian rhythms can also be modified by non-photic signals, such
as exposure to a novel running wheel or gentle interruption of sleep, in a variety
of nocturnal and diurnal species (Mrosovsky and Salmon 1987; Amir and Stewart
1996; Hastings et al. 1997; Klerman et al. 1998; Hut et al. 1999; Kas and Edgar
1999; Antle and Mistlberger 2000). The geniculohypothalamic tract (GHT),
extending to the SCN from the intergeniculate leaflet (IGL) of the lateral
geniculate nucleus (LGN) represents one source of non-photic input to the SCN
(reviewed in Harrington 1997). The subpopulation of IGL cells that contain
neuropeptide-Y (NPY) play an especially important role. This conclusion is based
on studies in hamsters that have looked at c-Fos expression in NPY cells, or at
effects of infusions of NPY or NPY antagonists into the SCN (Albers and Ferris
1984; Huhman and Albers 1994; Janik and Mrosovsky 1994; Wickland and Turek

1994; Biello 1995; Janik et al. 1995).

88

We recently investigated the relationship between NPY-containing cells in
the IGL and patterns of wheel running in the grass rat, Arvicanthis niloticus
(Smale et al. 2001b). The grass rat is endemic to equatorial Africa and exhibits
diurnal patterns of activity both in the laboratory and in the wild (Katona and
Smale 1997; Blanchong and Smale 2000). Although grass rats do not exhibit
phase shifts when given access to a novel wheel (L. Smale, unpublished
observations), some switch to a “nocturnal” pattern of activity when given
continuous access to a wheel (Blanchong et al. 1999). Several lines of evidence
suggest that this change reflects an unusual form of masking, rather than a
switch within the circadian timekeeping system. First, several basic features of
the activity patterns are the same in day- and night-active grass rats. Specifically,
both exhibit a short burst of activity prior to lights-on and a peak immediately after
lights-off (Blanchong et al. 1999). Second, night-active animals appear to
partition total sleep time equally between the light and dark periods, but they
experience more fragmented sleep bouts during the light period (L. Smale and M.
Schwartz, unpublished observations), suggesting that a signal promoting diurnal
wakefulness remains. Third, although hourly body temperature averages of night-
active grass rats are lower than those of day-active animals during the light
period, their lowest body temperatures still occur late in the night, as they do in
day-active grass rats (Blanchong et al. 1999). Finally, there are no transients
during switches from night- to day-active patterns following removal of a wheel,
or on the rare occasions that switches occur spontaneously (see below). If the

switch between patterns was driven by changes in the SCN, transients would be

89

expected (Redlin and Mrosovsky 2004). Thus running at night appears to result
in increased nocturnal arousal without a change in the fundamental underlying
diurnal circadian system. Using this animal model therefore allows us to examine
activity-induced arousal either in conjunction with circadian signals promoting
wakefulness (in day-active grass rats), or without them (in night-active grass
rats). In this model system, the dissociation between activity and circadian
influences emerges from the animal’s own behavior, running in a wheel that is
continuously available, rather than being triggered by periodic changes in the
animal’s environment.

As in other mammals, the SCN of the grass rat receives NPY input that
comes from the IGL (Smale and Boverhof 1999; Smale et al. 2001b). When
provided continuous access to running wheels, Fos immunoreactivity (Fos-IR)
within NPY cells is high during the day and low at night for day-active grass rats,
and low during the day but high at night in night-active ones. Grass rats housed
with running wheels show higher percentages of NPY cells expressing Fos than
do animals without running wheels (Smale et al. 2001 b). The reversed rhythm
within NPY cells of night— and day-active grass rats is therefore caused, at least
in part, by the differences in when these animals run.

Although projections to the IGL have been well-defined (Morin 1994;
Vrang et al. 2003), little is known about the specific route through which
information regarding arousal state reaches the NPY cells in the IGL. One
possibility involves the orexins (hypocretins), a recently described family of

peptides present in cells of the lateral hypothalamus (LH) (Sakurai et al. 1998; de

90

Lecea and Sutcliffe 1999). The orexins consist of two peptides, orexin A (OXA)
and B (OXB), derived from the same precursor protein (Sakurai et al. 1998; de
Lecea and Sutcliffe 1999). Orexins are thought to be involved in the regulation of
arousal, general activity and feeding (Lubkin and Stricker—Krongrad 1998;
Edwards et al. 1999; Hagan et al. 1999; Kunii et al. 1999; Piper et al. 2000;
Estabrooke et al. 2001; Hungs et al. 2001; Yoshimichi et al. 2001; Kotz et al.
2002). OXA appears to play a more important role in many of the reported
actions of the orexins than does OXB (Sakurai et al. 1998; Edwards et al. 1999;
Kunii et al. 1999).

Fibers containing orexin project to the IGL in several species, including the
hamster (McGranaghan and Piggins 2001; Mintz et al. 2001) and the grass rat
(Novak and Albers 2002), raising the possibility that orexin might mediate the
influence of arousal on NPY cells. To investigate this possibility, we first
examined patterns of OXA and OXB cell activation in relation to activity state (as
indicated by wheel running) and time of day in day— and night-active grass rats.
We then examined tissue processed for NPY and either OXA or OXB
immunoreactivity (IR) to determine whether orexin fibers could contact NPY cells

in the IGL.

Methods

Animal housing and determination of activity patterns

Thirty-nine adult male grass rats bred in a captive colony were used in this

study. Animals were kept on a 12:12 lightzdark cycle with a red light (< 5 lux) kept

91

on constantly and were provided food (Harlan Teklad 8640 rodent diet, Harlan
Teklad Laboratory, Madison, WI) and water ad libitum. All animal handling
procedures followed in this study were approved by the Michigan State University
All-University Committee for Animal Use and Care. All animals were individually
housed in cages (28 x 34 x 16 cm) that in some cases were equipped with
running wheels (26 cm diameter, 8 cm width). Each wheel revolution was
recorded when a magnetic bar on the wheel closed a switch mounted on the
wheel housing. Wheel running data were collected and quantified using the DSI
Dataquest 3 system (MiniMitter, Sunriver, OR). After stable wheel running
rhythms had been established for at least one week, animals were classified as
day- or night-active. A grass rat was classified as night-active if wheel running
continued for more than 4 hours after lights-out, while day-active animals were
defined as those that ceased running within 2 hours after lights-out. Animals that
exhibited ambiguous activity patterns were excluded from the study. Average
hourly numbers of wheel revolutions were calculated from the last 5 days in
running wheels, and animals were considered to be inactive during a given hour
if the number of wheel revolutions was less than 10% of the daily peak. Average

hourly activity levels to illustrate both patterns are presented in Figure 3.1.

Tissue processing and analysis

At the time of sacrifice, all grass rats were deeply anesthetized with
sodium pentobarbital (Nembutal; Abbot Laboratories, North Chicago, IN).
Animals sacrificed during the dark period were fitted with light-tight hoods to

prevent exposure to light during perfusion. Animals were perfused transcardially

92

Figure 3.1. (A) Actogram illustrating wheel running activity of an individual grass
rat. Although initially day-active, this individual exhibited a spontaneous switch to
a night-active pattern, illustrating both the two basic patterns and the lack of
transients during a switch. This spontaneous switch is atypical; most animals in
our colony establish stable day- or night-active rhythms shortly after introduction
of a wheel. (B) Line graph illustrating average hourly wheel revolutions over a 5
day monitoring period of all day-active (n = 18, open circles) and night-active (n =
18, closed circles) grass rats used in this study. Arrows at ZT 4 and ZT 16
indicate sampling times for animals used in this study. Bar at bottom indicates
light cycle.

93

 

 

 

 

 

 

 

 

-0— Day-Active
—o— Night-Active

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0000'

 

 

4000 ‘
3000
2000 ‘
1 000

mama .50: Log mco_S_o>o._ _ooc>>

Time (ZT)

94

with 0.01 M phosphate-buffered saline (PBS; pH 7.4, 150 — 200 mllanimal)
followed by 150 to 200 mL fixative (4% paraformaldehyde with 75 mM lysine and
10 mM sodium periodate, in 0.1 M phosphate buffer, pH 7.2) Brains were post
fixed for 8 h, then transferred into 20% sucrose. After 24 h in sucrose, brains
were stored at -20°C in cryoprotectant (Watson et al. 1986) until the time of
sectioning. All brains were sectioned at 30 pm in three series using a freezing
microtome.

