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This is to certify that the
dissertation entitled

DZ RECEPTOR-MEDIATED REGULATION OF GENE
EXPRESSION IN TUBEROINFUNDIBULAR DOPAMINE
NEURONS

presented by
YVONNE MARIE WILL-MURPHY

has been accepted towards fulfillment
of the requirements for the

PhD. degree in Pharmacology and ToxicologL

Wflaéx

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6/01 cJCIRC/DateDue.p65-p.15

D2 RECEPTOR-MEDIATED REGULATION
OF GENE EXPRESSION IN
TUBEROINFUNDIBULAR DOPAMINE NEURONS

By

Yvonne Marie Will-Murphy

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Pharmacology and Toxicology

2004

ABSTRACT

D2 RECEPTOR-MEDIATED REGULATION OF GENE EXPRESSION IN
TUBEROINFUNDIBULAR DOPAMINE NEURONS

By

Yvonne Marie Will-Murphy

Activation of DZ receptors with selective agonists, such as quinelorane,
stimulates the synthesis and release of dopamine (DA) from tuberoinfundibular
(Tl) DA neurons through an afferent neuronal system involving tonically active
dynorphin neurons via kappa opioid receptors. Is the DZ receptor activation of
TIDA neurons is accompanied by an increase in genomic expression in these
neurons?

The purpose of this dissertation was to characterize DZ receptor-mediated
regulation of gene expression in TIDA neurons located in the dorsomedial
arcuate nucleus (DM-ARC) using a combination of anatomical,
immunohistochemical, molecular and pharmacological experimental approaches
to answer the following questions: 1) Are there sexual differences in the
distribution of TIDA neurons and in F03 and related antigens (F RA) expression in
these neurons? 2) Do DZ receptors regulate immediate early gene expression in
TIDA neurons? 3) Do kappa opioid receptors mediate DZ receptor regulation of
Fos expression in TIDA neurons? 4) D0 D2 receptors regulate TH gene
expression in TIDA neurons? and 5) Does Fos play a role in the regulation of TH

gene expression in TIDA neurons?

1. There were no sexual differences in the number of TlDA neurons but the
the percentage of these neurons containing FRA-IR nuclei was 2-3 times higher
in females than in males.

2. Acute administration of the D2 agonist quinelorane caused a time-related
increase in the percentage of TlDA neurons expressing Fos. This effect was
blocked by administration of the D2 antagonist, raclopride.

3. The kappa receptor agonist U50-488 had no effect per se, but reversed
the stimulatory effects of quinelorane on Fos expression in TIDA neurons. This
suggests that DZ receptor-mediated regulation of Fos in these neurons involves
inhibition of endogenous dynorphin release and thus the loss of tonic inhibition of
gene expression.

4. Quinelorane significantly increased TH mRNA in TIDA neurons and this
effect was blocked by raclopride. Thus, DZ receptors regulate TH gene
expression in TIDA neurons.

5. c-fos antisense blocked the quinelorane-induced activation of TH mRNA
expression in TIDA neurons, whereas c-fos sense and nonsense were without
effect. These results reveal that Fos, at least in part, mediates the activation of

TH gene expression in TIDA neurons.

This work is lovingly dedicated to my husband Frank,
my son Conor,
my parents Kurt and Gerry Will and
my Godparents Lou and Karen Mihalyfy

ACKNOWLEDGEMENTS

First and foremost, I wish to thank my thesis advisor and mentor, Dr. Keith

Lookingland. I will be forever grateful for all 07 your time, advice, and support
This has been a very long road and I thank you for believing in me. I will always

remember your slogan, “You can do it!”

In addition, I would like to give a speCIal thanks to my guidance committee
Drs. Kenneth Moore, William Falls, Norbert Kaminiski, and John Thornburg. I am
grateful for their advice, support, and patience.

There have been several post-doctoral fellows and senior students in the
lab that I would like to thank: Chris McMahon, Sun Cheung, Rob Durham, Ken
Hentschel and John Morris. Even through the long daYs crouched over swirling

plates of brain tissue, you always found a way to make me laugh. Our "unofficial"

lab meetings will not be forgotten.

I would like to thank the technicians Erika Bronz, and Tom “Bubba” Ingram
for providing technical assistance. A special thanks goes to Mark Jacob fOr
going above and beyond the call of duty. From washing dishes to creating
computer artwork to making the HPLC room a 70’s haven, you are a man of a

many talents. I wish you luck in the medical field.

I would like to recognize the front office staff, Diane Hummel, Nelda
CarPenter, Mickie Vanderlip, Faye Spencer, and Jeff Nobis for assisting me with

all of my complicated administrative dilemmaS.

I would like to acknowledge Dr Joan Mattson. Thank YOU for introducing

me to the world of science and medicine.

Finally I would like to thank my husband Frank, my parents Kurt and
Gerry Will, my Godparents Karen and Lou Mihalyfy and the Murphy family for an

the love, support and encouragement to see me through to the end. Thank you

for always believing in me. I could never have done it without you.

vi

TABLE OF CONTENTS

List of Tables ..

.....xii

List of Figures -.........xiii

List of Abbreviations
CHAPTER ONE ,

General Introduction
Tuberoinfundibular Dopamine Neurons .. _

lncertohypothalamic Dopamine Neurons - 7

Neurochemical Events in Dopamine Nerve Terminals .. ...9

Regulation of Tuberoinfundibular and lncertohypothalamic Dopamine Neuronal

Activity............................. ,, .. ...12

Sexual Differences ..... . ”12

Effects ofPro/actin , -13

Dopamine Receptor Regulation . 15

Kappa Opioid Receptor Regulation 18

Regulation of Immediate Early Gene Expression ”.21

Regulation of Tyrosine Hydroxylase Expression in Dopamine Neurons ........... 28
Hypothesis .....35
SpeClfiCAlmS35
Materials and Methods.....................,...... .......36

Animals 36

vii

Anesthetic Agents -.

38
Oligonucleotides ---.....38
Stereotaxic Implantation of Intracerebroventricular Guide Cannu/a ,,,,,,, 39

Dual lmmunohistochemistry Using Fluorescent Rhodamine and Nickel

Intensified Diaminobenzidine .. - - ...40
Tissue Preparation 40
Detection of Fos Related Antigens and Tyrosine Hydroxylase 41

Slide preparation and Tissue Mounting 43
Quantification and Analysis .... . . ...43

Dual Immunohistochemistry Using DAB and Nickel lntensified DAB ,,,,,, 47

Characterization of Anti-Fos Antisera ..

...47
Characterization of Anti—TH Antisera __50
Tissue Preparation ”52
Detection of Fos and TH ..... ~ _ 53
Tissue Mounting ...-_54
Quantification and Analysis ...,54

Dual Immunohistochemistry Using Nickel lntensified DAB and Alkaline

Phosphatase .............................................................................. 56

Characterization of Anti-Fos Antisera ...56

Characterization of Alkaline Phosphatase lmmunohistochemical

Staining Technique . . ...58

Tissue Preparation ...60

Tissue Staining Using Nickel lntensified DAB and Alkaline

viii

Pho tase
spha 61
I'ss Mountin
‘ ue g ----..................-........'..63
‘ ' andAnal siswm...
Quantification y 63

In situ hybridization histochemica/ detection of TH mRNA 65

Sllde preparation - 65
T'ss P arat'on

Labeling the TH mRNA Probe 55
Extraction ofthe Labeled Probe 57

Counting Radioactivity 67

Hybridization and Postwash . .. .......... 68

Emulsion Dip . 69
Developing the Slides 69

Quantification and Analysis 70
StatisticalAnalyses

CHAPTER THREE

Sexual Differences in the Distribution of TH-IR Neurons and Expression of F03

and Related Antigens in Subdivision of the Arcuate Nucleus _73

Introduction . . .73

Materials and Methods ..........77

Results __.79

Discussion .. ...90

CHAPTER FOUR .. ...96

[)2 Receptor Regulation of Immediate Early Gene Expression in TH-IR Neurons
in SubdivisionsoitheArcuate Nucleusge
Introduction ..........96
Materials and Methods 99
Discussion 132
CHAPTER FIVE 139
The Role of Kappa Opioid Receptors in DZ Receptor Regulation of Fos
Expression in TH-IR neurons in Subdivisions of the Arcuate Nucleus .139
Introduction 139
Materials and Methods ............143
Results 144
Discussion.............................. 163
DZ Receptor Regulation of TH Gene Expression in Subdivisions of the Arcuate
Nucleus-173
Introduction 173
Materials and Methods 174
Results 175
Discussion 188
CHAPTER SEVEN 196
The Role of Fos in the Regulation of TH Gene Expression in TIDA neurons in

Subdivisions oftheArcuate Nudeus ,,,....195

Introduction .. .

Materials and Methods _,
Results
Discussion.................................
CHAPTER EIGHT-
GeneralSummary
VL-ARC

Concluding Discussion ..

DM—ARC............

BIBLIOGRAPHY ....

Xi

............196
......202
.....203
...221
...230
...230
...230
...232
...233
....234
...234
..240
..242

...243

LIST OF TABLES

 

Table 2.1 List of drugs used, and their pharmacological properties, doses
vehicles, routes of administration and sources. 37

Table 2.2 List of base sequences for C-fOS antisense, sense, and nonsense
Oligonucleotides. 39

xii

LIST OF. FIGURES
Figure 1-1 Sagittal section of the rat brain schematically depicting the location
of catecholamine—containing cell bodies... .. ,_ 2
Figure 1.2 Frontal section of the rat brain SChematically depicting the location
of TIDA neurons In the arcuate nucleus in the middle
hypothalamus. . ...4

Figure 1.3 Frontal section of the rat brain schematically depicting the
distribution of tyrosine hydroxylase immunoreactive neurons in the
dorsomedial and ventrolateral subdivisions of the arcuate
nucleus............................ _ 6

Figure 1.4 Schematic diagram of the neurochemical events in A. TIDA

neurons and B. lHDA neurons. _ ............10

Figure 1.5 Schematic representing a sagittal section Of the rat brain_ .14
Figure 1.6 Schematic diagram depicting intracellular events in TH neurons. .27

Figure 1.7 A schematic representation 01‘ the rat TH Proximal gene
promoter . ...........31

Figure 2.1 High power (4OOX) computer-captured image of a section through
the arcuate nucleus Immunohistochemically processed using

fluorescent rhodamine for the detection of TH and nickel Intensified
DAB for FRA. ‘ 44

Figure 2.2 Three representative frontal sections through the hypothala

depicting the rostral, middle, and caudal regions 0f the arcuate
nucleus. . ”...“..46

Figure 2.3 High power (4OOX) computer-captured image of a section through
the arcuate nucleus immunohistochemically processed using DAB
for the detection of TH and nickel intensified DAB for Fos under
brightfield microscopy. ..........55

Figure 2.4 High power (4OOX) computer-captured image of a section through
the arcuate nucleus immunohistochemically processed using
alkaline phosphatase for the detection of TH and nickel intensified
DAB for Fos under bright field microscopy. 64

F'Qure 2.5 High power (4OOX) computer—captured images of a section through

the arcuate nucleus processed for TH mRNA using in 5”“

hybridization. 71

xiii

Figure 3.1

.-
siltm \1
._ \m .

p
il‘li.

F{We

FVl-Iii

it

Figure 3.1 Distribution of TH-IR neurons throughout the rostro-caudal extent of

the ARC in male rats. 80
Figure 3.2 Distribution of TH-IR neurons throughout the rostro-caudal extent of
the ARC in female rats ............81
Figure 3.3 Percentages of TH-IR neurons expressing FRA throughout the
rostro—caudal extent of the ARC in male
ratsBZ

Figure 3.4 Percentages of TH-IR neurons expressing FRA throughout the
rostro-caudal extent of the ARC in female rats. ...83

Figure 3.5 Comparison of the numbers of TH-lR neurons in the rostral, middle,
and caudal regions of the DM-ARC in male and female rats. ...84

Figure 3.6 Comparison of the percent of TH-IR neurons expressing FRA in the
rostral, middle, and caudal regions of the DM-ARC in male and
female rats. 86

Figure 3.7 Comparison of the numbers of TH-IR neurons in the rostral, middle,
and caudal regions of the VL-ARC In male and female
rat387

Figure 3.8 Comparison of the percent of TH-IR neurons expressing FRA in the
rostral, middle, and caudal regions of the VL-ARC in male and
female rats. 88

Figure 3.9 Comparison of the numbers of TH-IR neurons and the percent of
these neurons expressing FRA in the MZI of male and female
ratsag

Figure 4.1 High power (4OOX) computer-captured images demonstrating
increased Fos expression in TH-IR neurons of the DM-ARC 2 h
following the administration of quinelorane... ...101

Figure 4.2 Time course effects of quinelorane on the percentage of TH-lR

neurons expressing Fos in the rostral, middle, and caudal regions
ofthe DM-ARC. 102

Figure 4.3 Time course effects of quinelorane on the numbers of TH-IR

neurons in the rostral, middle, and caudal regions of the DM-

xiv

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knit

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halite a

Fly,

Fir

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Fi

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

Figure 4.8

Figure 4.9

Figure 4.10

Figure 4.11

Figure 4.12

Figure 4.13

Figure 4.14

Figure 4.1 5

Time course effects of quinelorane on the percentage of TH-IR

neurons expressing Fos in the rostral, middle, and caudal regions
oftheVL-ARC. ..........105

Time course effects of quinelorane on the numbers of TH-IR
neurons in the rostral, middle, and caudal regions of the VL—