For all studies, tissue was processed for immunohistochemical staining
using the following general procedure. All sections were incubated in 5%
blocking serum in PBS with 0.3% Triton-X 100 (Research Products International,
Mount Prospect, IL) for 1 h. Tissue was then incubated in primary antibody for 24
h at 4°C in PBS with 0.3% Triton-X 100 (PBS-TX) and 3% serum. Next, tissue
was incubated in biotinylated secondary antibody (1:500; in PBS-TX with 3%
serum) for 1 h. Tissue was then placed in avidin-biotin complex (AB complex;
0.9% each avidin and biotin solutions, Vector Laboratories, Burlingame, CA; in
PBS-TX) for 1 h. Sections were then rinsed three times for 10 minutes in acetate
buffer, then reacted for 7 minutes with diaminobenzidine (DAB; 0.1 mg/ml,
Sigma) enhanced with nickel chloride (25 mg/ml; Sigma) in acetate buffer with
3% hydrogen peroxide. Following the nickel-enhanced DAB reaction, tissue was
rinsed 5 times for 2 minutes in PBS-TX. Nickel-labeled tissue was then blocked
using 5% serum in PBS-TX for 1 h, then allowed to react with primary antibody
for 24 h at 4°C (in PBS-TX and 3% serum). This step was followed by incubation

in biotinylated secondary antibody for 1 h (1:500; in PBS-TX and 3% serum).

95

Tissue was then incubated for 1 h in AB complex. Following the AB incubation,
tissue was reacted with DAB (0.5 mg/ml, Sigma) in a tris-hydrochloride buffer
(Trizma, Sigma; pH 7.2) with hydrogen peroxide (0.35 pl 30% hydrogen
peroxide/ml buffer). For all reactions, primary antibodies were omitted in control
sections. During immunohistochemical processing, all issue was rinsed three
times for 10 minutes in PBS between each step, with the exception of the PBS-
TX rinses between AB complex incubation and nickel-DAB reaction.

To evaluate relationships between orexin fibers and NPY cells, tissue was
blocked using normal horse serum (NHS; Vector), then reacted with either goat
anti-OXA or goat anti-OXB primary (Santa Cruz Biotechnology, Santa Cruz, CA;
1:10,000) and horse anti-goat secondary (Vector). Following PBS-TX rinses after
the orexin nickel-DAB reaction, tissue was rinsed for 24 h in PBS at 4°C. For the
NPY reaction, tissue was blocked using normal donkey serum (NDS; Jackson
Laboratories, West Grove, PA), then incubated in rabbit anti-NPY primary
(Peninsula Labs, Belmont, CA; 112,000) and donkey anti-rabbit secondary
(Jackson). For the orexin-Fos study, tissue was blocked using NDS then nickel-
labeled for Fos using a rabbit anti-Fos primary antiserum (Santa Cruz; 1:2000)
and donkey anti-sheep secondary (Jackson). Fos-labeled tissue was then
reacted for either OXA or OXB antisera using the same serum and antibodies
described above. The supplier reports no cross-reactivity between the OXA and
OXB antibodies used in this study; preabsorption tests using blocking peptides

supplied by Santa Cruz confirm this in our tissue.

96

After processing for orexin and NPY or for orexin and Fos, all tissue was
mounted on gelatin coated slides, dehydrated, and coverslipped. Tissue was
examined using a light microscope equipped with a drawing tube (Leitz, Laborlux
S; Wetzlar, Germany). High-resolution digital photographs of representative
sections were taken using a digital camera (Carl Zeiss, AxioCam MRc;
Gdttingen, Germany) attached to a Zeiss light microscope (Axioskop 2 plus).
Image contrast and color balance were optimized using Zeiss AxioVision
software (Carl Zeiss Vision). Graphs were prepared using SigmaPlot (SPSS;
SPSS, Chicago, IL). Final figures were assembled using Adobe Illustrator and

Adobe Photoshop (Adobe Systems, San Jose, CA).

Patterns of Fos Expression in Orexin Cells

For wheel running studies, tissue from a total of 36 animals (n = 18 day-
active, 18 night-active) was collected as follows: Six day-active and six night-
active animals were housed with running wheels and sacrificed at Zeitgeber time
(ZT) 4. A second group of six day-active and six night-active animals were
housed with running wheels and sacrificed at 2T 16. The final twelve day- and
night-active grass rats were initially housed with running wheels, but wheels were
removed 5 days prior to sacrifice. Day-active animals in this final group were
sacrificed at ZT 4, while the night-active animals without running wheels were
sacrificed at ZT 16. We did not examine effects of running wheels on day-active
animals at ZT 16 or night-active animals at ZT 4, because these animals do not

run at these times.

97

Following immunohistochemical staining for orexin and Fos, three
representative sections per animal were chosen for analysis, following the
method of Martinez et. al. (2002). Briefly, a box 1200 pm by 700 pm was placed
with one side against the third ventricle, and the bottom of the box aligned with
the ventral border of the fornix (Figure 3.2). The box was subdivided into three
equal regions, referred to here as the medial, central, or lateral regions. Although
this box does not include all areas of the grass rat brain containing orexin cells,
examination of several animals revealed that the majority of orexin-
immunoreactive cell bodies in the lateral hypothalamus and perifornical area fall
within the boundaries of the box. The number of orexin, Fos, and double-labeled
cells in the box were counted unilaterally in each animal by an observer blind to
the experimental group of the animal. The hemisphere chosen for analysis was
randomized between subjects. All double-labeled cells were confirmed using a
high-power (50x) objective. Camera lucida drawings were prepared for each
section examined.

To determine whether differences in wheel running patterns are correlated
with differences in orexin cell activity, we compared day- and night-active animals
housed with running wheels and sacrificed at either ZT 4 or ZT 16. Percentages
of Fos-labeled OXA and OXB cells for each animal were calculated and analyzed
using SYSTAT (SPSS). Percentages were arc sine transformed and then
analyzed using a two-way analysis of variance (ANOVA), with phenotype (day-
vs. night-active) and time of sacrifice (ZT 4 vs. ZT 16) as independent variables.

To determine whether heightened Fos-IR in orexin cells was caused by access to

98

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3.2. Drawing of representative sections through (A) rostral, (B) middle
and (C) caudal sections of the grass rat LH. Boxes representing sampling area
are 1200 x 700 pm, divided from left to right into medial, central, and lateral
divisions. Dots represent cells stained for OXA. Distribution of OXB cells were
similar (not shown). 3V = 3rd ventricle; f = fornix.

 

99

the running wheel, we compared animals with running wheels to animals that had
running wheels removed 5 days prior to sacrifice. Percentages of Fos-labeled
OXA and OXB cells were arc sine transformed, and then effects of the wheels
were examined with paired t tests for day-active animals at ZT 4 and night-active
animals at ZT 16, respectively. Potential differences in distribution of double-
labeled cells were analyzed using multivariate repeated measures ANOVA, using
presence of wheel as a grouping factor, orexin type (OXA or OXB) as an
independent variable, and region of analysis (medial, central, or lateral) as
dependent variables. For all statistical analyses, the null hypothesis was

accepted when P > 0.05.

IGL Anatomy

For IGL anatomy studies, tissue from 3 grass rats was collected and
alternate series were processed for NPY and either OXA or OXB to determine
whether orexin fibers were in proximity to, and appeared to contact, NPY cells in
the IGL. Tissue was then examined at high power (100x) and relationships
between NPY cells and either OXA or OXB fibers in the IGL were examined and
photographed. Total numbers of NPY cells that did or did not appear to be in
contact with OXA and OXB fibers were counted for six representative sections

through the IGL in each animal.

100

Results

Patterns of Fos Expression in Orexin Cells

Orexin, Fos, and double-labeled cells were clearly visible and
distinguishable following immunohistochemical processing of tissue (Figure 3.3).
Although absolute numbers of OXA vs. OXB cells did not significantly differ
among treatment groups (data not shown), there were significant differences in
the percentage of orexin cells expressing Fos. Overall, the percentages of OXA
and OXB cells expressing Fos were higher when animals were actively running
at the time of sacrifice, and peak Fos expression in orexin cells was reversed
among day- and night-active phenotypes (Figure 3.4). Day-active animals
showed higher percentages of OXA and OXB cells expressing Fos in at ZT 4,
while night-active grass rats had higher percentages at ZT 16. Although OXA
cells appeared to be more likely to express Fos than did OXB cells at the time of
peak wheel running, the difference was not significant. ANOVA for both OXA and
OXB shows a significant interaction between phenotype and time (OXA: F =
15.461, df= 1, p = 0.001; OXB: F= 21.559, df= 1, p < 0.001).

Access to a running wheel significantly affected Fos percentages in OXA
and OXB cells in night-active grass rats only (Figure 3.5). No significant
differences were observed in OXA or OXB cells in day-active animals. Night-
active animals with wheels had significantly higher percentages of OXA cells
expressing Fos than animals without wheels (OXA: t = -2.861, p = 0.032; OXB: t
= -3.412, p = 0.019). Levels of double labeling in day-active grass rats with

wheels appeared to be higher than in those without wheels, but the difference

101

.3 ‘

 

Figure 3.3. Photomicrograph (40x) of orexin A and B cells in the grass rat LH.
Cells marked * are expressing Fos-IR only; Open arrows indicate cells
expressing only OXA (Panel A) or OXB (Panel B); Solid arrows point to double-
labeled cells. Scale bar = 50 pm.

102

 

(A)
01

Orexin A

00
O
l——I

 

N
01
J

N
O
l

.3
01

_L
O
l

U!

 

 

 

 

 

 

|:| Day-active Orexin B
all Night-active

l

 

(JO
0
l

N
01

 

N
O

 

Percent orexin cells expressing Fos
O

_x .3
O 01
1 1

0|
1

 

 

 

 

   

fl

 

 

 

 

O

ZT 04 ZT 16
Time

Figure 3.4. Temporal patterns of mean (:I: SEM) levels of Fos expression in OXA
(top) and OXB (bottom) cells in day- and night-active animals sacrificed at ZT 4
and ZT 16. Light bars represent day-active animals; dark bars represent night-
actlve grass rats. Significant differences (p < 0.05) noted by different letters.

103

 

(JD
01

Day-active grass rats - ZT 4

l

00
O
l

N
01
l

 

N
O
J

.3
U1
1

 

_L
O
1

U1
1

 

 

 

 

 

 

- OXA Night—active g'rass'rats - ZT 16
, I::l oxe

00
O

 

N N
O 01
l 41

Percent orexin cells expressing Fos
O

 

A
01
l
l
I
=4

 

.x
O
1

 

01
1

 

 

 

 

 

 

 

O

I j

+

 

Wheels

Figure 3.5. Fos-IR in orexin cells in grass rats housed with (+) and without (-)
running wheels. Day-active grass rats (top) were sacrificed at ZT 4, night-active
animals (bottom) at ZT 16. Dark bars = OXA; light bars = OXB. Significant
differences (p < 0.05) noted by different letters. Removal of running wheel
caused significant reductions in Fos-IR only in night-active animals. Effects of
wheels were not significant in day-active animals, although trends were similar (p
= 0.06 and p = 0.08 for OXA and OXB, respectively).

104

was not statistically significant (OXA: t = -2.230, p = 0.060; OXB: t = -1.940, p =
0.081).

In addition to differences in total percentages of orexin cells expressing
Fos-IR, significant differences in distribution of double-labeled cells were
observed in night-active grass rats (Figure 3.6). For animals with wheels, ANOVA
revealed significant effects of area (F = 14.698, df = 2, p < 0.001), with no
interaction with type of orexin (OXA vs. OXB). In animals without wheels, there
was an interaction between orexin type and area of analysis (F = 5.876, df = 2, p
= 0.010). Night-active grass rats with running wheels showed significantly higher
percentages of double-labeled cells in medial regions, and decreased
percentages in central and lateral portions of the area of study. Although the
overall activation of OXA cells was much lower in night-active animals without
wheels (Figure 3.6), the spatial gradient was still apparent. In contrast, OXB cells
in this group expressed virtually no Fos (Figure 3.6). In OXA and OXB cells of
day-active animals, no significant difference in Fos-IR was observed across
medial, central or lateral regions of the LH in any treatment group (day-active
with wheels: F = 2.401, p = 0.116; day-active without wheels: F = 1.190, p =
0.325).

IGL Anatomy
OXA and OXB fibers were both present in the IGL of all grass rats

examined, and clearly distinguished it from surrounding tissue (Figure 3.7).
These OXA and OXB fibers appeared to terminate on or follow the borders of

NPY cells throughout the rostro-caudal extent of the IGL. In some cases, orexin

105

 

50
Night-active, with wheels

.A
O

(a)
O
l

N
O
I

.r.
O
l

 

 

 

 

 

   

Percent orexin cells expressing Fos
O

 
   

     

 

 

 

- OXA Night-active, without wheels
II] OXB
40 .
30 .
20 -
10 ~ 3
b C
C c ' '
o

Medial Central Lateral

Figure 3.6. Regional patterns of orexin Fos-IR in night-active grass rats housed
with (top) or without (bottom) running wheels at ZT 16 demonstrating both the
medial to lateral gradient in orexin cell activity, and the differences and
Similarities between OXA- and OXB-labeled cells. Significant differences (p <
0-05) noted by different letters. Dark bars = OXA; light bars = OXB.

106

 

Figure 3.7. Photomicrographs of OXA (Panels A and B) and OXB (Panels C and
D) fibers and NPY cells in the grass rat IGL. Arrows indicate where contacts
between NPY cells and orexin fibers appear to be present. Panels (A) and (C)
are 40x; scale bar is 50 um. Panels (B) and (D) are 100x close-ups of regions
marked with " in panels (A) and (C), respectively; scale bar is 20 pm.

107

fibers appeared to wrap around the NPY cells. These fibers exhibited numerous
varicosities, suggesting the potential for synapses between the orexin fibers and
the NPY cells. In all animals, approximately half of the NPY cells appeared to be
contacted by OXA or OXB fibers (55.90 :I: 6.52% and 42.58 :I: 6.47%,
respectively). Boutons were also present on fibers that were not near NPY cells,
suggesting that orexin fibers may form synapses with other cell populations in
this region. Although synapses between orexin fibers and NPY cells can not be
observed using light microscopy, the nature of the spatial relationship between
these cells and fibers clearly suggest the potential for functional contacts
between NPY cells of the IGL and orexin fibers originating from the LH in the

grass rat.

Discussion

IGL Anatomy

Orexin fibers have been documented in the IGL of several species
(McGranaghan and Piggins 2001; Mintz et al. 2001), including the grass rat
(Novak and Albers 2002). The data presented here show that both OXA and
OXB fibers are not only present in the IGL but that they are closely associated
with NPY cells in this structure. While investigation of what appear to be contacts
at the light level cannot provide direct information about synapses, it does
suggest that the fibers communicate with a particular cell. Light-level
investigation of tissue reacted for either OXA or OXB and NPY suggest that

orexin fibers in the grass rat IGL may contact about half of the NPY cells visible

108

in the tissue (Figure 3.7). A greater percentage of NPY cells appeared to be
contacted by OXA fibers than by OXB fibers, but the variability was high, and
may simply reflect the small sample size or the denser distribution of OXA fibers
in the grass rat IGL (J. Nixon, unpublished observations) rather than a functional
difference. In many cases, the labeled fibers appeared to be wrapped around or
to closely follow the contours of the NPY cells. Numerous varicosities suggestive
of synapses were seen on orexin fibers where they appeared to contact NPY
cells. Varicosities were also present on orexin fibers that were not near NPY
cells, suggesting that they form synapses with other cell populations in the IGL
as well. Both the hamster and grass rat IGL contain enkephalin neurons that
project to the SCN (Morin and Blanchard 1995; Smale and Boverhof 1999), and
neurotensin- and GABA-positive neurons that project to the SCN have recently
been identified in the hamster IGL (Morin and Blanchard 2001). The varicosities
and boutons that were not near NPY neurons raise the possibility that orexin
projections might also be able to influence the circadian system through these
populations of neurons. Associations between orexin fibers and NPY cells
elsewhere in the brain set a precedent for orexin activation of NPY cells. Orexin
fibers form functional synaptic contacts with NPY-containing neurons in the
arcuate nucleus (Horvath et al. 1999a), and intraventricular administration of
OXA increases Fos expression in this population of NPY cells (Yamanaka et al.
2000). The present double-labeling data suggest that similar interactions

between NPY cells and orexinergic inputs could occur in the IGL.

109

Fos expression In orexin cells: general patterns

Orexin cell function has been correlated with activity state in a variety of
contexts, and direct experimental induction of arousal induces Fos in orexin cells
(Chemelli et al. 1999; Estabrooke et al. 2001). Here we used day- and night-
active grass rats as a model system with which to further examine orexin cell
function in relation to time of day and arousal state. Rather than directly arousing
the animals, we created conditions that, in some individuals, dramatically alter
the temporal patterns of spontaneous rhythms in arousal state by providing them
with running wheels (Blanchong et al. 1999). We found that with continuous
access to wheels day- and night-active grass rats showed the same general
relationship between locomotor activity and Fos expression in orexin cells, but
patterns of orexin cell function were reversed with respect to time of day.
Specifically, in both groups of animals, Fos-IR percentages in OXA- and OXB-
Iabeled cells were higher when the animals were actively running in wheels,
either at ZT 4 in day-active animals or at ZT 16 in night-active individuals (Figure
3.4). This difference between the day- and night- active animals could
theoretically reflect differences in direct circadian control over these cells, or they
could be consequences of the differences in activity. The latter hypothesis is
consistent with the data on how an animals’ access to a wheel influenced
patterns of Fos expression in orexin neurons. The effects of wheels on day-active
animals approached significance (p = 0.06 and 0.08 for OXA and OXB,
respectively), and they were significant in night-active animals for both OXA and
OXB (Figure 3.5). That is, continuous access to a wheel, which maintains arousal

at a time when these animals would otherwise sleep, induced orexin cell activity

110

in these night-active animals. These data provide further evidence that rhythms
in orexin cells emerge from mechanisms more directly associated with arousal,
rather than being directly driven by the circadian system (Taheri et al. 2000;
Estabrooke et al. 2001; Martinez et al. 2002). We also view this hypothesis as
more plausible for the same reasons that the effects of the running wheels on
circadian rhythms reflect masking, rather than effects on the circadian time-

keeping system, in our animals (see above).