Time course effects of quinelorane on the numbers of TH-IR

neurons and the percentage of these neurons expressing Fos in
the MZI. 107

Effects of quinelorane on the percentage of TH-IR neurons
expressing Fos in the rostral, middle. and caudal regions of the
DM-ARC of vehicle- or raclopride—treated rats. ..108

Effects of quinelorane on the numbers of TH-IR neurons in the
rostral, middle, and caudal regions of the DM—ARC of vehicle- or
rac|0pride-treated rats. 109

Effects of quinelorane on the percentage 0f TH-IR neurons
expressing Fos in the rostral, middle, and caudal regions of the VL-
ARC of vehicle- or raclopride—treated rats. ....1 10

Effects of quinelorane on the numbers of TH-IR neurons in the
rostral, middle, and caudal regions of the VL-ARC of vehicle- or
raclopride—treated rats. .....1 11

Effects of quinelorane on the numbers of TH-IR neurons and the
percentage of these neurons expressing Fos in the MZI of vehicle-
or raclopride-treated rats. ..1 12

Time course effects of saline of the percentage of TH-IR neurons

expressing Fos in the rostral, middle, and caudal regions of the VL-

Time course effects of saline of numbers of TH-IR neurons in the
rostral, middle, and caudal regions of the VL-ARC. ..1 15

Effects of time of day on the percentage of TH-IR neurons
expressing Fos ofthe rostralVL—ARC. 117

Effects of time of day on the percentage of TH-IR neurons
expressing Fos ofthe middle VL-ARC. 118

XV

iqarc’i (6'

:litre 4.17

Figure 4.16

Figure 4.17

Figure 4.18

Figure 4.19

Figure 4.20

Figure 4.21

Figure 4.22

Figure 4.23

Figure 4.24

Figure 4.25

Figure 4.26

Figure 4.27

Figure 5.1

Effects of time of day on the percentage of TH-IR neurons
expressing Fos ofthe caudal VL-ARC. 119

Time course effects of saline of the percentage of TH-IR neurons

expressing Fos in the rostral, middle, and caudal regions of the

Time course effects of saline of numbers of TH-IR neurons in the
rostral, middle, and caudal regions of the DM-ARC. ....121

Effects of time of day on the percentage of TH-IR neurons
expressing Fos of the rostral DM-ARC. 122

Effects of time of day on the percentage of TH-IR neurons
expressing Fos of the middle DM-ARC. 126

Effects of time of day on the Percentage 0t TH-IR neurons
expressing Fos ofthe caudal DM-ARC. 124

Time course effects of saline of numbers of TH-IR neurons and the
percentage of these neurons expressrng Fos In the MZI. 125

Effects of time of day on the percentage 0f TH-IR neurons
expressing Fos ofthe MZI. 126

Medium power (ZOOX) computer-captured image demonstrating c-
Jun-IR nuclei, TH—IR neurons, and colocalization of Jun-IR nuclei in
TH-IR neurons of the middle DM-ARC following administration of
quinelorane. 128

Time course effects of quinelorane on the numbers of TH-IR
neurons and the percentage of these neurons expressing c-Jun in
the middle region of the DM-ARC. ......129

Time course effects of quinelorane on the numbers of TH-IR
neurons and the percentage of these neurons expressing c-Jun in
the middle region of the VL-ARC. ...130

Time course effects of quinelorane on the numbers of TH-IR

neurons and the percentage of these neurons expressing c-Jun in

Frontal section schematic depicting the pr0posed neuronal pathway

that mediates the stimulatory effects of DZ receptors on TIDA
neuronalactivity. 142

xvi

titre 5.2

'
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fol/re .1

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n

Figure 5.2 High power (4OOX) computer-capturedI images demonstrating

increased Fos expression in TH- IR neurons of the DM-ARC 2 h
following administration of nor-BNI.. 145

Figure 5.3 Time course effects of nor-BNI on the percentage of TH-IR neurons

expressing Fos in the rostral, middle and caudal regions of the DM-

Figure 5.4 Time course effects of nor-BNI on the numbers of TH-IR neurons in
the rostral, middle and caudal regions of the DM—ARC. 147

Figure 5.5 Time course effects of nor-BNI on the percentage of TH-IR neurons

expressing Fos in the rostral, middle and caudal regions of the VL-

Figure 5.6 Time course effects of nor-BNI on the numbers of TH-IR neurons in
the rostral, middle and caudal regions of the VL-ARC. .150

Figure 5.7 Time course effects of nor-BNI on the numbers of TH-IR neurons
and the percentage of these neurons expressing Fos in the

Figure 5.8 Effects of nor-BNI on the percentage of TH-IR neurons expressing
Fos in the rostral, middle and caudal regions of the DM-ARC of
vehicle- or U50-488-treated rats. ..1 52

Figure 5.9 Effects of nor-BNI on the numbers of TH -lR neurons in the rostral,

middle and caudal regions of the DM-ARC of vehicle— or U50-488-
treated rats. ..153

Figure 5.10 Effects of nor-BNI on the percentage of TH-IR neurons expressing
Fos in the rostral, middle and caudal regions of the VL-ARC of
vehicle- or U50-488—treated rats. __154

Figure 5.11 Effects of nor-BNI on the numbers of TH- IR neurons in the rostral,

middle and caudal regions on the VL-ARC of vehicle— or USO-488-
treated rats. 155

Figure 5.12 Effects of nor-BNI on the numbers 0t TH'IR neurons and the

percentage of these neurons expressing Fos in the MZI of vehicle-
orU50-488—treated rats- 156

Figure 5.13 Effects of quinelorane on the percentage of TH-IR neurons

expressing Fos in the rostral, middle and caudal regions of the DM-
ARC of vehicle- or U50-488-treated rats. ..........158

xvii

WM

“11.“

$1

figure 6

Figure 5.14 Effects of quinelorane 0n the numbers of TH-IR neurons in the

rostral, middle and caudal regions of the DM-ARC of vehicle- or
U50-488-treated rats. .........159

Figure 5.15 Effects of quinelorane on the percentage of TH- IR neurons

expressing Fos in the rostral middle and caudal regions of the VL-
ARC of vehicle- or USO-488-treated rats .. ..160

Figure 5.16 Effects of quinelorane on the numbers of TH- IR neurons in the
rostral, middle and caudal regions of the VL-ARC of vehicle— or
USO-488-treated rats” ..161

Figure 5.17 Effects of quinelorane on the numbers of TH- IR neurons and the
percentage of these neurons expressing Fos in the MZI of vehicle-
orU50-488-treated rats .162

Figure 5.18 Schematic depicting the proposed neuronal pathway following DZ
receptor activation. 163

Figure 6.1 High power (4OOX) computer-captured images demonstrating
increased TH mRNA expression in neurons of the DM-ARC 4 h
following administration of quinelorane .. . 176

Figure 6.2 Time course effects of quinelorane on the labeling density ratio of
TH mRNA expression per neuron in the DM-ARC. ...1 77

Figure 6.3 Time course effects of quinelorane on the number of neurons
containing TH mRNA in the DM-ARC” .. ..1 78

Figure 6.4 Time course effects of quinelorane on the labeling density ratio of
TH mRNA per neuron in the VL-ARC. .179

Figure 6.5 Time course effects of quinelorane on the numbers of neurons
containing TH mRNA in the VL-ARC. 180

Figure 6.6 Time course effects of quinelorane on the numbers of neurons
containing TH mRNA and the labeling density ratio of TH mRNA
perneuronintheMZln . ..182

FiSlure 6.7 Effects of quinelorane on the labeling density ratio of TH mRNA per
neuron in the DM-ARC of vehicle- or raclopride-treated rats. ..183

Figure 6.8 Effects of quinelorane on the numbers of neurons containing TH

mRNA in the DM-ARC of vehicle- or raclopride-treated rats. ..... 184

xviii

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Flg‘IIE W

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722

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Figure 6.9

Figure 6.10

Figure 6.1 1

Figure 6.1 2

Figure 7.1

Figure 7.2

Figure 7.3

Figure 7.4

Figure 7.5

Figure 7.6

Figure 7.7

Figure 7.8

Effects of quinelorane on the labeling density ratio of TH mRNA per
neuron in the VL-ARC of vehicle- or raclopride-treated rats. ...1 85

Effects of quinelorane on the numbers of neurons containing TH
mRNA in the VL-ARC of vehicle- or raclopride—treated rats. 186

Effects of quinelorane on the numbers of neurons containing TH
mRNA and the labeling density ratio of TH mRNA per neuron in the
MZI of vehicle- or raclopride-treated rats. ......187

Sequence of TIDA neuronal activity and gene expression following
activation of D2 receptors. ....193

A schematic representation of the rat TH proximal gene
promoter197

A schematic representation of the process of normal gene
expression and two of the possible mechanisms by which antisense
can attenuate translation of protein from mRNA. ...199

Time course effects of c-fos antisense oligonucleotide on the
percentage of TH-IR neurons expressmg Fos In the rostral, middle
and caudal regions of the DM-ARC In quIneIorane-treated rats. .204

Time course effects of c—fos antisense oligonucleotide on the
numbers of TH-IR neurons in the rostral, middle and caudal regions
of the DM-ARC in quinelorane-treated rats. ......206

Time course effects of c-fos antisense oligonucleotide on the
percentage of TH-IR neurons expressing Fos in the rostral, middle
and caudal regions of the VL-ARC in quinelorane-treated rats. ..207

Time course effects of c—fos antisense oligonucleotide on the
numbers of TH-IR neurons in the rostral, middle and caudal regions
of the VL-ARC in quinelorane-treated rats. .208

Time course effects of c-fos antisense oligonucleotide on the
numbers of TH-IR neurons and the percentage of these neurons
expressing Fos in the MZI in quinelorane-treated rats. .209

Effects of c-fos antisense, sense and nonsense oligonucleotides on
the percentage of TH-IR neurons expressing Fos in the rostral,
middle and caudal regions of the DM-ARC in quinelorane-treated
rats. 210

xix

fiqliff (Ti

“filth ii

50%

fame

Rt

Figure 7.9 Effects of c-fos antisense, sense and nonsense Oligonucleotides on
the numbers of TH-IR neurons in the rostral, middle and caudal
regions of the DM-ARC In quinelorane-treated rats. .21 1

Figure 7.10 Effects of c-fos antisense, sense and nonsense Oligonucleotides on
the percentage of TH-IR neurons expressing Fos in the rostral,
middle and caudal regions of the VL-ARC in quinelorane-treated
rats. 212

Figure 7.11 Effects of c-fos antisense, sense and nonsense Oligonucleotides on
the numbers of TH-IR neurons in the rostral, middle and caudal
regions of the VL-ARC in quinelorane-treated rats. ..213

Figure 7.12 Effects of c-fos antisense, sense and nonsense oligonucleotides on
the numbers of TH-IR neurons and the percentage of these
neurons expressing Fos in the MZI in quinelorane-treated rats. .214

Figure 7.13 Effects of c-fos antisense sense and nonsense Oligonucleotides on
the labeling density ratio of TH mRNA per neuron in the DM-ARC in
quinelorane-treated rats ...216

Figure 7.14 Effects of c-fos antisense, sense and nonsense oligonucleotides on
the numbers of neurons expressing TH mRNA in the rostral, middle
and caudal regions of the DM-ARC in qumelorane-treated rats. .217

Figure 7.15 Effects of c-fos antisense, sense and nonsense Oligonucleotides on
the labeling density ratio of TH mRNA per neuron in the rostral,
middle and caudal regions of the VL-ARC in quinelorane-treated
rats. 218

Figure 7.16 Effects of c-fos antisense, sense and nonsense oligonucleotides on
the numbers of neurons expressing TH mRNA in the rostral, middle
and caudal regions of the VL-ARC in quinelorane-treated rats. ..219

Figure 7.17 Effects of c-fos antisense, sense and nonsense oligonucleotides on
the numbers of neurons expressing TH mRNA and the labeling
density ratio of TH mRNA of these neurons in the MZI in
quinelorane-treated rats. 220

XX

2-DG
3V
ABC
AL
AMPA
AP
AP-1
AP-2
ARC
AS
ATF-1
CAMP
CC
CaRE
CRE
ORE-2
CREB
CREM
CSF
DA
DAB
DD
ddHZO
DEPC
DM
DMN
DNA
DOPA
DOPAC
DYN
Egr1
EtOH

FRA
GABA
GBL

HEPT
HIF
HIPP
ic

ICV
IEG
lHDA

ABBREVIATIONS

 

2-deoxyglucose

third ventricle

avidin—biotin complex

anterior lobe of the pituitary
alpha-amino-3-hydroxy—5-methylisoxazole-4-proprionic acid
alkaline phosphatase

activated protein 1

activated protein 2

arcuate nucleus

anusense

activating transcription factor-1
cyclic adenyl monophosphate
corpus callosum

calcium response element
cyclic AMP response element
cyclic AMP response element 2
cyclic AMP response element binding protein
CAMP response element modulator
cerebrospinal fluid

dopamine
3,3’-diaminobenzidine

DOPA decarboxylase

double distilled water
diethylpyrocarbonate
dorsomedial

dorsomedial hypothalamic nucleus
deoxyribonucleic acid
3,4-dihydroxyphenylalanine
3,4-dihydroxyphenylacetic acid
dynorphin

early growth response 1
ethanol

fornix

Fos and its related antigens
gamma aminobutyric acid
gamma butyrolactone

hour(s)

heptamer

hypoxia-inducible factor
hippocampus

internal capsule
intracerebroventricular
immediate early gene
incertohypothalamic dOpamine

xxi

. [4 if, (n

IL
lnfS
in
IR
K+
K9
MAO
ME
mg
min
ml
mm
mRNA
mt
MZI
NIH
NL
NMDA
nmol
nor—BNI
Norepi
NS
OCT
Opt
OX
PBS
PC-12
PF
PKA
PKC
Ppit
PRL
RNA
S
sc
SCN
SEM
SOX
Sp
SP-1
SRE
SRF
ST
TBS
TF’s
TH

intermediate lobe of the pituitary
infundibular stalk
intraperitoneal
immunoreactive

potassium

kilogram(s)

monoamine oxidase
median eminence
milligram(s)

minute(s)

milliliters

millimeters

messenger ribonucleic acid
mammillothalamic tract
medial zona incerta
National Institutes of Health
neural lobe of the pituitary
N-methyI-D-aspartate
nanomoles
nor-binaltorphimine
norepinephrine

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CHAPTER ONE
GENERAL INTRODUCTION

The mammalian brain contains several different dopaminergic neuronal
systems classified alphanumerically based on anatomical distribution of their
perikarya (Dahlstrdm and Fuxe, 1964; Figure 1.1). These systems differ in their
function and how they are regulated (e.g. via autoreceptors, long loop neuronal
circuits or hormones). All dopamine (DA) neurons, however, have the same
intracellular enzyme systems involved in the synthesis and metabolism of DA
(Lookingland and Moore, 1995).