Fos expression in orexin cells: heterogeneity of orexin cell function

Relatively little is known about potential functional differences among
orexin cell populations. Here, we found clear regional differences in the
distribution of orexin cells that were activated in night-active grass rats, but not in
day-active ones (with or without wheels). In night-active animals housed with
running wheels there was a medial- to lateral gradient of Fos expression at ZT 16
in both OXA and OXB cells such that these cells were more likely to express Fos
in the medial and central regions of the LH (Figure 3.6). This medial to lateral
gradient was not seen in any group of day-active animals.

Another surprising finding involved the difference between cells stained for
OXA and those stained for OXB in night-active animals without running wheels.
In these animals percentages of OXB cells with Fos-IR were extremely low
across the entire sampling area. In the same animals a clear medial to lateral
gradient was present in OXA cells (Figure 3.6). Thus, in the medial and central

regions OXA cells were considerably more likely to express Fos than OXB cells.

111

This finding was unexpected and may represent the first evidence of a functional
difference between cells stained for these two peptides. OXA and OXB originate
from the same precursor protein, suggesting that any cell producing OXA should
also produce OXB. Although OXA staining has been reported in various studies
to be higher than OXB-IR (Cutler et al. 1999; Date et al. 1999; Nambu et al.
1999), the assumption has been that this is because OXB degrades more rapidly
than OXA (Sakurai et al. 1998). In the current study it is not the absolute
numbers of OXA vs. OXB cells that is at issue, but, rather, the likelihood that
these cells express Fos. Percentages of OXA and OXB cells expressing Fos
were similar in all groups of animals studied except in night-active grass rats
housed without wheels. In these animals, the proportionally high levels of Fos
seen in the medial and central groups of OXA compared to OXB cells could
reflect either differences in rates of production or degradation of these two
proteins. For example, the relative rates of degradation of OXA vs. OXB could be
different in these medial and central cells than in orexin cells in the lateral region
of the LH. Alternatively, it is possible that some cells may produce OXA but not
OXB. Although both peptides come from the same precursor protein, there may
be some cellular level of regulation over the final processing such that one
peptide is produced in higher quantities than the other. Cells producing other
precursor proteins, such as pro-opiomelanocortin, do not always make every
peptide that can be derived from the precursor (Raffin-Sanson et al. 2003). Some
difference in the function of each orexin peptide is suggested by the reported

differences in binding affinity of OXA vs. OXB to each receptor (Sakurai et al.

112

1998; Smart et al. 1999) coupled with the differential and often non-overlapping
distribution of each orexin receptor subtype in many parts of the rodent brain,
including the IGL (Trivedi et al. 1998; Marcus et al. 2001). The data presented
here therefore raise intriguing questions about the functional impact of
heterogeneity among orexin cell populations with respect to possible effects on
their target cells.

Interestingly, the conditions under which the OXA, but not OXB cells
expressed Fos are the conditions in which behavioral differences between the
day- and night-active “wheel-runners” are not apparent (Blanchong et al. 1999).
That is, when a wheel is removed from the cage of a night-active grass rat its
activity pattern immediately becomes indistinguishable from that of a diurnal
grass rat. The tendency to become night-active when presented with a running
wheel is heritable (Blanchong et al. 1999), but neural mechanisms underlying the
predisposition are unknown. The unexpected finding of Fos expression in some
medial OXA neurons of animals pre-disposed to become night-active raises the
intriguing possibility that inter-individual differences in the medial OXA cells could
actually influence whether animals run at night when a wheel is made available

to them.

General conclusions

Orexin cells are uniquely positioned to communicate arousal state
information to cell populations elsewhere in the brain because of their

widespread fiber distribution and well-established association with sleep-related

113

nuclei such as the locus coeruleus (Peyron et al. 1998; Cutler et al. 1999; Date et
al. 1999; Nambu et al. 1999; McGranaghan and Piggins 2001; Mintz et al. 2001;
Novak and Albers 2002). Both the physical contacts we observed between orexin
fibers and NPY neurons, and the temporal patterns of orexin cell function,
provide some support for the hypothesis that the orexins represent a candidate
system for relaying arousal state information to the NPY cells in the IGL.
Temporal patterns of change in Fos-IR in orexin cells of grass rats housed with
running wheels were very similar to those found previously in NPY cells of
animals under the same housing conditions (Smale et al. 2001b), and both
populations of cells respond with an increase in Fos to input signals associated
with locomotor activity. Although these patterns could reflect similar input signals
to these two populations of cells (eg. from the raphé nucleus), they might reflect
a direct functional link through which orexin cells relay activity state information to
the NPY cells in IGL.

A second basic theme that emerged from the data involves the functional
heterogeneity of the orexin cell population. In night-active grass rats orexin cells
located in different regions of the hypothalamus exhibited different patterns of
activation, and patterns of Fos activation among OXA cells did not always
parallel those of OXB cells. Heterogeneity with respect to patterns of Fos
expression in different populations of orexin cells could theoretically reflect
differences in the nature of the inputs these cells receive, or in how these cells
respond to incoming signals. The heterogeneity of the OX cell population is a

subject that warrants further investigation.

114

The act of running in a wheel has been described as abnormal obsessive
behavior, a self-reinforced act that persists in part due to feedback that overrides
normal behavioral and physiological signals (reviewed in Sherwin 1998). In night-
active grass rats, wheel-running at the beginning of the dark period, perhaps
initiated by circadian signals, may feed back on itself in a positive way,
overwhelming the influence of physiological systems that would ordinarily lead
the animal to sleep. The present results raise the possibility that activation of the
orexin system by wheel running could provide a signal that feeds back in some
individuals to prolong the period of running well into the night, overwhelming
circadian signals that ordinarily promote sleep at this time. Previous studies have
linked orexin cell activation to high-arousal states, such as stress (Espafla et al.
2003). It is interesting to note that stress-related arousal has been implicated as
a possible cause of insomnia (Bonnet and Arand 2000; Drake et al. 2003). Night-
active grass rats, with their obsessive wheel-running, may thus represent an
interesting animal model of how arousal systems, such as orexin pathways,

might be related to insomnia.

115

Chapter 4

Nixon, J. P. and L. Smale (2005). "Orexin fibers form appositions with fos
expressing neuropeptide-Y cells in the grass rat intergeniculate leaflet." Brain
Research 1053(1-2): 33-37.

116

CHAPTER 4
Orexin fibers form appositions with Fos expressing

neuropeptide-Y cells in the grass rat intergeniculate leaflet

Introduction

In mammals, circadian rhythms in physiology and behavior are generated
by the suprachiasmatic nucleus (SCN) (Ralph et al. 1990) and entrained by the
light-dark cycle through the retinohypothalamic tract (Johnson et al. 1988). In
several nocturnal and diurnal species, circadian rhythms can also be modified by
non-photic signals, such as access to a novel running wheel (Mrosovsky and
Salmon 1987; Hastings et al. 1997; Hut et al. 1999; Kas and Edgar 1999; Antle
and Mistlberger 2000). One important pathway through which both photic and
non-photic information reaches the SCN is the geniculohypothalamic tract (GHT),
which originates in the intergeniculate leaflet (IGL) of the lateral geniculate
nucleus (Harrington 1997). Although most IGL afferents arise from structures that
are part of the visual system (Morin and Blanchard 1998; Vrang et al. 2003;
Horowitz et al. 2004), including the retina (Smale et al. 1991; Morin et al. 1992;
Moore and Card 1994; Goel et al. 1999; Smale and Boverhof 1999), neural
structures and cell groups associated with arousal (such as the raphé nuclei)
also project to the IGL (Harrington 1997; Vrang et al. 2003). Lesions of the IGL
as well as peptide infusion and blocking studies in several rodent species have
shown that the subpopulation of IGL cells that contain neuropeptide-Y (NPY) play

an important role in the integration of photic and non-photic effects on circadian

117

rhythmicity (Albers and Ferris 1984; Johnson et al. 1989; Huhman and Albers
1994; Wickland and Turek 1994; Biello 1995; Marchant et al. 1997).

Although projections to the IGL have been well-defined (Morin 1994;
Vrang et al. 2003), little is known about the specific route through which
information regarding arousal state reaches the NPY cells in this region. Several
lines of evidence suggest that one source of input might be cells in the lateral
hypothalamus (LH) that contain orexins (hypocretins), a family of peptides
involved in the regulation of arousal, general activity and feeding (Hagan et al.
1999; Estabrooke et al. 2001; Hungs et al. 2001; Kotz et al. 2002). First, fibers
containing orexin project to the IGL in several nocturnal and diurnal species
(McGranaghan and Piggins 2001; Mintz et al. 2001; Novak and Albers 2002). We
have recently shown that in one species these fibers appear to contact NPY cells
within the IGL (Nixon and Smale 2004). Second, increases in physical activity
associated with sleep deprivation or forced exercise are positively correlated with
increased release of orexin into cerebrospinal fluid of several species (Wu et al.
2002; Martins et al. 2004). Finally, orexin release is known to stimulate NPY cells
in other brain regions (Yamanaka et al. 2000), raising the possibility that NPY-
containing cells might be capable of responding to orexins more generally.

Neuropeptide-Y and orexin have both been associated with non-photic
influences on rhythms in the grass rat, Arvicanthis niloticus, a murid rodent that
exhibits diurnal patterns of general activity both in the laboratory and in the wild
(Katona and Smale 1997; Blanchong and Smale 2000). When given continuous

access to a novel wheel, some individual grass rats exhibit an unusual form of

118

masking, switching from a day-active to a night-active pattern of activity
(Blanchong et al. 1999). In our colony, this shift from day- to night-active occurs
in approximately 30% of animals given free access to a wheel (Mahoney et al.
2001). This switch to a night-active pattern is not associated with transients
(Nixon and Smale 2004; Redlin and Mrosovsky 2004), and appears to reflect a
masking effect of the wheel rather than a shift in the internal clock (Nixon and
Smale 2004).

Recent investigations in our laboratory suggest a relationship between
wheel running patterns, orexin cells and NPY-containing cells in the grass rat
IGL. Specifically, individual differences in patterns of wheel running in the grass
rat are correlated with patterns of Fos expression in both neuropeptide-Y cells in
the intergeniculate leaflet (Smale et al. 2001b) and in orexin cells in the lateral
hypothalamus (Nixon and Smale 2004). We have also shown that orexin-
containing fibers appear to contact NPY cells in the grass rat IGL (Nixon and
Smale 2004). Taken together, these data suggest that the orexin fiber-NPY cell
appositions may represent a pathway through which signals associated with
arousal reach NPY cells in the IGL. To evaluate this hypothesis further, we used
day- and night-active grass rats housed with running wheels to determine
whether orexin A (OXA) fibers contact NPY cells that express c—Fos, and whether
NPY cells with Fos are more likely to receive input from OXA fibers than are NPY

cells without Fos.

119

Materials and Methods

Tissue used in this study was collected during a previous experiment
(Smale et al. 2001b). Briefly, adult male grass rats bred in a captive colony were
kept on a 12:12 lightzdark cycle, with food and water provided ad libitum. All
animals were individually housed in cages equipped with running wheels; a
magnetic bar on each wheel closed a switch affixed to the cage. Wheel running
data were collected and quantified using the DSI Dataquest 3 system (MiniMitter,
Sunriver, OR). After establishment of stable wheel running rhythms, animals
were classified as day-active (n=3) or night-active (n=4). Tissue was collected at
Zeitgeber time (ZT) 4 for day-active animals, and at ZT 16 for night-active grass
rats. We did not look at night-active animals during the day or at day-active
animals during the night because Fos expression in NPY cells is extremely low in
these animals (Smale et al. 2001 b). At the time of sacrifice, grass rats were given
an overdose of sodium pentobarbital and transcardially perfused using 4%
paraformaldehyde in 0.01 M phosphate-buffered saline (PBS). Tissue was
sectioned on a freezing microtome at 30 micrometers and stored in
cryoprotectant at -20°C. All animal handling procedures in this study followed
National Institutes for Health guidelines and were approved by the Michigan
State University All-University Committee for Animal Use and Care.

Every third section from each animal was labeled for Fos, OXA, and NPY
immunoreactivity using the following procedure: All sections were incubated in
5% normal donkey serum (NDS; Jackson Laboratories, West Grove, PA) in 0.01

M PBS with 0.3% Triton-X 100 (TX; Research Products International, Mount

120

Prospect, IL) for 1 h. Tissue was then incubated in rabbit anti-Fos antibody
(Santa Cruz Biotechnology, Santa Cruz, CA; 1:25,000) for 24 h at 4°C, in PBS
with 0.3% TX (PBS-TX), 0.01% sodium azide (Sigma-Aldrich, St. Louis, MO)
and 3% NDS. Next, tissue was incubated in biotinylated donkey anti-rabbit
antibody (Jackson; 1:500, in PBS-TX with 3% N08) for 1 h. Tissue was then
placed in avidin-biotin complex (AB complex; 0.9% each avidin and biotin
solutions, Vector Laboratories, Burlingame, CA; in PBS-TX) for 1 h. Sections
were then rinsed in PBS, and reacted for 7 minutes with diaminobenzidine (DAB;
0.1 mg/ml, Sigma) enhanced with cobalt chloride (2 mg/ml; Sigma) in 0.1 M
phosphate buffer with hydrogen peroxide (15 pl 0.3% H202/ml buffer). Following
the cobalt-enhanced DAB reaction, tissue was rinsed in PBS with 0.02% TX.
Cobalt-labeled tissue was then blocked using 5% normal horse serum (NHS;
Vector) in PBS-TX for 1 h, then incubated in goat anti-orexin A primary antibody
for 24 h at 4°C (Santa Cruz; 1210,000, in PBS-TX, 3% NHS, and 0.01% sodium
azide). This step was followed by incubation in biotinylated horse anti-goat
antibody for 1 h (Vector; 1:200, in PBS-TX and 3% NHS). Tissue was then
incubated for 1 h in AB complex. Following the AB incubation, tissue was rinsed
in PBS, then in tris-hydrochloride (Trizma, Sigma; 15.4 g/L, pH 7.2). Tissue was
next reacted for 8 minutes with DAB (0.5 mg/ml, Sigma) in tris-hydrochloride with
hydrogen peroxide (0.35 pl 30% H202/ml buffer). Tissue was then rinsed in tris-
hydrochloride, followed by PBS, and then incubated at 4°C with gentle agitation
in PBS with 0.01% sodium azide for 24 hours. Tissue was then blocked for 1 h

using 5% NHS in PBS-TX, then incubated with rabbit anti-NPY antibody

121

(Peninsula Laboratories, Belmont, CA; 121000, in PBS-TX, 3% NDS, and 0.01%
sodium azide) for 24 h at 4°C, followed by incubation in biotinylated donkey anti-
rabbit antibody (Jackson, 1:200; in PBS-TX and 3% serum) for 1 h. Tissue was
then incubated for 1 h in AB complex, rinsed in PBS with 0.02% TX and then in
PBS. Sections were reacted for 45 seconds using the Vector Vectastain VIP kit,
following kit directions. Sections were then mounted on gelatin coated slides,
dehydrated, and coverslipped. Control sections were obtained by omitting one of
the three primary antibodies (anti-Fos, anti-OXA, or anti-NPY) prior to reacting
tissue. In these control sections, deletion of the primary antibody produced tissue
that was double-labeled for the remaining two antigens (either Fos and OXA, Fos
and NPY, or OXA and NPY).

Twelve representative sections from each animal were examined using a
light microscope equipped with a drawing tube (Leitz, Laborlux S; Wetzlar,
Germany). For each section, bilateral counts of all Fos nuclei in NPY cells that
did or did not exhibit appositions with orexin fibers (“NPY+OXA” or “NPY-Only”
cells, respectively) within the IGL were performed by an individual blind to the
identity of the animal. All Fos nuclei and OXA fiber contacts were verified under
high-power (100x) magnification. An NPY cell was counted as Fos-positive only if
the nucleus and cell boundary were clearly in the same plane of focus. Each
NPY-Fos cell also exhibiting one or more apposition with an OXA fiber was
counted as one NPY+OXA cell. In all cases, both Fos-positive nuclei and fiber
appositions that were unclear at high-power magnification were not included in

the analysis. The percentages of NPY-Only and of NPY+OXA cells that

122

expressed Fos were determined for each animal. Percentages were arc sine
transformed and compared using dependent t-tests. High-resolution digital
photographs of representative sections were taken using a digital camera (Carl
Zeiss, AxioCam MRc; Gottingen, Germany) attached to a Zeiss light microscope
(Axioskop 2 Plus). Image contrast and color balance were optimized using Zeiss
AxioVision software (Carl Zeiss Vision). Graphs were prepared using SigmaPlot
(SPSS; SPSS, Chicago, IL). Final figures were assembled using Adobe Illustrator

and Adobe Photoshop (Adobe Systems, San Jose, CA).

Results

Appositions between NPY-containing cells and OXA fibers were present
throughout the rostro-caudal extent of the IGL. Orexin A fibers within the IGL
exhibited numerous varicosities visible as distinct thickenings in orexin fibers.
Varicosities and boutons were especially apparent when fibers were in close
proximity to NPY cells. In many cases OXA fibers closely followed the borders of
NPY cell bodies, clearly suggesting that contact between the fibers and NPY
cells were not incidental. The NPY cells did not appear to exhibit appositions with
more than one OXA fiber, although in many cases individual OXA fibers
contacted more than one NPY cell. Orexin A fiber appositions with NPY cells
(Figure 4.1) were seen in both day- and night-active animals. In control sections,
deletion of each primary antibody eliminated all staining for its antigen (not

shown).