The mesotelencephalic DA system, which consists of the nigrostriatal and
the mesolimbic systems, is the best studied of the DA neurons. Neurons of the
nigrostriatal system originate from perikarya in the substantia nigra (A8 and A9)
and project to the striatum, which consists of the caudate nucleus and putamen.
These neurons are involved in the modulation of muscle movement; destruction
of these neurons can lead to Parkinson’s disease. The cell bodies of the
mesolimbic system are located in the ventral tegmental area (A10) and terminate
in the nucleus accumbens and other subcortical structures such as the lateral
septum and olfactory tubercle. These neurons are involved in mediating
emotionally-driven affective behaviors and dysfunction in this system is proposed
to play a role in schizophrenia. Treatments of Parkinson’s disease and
schizophrenia have typically utilized agonists and antagonists that act at specific

DA receptors. However, DA receptors regulate other neurological functions

 

 

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Figure 1.1 Sagittal section of the rat brain schematically depicting the location of
catecholamine-containing cell bodies (Moore, 1987). Cell groups A1-A7 are
noradrenergic and groups A8-A16 are dopaminergic (Dahlstrom and Fuxe,
1964). Abbreviations: CC, corpus callosum; HIPP, hippocampus; Norepi,
norepinephrine; ST, striatum.

 

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outside the mesotelencephalic system, which can result in unwanted side effects
by acting at non-targeted DA neuronal systems such as those in the
diencephalon.

The diencephalic DA systems consist of neurons located in the
subthalamus (A11 and A13) and the hypothalamus (A12, A14 and A15). Much
less is understood about the regulation of these neuronal systems that originate
in the diencephalon compared to those that originate in the mesotelencephalic
DA systems. Interestingly, the numbers of DA neurons in the diencephalon are
similar to the numbers in the mesotelencephalic system (Van Den Pol et al.,
1984). While much is still to be learned about the regulation of various DA
neuronal systems in the diencephalon, it is known that some of these neurons
play an important role in regulating the secretion of pituitary hormones. The
focus of the experiments described in this thesis is the regulation of
tuberoinfundibular (T I) DA neurons by DA receptors. The incertohypothalamic
(IH) DA system is incorporated to highlight the differences in the regulation of

TIDA neurons compared to DA neurons that are regulated by DA autoreceptors.

Tuberoinfundibular Dopamine Neurons

Anatomy and Function

The perikarya of the TIDA neurons (A12) are located in the arcuate
nucleus (ARC) and their axons project ventrally and terminate in the median

eminence (ME) (Figure 1.2). The cytoarchitecture consists of various layers:

the subependymal and internal and external layers (LefstrOm et al., 1976; Mezey

 

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Figure 1.2 . Frontal section of the rat brain schematically depicting the location
of TIDA neurons in the arcuate nucleus and the lHDA neurons of the medial zona
incerta in the middle hypothalamus (Chan-Palay et al., 1984). The left side of the
figure shows the topography of TH-IR fibers and terminals; the right side shows
the location of the TH-IR cell bodies. Abbreviations: DM, dorsomedial
hypothalamic nucleus; f, fornix; ic, internal capsule; mt, mammillothalamic tract;
VMIp, lateral posterior part of the ventromedial nucleus; VMmp, medial posterior
Part of the ventromedial nucleus; ZI, zona incerta.

 

and Palkovits, 1982). The subependymal layer contains noradrenergic axon

terminals while the external layer is comprised of those containing DA (Lefstrom

et al., 1976). The external layer is further divided into the medial and lateral
palisade zones. From there, DA is released, diffuses into the hypophysial portal
blood system, and is carried to the anterior pituitary. DA acts on 02 receptors
located on pituitary Iactotrophs to inhibit the secretion of prolactin (PRL).

The ARC is a relatively long structure (approximately 1.5 to 2.0 mm) in the
rat (Paxinos and Watson, 1986) with a majority of the DA neurons located in the
rostral half of the ARC and some scattered in the caudal half (BjOrklund and
Nobin, 1973). Because of this, the ARC can be divided into rostral, middle and
caudal regions. These neurons have a regular dorsoventrally oriented
projections and connect each portion of the ARC to a corresponding part of the
ME (Lindvall and BjOrkIund, 1978). The axons originate from one of the main
dendritic shafts or from the cell bodies and course ventromedially towards the
ME (Chan-Palay et al., 1984).

In addition to dividing the ARC rostrocaudally, the neurons are distributed
in such a way that they can also be located in either the dorsomedial (DM) or
ventrolateral (VL) ARC (Figure 1.3). Anatomical, histochemical, and biochemical
evidence suggest that these regions contain separate populations of TIDA
neurons. The perikarya of TIDA neurons in the DM—ARC are small and display
dark immunostaining of tyrosine hydroxylase (TH), indicative of abundant TH
protein (the rate limiting enzyme in DA synthesis). The dendrites have a

dorsoventral orientation, and the terminals project to both medial and lateral

 

   
     
    

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Ventricle

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Figure 1.3. Frontal section of the rat brain schematically depicting the
distribution of tyrosine hydroxylase immunoreactive neurons in the dorsomedial
and ventrolateral subdivisions of the arcuate nucleus (Fuxe et al., 1985).

 

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portions of the ME (Selmanoff, 1981; Van den Pol et al., 1984). The neurons of
the VL-ARC, however, are larger and have pale TH immunostaining. These
neurons have dendrites with mediolateral orientations (Van den Pol et al., 1984)
and their axon terminals extend only to the lateral portion of the ME (Everitt et al.,
1986). In the DM-ARC, the TIDA neurons contain both TH and DOPA
decarboxylase (DD) enzymes, which are necessary to synthesize DA. However,
the neurons of the VL-ARC contain TH but lack DD. Thus under normal
circumstances these neurons are capable of producing only the inactive DA
precursor 3,4-dihydroxyphenylalanine (DOPA) rather than DA (Meister et al.,
1988; Komori et al., 1991; Balan et al., 2000). Therefore these neurons can be
considered “DOPA-ergic” neurons, as opposed to DA neurons, and their function

is unknown.

lncertohypothalamic Dopamine Neurons

Anatomy and Function

The cell bodies of the lHDA system (A13) are located in the rostral portion
of the medial zona incerta (MZI) just ventral to the mammillothalamic tract
(Figure 1.2). Early studies using glyoxylic acid histochemical fluorescence
techniques reported that this system consists of short DA neurons that project to
the anterior, dorsomedial and posterior regions of the hypothalamus (Bjdrklund et
al., 1975). Later studies using tract-tracing (Wagner et al., 1995; Cheung et al.,
1998) and neurochemical techniques (Eaton et al., 1994b) revealed that lHDA

neurons project to various areas within the hypothalamus, such as the

 

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paraventricular nucleus, as well as outside the hypothalamus, specifically the
central amygdala and horizontal diagonal band of Broca. The DA terminals in
the paraventricular nucleus originate exclusively from the lHDA neurons;
however, these neurons provide only a portion of the DA innervation to the
central amygdala and horizontal diagonal band (Cheung et al., 1998). These
studies also revealed that the cell bodies of the lHDA neurons projecting the
paraventricular nucleus, central amygdala, and horizontal diagonal band are not
organized into distinct groups within the MZI (Cheung et al., 1998).

There is very little information regarding the function of lHDA neurons, but
their widespread projections to various regions in the brain suggest that these
neurons have an integrative function. The rostral zona incerta is involved in
processing afferent sensory information from such brain regions as the
hypothalamus, thalamus, and brain stem, and coordinating specific biological
responses (Ma et al., 1997). In addition examination of projection sites of IHDA
neurons may offer some insight to their function.

The paraventricular nucleus contains corticotropin-releasing hormone
perikarya and is involved with neuronal regulation of hypothalamic-pituitary-
adrenal axis. Activation of DA receptors stimulates corticotropin-releasing
hormone mRNA expression in the paraventricular nucleus (Eaton et al., 1996)
while central administration of corticotropin-releasing hormone increases DA
metabolism in the paraventricular nucleus (Pan et al., 1995). This suggests that

lHDA neurons participate in neuronal regulation of the hypothalamic-pituitary-

 

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neurons that ma

implicated in the regulation of luteinizin
he MZ‘ increases Iuteir‘IiZing hormone secretion. which in turn

9 hormone and ovulation. Direct injection

of DA into 1'.
regulates ovulation (MacKenzie et al. , 1984; James at al., 1987). In contrast,

lesions of the MZI block the proestrous surge of luteinizing hormone (Sanghera
et al., 1991) and disrupt the estrous cyde (MacKenzie et al., 1984)

Neurochemical Events in Dopamine Nerve Terminals

“9““ 1A Tepfesents the neurochemical events in DA nerve termli‘a\s'
s nthes‘is be he ‘ ' ' -
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y the rate-limitin
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majority of newly synthesized cytosolic DA is packaged into syn aptic v
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XidaSe

(MAO) to form 3,4-dihydroxyphenylacetic acid (DOPAC) Which reflacts DA

release from DA neurons.
ism due to the

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aintai'ns a steady state of

regUIation 0f TH by end product inhibition that m
the end

releasable DA in the terminals. Once DA is released, there is a loss or

product inhibition and the synthesis of DA increases. The coupling between DA

 

 

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Figure 1.4. Schematic diagram of the neurochemical events in A. TIDA neurons
and B. lHDA neurons. Dietary tyrosine is transported into the nerve terminal
where it is converted by the rate-limiting enzyme, TH to DOPA. DOPA is rapidly
decarboxylated by DD to form DA. Newly synthesized DA is stored within
vesicles until release upon arrival of action potentials. Excess non-vesicular DA
is metabolized by MAO to DOPAC. Abbreviations: DA, dopamine; DD, DOPA
decarboxylase; DOPA, 3,4-dihdroxyphenylalanine; DOPAC 3,4-
dihydroxyphenylacetlc acid; MAO, monoamine oxidase; TH, tyrosine
hydroxylase.

release, synthesis and metabolism allows two methods to neurochemically
estimate DA neuronal activity. The first method measures the concentration of
the DOPAC in terminal regions of DA neurons as an index of neuronal activity
(Lookingland et al., 1987). The second method uses the synthesis of DA as an
estimate of activity by measuring DOPA accumulation following the
administration of a DD inhibitor (Carlsson et al., 1972).

There are three major differences between the TIDA neuronal system and
the other dopaminergic neuronal systems such as the lHDA system following the
release of DA. These differences are related to the unique architecture of TIDA
neurons. First in the lHDA system, DA is released into a synapse where it can
bind to post-synaptic receptors, unlike the TIDA system where DA is released
and diffuses into the hypophysial portal blood system. The synaptic actions of
DA released from lHDA neurons are terminated predominately via high affinity
reuptake pumps on the DA nerve terminals. DA can be repackaged into synaptic
vesicles for re-release, metabolized to DOPAC, or can feedback to inhibit DA
synthesis. TIDA neurons lack high affinity reuptake pumps; however, there is
evidence that these neurons have a low affinity reuptake system. The functional
significance of this system is not known since little to no DA is taken-up and
metabolized to DOPAC in the median eminence (Demarest and Moore, 1979b;
Annunziato et al., 1980). Finally, in addition to acting at post-synaptic DA
receptors, DA released from lHDA neurons also acts at pre-synaptic
autoreceptors which further inhibits the synthesis and release of DA from these

neurons. In contrast, TIDA neurons lack pre-synaptic autoreceptors as

11

demonstrated by neurochemical (Lookingland and Moore, 1984; Gunnet et al.,
1987), in situ hybridization (Weiner et al., 1991; Fox et al., 1993) and
microdialysis studies (Timmerman et al., 1995) and are unresponsive to first
generation DA agonists that act pre-synaptic DA receptors (Gudelsky and Moore,

1976; Demarest and Moore, 1979a).