123

 

Figure 4.1. Photomicrographs (100x) of orexin A (OXA) fiber appositions with
neuropeptide—Y (NPY) cells (Panels A, B, C) or NPY cells expressing Fos
(Panels D, E, F) in the grass rat IGL. Arrows indicate appositions between OXA
fibers and NPY cells; Scale bar is 50 pm.

124

The percentage of all the NPY cells in the IGL that were contacted by
OXA fibers appeared to be higher in night-active grass rats than in day-active
animals, but the difference was not significant (Night-active mean=26.84:l:3.49%;
Day-active mean=14.69:l:3.77%; t=2.331, df=5, p=0.067). In day-active animals
there was no difference between levels of Fos expression within NPY cells with
OXA fiber appositions (“NPY+OXA cells”) than in NPY cells that were not
contacted by OXA fibers (“NPY-Only cells”) (t=-1.879, df=2, p=0.201). In night-
active animals, NPY+OXA cells were significantly more likely to express Fos than

were NPY-Only cells (t=-43.814, df=3, p<0.001) (Figure 4.2).

Discussion

The NPY cells of the grass rat IGL represent one of a very limited number
of cell groups directly involved in the circadian system in which day- and night-
active grass rats have been shown to differ. Specifically, Fos expression in NPY
cells is highest when animals are actively running in wheels, either during the day
(in day-active grass rats) or at night (in night-active grass rats) (Smale et al.
2001b). In contrast, other studies examing day— and night-active grass rats have
demonstrated no significant difference with respect to overall expression of c-
Fos, or of c-Fos expression rhythms in vasopressin, vasoactive intestinal
polypeptide, or calbindin cells within the SCN (Rose et al. 1999; Mahoney et al.
2000; Smale et al. 2001a). Furthermore, grass rats placed in constant conditions
continue to show stable day- or night-active activity rhythms, respectively

(Mahoney et al. 2001). We have also seen little indication that the switch to a

125

 

- NPY+OXA
[:1 NPY-Only *

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Day-Active (ZT 04) Night-Active (ZT 16)

Figure 4.2. Percentages (:tSEM) of Fos expression in neuropeptide-Y (NPY)
cells that do (NPY-OXA) or do not (NPY-Only) exhibit appositions with orexin A
(OXA) fibers in the IGL of day- and night-active grass rats. Asterisk indicates
significant difference between columns (p < 0.0001). Dark bars = NPY+OXA
cells; light bars = NPY-Only cells.

126

night-active running pattern involves a change in responsiveness to light; phase-
response curves and light-induced c-Fos within the SCN are quite similar in day-
and night-active animals (Mahoney et al. 2001). Taken together, these data
suggest that differences in activity rhythms of these grass rats do not simply
reflect individual variation in a masking response to light (but see (Redlin and
Mrosovsky 2004)). The similarities in SCN characteristics and responsiveness to
light suggest that the difference in activation patterns of NPY cells in day- and
night-active grass rats is more likely the result of non-photic influences, such as
arousal, rather than photic input to the IGL.

The data presented in this study suggest that orexin A fibers could
mediate the effects of activity state on NPY cells in the IGL. In the grass rat,
patterns of activation of orexin cells in the LH during wheel running parallel
patterns of activation of NPY cells in the IGL (Nixon and Smale 2004). Wheel
running is a self-motivated behavior involving high levels of physical activity
(Sherwin 1998), and previous studies have shown that periods of vigorous
activity are associated with increased release of OXA into cerebrospinal fluid (Wu
et al. 2002; Martins et al. 2004). Orexin release is capable of inducing Fos in
NPY-containing cells in the arcuate nucleus (Yamanaka et al. 2000), raising the
possibility that the same might be true in the IGL as well. Taken together, these
data provide support for the hypothesis that orexin cells in the LH may transmit
information regarding the activity state of an animal directly to NPY cells in the
IGL. These NPY cells may then integrate orexin signals with inputs from other

brain regions and transmit this information to the SCN.

127

The relationship between OXA fiber contacts and c-Fos induction in NPY
cells was only significant in night-active grass rats. The current data do not allow
us to clearly determine whether OXA input to NPY cells is associated with Fos
expression in these cells in day-active animals. The differences observed
between the day- and night-active animals may reflect differences in the relative
importance of other inputs to NPY cells, or differences in the time of day at which
they were sampled. The first of these possibilities is supported by the general
trend for a greater proportion of NPY cells to be contacted by OXA fibers in the
IGL of night-active compared to day-active grass rats. Interestingly, we have
recently shown that a medially-located subset of OXA cells is more likely to
express Fos in night-active than in day-active grass rats, even in the absence of
running wheels (Nixon and Smale 2004). It is possible that individual differences
in OXA cell group activation are directly associated with different activity patterns
in this animal. That is, feedback from this medial group of OXA cells, perhaps
through direct projections to NPY cells in the IGL, might be important in
determining whether or not an individual grass rat will establish a nocturnal
pattern of wheel running.

This study has two primary findings. First, many NPY cells that express
Fos in animals that are running in wheels have appositions with OXA fibers.
Second, in night-active grass rats, NPY cells that exhibit OXA fiber appositions
are significantly more likely to express Fos during wheel running than NPY cells
without such contacts. These data support the hypothesis that orexins may

function as an important source of activity state feedback to the circadian system.

128

It is tempting to speculate that individual differences in this system may
contribute to differences in temporal patterns of daily activity, such as those of

day- vs. night-active grass rats.

129

CHAPTER 5
Retrograde labeling of orexin neurons projecting to the grass rat

intergeniculate leaflet

Introduction

The lateral hypothalamus (LH) in mammals is thought to be involved in the
mediation of general arousal, ingestive behavior, and modulation of brainstem
nuclei in the control of motivated behaviors (reviewed in Simerly 1995). Recent
investigations suggest that the orexins (hypocretins), a family of hypothalamic
peptides found in neurons primarily restricted to the perifornical region (PeF) of
the LH, might be involved in hypothalamic modulation of some or all of the
functions ascribed to this region (Sakurai et al. 1998; Date et al. 1999; de Lecea
and Sutcliffe 1999). Orexin has been strongly implicated in the regulation of
arousal state (Chemelli et al. 1999; Hagan et al. 1999; Lin et al. 1999; Piper et al.
2000), feeding and drinking behaviors (Sakurai et al. 1998; Kunii et al. 1999;
Sweet et al. 1999), and the modulation of autonomic functions such as breathing,
blood pressure and heart rate (Lin et al. 2002; Shirasaka et al. 2002; Young et al.
2005; Zhang et al. 2005b; Zheng et al. 2005). Although a relatively small number
of neurons contain orexin, these cells extend projections to a wide number of
brain regions in rats and hamsters (Sakurai et al. 1998; Date et al. 1999; de
Lecea and Sutcliffe 1999). Understanding the afferent and efferent connections
of these neurons could thus prove to be important in our understanding of LH

control of an important suite of physiological and behavioral actions as a whole.

130

Orexin neurons do not form compact clusters; instead, they are diffusely
scattered throughout the PeF (Sakurai et al. 1998; Date et al. 1999; de Lecea
and Sutcliffe 1999). Currently, very little is known about the topographical
organization of this loose grouping of cells. The orexin neurons could represent a
homogenous population of cells, or they might be organized into distinct
subgroups with specific functions, either with respect to afferent connections,
efferent projections, or both. In other phenotypically distinct populations of
neurons that are diffusely distributed, such as those containing gonadotropin
hormone releasing hormone, there is evidence for spatial organization that maps
onto differences in their inputs, outputs and functions (King et al. 1998). Although
there is some evidence from experiments utilizing retrograde tract tracers for
spatial partitioning of orexin cells with respect to their efferent connections for
some brain regions, such as the locus coeruleus (LC) (Espai‘la et al. 2005),
orexin neurons extending projections to other brainstem nuclei do not display
similar spatial partitioning (Ciriello et al. 2003b; Zheng et al. 2005).

While it is clear that orexin efferents do not always originate from
topographically isolated groups of neurons, identification of brain regions that do
receive input from small groups of orexin cells could be important in
understanding the behavioral effects of orexins on specific brain regions.
Widescale lesions of the LH (Hetherington and Ranson 1940; Anand and
Brobeck 1951) affect more neuron phenotypes than just those containing orexin,
while selective destruction of all orexin-containing neurons (Chemelli et al. 1999)

affects all brain regions receiving orexin input at once. Orexin infusion studies

131

have been useful (Mullett et al. 2000; Sato-Suzuki et al. 2002; Kiwaki et al.
2004), but interpretation of results is potentially complicated by the diffusion of
orexin into adjacent regions. Finally, although targeting of individual orexin
neurons is possible (Mileykovskiy et al. 2005), the technical problems associated
with doing so on a large scale are daunting. With the identification of at least
some functionally isolated subgroups of orexin neurons, examination of the
behavioral effects of orexins might be easier. For example, a targeted lesion
could potentially interrupt orexin projections to the LC while leaving input to other
nuclei largely intact.

One potential link between orexin and behavior involves arousal state
feedback to the intergeniculate leaflet (IGL). The IGL is a subdivision of the
lateral geniculate complex of the thalamus. The IGL is best known for its role in
the integration of photic and non-photic input to the mammalian circadian system
(Morin et al. 1992; Moore and Card 1994; Morin and Blanchard 1995). Neurons
containing neuropeptide-Y (NPY) within the IGL are important in the modulation
of behavioral responses to arousal (Albers and Ferris 1984; Harrington et al.
1985; Johnson et al. 1989; Huhman and Albers 1994; Wickland and Turek 1994;
Biello 1995; Marchant et al. 1997). The IGL receives input from orexin neurons in
several species, including rats (Sakurai et al. 1998; de Lecea and Sutcliffe 1999),
hamsters (Mintz et al. 2001; Khorooshi and Klingenspor 2005), and the diurnal
grass rat (Arvicanthis niloticus) (Novak and Albers 2002). In the grass rat, orexin
input is potentially involved in relaying arousal state feedback to the IGL.

Specifically, both NPY cells and orexin neurons exhibit similar patterns of Fos

132

expression in response to running wheel-induced arousal (Nixon and Smale
2004). The orexin neurons that project to the grass rat IGL form close
appositions with NPY cells, including those that express Fos in response to
arousal (Nixon and Smale 2005). Additionally, in some grass rats orexin cells in
medial portions of the LH were more likely to express Fos than those located
farther from the midline (Nixon and Smale 2004). In this study, we sought to
determine whether orexin cells projecting to the IGL represent a spatially
segregated subgroup of orexin cells, or are randomly distributed within the orexin
cell population. To do this, we injected a retrograde tract-tracer (cholera toxin
subunit B; CTB) into the grass rat IGL, then examined sections labeled for both

orexin and CTB.

Methods

Animal handling

Seventeen adult male grass rats bred in a captive colony were used in this
study. Animals were kept on a 12:12 lightzdark cycle with a red light (< 5 lux) that
was kept on constantly, and were provided food (Harlan Teklad 8640 rodent diet,
Harlan Teklad Laboratory, Madison, WI) and water ad libitum. Animals were
individually housed in clear plastic cages (28 x 34 x 16 cm) with wire lids. All
animal handling and surgical procedures used in this study follow National
Institutes of Health guidelines and were approved by the Michigan State

University All-University Committee for Animal Use and Care.

133

Surgical procedures

All animals received unilateral stereotaxic injections of a retrograde tract
tracer directed at the IGL. At the time of surgery, animals were anesthetized with
sodium pentobarbital (Nembutal; Abbot Laboratories, North Chicago, IN; 0.1
cc/100 g body weight, i.p.). The top of the head was shaved and covered with
iodine (NovaPlus Povidone-Iodine, 10%; Novation Inc, Irving, TX). Artificial tears
(Butler Company, Columbus, OH) were applied to protect the eyes. Each animal
was then given a 30 pl injection of local anesthetic (Lidocaine; Elkins-Sin Inc,
Cherry Hill, NJ) and mounted in a Stoelting stereotaxic frame. Injections were
made using a Hamilton 701N microliter syringe filled with 0.04 pl of either a 1%
solution of CTB (Sigma-Aldrich, St. Louis, MO) dissolved in phosphate buffered
saline (PBS; 0.01 M, pH 7.4) or of CTB conjugated with AlexaFluor 488 (CTB-
FITC; Molecular Probes, Eugene, OR). Injection of tracer was performed using a
Starrett 262M manual microstepper over a period of two minutes, with the
injection needle left in place for an additional 5 minutes to avoid spread of CTB
upon its removal from the brain. Seven animals received injections of CTB, and
ten animals were given injections of the CTB-FITC conjugate. For all injections
into the IGL, the incisor bar was set at 0.0 mm, and injection coordinates were in
the range of 0.3 - 0.5 mm anterior to the bregma, 2.9 - 3.0 mm lateral to the
midline and 4.5 mm below the dura. Post-surgically, the wound was closed using
autoclips and coated with either Nolvasan antiseptic ointment (Fort Dodge
Animal Health, Fort Dodge, IA; 1% chlorihexidine acetate in 10% sterile alcohol

base) or iodine. All animals then received 1 cc of sterile saline (so) to prevent

134

dehydration and 0.06 cc of Bupranex (buprenorphine hydrochloride; Reckitt and

Colman, Hull, England, i.m.) to alleviate pain.

Tissue collection and processing

Two days after surgery, animals were given a lethal dose of sodium
pentobarbital and perfused transcardially with PBS (150 — 200 mllanimal)
followed by 150 to 200 mL fixative (4% paraformaldehyde with 75 mM lysine and
10 mM sodium periodate, in 0.1 M phosphate buffer, pH 7.2) Brains were post
fixed for 4 h, then transferred into 20% sucrose. After 24 h in sucrose, brains
were sectioned at 30 pm in three series using a freezing microtome.

For animals receiving CTB injections, one series of tissue was processed
for visualization of CTB. All sections were incubated in 5% normal horse serum
(NHS; Vector Laboratories, Burlingame, CA) in PBS with 0.3% Triton-X 100
(PBS-TX; Research Products International, Mount Prospect, IL) for 1 h. Tissue
was then incubated in goat anti-choleragenoid antibody (List Biologicals,
Campbell, CA; 1:5000, in PBS-TX and 3% NHS) for 24 h at 4°C. Next, tissue was
incubated in biotinylated horse anti-goat secondary antibody (Vector; 1:200; in
PBS-TX with 3% NHS) for 1 h. Tissue was then placed in avidin-biotin complex
(AB complex; 0.9% each avidin and biotin solutions, Vector; in PBS-TX) for 1 h
and then reacted with DAB (0.5 mg/ml, Sigma) in a tris-hydrochloride buffer
(Trizma, Sigma; pH 7.2) with hydrogen peroxide (0.35 pl 30% hydrogen
peroxide/ml buffer). Tissue was then mounted on gelatin-coated slides,

dehydrated, and coverslipped.

135

For animals receiving CTB-FITC injections, tissue was processed for
orexin using the same general procedure as described above, with the following
changes: Blocking steps were performed using normal donkey serum (NDS,
Jackson Laboratories, West Grove, PA), and tissue was incubated in goat anti-
orexin A antibody (Santa Cruz; 1:10.000, in PBS-TX, with 3% NDS) followed by
Texas Red sulfonyl chloride-conjugated donkey anti-goat secondary (AbCam Inc,
Cambridge, MA; 1:200, in PBS-TX with 3% NDS). Following incubation with the
secondary antibody, tissue was mounted on gelatin-coated slides and
coverslipped using an anti-fading reagent for fluorescent labeling (ProLong Gold;

Molecular Probes).

Cell counts and analysis

Tissue was examined using either a light microscope (Leitz, Laborlux S;
Wetzlar, Germany) for CTB staining, or a fluorescent microscope (Zeiss
Axioskop 2 plus; Carl Zeiss, Gottingen, Germany) equipped with a high-
resolution digital camera (AxioCam MRc) for CTB-FITC / orexin colocalization.
To evaluate the distibution of double-labeled cells, three representative sections
ipsilateral to the site of injection were examined. Images were taken under 20x
magnification at several planes of focus through the lateral hypothalamus using
green channel filters for visualization of CTB-FITC, and red channel filters for
visualization of Texas Red-labeled orexin neurons. Additional images were taken
at 40x for confirmation of labeling in individual neurons. Composite images were

obtained by combining red and green color channels using Adobe Photoshop

136

(Adobe Systems, San Jose, CA). Individual composite images were overlaid in
Photoshop to create a multi-layer mosaic for use in performing cell counts.

For all tissue labeled with CTB-FITC, the average number and distribution
of cells labeled for both CTB-FITC and orexin were counted. The area sampled
was within a 700 pm by 1600 pm rectangle aligned to the edge of the third
ventricle and ventral border of the fornix. The sampling area was divided into four
equal 400 pm wide divisions, referred to as “medial”, “central”, “lateral 1”, and
“lateral 2” (Figure 5.1). The regions labeled “medial”, “central” and “lateral 1” in
this study corresponds to regions labeled “medial”, “central”, and “lateral” used in
Martinez et al. (2002). In all areas sampled, numbers of cells clearly labeled with
orexin alone or with both CTB-FITC and orexin were counted using the
composite image mosaic described above. Cells labeled with orexin were only
counted as double-labeled if they expressed CTB-FITC labeling and the two
labels were clearly in the same plane of focus. Total percentages of neurons
expressing both orexin and CTB-FTC labeling were compiled, arcsine
transformed, and analyzed using a two-way ANOVA to test for effects of area
(medial, central, lateral 1, and lateral 2) and section examined (rostral, middle, or
caudal). Analyses were calculated using Statistica, (StatSoft, Tulsa, OK). Graphs
were prepared using SigmaPlot (SPSS, SPSS, Chicago, IL). Final figures were

prepared using Adobe Photoshop and Adobe Illustrator (Adobe Systems).