Regulation of Tuberoinfundibular and lncertohypothalamic Dopamine
Neuronal Activity

Sexual Differences

There are marked differences in the activities of TIDA neurons of male
and female rats. Although there are no sexual differences in the density of TIDA
nerve terminals (as reflected in the concentration of DA in the ME; Gunnet et al.,
1986b), the rate of turnover, synthesis and metabolism of DA in this region and
the concentration of DA in the hypophysial portal blood is 2-3 times higher in
female than in male rats (Demarest et al., 1981; Gudelsky and Porter, 1981).
This difference is due in part to a greater responsiveness of TIDA neurons to
positive feedback effects of circulating PRL in females versus males (Demarest
and Moore, 1981) and the effects of gonadal steroids (Gunnet et al., 1986b).
TIDA neuronal activity is decreased in the female and increased in the male after
castration, and these effects are reversed by replacement with estrogen or with
testosterone, respectively (Gunnet et al., 1986b; Toney et al., 1991). The
stimulatory effect of estrogen on TIDA neuronal activity in female rats is mediated
through its positive influence on circulating PRL concentrations (Toney et al.,

1992). In males, however, the inhibitory effects of testosterone on TIDA neurons

12

 

 

 

a\d

 

 

is PRL independent (Toney et al., 1991) and may possibly occur through an
indirect neuronal mechanism involving dynorphin neurons (Manzanares et al.,
1992a)

Unlike the TIDA neurons, lHDA neurons are not responsive to
experimentally-induced changes in circulating levels of gonadal steroids. There
is no sexual difference in the basal activity of the lHDA neurons, and neither
castration nor steroid hormone treatment alters the activity of these neurons in

male or female rats (Gunnet et al., 1986a).

Effects of Prolactin

As depicted in Figure 1.5, DA released from TIDA neurons in the ME
diffuses into the portal blood and is transported to the anterior pituitary where it
tonically inhibits the secretion of PRL from anterior pituitary Iactotrophs. PRL, in
turn, can feed back to activate TIDA neurons. Under normal physiological
situations, the activity of TIDA neurons is regulated to large extent by circulating
levels of PRL. TIDA neuronal activity is reduced during periods of
hypoprolactinemia caused by either hypophysectomy, or administration of DA
agonists or PRL antibody (Gudelsky and Porter, 1980; Demarest and Moore,
1981; Arbogast and Voogt, 1991; Hentschel et al., 20003). During periods of
increased circulating levels of PRL following the administration of exogenous
PRL, DA antagonists, estrogen or implantation of pituitary tissue, TIDA neuronal
activity is increased (Moore et al., 1980; Selmanoff, 1981; Morgan et al., 1982;

Moore et al., 1985).

13

 

 

Figure 1.5. Schematic representing a sagittal section of the rat brain (Modified
from Fuxe et al., 1985). TIDA neurons release DA in the ME, where it diffuses
into the hypophysial portal blood system and is transported to the anterior
pituitary. DA binds to DZ receptors on Iactotrophs and inhibits prolactin
secretion. Systemic prolactin can also feedback and activate these neurons,
thus inhibiting its own secretion. Abbreviations: AL, anterior lobe of the pituitary;
DA, dopamine; DZ-R, D2 receptor; IL, intermediate lobe of the pituitary; NL,
neural lobe of the pituitary, TIDA, tuberoinfundibular dopamine neurons.

14

 

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There are two components to the activation of TIDA neurons by PRL: a
rapid tonic component and a delayed inductive component (Demarest et al.,
1984, 1985, and 1986). The tonic component, observed in female rats,
determines the basal rate of PRL secretion by regulating short-term changes in
TIDA neuronal activity in response to acute increases or decreases in circulating
PRL concentrations. The delayed inductive component is observed in both
females and males, and involves a change in capacity of TIDA neurons in
response to long-term changes in circulating PRL involving, at least in part, a
neurotensin-dependent mechanism (Hentschel et al., 1998).

The lHDA neurons, unlike TIDA neurons, do not respond to circulating
levels of PRL. They also do not respond to chronic elevations of PRL (Arbogast
and Voogt, 1991; Selmanoff et al., 1991), suggesting that these neurons are not
involved in the regulation of basal PRL secretion and do not mediate the effects

of increased PRL in reproductive function.

DA Receptor Regulation

Major ascending mesotelencephalic DA neurons are regulated by DA
receptor-mediated mechanisms consisting of autoreceptors and long loop
neuronal feedback circuits. Autoreceptors are located on cell membranes of
nerve terminals, cell bodies, and dendrites (Lookingland and Moore, 1984; Fuxe
et al., 1985). Somatodendritic autoreceptors regulate neuronal firing whereas
terminal autoreceptors exert effects on DA synthesis and release. Activation of

these receptors leads to inhibition, while blockade results in stimulation. Long

15

loop neuronal feedback comes into play following stimulation of postsynaptic
receptors. For example, GABA neurons project from the striatum to the
substantia nigra where they communicate with the DA cell bodies of the
nigrostriatal system. As DA is released and stimulates the post-synaptic
receptors, the striatal GABA neurons exert a negative feedback effect on the DA
cell firing (Roth and Elsworth, 1995).

In addition to the mesotelencephalic system, lHDA neurons of the MZI are
regulated by DA receptor-mediated mechanisms as demonstrated by
neurochemical studies using DA receptor agonists and antagonists (Lookingland
and Moore, 1984). DA agonists (such as apomorphine) decrease and DA
antagonists (such as haloperidol and raclopride) increase lHDA neuronal activity
(Tian et al., 1991; Eaton et al., 1992).

TIDA neurons, however, are different in that they are primarily regulated
by PRL. Early studies demonstrated that acute administration of first generation
DA agonists (apomorphine and bromocriptine) and antagonists (haloperidol) had
no direct effect on TIDA neurons (Gudelsky et al., 1978; Demarest and Moore,
1979) suggesting that TIDA neurons do not have autoreceptors (Moore, 1987).
However, these neurons are responsive to these drugs in a delayed manner as a
result of their ability to alter circulating concentrations of PRL (Moore and
Lookingland, 1995).

The discovery of multiple DA receptor subtypes has led to research

identifying their specific roles and function in the mesotelencephalic DA system.

This research has, in turn, initiated the development of more selective drugs with

16

the goal of more effective clinical management of the treatment of
neuropsychiatric diseases (such as Parkinson’s disease and schizophrenia) with

reduced unwanted side effects. The DA receptor subtypes have been divided
into two main categories, D1 and D2. The D1 family consists of 01 and 05

receptors, which are found post-synaptically. Activation of these receptors

stimulates the production of cAMP through a Gs protein. The D2 family of

receptors includes D2, D3, and D4 receptors. These receptors are found both
pre- and post-synaptically and are linked to G proteins, inhibiting cAMP (Spano
et al., 1978; Kebabian and Calne, 1979; Vallone et al., 2000).

DA autoreceptors are inhibitory and thus have been categorized as D2
receptors. Systemic administration of selective DZ agonist quinelorane
decreases neuronal activity in DA neuronal systems regulated by DA receptors
(Foreman et al., 1989; Eaton et al., 1994a). However, quinelorane stimulates
TIDA neuronal activity (Berry and Gudelsky, 1991; Eaton et al., 1993).
Considering the lack of response by TIDA neurons following the administration of
classical DA agonists, and that D2 receptors typically exert inhibitory effects, the
results were unexpected. The D2 receptor antagonist raclopride is able to
reverse the effects of quinelorane, leading to the conclusion that this stimulatory
effect is mediated through DZ receptors (Eaton et al., 1993). If this effect is
mediated through pre-synaptic autoreceptors then TIDA neuronal activity should
decrease as seen in other DA neuronal systems. DZ agonists can also act on DZ

receptors on pituitary Iactotrophs to decrease PRL secretion; however,

17

decreases in PRL would feedback on TIDA neurons and decrease, not increase
neuronal activity.

This response is different from what is seen with autoreceptor regulation,
raising the question of how DZ receptors mediate these effects? If this is not
mediated through autoreceptors or changes in PRL secretion, then it must be
through an indirect neuronal system. Medial basal hypothalamic deafferentation
lesions, designed to surgically isolate TIDA neurons from the rest of the brain,
were able to block the stimulatory effects of quinelorane on these neurons
(Durham et al., 1996). These studies demonstrated that the stimulatory effect of
D2 receptor activation on TIDA neuronal activity is mediated through a neuronal
system originating outside the mediobasal hypothalamus. This neuronal system
must involve projection neurons that are acting through the activation of a
quiescent stimulatory neuronal system or through the suppression of a tonically
active inhibitory neuronal system. Because DZ receptors exert inhibitory effects
on their target cells, it is more likely that DZ receptors would inhibit or turn off an

active system as opposed to turning on a stimulatory system.

Kappa Opioid Receptor Regulation

There are three classes of endogenous opioid peptides: B-endorphin,
enkephalin, and dynorphin. Dynorphins were originally isolated and characterized
from porcine hypothalamus and pituitary (Goldstein et al., 1979; Goldstein et al.,
1981 ) and consist of a group of peptides, dynorphin A, dynorphin B and

dynorphin”, (the amino-terminal octapeptide fragment of dynorphin A), that are

18

all derived from the preprodynorphin gene. Early studies using
radioimmunoassay detected dynorphin A and dynorphinie throughout the brain

(Cone et al., 1983) including the hypothalamus (Zamir et al., 1984). These
studies examined microdissected regions of the brain rather than individual
neurons. Later studies used in situ hybridization to achieve cellular resolution
and demonstrated prodynorphin mRNA throughout the hypothalamus including
the ARC, dorsomedial nucleus, paraventricular nucleus, and ventromedial
nucleus (Merchenthaler et al., 1997), suggesting a neuroendocrine function of
dynorphin. One area found to be devoid of prodynorphin mRNA was the MZI
(Merchenthaler et al., 1997).

Dynorphin is involved in the regulation of nociception, diuresis, feeding,
and secretion of hormones including vasopressin, alpha-melanocyte—stimuIating
hormone, and PRL (Mansour et al., 1988; Dhawan et al., 1996). Dynorphin acts
at kappa opioid receptors (Chavkin et al., 1982) which are members of the seven

transmembrane G protein coupled receptors (Mansour et al., 1995). Specifically

these receptors are linked to inhibitory Gi/o proteins which when activated leads

to decreases in adenylate cyclase activity and CAMP, and increases in K+
conductance (Childers, 1991; Childers et al., 1998b; Takekoshi et al., 2000).
Kappa receptor mRNA (Merchenthaler et al., 1997) and binding sites (Mansour
et al., 1988) have been detected in many regions of the diencephalon including
the ARC, dorsomedial nucleus, paraventricular nucleus, and ventromedial

nucleus, but not in the MZI.

19

There is some debate concerning subtypes of kappa opioid receptors.

Early pharmacological studies have suggested the existence of several kappa
receptor subtypes, K1, K2 and K3 (lyengar et al., 1986; Zukin et al., 1988; Cheng
et al., 1992). The pharmacology of the cloned kappa opioid receptor
corresponds to the K1 receptor with high affinity for agonists such as U50-488

and dynorphin, and antagonists like nor-binaltorphimine (nor-BNI) (Dhawan et al.,
1996; Childers et al., 1998a). Nonselective ligands have been shown to bind to

K2 receptors, but these ligands also have mu receptor antagonist properties
(Fowler and Fraser, 1994). The pharmacological profiles of K2 and K3 receptors
are still poorly defined and molecular cloning data have yet to provide evidence
of kappa receptor subtypes. Thus to date no specific K2 or x3 agonists or

antagonists have been developed.

Drugs that activate kappa receptors exert only inhibitory actions on
diencephalic DA neurons, but the degree of inhibition is generally dependent
upon the level of activity of these DA neurons. Kappa receptor agonist USO-488
decreases elevated TIDA neuronal activity found under basal conditions in
female rats, but has no effect on the activity in male rats (Manzanares et al.,
1992a). Conversely, the kappa opioid receptor antagonist nor-BNI increases
TIDA neuronal activity in gonadally—intact male rats, but not females. Similarly,
central administration of dynorphin antibodies also leads to increases in neuronal
activity in male rats (Manzanares et al., 1992b). These studies concluded that
dynorphin tonically inhibits TIDA neurons via kappa opioid receptors in male rats,

but not in females (Manzanares et al., 1992a). This sexual difference is due to

20

the effects of estrogen. Estrogen acts in females via a prolactin-independent
mechanism to suppress kappa opioid receptor mediated inhibition of TIDA
neurons, possibly by decreasing the release of endogenous dynorphin (Wagner
et al., 1994).

Because dynorphin acting via kappa opioid receptor tonically inhibits TIDA
neurons, further studies were carried out to determine if these neurons played a
role in D2 receptor regulation of TIDA neurons. USO-488 reverses the
stimulatory effects of quinelorane on TIDA neurons demonstrating that dynorphin
neurons via kappa opioid receptors mediate the effects of DZ receptor regulation
of TIDA neurons (Durham et al., 1996).

The lHDA neurons, unlike the TIDA neurons, do not respond to kappa
receptor agonists or antagonists (Tian et al., 1992). This is consistent with
studies demonstrating a lack of dynorphin-IR and kappa receptor mRNA and
binding in the MZI. These results demonstrate that lHDA neurons are not
regulated by kappa receptors. Instead, these neurons are regulated by mu

receptors (Tian et al., 1992).