Results

In six grass rats, the tracer was successfully deposited in or near the IGL,

with variable spread to adjacent regions of the hippocampus and geniculate

137

 

 

 

 

 

 

 

 

 

 

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Figure 5.1. Line drawing of the lateral hypothalamic area at low magnification
(5x) depicting the 1600 um x 700 pm sampling area used in this study. The
sampling box is aligned to the third ventricle and ventral border of the fornix, and
divided from left to right into 40 pm divisions labeled Medial (M), Central (C),
Lateral 1 (L1), and Lateral 2 (L2), respectively. Circles represent cells containing
orexin A. Scale bar = 500 pm; f= fornix, mt = mammalothalamic tract, 3V = third
ventricle.

138

complex (CTB, n = 1; CTB-FITC, n = 5; Figure 5.2). For CTB injections that were
centered on these regions rather than on the IGL, the majority of backfilled cells
in the hypothalamus were located in the ventromedial hypothalamus and arcuate
nucleus rather than in the perifornical region (not shown). In the animal with a
CTB injection centered on the IGL, neurons backfilled with CTB were visible in
the perifornical region of the LH. Very few CTB-positive neurons were observed
in the perifornical region contralateral to the injection site in this animal.

Ipsilateral to the injection site of CTB-FITC injections, individual neurons
expressing both CTB-FITC and orexin were visible throughout the LH (Figure
5.3). On average, approximately 20 percent (range = 17.29 to 27.23%; mean =
18.54 :I: 1.77%, n = 5) of all orexin neurons in the area of analysis also expressed
CTB-FITC (Mean by area: medial = 30.80i8.15%; central = 19.0612.05%; lateral
1 = 16.93zt2.14%; lateral 2 = 17.3513.99%). The percentage of orexin neurons
that were double-labeled did not vary significantly across regions (Two-way

ANOVA; df=9, F= 1.469, p = 0.199).

Discussion

We have shown here that the orexin cells that project to the grass rat IGL
represent a diffusely distributed subpopulation of orexin neurons, rather than a
topographically distinct subset of cells. Although the CTB injections were not
restricted to the IGL, we are confident that cells double—labeled for CTB and OXA
accurately reflect the neurons that project to the IGL, as the hippocampus and

other regions of the geniculate complex receive little or no input from orexin

139

Figure 5.2. Line drawings depicting injection sites for animals described in this
study. Darkly shaded area indicates placement of needle tip; lighter shaded area
indicates spread of cholera toxin tracer from injection site. All drawings were
traced under low magnification (10x). Drawings are arranged rostrally to caudally
through the intergeniculate leaflet. Scale bar = 300 pm. APT: anterior pretectal
nucleus; ar: acoustic radiation; DLG: dorsolateral geniculate nucleus; IGL:
intergeniculate leaflet; LP: lateral posterior thalamic nucleus; ml: medial
lemniscus; opt: optic tract; Po: posterior thalamic nuclear group; SubG:
subgeniculate nucleus; VLG: ventrolateral geniculate nucleus; VPM: ventral
posteromedial thalamic nucleus; ZID: zona inserta, dorsal; ZIV: zona incerta,
ventral.

140

 

 

 

 

 

 

 

 

 

 

 

 

 

141

 

Figure 5.3. Photomicrographs depicting cells in the lateral hypothalamus after
staining with Texas Red labeled orexin (red fluorescence, panels A, D, and G)
and injection of FlTC-conjugated cholera toxin B (green fluorescence, panels C,
F, and I). Panels B, E, and H are composite images showing both labels.
Neurons exhibiting immunoreactivity for both orexin and cholera toxin B are
marked with an asterisk (*). Arrows indicate cells labeled with cholera toxin B

only. Scale bar = 25 pm.

142

 

 

143

Figure 5.3
(continued)

 

144

Figure 5.3
(continued)

 

145

neurons in the grass rat (see Chapter 2). In animals with injections centered on
the medial geniculate nuclei or hippocampus, CTB-labeled cells were more
prevalent in the arcuate nucleus and ventral hypothalamus; few or no neurons
were backfilled in the perifornical region in these animals (not shown).

Because the IGL extends the entire rostral-caudal length of the lateral
geniculate complex, no single injection in this experiment covered the IGL as a
whole. The 20% of orexin neurons labeled with CTB in this study therefore
represents a minimum estimate. In other investigations utilizing retrograde tract-
tracing in rats, a similar percentage of orexin cells has been shown to project to
the dorsal vagal complex (20%; Zheng et al. 2005) and nucleus ambiguus (16%;
Ciriello et al. 2003b), while 8 to 12% of orexin neurons exhibit projections to locus
coeruleus and basal forebrain structures (Espafia et al. 2005). Experiments
examining colocalization of multiple retrograde tracers have shown that single
orexin neurons in rats often project to more than one structure (Ciriello et al.
2003b; Espafia et al. 2005). Given the large number of brain regions innervated
by orexin fibers and the relatively small number of orexin neurons in the grass rat
brain, it is likely that axonal projections from orexin neurons in the grass rat
bifurcate to innervate multiple structures as well. Although the orexin cells
projecting to the IGL do not form a topographically isolated cluster, they may still
differ from the other orexin neurons with respect to their afferent projections as a
whole. That is, the orexin neurons examined in this study may project to the IGL
as well as to other arousal-related areas, while other orexin neurons may

preferentially project to regions important in the regulation of ingestive behavior.

146

There is some evidence for such a segregation of orexin neurons by efferent
pathways; dual tract-tracing experiments have identified individual orexin
neurons that project to two different feeding- or arousal-related brain regions
(Ciriello et al. 2003b; Espafla et al. 2005).

Overall, it appears that parcellation of orexin neurons into groups
projecting to specific brain regions is unlikely to be an easy task. In this study we
did not find evidence that neurons projecting to the IGL represent a spatially
distinct subset of the orexin cell population, similar to findings in some other
studies examining retrogradely labeled orexin neurons. A similar conclusion was
reached for populations of orexin neurons that project to basal forebrain
structures, the dorsal vagal complex, and the nucleus of the solitary tract are
randomly distributed within the larger orexin cell population (Ciriello et al. 2003b;
Espafla et al. 2005; Zheng et al. 2005). However, orexin neurons projecting to
locus coeruleus are predominantly located in the dorsal half of the orexin cell
population (Espafia et al. 2005), while those projecting to the nucleus ambiguus
are clustered lateral to the fornix (Ciriello et al. 2003b). It is interesting to note
that although examination of single injections did not consistently identify specific
isolated groups of orexin neurons, in at least one study in rats utilizing multiple
retrograde tracers, orexin cells that expressed both retrograde labels did form
two distinct clusters (Ciriello et al. 2003b). These clusters of cells represent only
a small proportion of the orexin neurons projecting to one specific region, but the
clusters of neurons identified may be different with respect to their afferent

inputs. (that statement above was a little unclear) For example, although many

147

orexin neurons project to the IGL, not all of these neurons may be activated
under the same conditions; those located closer to the third ventricle may be
more responsive to arousal-induced activity, while those farther from the midline
may receive input from other areas. It is thus possible that future identification of
functional subgroups of orexin cells could be revealed by injection of retrograde
tract-tracers into multiple rather than single brain regions.

Orexins have been implicated in the control or modulation of a wide
variety of physiological or behavioral actions, including arousal (Hagan et al.
1999; Hara et al. 2001; Yoshimichi et al. 2001; Sato-Suzuki et al. 2002;
Yamanaka et al. 2003), sexual behavior (Iqbal et al. 2001; Russell et al. 2001;
Gulia et al. 2003), feeding (Sweet et al. 1999; Kotz et al. 2002; Thorpe and Kotz
2005), and autonomic functions (Shirasaka et al. 2002; Voisin et al. 2003; Young
et al. 2005; Zheng et al. 2005). Currently, our understanding of orexin actions in
the brain has largely been based upon experiments involving the infusion of
orexins into discrete brain regions (Sweet et al. 1999; Bourgin et al. 2000;
Methippara et al. 2000; Sato-Suzuki et al. 2002; Kiwaki et al. 2004). However,
these experiments are not likely to reflect the physiological effects of orexins
under normal circumstances. Because orexin neurons can project to multiple
brain regions orexin would ordinarily be released at a number of target sites at
the same time. The extensive projections of orexin neurons to structures involved
a wide range of functions (Peyron et al. 1998; Cutler et al. 1999; Date et al. 1999)
are thus likely to provide signals synchronously to multiple functional systems to

aid in the coordination of behavioral state with state-dependent processes.

148

Identification of specific regions receiving concurrent input from subgroups of
orexin neurons may prove important in understanding the function of orexin in the

integration of these behaviors.

149

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