Regulation of Immediate Early Gene Expression

For over a decade, inducible transcription factors have been studied and
have become a tool for mapping neuronal activity. Using immunohistochemistry
to identify neurons expressing specific transcription factors gained popularity
because other techniques could not differentiate the activity of individual

neurons. Neurochemistry, as described earlier, uses levels of neurotransmitters

21

and/or their metabolites to examine the changes in activity of neuronal
populations or subpopulations. Z-Deoxyglucose (Z-DG) involves glucose
utilization as an index of metabolic requirements, but as with neurochemistry, this
technique reveals brain regions that are activated by specific stimuli. The
disadvantage of these techniques is the lack of cellular resolution.
Electrophysiology studies allow for the study of individual neurons, however the
neurons are not typically identified neurochemically. Transsynaptic stimulation of
neurons can activate two different mechanisms by which they process and
transmit information. First is electrophysiological activity, which occurs in
milliseconds to immediately process and convey information about the stimulus.
Secondly, is the longer-acting second messenger signal cascade which occurs
over minutes to hours. This mechanism evokes the production of transcription
factor proteins which initiate transcription and/or repression of other genes,
thereby altering the responses of neurons to subsequent stimuli (Biguet et al.,
1991; Sheng and Greenberg, 1990; Herdegen and Leah, 1998).

So what are these transducible transcription factors? The discovery and
subsequent research began in 1911, when Peyton Rous isolated a factor (later
named the Rous sarcoma virus) from tumor infiltrate that caused sarcoma in
chickens when they were injected with it (Wyke, 1983). In 1966 other
researchers discovered another factor from osteosarcoma in a mouse that could
induce osteosarcomas in younger mice when injected with it. This factor was
later identified as viral particles and named FBJ—MSV (Finkel et al., 1966). Later

in the 1960’s the virus was analyzed and a gene was identified that was believed

22

to allow the virus to cause sarcomas in vivo. The term, “oncogene,” meaning
cancer-causing gene, was coined. It was discovered in the early 1970’s the gene
was found in all DNA of vertebrates (Stehelin et al., 1976). Oncogenes were
derived from normal cellular genes, termed proto-oncogenes through
recombination with viruses (Bishop, 1985). The invading virus “captures” the
gene from the DNA of the infected cell. Under normal conditions, proto-
oncogenes do not cause cancer because they are in a restrictive environment.
However, there can be overexpression of oncogenes when they are in virally
infected cells because there usually are not the regulatory elements present that
can regulate growth. These are retroviral oncogenes that induce cellular
transformation by RNA tumor viruses (Curran and Morgan, 1995). Tumors result
from unregulated and uncontrolled growth and cell division from overexpression
of oncogenes. It wasn’t until 1982 that the FBJ-MSV was determined to be v-fos
(Curran and Teich, 1982). Its cellular component (c-fos) was identified a short
time later, and discovered to have DNA binding properties and gene activator
properties (Curran et al., 1983).

Around the same time, Jun was first described as the oncogene of the
avian sarcoma virus 17 (ASV 17). It is a retrovirus capable of causing
fibrosarcomas in chickens. The name Jun came from ju-nana which is Japanese
for number 17. The cellular component was isolated from human cells and found
to be a major part of the AP-1 site in mouse rat and chicken (Herdegen and

Leah, 1998).

23

Immediate early genes were first characterized in non-neuronal cells when
researchers were trying to identify genes responsive to growth factors that induce
cells to re-enter the cell cycle from the resting G0 phase. This lead to the
discovery of genes that were induced within minutes of growth factor stimulation
(Sheng and Greenberg, 1990) including the Fos and Jun protein families. Fos
family consists of Fos, FosB, FRA-1, and FRA-Z proteins. The Jun family
consists of c-Jun, JunB and JunD. All immediate early genes share some
common characteristics. Under basal conditions, immediate early genes have
low expression, but are rapidly induced, sometimes within minutes, following
stimulation. The induction of transcription is transient and independent of protein
synthesis indicating that the factors responsible for inducing the immediate early
gene preexist in the cell. However, new protein synthesis is required to turn off
transcription of immediate early genes. The fact that immediate early gene
expression is tightly controlled at the level of transcription and translation and by
post-translational modification suggests a regulatory role for protein products in
cellular response to external stimuli.

Fos family members differ in their time frame of induction. Fos is the first
to be induced by acute challenges with the mRNA expression peaking at 15
minutes and the protein at a maximal level at 1-2 h and returning to basal levels
by 4-6 h (Kovécs, 1998). F033 is also induced following acute stimuli, but has a
delayed induction and a longer half-life (approximately 9 h) (Kovacs, 1998;
Herdegen and Leah, 1998). FRA-1 and FRA-Z also have a delayed induction

with their proteins gradually accumulating in the nucleus, especially after

24

repeated and chronic stimulation (Kova'cs, 1998; Herdegen and Leah, 1998).
Like the Fos proteins, the Jun proteins also differ in their half lives and induction.
c-Jun and JunB are similar to Fos in that they are both induced quickly and their
protein half lives are 1 1/2 to 2 h (Herdegen and Leah, 1998). Interestingly c-Jun
expression can last up to 6 h due to cis activation. JunD has a higher basal
expression and longer half life compared to c-Jun and JunB with the half-life of
the mRNA of 6 h.

All Fos and Jun proteins have a leucine zipper, the conserved dimerization
domain that allows strong protein-protein interactions (Kouzarides and Ziff, 1988
and 1989). All Fos and Jun proteins form heterodimers, and Jun proteins can
form homodimers but this combination has less affinity for the DNA binding site
compared to the heterodimers (Vogt and 805, 1990). However, Fos proteins can
not form homodimers because the structure of their leucine zippers and flank
regions (Kouzarides and Ziff, 1988; Herdegen and Leah, 1998) . The Fos/Jun
dimers contain a DNA binding region that lies adjacent to the leucine zipper
(Abate et al., 1990) which binds directly to DNA elements that contain the
conserved sequence ATGACTCA (Dragunow and Hughes, 1995). This
sequence was first identified as phorboI-ester inducible promoter element and
binding site for transcription factor named activator protein-1 (AP-1) which was
later determined to consist of Fos/Jun dimers (Curran and Franza, 1988; Hirai et
al., 1990; Ryder et al., 1988; Zerial et al., 1989).

There are two signaling pathways for Fos induction following the activation

of receptors, the inositol phosphate-protein kinase C (PKC) pathway and the

25

 

 

calcium pathway. Stimulation by serum, growth factors and PKC activators leads
to the phosphorylation of the constitutive transcription factor serum response
factor (SRF; Treisman, 1986 and 1992). SFR forms a homodimer and binds to
the serum response element (SRE) site on the c-fos gene promoter. Following
exposure to calcium or stimulators of CAMP formation (such as forskolin), the
constitutive transcription factor CAMP response element binding protein (CREB)
becomes activated (Fisch et al., 1987) through phosphorylation by CaM kinase l
and II (Sheng et al., 1991) and protein kinase A (PKA; Gonzalez and Montminy,
1989). CREB forms a homodimer and binds to the CAMP/Ca+2 response element
(CREICaRE) site on the c-fos gene promoter. Once bound to the DNA, SRE or
CREB leads to the induction of c—fos mRNA, which can be seen as early as 15
minutes after stimulation. c-fos mRNA is translated to Fos protein in the
cytoplasm of the cell and translocates to the nucleus. Fos then dimerizes with a
Jun protein and binds to the AP-1 site on the long term gene promoter
(Dragunow and Hughes 1995). These intracellular events are summarized in
Figure 1.6.

All Fos and Jun proteins can dimerize with one another, but each
heterodimer has different effects on gene expression and different
transactivational abilities. Fos/c—Jun heterodimers increase whereas Fra-1/c-Jun
decrease DNA stability and have low or no transactivational activity (Suzuki et
al., 1991 ). There are many stimuli of Fos expression in neurons such as
seizures, hypoxialischemia, memory formation, stress, sensory stimulation,

Circadian rhythms (Dragunow and Hughes, 1995). Expression of Fos and its

26

 

Figure 1.6. Schematic diagram depicting intracellular events in TH neurons
(Modified from Hughes and Dragunow, 1995). Stimulation of a receptor leads to
the activation of second messengers and a phosphorylation cascade.
Constitutive transcription factors are covalently modified and translocate to the
nucleus where they stimulate the synthesis of immediate early genes such as c-
fos. Fos protein translocates into the nucleus, dimerizes with a Jun protein and
binds to the AP-1 site located within the TH gene promoter. TH protein is then
transported to its site of action in the axon terminal. Abbreviations: AP-1,
activator protein-1; DA, dopamine; IEG, immediate early gene; TF’s, transcription
factors; TH, tyrosine hydroxylase.

27

 

9'-

[Egg

"'2
"I

iiiih’l‘h a

Ill/A,» If
’ v

related antigens has been correlated with neuronal activity (Hoffman et al.,
1993). Utilizing IEG as an index of neuronal activity it is possible to glean insight
into the anatomical identification of populations of neurons responsive to
pharmacological and physiological manipulations (Yang et al., 1999). Using this
approach subpopulations of Chemically similar neurons can be identified within a
given brain region (Hoffman et al., 1993; Cheung et al., 1997) and then changes
in the number of active neurons within that subpopulation can be determined

(Hoffman et al., 1993; Hentschel et al., 2000b).

Regulation of Tyrosine Hydroxylase Expression in Dopamine Neruons

Immediate early genes have been shown to be responsive to
transsynaptic stimulation of neurons. It has been proposed that immediate early
genes, such as Fos, encode regulatory proteins that control the expression of
long term or late response genes, such as the TH gene. The products of these
long term genes are believed to serve more specific functions in the neuronal
response, such as the synthesis of DA.

TH is the rate-limiting enzyme in DA synthesis. The catalytic activity of
this molecule determines the synthetic rate of DA in the TIDA system (Wang et
al., 1993). It can be regulated in the short term by altering the enzyme itself or in
the medium or long term through the regulation of gene expression and/or
synthesis of new protein (Biguet et al., 1991; Herdegen and Leah, 1998).

The short term regulation of the TH enzyme consists of feedback

inhibition, allosteric regulation by polyanions, enzyme phosphorylation and

28

 

enzyme stability. TH utilizes tyrosine, oxygen and tetrahydrobiopterin to begin
the synthesis of DA. One form of feedback inhibition is reversible and involves
DA binding to TH, thus preventing tetrahydrobiopterin from binding to TH and
inhibiting further DA synthesis. TH enzymatic activity can also be decreased
when the active site of TH is bound by a DAlferric iron complex (Okuno and
Fujisawa, 1991). Allosteric regulation is the modulation of enzyme activity at a
site outside the active site of the protein. Heparin, phospholipids and polyanions
have been demonstrated to increase TH activity by binding to the N-terminal
regulatory domain of the TH protein and produciing a conformational change that
activates the enzyme (Daubner and Fitzpatrick, 1993; Daubner and Piper, 1995).
In addition to allosteric regulation, TH is a substrate for direct phosphorylation by
different protein kinases that can regulate its activity (Porter 1986; Arbogast and
Voogt, 1995). In vitro studies have found at least seven different kinases that

can phosphorylate TH (e.g. PKA, PKC, protein kinase G, and mitogen-activated

protein kinase) at serine residues on the amino-terminal (Sere, Ser19, Ser31, and

Ser40) resulting in increased activity of the enzyme (Campbell et al., 1986;

Haycock, 1990). While the activity of TH increases following phosphorylation,
the stability actually decreases resulting in a shorter half-life (Gahn and
Roskoski, 1995). In contrast, tyrosine, oxygen or inhibitors (e.g. DA) increase TH
stability. One explanation is the inactive enzyme could be available for
recruitment into the active pool. Reducing stability would provide a mechanism
for readily reversing the phosphorylation activation of TH (Okuno and Fujisawa,

1991 ).

29

The long term regulation of TH consists of transcriptional regulation,
alternative RNA processing, regulation of RNA stability and translational
regulation. Alterations in TH gene expression have been reported in response to
the administration of various drugs or manipulation of several endogenous
hormone systems (Wang et al., 1993; Kedzierski et al., 1994; Rodriguez-Gomez
et al., 1997; Najimi et al., 2002). During the past several years, the focus has
become the study of promoter/enhancer sequences of the TH gene promoter
(Figure 1.7) and the proteins that bind them to better understand transcriptional
regulation of TH. The AP-1 site is found at base pairs (bp) -204 to -198 on the
TH gene proximal promoter and is the binding site for Fos/Jun heterodimers.
Various studies utilizing PC12 cells (a cell line derived from rat
pheochromocytoma which produces DA and norepinephrine) have demonstrated
the AP-1 site is responsive to depolarization (Nagamoto-Combs et al., 1997),
calcium (Nagamoto-Combs et al., 1997), phorbol ester (e.g. 1Z-O-tetradecanoyl-
phorbol-13-acetate (TPA); lcard-Liepkalns et al., 1992; Piech-Dumas et al.,
2001), nerve growth factor (Gizang—Ginsberg and Ziff, 1990), hypoxia (Ringstedt
et al., 1995; Norris and Millhom, 1995), and muscarinic cholinergic agonists
(Chen et al., 1996) leading to increases in TH mRNA levels. Preceding the
increases in TH mRNA, phorbol ester, nerve growth factor, depolarization, and
calcium lead to stimulation of Fos, c-Jun and sometimes JunB and JunD (lcard-
Liepkalns et al., 1992; Gizang-Ginsberg and Ziff, 1990; Nagamoto-Combs et al.,

1997; Sun and Tank, 2003; Nakashima et al., 2003).

30

 

 

TH Gene Promoter

 

Figure 1.7. A schematic representation of the rat TH proximal gene promoter.
The putative and functional elements involved in regulation of TH transcription
are shown. HIF: Binding site for hypoxia-induced protein. AP-2: Binding site for
AP-Z transcription factor found in adrenergic and noradrenergic neurons. AP-1:
Highlighted in red; functional binding site for Fos/Jun; involved in stimulated
expression. E box: Overlaps with AP-1 site and is involved with cell specificity.
OctlHept: Binding site for Oct-Z; represses expression. Sp1/Egr1: Binding sites
for Sp1 and Egr1; involved in basal and stimulated expression. CRE-2: Binding
site for CREB, involved in stimulated expression, works as a modulator possibly
with CRE. CRElCaRE: Binding site for cyclic AMP response element binding
protein (CREB), regulates basal and cyclic AMP induced expression. TATA
box: Conserved AoT-rich core promoter site. Abbreviations: AP, activated
protein; CaRE, calcium response element; CRE, CAMP response element; Egr,
early growth response; Hept, heptamer; HIF, hypoxia-inducible factor; Oct,
octamer; Sp, specificity protein. Modified from Papanikolaou and Sabban, 2000.

31

The CRE site (bp -45 to -38) is involved in the basal transcription of the
TH gene as well as CAMP-mediated gene induction in PC12 and neuroblastoma
cell lines (Kim et al., 1993; Yang et al., 1998). This was determined to be
regulated by PKA, but not phorbol ester-mediated induction which increase PKC
(Kim et al., 1994). PKA phosphorylates and activates the constitutive
transcription factor CREB, which can form homodimers or heterodimers with its
protein family members CAMP response element modulator (CREM) or activating
transcription factor-1 (ATF-1) and bind to the CRE site (reviewed in Herdegen
and Leah, 1998). Further studies utilizing CREB antisense RNA expression
vectors demonstrated that CREB, and not CREM and ATF, is essential for
CAMP-mediated regulation of TH gene expression (PieCh-Dumas and Tank,
1999). Interestingly, more recent experiments showed that TPA stimulates the
TH gene promoter via the CRE, in addition to AP—1 (PieCh-Dumas et al., 2001).
The CRE is also responsive to calcium (Nagamoto-Combs et al., 1997), and
nicotinic and muscarinic cholinergic agonists (Chen et al., 1996) leading to
increases of TH mRNA in cell lines.

Like the CRE site the CRE-Z site (bp -97 to -90) is responsive to CAMP
and TPA (Best et al., 1995; Best and Tank, 1998). However, this site plays a
modulatory role and requires a downstream element (possibly CRE). The
transcription factors that bind to this site have yet to be determined.

The specific protein-1 (Sp-1) and early growth response-1 (Egr—1)
response elements overlap (bp -127 to -107) and bind Sp-1 and Egr—1 proteins.

This site is interesting in that Sp-1 and Egr—1 play different roles. Sp-1 binds to

32

 

the promoter and participates in the basal expression of the TH gene, possibly
along with the CRE site, but is not affected by CAMP or TPA (Yang et al., 1998).
However, after the stimulation of PC12 cells with TPA, Egr-1 is induced, binds to
the Sp—1lEgr-1 binding site (displacing Sp—1) and leads to the stimulation of TH
mRNA. Like the CRE-Z site, Egr—1 is believed to play a modulatory role, possibly
with the AP—1 site (Nakashima et al., 2003).

The hypoxia-inducible factor (HIF) response element is the binding site for
hypoxia-induced protein (HIP) in response to hypoxia and may work in
conjunction with AP-1 (Norris and Millhom, 1995). The heptamer (Hept)
response element can bind octamer-2 (Oct-Z) protein and repress TH gene
expression (Yoon and Chikaraishi, 1992; Dawson et al., 1994). Subsequently,
deletion or mutation of the Hept site results in increased TH promoter activity.
Adjacent to Hept is the Oct response element, which also binds the Oct-Z
protein. Interestingly mutation of this binding site has no effect on TH gene
expression (Goc and Stachowiak, 1994).

In humans, alternative splicing results in 4 isoforms of TH in the brain
which may confer specificity for axonal transport proteins (Lewis et al., 1993).
These isoforms may also be regulated by different signaling pathways (Haycock
and Wakade, 1992). More research needs to be carried out to fully understand
the functional role of these isoforms.

The stability of TH mRNA and translational regulation are two other areas
that are being explored. Various stimulators of TH gene expression such as TPA

and hypoxia can increase the half-life of TH mRNA or increase mRNA stability

33

 

 

(Fossom et al., 1992; Vyas et al., 1990; Czyzyk-Krzeska et al., 1994b). The
increased stability may be due to a RNA-protein interaction (Czyzyk-Krzeska et
al., 1994a). A 66 kDA protein has been shown to bind to a sequence in the 3’-
untranslated region of TH mRNA that could protect the mRNA from degradation
by nucleases. While increases in TH mRNA can lead to increases in the
amounts of protein and in enzyme activity, this is not always true. An increase of
five to fifty-fold of TH mRNA content following experimental stimulation may only
lead to a two to three-fold increase or even no Change in TH-IR or enzyme
activity (Baruchin et al., 1990; Kaneda et al., 1991). One hypothesis is while
there is an increase in TH mRNA content and stability, not all of the mRNA is

“loaded” onto ribosomes for translation to protein (Kumer and Vrana, 1996).

34

HYPOTHESIS

The overall goal of studies described in this dissertation is to test the
hypothesis that acute changes in the activity of TIDA neurons following
stimulation of DZ receptors, via a mechanism involving kappa opioid receptors,
leads to changes in immediate early and long term gene expression in these
neurons. In addition, the expression of long term genes is preceded by and

dependent on the expression of these immediate early genes.

SPECIFIC AIMS

1. Determine if there are sexual differences in the distribution of TH-IR
neurons and the basal level of FRA expression in subdivisions of the ARC.

2. Determine if DZ receptors regulate immediate early gene expression in
TIDA neurons

3. Determine the role of kappa opioid receptors in DZ receptor regulation of
Fos expression in TIDA neurons.

4. Determine if DZ receptors regulate TH gene expression in TIDA neurons.

5. Assess the role of Fos in the regulation of TH gene expression in TIDA

neurons.

35

CHAPTER TWO
MATERIALS AND METHODS
A. Animals

All experiments were performed using gonadally-intact male and female
Long-Evans rats weighing 175-225 g purchased from either Harlan Breeding
Laboratories (Indianapolis, IN) or Charles River Laboratories (Wilmington, MA).
Animals were housed Z — 3 per cage in a temperature- (22 i 1°C) and light-
(lights on between 06:00h and 20:00h) controlled environment with food (Harlan
Teklad Rodent Diet; Bartonville, IL) and tap water provided ad libitum. In
experiments involving female rats, estrous cycles were monitored daily by
vaginal lavage, and only diestrous females exhibiting two consecutive cycles

were used.

B. Drugs

Dosages of all drugs were based on their respective salts. Quinelorane
(Research Biochemicals International; Natick, MA) and S-(-)-rac|opride L-tartrate
salt (raclopride; Sigma Chemical Company; St. Louis, M0) were dissolved in
0.9% saline. Nor-binaltorphimine dihydrochloride (Nor-BNI; Sigma Chemical
Company) and trans-(i)-3,4—dichloro-N-methyl-N-(Z-[1-pyrrolidinyl]cyclohexyl)-
benzeneacetamide methanesulfonate salt (U-50488H; Sigma Chemical
Company) were dissolved in distilled water. Doses and routes of administration

of drugs are indicated in Table 2.1 and the legends of the appropriate figures.

36

 

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37

C. Anesthetic Agents

Ketamine hydrochloride (Fort Dodge Animal Health; Fort Dodge, IA) and
xylazine (The Butler Company; Columbus, OH) were used as an anesthetic and
muscle relaxer, respectively, prior to intracerebroventricular (icv) cannulation
surgery. This solution was prepared by combining 8 ml of 100 mglml ketamine
(final concentration 80 mglml), 1 ml of 100 mglml xylazine (final concentration 10
mglml) and 1 ml of 0.9% saline. Equithesin was used euthanize rats prior to
perfusion in the immunohistochemistry studies. Equithesin was prepared by first
dissolving 42.519 chloral hydrate, 9.7Zg pentobarbital, and 21.269 magnesium
sulfate in 443.4 ml propylene glycol, followed by 120 ml 95% EtOH. The total

volume was brought to 1000 ml with ddH20 (Nielsen and Moore 1982).

D. Oligonucleotides

c-fos antisense, sense, and nonsense oligonucleotides were made,
purified, and lyophilized by Macromolecular Facilities in the Department of
Biochemistry at Michigan State University. They were reconstituted with sterile
0.9% saline, aliquoted and frozen at —ZO°C. All 15 mer sequences were
phosphorothioated, which replaces internucleotide oxygen molecules with sulfur
to ensure stability and resistance to nucleases once injected into the brain
(Wahlestedt 1994). c-fos oligonucleotides that are completely phosphorothioated
and complimentary to the translation start site of c-fos mRNA are the most
effective (Hooper et al 1994). The sequences are listed in Table 2.2. The sense

oligonucleotide had the same base sequence found in the targeted mRNA and

38

therefore could not bind with it. The nonsense oligonucleotide was made by
using the same G:C:T:A content of the antisense probe and switching the

position of the bases.

Table 2.2 List of base sequences for c-fos antisense, sense, and nonsense
oligonucleotides (Moller et al 1994).

 

 

 

 

 

 

 

 

Oligonucleotide Sequence
c-fos antisense 5’-GAA CAT CAT GGT CGT-3’
c-fos sense 5’—ACG ACC ATG ATG TTC-3’
c-fos nonsense 5’-GTA CCA ATC GGG A1T-3’
E. Stereotaxic Implantation of lntracerebroventricular Guide Cannula

Rats in studies requiring intracerebroventricular (icv) injection were
anesthetized with ketamine/xylazine (80:10 mg/kg; ip). Rats were assessed for
surgical anesthesia by pinching a back toe. The absence of a reflex movement
was determined as sufficient anesthesia to begin the procedure. The surgical
site was shaved and cleaned with betadine scrub. The rats were placed in a
stereotaxic frame (David Kopf Instruments; Tujunga, CA) with the incisor bar set
2.4 mm below the horizontal plane. A 23-gauge stainless steel guide cannula
was implanted into the right lateral ventricle (1.4 mm lateral to the midline at
bregma and 3.2 mm below the dura mater surface) and anchored to the skull
with stainless steel screws and dental cement. Animals were allowed to recover

for 5 to 7 days prior to the experiment. On the day of the experiment, 3 ul of the

compound or its respective vehicle was injected icv with a 5 pl Hamilton syringe

39

connected to a 30 gauge stainless steel injector needle which protruded 1 mm
beyond the end of the cannula guide and into the right lateral ventricle.
Placement of each cannula was verified histologically, and only those animals

with correct cannula placement were included in the study.

F. Dual Immunohistochemistry Using Fluorescent Rhodamine and
Nickel Intensified Diaminobenzidine (DAB)

1. Tissue Preparation

Rats were anesthetized with Equithesin (4 ml/kg; ip) and perfused through
the aorta with 0.9% saline at 4°C for 2 min, followed by 4% paraformaldehyde
(PF) in 0.1M phosphate buffer saline (PBS; pH 7.4) at 4°C for 15 min. The brains
were removed from the skull and postfixed in 4% PFIPBS at 4°C overnight. The
brains were then placed in 0.1M PBS containing 20% sucrose for 24-48 h and
finally in 0.1M PBS containing 40% sucrose for cryoprotection at 4°C for at least
24 h prior to sectioning.

A cryostat (lEC Microtome, International Equipment; Needham Heights,
MA) was used to slice 30 um sections through the frontal plane of the arcuate
nucleus (ARC), beginning at approximately 2.0 mm posterior from bregma. One
plate of 10 wells containing 0.05M Tris buffer saline pH 7.6 (TBS) at 4°C were
prepared for each brain. Six to eight consecutive sections were collected into
each of the 10 wells. All of the sections were then processed for dual

immunohistochemistry to detect Fos and related antigens (FRA’s) and TH.

40

2. Detection of Fos Related Antigens (FRA) and Tyrosine Hydroxylase
(TH)

Sections were initially rinsed 3 times for 5 min each in TBS with 0.3% of
the detergent Triton X 100 (TX; Research Products International Corp, Elk
Grove Village, IL) with mild agitation on a Gyrotory Shaker (model 62; New
Brunswick Scientific Company; Edison, NJ). Unless othen/vise specified, tissue
sections were lightly agitated at all steps at room temperature. Sections were
incubated in 3% normal donkey serum (Sigma) in TBS/T X for 30 min to block
non-specific binding of the secondary antiserum.

Anti-FRA antiserum (Genosys Biotechnologies; The Woodlands, TX) was
generated in sheep against a synthetic peptide derived from a conserved region
of mouse and human c-fos gene common to all FRA genes including Fos, Fos B,
FRA1, and FRAZ. The sections were incubated in the anti-FRA antiserum
diluted to 12500 with TBS/T X containing 1.5% normal donkey serum for 40 h at
4°C. Sections were rinsed in TBS/T X 3 times for 5 min each to remove excess
primary antibody and incubated with secondary biotinylated donkey anti-sheep
lgG antiserum (Accurate Chemical and Scientific Corp; Westbury, NY) diluted
1:200 in TBS/T X for 1 h. Excess secondary antibody was removed with 3
TBS/T X rinses 5 min each. Sections were then labeled with an avidin-biotin
complex (ABC Elite kit; Vector Laboratories; Burlingame, CA) consisting of avidin
D and biotinylated horseradish peroxidase. Sections were rinsed of excess
avidin-biotin complex with 3 rinses of TBS/T X. FRA protein was localized with a
DAB substrate kit (Vector Laboratories) consisting of 3,3’-diaminobenzidine

(DAB), nickel sulfate, and hydrogen peroxide. Cells positive for FRA proteins

41

contained a black insoluble precipitate localized in the nucleus. Sections were
washed in TBSfT X 6 times for 5 min each and then blocked with 3% normal
horse serum (Sigma) in TBS/T X for 30 min to prevent non-specific binding with
the secondary antibody.

The primary antiserum used to detect tyrosine hydroxylase (TH) was a
monoclonal anti-TH antiserum (Incstar Corp; Stillwater, MN) generated in mouse
against a fragment of purified TH isolated from rat PC12 cells. The epitope was
derived from the midportion of the TH molecule where extensive species
homology exists. There was no cross reactivity with dihydropterdine reductase,
dopamine B-hydroxylase, tryptophan hydroxylase, phenylalanine hydroxylase, or
phenylethanolamine-N-methyltransferase.

Sections were incubated in the primary anti-TH antiserum diluted 1:1000
in TBSfl' X containing 1.5% normal horse serum for 40 h at 4°C. Excess primary
antibody was washed from the tissue with 3 rinses of TBS/T X. The primary
antibody was labeled with secondary biotinylated horse anti-mouse lgG antisera
(Vector Laboratories) diluted 1:200 with TBS/T X for 1 h. Sections were rinsed 3
times with TBS/T X. The chromogen solution to label the TH was tetramethyl-
rhodamine isothiocyanate-tagged avidin D (Vector Laboratories) diluted 1:500 in
TBS/T X. The sections were incubated in this solution for 1 h in darkness, and
the excess chromogen was washed away with 3 rinses of TBS/T X. Neurons
positive for TH contained a fluorescent red-orange cytoplasm. Tissue sections
were stored in 24 well plates with TBS at 4°C until mounted onto gelatin-coated

glass microscope slides.

42

3. Slide Preparation and Tissue Mounting

Before sections were mounted onto glass microscope slides, the slides
were cleaned and gelatin coated (or subbed). Slides were soaked in soapy
distilled water for 1 h and rinsed thoroughly with ddH20. Slides were dipped
once in subbing solution consisting of 1.889 gelatin and 0.1889 chromium
potassium sulfate dissolved in 750 ml ddH20 at 60°C. Slides were allowed to dry
for approximately 1 h and dipped again into the subbing solution. The slides
were air dried overnight before use.

Sections were free-floated in a petri dish of TBS and mounted onto
gelatin-coated slides. The slides were allowed to air-dry overnight, dehydrated in
a series of ethanols (70%, 95%, and 100%) for 10 min each, and delipidated in

xylene for 15 min. Slides were coverslipped using DPX mounting media and air

dried overnight.

4. Quantification and Analysis

Each slide was coded to eliminate bias during counting. Sections were
viewed using a Leitz Laborlux S microscope under bright field microscopy (Leica;
Wetzlar, Germany) to identify DAB-nickel labeled FRA nuclei, and under dark
field fluoromicroscopy (model 103 mercury lamp housing, Leitz) to visualize the

red-orange rhodamine labeled TH neurons (Figure 2.1).

43

 

Figure 2.1 High power (4OOX) computer-captured image of a section through
the arcuate nucleus (ARC) immunohistochemically processed using fluorescent
rhodamine for the detection of TH and nickel intensified DAB for FRA.
Fluorescent TH-IR neurons under dark field microscopy (Left Panel) and the
same image under bright field microscopy (Right Panel) showing black FRA-IR
nuclei. Arrows demonstrate TH-lR neurons containing FRA-IR nuclei. 3V, third
ventricle.

44

Figure 2.2 Three representative frontal sections through the hypothalamus
depicting the rostral, middle, caudal regions of the arcuate nucleus (ARC).
Distances posterior to bregma for these regions were approximately 2.3 mm for
rostral ARC, 2.8 mm for middle ARC, and 3.3 for caudal ARC (Paxinos and
Watson 1998). 3V, third ventricle; A13, incertohyphothalamic dopamine neurons;
ARC, arcuate nucleus; DMN, dorsomedial hypothalamic nucleus; ME, median
eminence; opt, optic tract; VMN, ventromedial hypothalamic nucleus; Zl, zona
incerta.

45

ROSTRAL

 

 

       
 
  
  

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c..- ‘3‘ .

“r7" “3.
I.-

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I

46

The total number of TH-lR neurons and the number of TH-IR neurons
expressing FRA-IR nuclei were counted bilaterally for each section of the rostral,
middle, and caudal regions of the dorsomedial (DM) and ventrolateral (VL) ARC
(Figure 2.2). The mean values for each animal were calculated, and the
numbers of TH-IR neurons with F RA-IR nuclei were expressed as a percentage

of the total number of TH-IR neurons in each region.

G. Dual Immunohistochemistry Using DAB and Nickel lntensified DAB

1. Characterization of Anti-Fos Antisera

Previous immunohistochemical studies performed in our laboratory utilized
a nonspecific antibody that identified not only Fos, but also related transcription
factors FosB, Fra-1 and Fra-Z. While it is possible to identify increases or
decreases in neuronal responsiveness by measuring FRA, it is not possible to
determine which transcription factor(s) is (are) being expressed since each gene
is induced by different types of stimuli and has a different half-life. Activation of
D2 receptors increases TIDA neuronal activity within 30 min. If acute stimulation
of these neurons can induce a Fos family member, it would seem logical that it
would be one that is induced following acute challenges. c—fos is the most rapidly
induced and most transient of the Fos family, thus it was chosen as the
immediate early gene to be studied. An antisemm (c-Fos 4) from Santa Cruz
Biotechnology (Santa Cruz, CA) has been used successfully to specifically label
Fos while not cross reacting with Fra-1, Fra-Z, or Fos B (Moller et al., 1994; Li
and Rowland, 1996; Grzanna et al., 1998; Yang et al., 1999). A second antisera

(c-Fos H125) specific for Fos was also offered by Santa Cruz Biotechnology.

47

Both anti—Fos antisera were characterized to determine the optimal labeling of
Fos. These are affinity-purified rabbit polyclonal antibodies that react with Fos of
mouse, rat and human, but are non cross-reactive with Fos B, Fra-1, or Fra-Z.
The epitope for c-Fos 4 antibody corresponds to an amino acid sequence
mapping at the amino terminus of c-Fos p62 of human origin. The epitope for c-
Fos H125 antibody corresponds to amino acids 210-335 mapping at the carboxy
terminus of c-Fos of human origin.

Cryoprotected, quinelorane-treated tissue sections were removed from the
-20°C freezer and allowed to warm to room temperature prior to processing.
Sections were rinsed in 3% H202 to reduce endogenous peroxidase activity and
in 1% sodium borohydride to neutralize aldehydes. Sections were incubated in
3% normal goat serum to prevent nonspecific binding of the secondary antibody,
followed by an avidin-biotin blocking kit (Vector Laboratories) to block
endogenous biotin sites.

A series of dilutions (121000, 113000, 1:10,000, 1:30,000, 1:100,000,
1:300,000) for each Fos antisera were prepared in TBS/T X containing 1.5%
normal goat serum. Sections were incubated in their respective antibody dilution
with mild agitation for 40 h at 4°C. Sections were washed in TBS 6 times for 10
min each. Sections were then incubated with secondary antibody biotinylated
goat anti-rabbit lgG (Jackson lmmunoresearch Laboratories; West Grove, PA)
diluted 1:500 in TBS/T X for 2 h at room temperature. Excess antibody was
rinsed away with TBS 6 times for 10 min each. Sections were incubated with an

avidin-biotin complex (ABC Elite Kit; Vector Laboratories) consisting of avidin D

48

and biotinylated horseradish peroxidase and rinsed 3 times with TBS and 3 times
with 0.175M sodium acetate. The chromogen solution consisted of 0.01%
hydrogen peroxide, 0.05% DAB, and nickel sulfate in 0.175M sodium acetate
(Hsu et al 1981); (Hsu and Soban 1982). Sections were incubated for 3 min in
the chromogen solution, then rinsed 3 times in 0.175M sodium acetate and 3
times in TBS. Cells positive for Fos protein contained a black insoluble
precipitate localized within the nucleus. Sections were stored in TBS at 4°C until
mounted onto gelatin-coated slides.

Sections were free-floated in a petri dish of TBS, mounted onto gelatin-
coated slides, and allowed to air-dry overnight. The sections were then
dehydrated in a series of ethanols (70%, 95%, 100%) and delipidated in 2 xylene
baths for 10 min each. Slides were coverslipped using DPX mounting media and
air dried overnight.

The background of tissue sections from the 1:1000 and 1:3000 dilutions of
both Fos antibodies was very dark, making it difficult to visualize the cells in the
ARC. There was distinct nuclear labeling with c-Fos 4 antibody, but there was
also nonspecific cytoplasmic staining throughout the brain sections with c-Fos
H125 antibody. At the 1:10,000 dilution, there were changes in staining intensity
of the background and cells for both Fos antibodies. The background was lighter
and it was easier to visualize Fos-IR cells. However there was a decrease in
staining intensity in cells located in the ARC and in the number of labeled cells.
At the 130,000 dilution, these changes were even more dramatic with a

significant decrease in the numbers of cells labeled and staining intensity. There

49

was no Fos-IR at the 12100000 or 12300000 dilutions with either anti-Fos
antiserum. Upon reviewing these slides, the c-Fos 4 antibody was chosen for
future immunohistochemical studies since it resulted in specific and distinct
labeling of nuclei while c-Fos H125 resulted in nonspecific cytoplasmic labeling.
Because the background was too dark at the 123000 dilution and the numbers of
Fos-IR nuclei decreased in the ARC at the 1210,000 dilution, a compromise was

made and the dilution of 125000 was chosen for future studies.

2. Characterization of Anti- TH Antiserum

Anti-TH antiserum (Chemicon lntemational Inc, Temecula, CA) is an
affinity-purified rabbit polyclonal antibody produced in rat adrenal medullary
tumor tissue. It reacts with TH of monkey, rat, hamster, human and bovine and
has no cross reactivity with dopamine-B-hydroxylase or DOPA-decarboxylase.

A series of dilutions (121000, 1:3000, 1210,000, 1230,000, 1:100,000,
1:300,000) of anti-TH antibody was prepared in TBS/T X containing 1.5% normal
goat serum. Sections were incubated in their respective antibody dilution with
mild agitation for 40 h at 4°C. Sections were washed in TBS 6 times for 10 min
each to remove excess primary antibody and then incubated with secondary
antibody biotinylated goat anti-rabbit lgG (Jackson lmmunoresearch
Laboratories) diluted 1:500 in TBS/T X for 2 h at room temperature. Sections
were washed in TBS 6 times for 10 min each to remove excess primary antibody,
labeled with an avidin-biotin complex (ABC Elite Kit; Vector Laboratories) to

amplify the signal, and rinsed 3 times with TBS and 3 times with 0.05M TBS (pH

50

7.2). Sections were incubated in a chromogen solution containing 0.01% H202
and 0.05% DAB in 0.05M TBS (pH 7.2) for 5 min. The reaction was quenched
with 3 rinses of 0.05M TBS (pH 7.2) and 3 rinses of TBS. Cells positive for TH
contained a brown insoluble precipitate in the cytoplasm. Sections were stored
in 24 well plates containing TBS at 4°C until mounted onto gelatin-coated glass
microscope slides.

Sections were free-floated in a petri dish of TBS, were mounted onto
gelatin-coated slides, and allowed to air-dry overnight. The sections were then
dehydrated in a series of ethanols (70%, 95%, 100%) and delipidated in Z xylene
baths for 10 min each. Slides were coverslipped using DPX mounting media and
air dried overnight.

The background of sections from 121000 and 123000 dilutions was very
dark making it difficult to differentiate specifically labeled TH cells from
background. At the 1210,000 dilution, the TH-lR cells in the ARC were countable,
but the background was still not optimal for dual immunohistochemistry. The
staining intensity and numbers of TH-lR cells decreased at the 1230,000 dilution.
TH-IR was very pale at 12100000 and completely disappeared at the 1:300,000
dilution. Because the 1210,000 dilution was too dark for dual
immunohistochemistry and there was a decrease in the numbers of labeled cells

at 1230,000, the dilution 120,000 was chosen for future experiments.

51

3. Tissue Preparation

Following appropriate treatments, rats were anesthetized with Equithesin
(4 mllkg; ip) and perfused through the aorta with 0.9% saline at 4°C for 2 min,
followed by 4% PFIPBS (pH 7.4) at 4°C for 15 min. The brains were removed
from the skull and postfixed in 4% PFIPBS at 4°C overnight. The brains were
then placed in 0.1M PBS containing 20% sucrose for cryoprotection at 4°C until
slicing.

A cryostat (IEC Minotome, International Equipment; Needham Height, MA)
was used to prepare 30 um sections through the frontal plane of the ARC.
Sections were placed in 24 well culture plates containing TBS at 4°C. Four
sections each representing the rostral, middle, and caudal ARC (12 sections
total) were chosen macroscopically (Figure 2.2) from each rat and prepared for
dual immunohistochemistry.

Sections were rinsed in 3% H202 to reduce endogenous peroxidase
activity and in 1% sodium borohydride to neutralize aldehydes. Sections were
incubated in 3% normal goat serum to prevent nonspecific binding of the
secondary antibody. Sections were incubated in 1% avidin D solution to block
endogenous biotin binding sites, followed by a 1% biotin solution to block the
remaining biotin sites on the avidin (Avidin—Biotin Blocking Kit, Vector

Laboratories).

52

4. Detection of Fos and TH

Neurons containing Fos were identified by incubating sections for 40-64 h
at 4°C in primary rabbit anti-c-Fos antiserum diluted to 125000 in TBSfl' X
containing 1.5% normal goat serum. Sections were washed in TBS 6 times for
10 min each. Sections were then incubated with secondary antibody biotinylated
goat anti-rabbit lgG (Jackson lmmunoresearch Laboratories; West Grove, PA)
diluted 1:500 in TBS/T X for 2 h at room temperature. Excess antibody was
rinsed away with TBS 6 times for 10 min each. Sections were incubated with an
avidin-biotin complex (ABC Elite Kit; Vector Laboratories) consisting of avidin D
and biotinylated horseradish peroxidase, rinsed 3 times with TBS and 3 times
with 0.175M sodium acetate. The chromogen solution consisted of 0.01 %
hydrogen peroxide, 0.05% DAB, and nickel sulfate in 0.175M sodium acetate
(Hsu, Raine, and Fanger 1981); (Hsu and Soban 1982). Sections were
incubated for 3 min in the chromogen solution and then rinsed 3 times in 0.175M
sodium acetate and 3 times in TBS. Cells positive for Fos protein contained a
black insoluble precipitate localized within the nucleus. Sections were incubated
in 3% normal goat serum in TBS/T X for 40 min to prevent non-specific binding by
the secondary antibody.

Sections were incubated in primary rabbit anti-TH (Chemicon) for 40-64 h
diluted 1220,000 in TBS/1' X containing 1.5% normal goat serum. Sections were
then incubated with secondary antibody biotinylated goat anti-rabbit lgG
(Jackson lmmunoresearch Laboratories) diluted 1:500 in TBS/T X for 2 h at room

temperature. Sections were washed in TBS 6 times for 10 min each to remove

53

excess primary antibody, labeled with an avidin-biotin complex (ABC Elite Kit;
Vector Laboratories) to amplify the signal, and rinsed 3 times with TBS and 3
times with 0.05M TBS (pH 7.2). Sections were incubated in a chromogen
solution containing 0.01 % H202 and 0.05% DAB in 0.05M TBS (pH 7.2) for 5 min.
The reaction was quenched with 3 rinses of 0.05M TBS (pH 7.2) and 3 rinses of
TBS. Cells positive for TH contained a brown insoluble precipitate in the
cytoplasm (Figure 2.3). Sections were stored in 24 well plates containing TBS at

4°C until mounted onto gelatin-coated glass microscope slides.

5. Tissue Mounting

Sections were free-floated in a petri dish of TBS, mounted onto gelatin-
coated slides, and allowed to air-dry overnight. The sections were then
dehydrated in a series of ethanols (70%, 95%, 100%) and delipidated in 2 xylene
baths for 10 min each. Slides were coverslipped using DPX mounting media and

air dried overnight.

6. Quantification and Analyses

Each slide was coded to eliminate bias during counting. Sections were
viewed using a Leitz Laborlux S microscope under bright field microscopy (Leica;
Wetzlar, Germany). Neurons containing Fos protein had black stained nuclei and

neurons with TH immunoreactivity had a brown cytoplasm (Figure 2.3).

54

 

Figure 2.3 High power (400x) computer-captured image of a section through the
arcuate nucleus (ARC) immunohistochemically processed using DAB for the
detection of TH and nickel intensified DAB for Fos under bright field microscopy.
TH-IR is identified by cells containing brown cytoplasm and Fos-IR is identified
by black stained nuclei. Arrow represents a TH-lR neuron colocalized with Fos-
lR nuclei. 3V, third ventricle.

55

The total numbers of TH-lR neurons and TH-IR neurons containing Fos-IR
nuclei were counted bilaterally in the DM and VL subregions of the rostral,
middle, and caudal ARC. Mean values for each animal were calculated, and the
numbers of TH-IR neurons with Fos-IR nuclei were expressed as a percentage of

the total number of TH-IR neurons in each region.

H. Dual Immunohistochemistry Using Nickel lntensified DAB and
Alkaline Phosphatase
1. Characterization of Anti-Fos Antisera

The staining with Anti-Fos antisera from Santa Cruz was variable between
lot numbers so it was difficult to achieve consistent, distinct nuclear labeling with
a pale transparent tissue background. It was decided to test another Fos
antibody also commonly used in several other studies (Hoffman et al., 1992;
Dragunow et al., 1994; Carr et al., 1999). The antiserum is affinity-purified rabbit
polyclonal antibody that reacts with Fos of mouse, rat and human, but is non
cross-reactive with the 39 kDa Jun protein. The epitope corresponds to residues
4 to 17 of human fos.

To characterize this antisera, cryoprotected, quinelorane-treated tissue
sections were removed from the -20°C freezer and allowed to warm to room
temperature prior to processing. Sections were rinsed in 3% H202 to reduce
endogenous peroxidase activity and in 1% sodium borohydride to neutralize
aldehydes. Sections were incubated in 3% normal goat serum to prevent
nonspecific binding of the secondary antibody. Sections were incubated in 1%

avidin D solution to block endogenous biotin binding sites, followed by a 1%

56

biotin solution to block the remaining biotin sites on the avidin (Avidin-Biotin
Blocking Kit, Vector Laboratories).

A series of dilutions (121000, 123000, 1210,000, 1230,000, 12100000,
12300000) of Fos antiserum were prepared in TBSfl' X containing 1.5% normal
goat serum. Sections were incubated with mild agitation for 40 h at 4°C.
Sections were washed in TBS 6 times for 10 min each and then incubated with
secondary antibody biotinylated goat anti-rabbit lgG (Jackson lmmunoresearch
Laboratories; West Grove, PA) diluted 1:500 in TBSfl' X for 2 h at room
temperature. Excess antibody was rinsed away with TBS 6 times for 10 min
each. Sections were incubated with an avidin-biotin complex (ABC Elite Kit;
Vector Laboratories) consisting of avidin D and biotinylated horseradish
peroxidase and rinsed 3 times with TBS and 3 times with 0.175M sodium
acetate. The chromogen solution consisted of 0.01% hydrogen peroxide, 0.05%
DAB, and nickel sulfate in 0.175M sodium acetate (Hsu, Raine, and Fanger
1981); (Hsu and Soban 1982). Sections were incubated for 3 min in the
chromogen solution and rinsed 3 times in 0.175M sodium acetate and 3 times in
TBS. Cells positive for Fos protein contained a black insoluble precipitate
localized within the nucleus.

Sections were free-floated in a petri dish of TBS, were mounted onto
gelatin-coated slides, and allowed to air-dry overnight. The sections were then
dehydrated in a series of ethanols (70%, 95%, 100%) and delipidated in 2 xylene
baths for 10 min each. Slides were coverslipped using DPX mounting media and

air dried overnight.

57

The background of the tissue sections was very dark in the 121000 and
1:3000 dilutions. ' Nonspecific cytoplasmic labeling was present and it was
difficult to identify the labeled cells in the ARC. Sections incubated in the
1210,000 dilution had specific nuclear labeling of Fos and the background was
pale enough to count the numbers of labeled cells. The background of the tissue
continued to become lighter at the 130,000 dilution and the numbers of labeled
nuclei decreased especially in the ARC. Fos-IR was not present in the ARC at
12100,000 and 12300000 dilutions. Taken together, these observations
determined that the 1210,000 dilution would yield the best results for dual

immunohistochemical studies.

2. Characterization of Alkaline Phosphatase Immunohistochemical
Staining Technique

While the IHC protocol utilizing DAB with nickel intensification and DAB is
widely and successfully used, there were some instances where the same lot
and dilution of antibodies resulted in blackish-brown cells that were too dark to
differentiate. Under these conditions it was difficult to identify which cells were
specifically labeled with TH, which had nonspecific cytoplasmic labeling of Fos,
and which cells were truly dual labeled. Another immunohistochemical method
employing the avidin/biotin complex amplification method offered by Vector
Laboratories utilizes alkaline phosphatase as the enzyme marker instead of
hydrogen peroxidase. There are four different substrate kits available as
adjunctive reagents to the ABC kit, producing a red, black, blue, or purple/blue

precipitate. Using the red precipitate as the chromogen to identify TH would offer

58

greater contrast to the black precipitate of localized Fos nuclei, thereby
facilitating better differentiation.

When the DAB protocol was optimized by Dr. Chris McMahon, various
studies were carried out using different dilutions of the avidin/biotin complex
reagents and DAB reagents. The final recipe for the avidin/biotin complex is one
quarter of the recommended recipe by Vector. In addition, the DAB reagent kit
was no longer used and instead reagents were made fresh. Therefore, in order to
optimize the alkaline phosphatase protocol, a dilution study of the avidin/biotin-
AP and alkaline phosphatase reagent kits was carried out.

Sections were incubated in primary rabbit anti-TH (Chemicon International
Inc.) for 40 h diluted 1220,000 in TBS/T X containing 1.5% normal goat serum.
Excess primary antibody is washed away with TBS 6 times 10 min each.
Sections were then incubated with secondary antibody biotinylated goat anti-
rabbit lgG (Jackson lmmunoresearch Laboratories) diluted 1:500 in TBS/T X for 2
h at room temperature and rinsed with TBS 6 times for 10 min each. Three
dilutions of avidin/biotin complex (ABC-AP Kit; Vector Laboratories) consisting of
avidin DH and biotinylated alkaline phosphatase H were prepared. The first was
made according to the manufacturer’s protocol in a dilution of 1:1. The next two
solutions were 122 and 1:4 dilutions of the original. Sections were incubated the
appropriate avidin-biotin complex dilution for 2 h, and rinsed 3 with TBS and 3
times with 0.1M TBS (pH 8.5) 10 min each. Three sets of the alkaline
phosphatase substrate kits (Vector Laboratories) were prepared. The first

solution was prepared according to the manufacturer’s protocol (1 :1 ), the second

59

was a 1:2 dilution and the third was a 1:4 dilution of the original. The TH protein
was localized using the appropriate substrate dilution and the endogenous
alkaline phosphatase inhibitor levamisole in 0.1M TBS (pH 8.5) for 30 min in the
dark. The reaction was quenched with 3 washes in 0.1M TBS (pH 8.5) and 3
washes in TBS. A pinkish-red precipitate formed in the cytoplasm of cells
positive for TH.

Sections were free-floated in a petri dish of TBS, were mounted onto
gelatin-coated slides, and allowed to air-dry overnight. The sections were then
dehydrated in a series of ethanols (70%, 95%, 100%) and delipidated in 2 xylene
baths for 10 min each. Slides were coverslipped using DPX mounting media and
air dried overnight.

While the background staining of the tissue sections was low in all 9 nine
dilutions, the TH-IR was best visualized using the avidin/biotin complex at a
dilution of 1:1 and alkaline phosphatase reagent kit at a dilution of 1:1. The
staining intensity of TH was the lightest with the 1:4 dilution of avidin/biotin

complex and the 1:4 dilution of the reagent kit.

3. Tissue Preparation

Following appropriate treatments, rats were anesthetized with Equithesin
(4 mllkg; ip) and perfused through the aorta with 0.9% saline at 4°C for 2 min,
followed by 4% PFIPBS (pH 7.4) at 4°C for 15 min. The brains were removed

from the skull and postfixed in 4% PFIPBS at 4°C overnight. The brains were

60

 

then placed in 0.1M PBS containing 20% sucrose for cryoprotection at 4°C until
slicing.

A cryostat (lEC Minotome, lntemational Equipment; Needham Height, MA)
was used to prepare 30 pm sections through the frontal plane of the ARC
beginning at approximately 2.0 mm posterior to bregma. Sections were placed in
24 well culture plates containing TBS at 4°C. Four sections each representing
the rostral, middle, and caudal ARC (12 sections total) were chosen
macroscopically from each rat and prepared for dual immunohistochemistry.

Sections were rinsed in 3% H202 to reduce endogenous peroxidase
activity and in 1% sodium borohydride to neutralize aldehydes. Sections were
incubated in 3% normal goat serum to prevent nonspecific binding of the
secondary antibody. Sections were incubated in 1% avidin D solution to block
endogenous biotin binding sites, followed by a 1% biotin solution to block the
remaining biotin sites on the avidin (Avidin-Biotin Blocking Kit, Vector

Laboratories).

4. Tissue Staining Using Nickel Intensified DAB and Alkaline
Phosphatase

Neurons containing Fos were identified by incubating sections for 40—64 h
at 4°C in primary rabbit anti-c-Fos antiserum (Oncogene Research Products)
diluted to 1210000 in TBS/T X containing 1.5% normal goat serum. Sections
were washed 6 times for 10 min each in TBS with mild agitation. Sections were
incubated with secondary antibody biotinylated goat anti-rabbit IgG (Jackson

lmmunoresearch Laboratories) 1:500 in TBS/T X for 2 h at room temperature and

61

rinsed 6 times in TBS for 10 min each. Secondary antibody was labeled with an
avidin-biotin complex (ABC Elite Kit; Vector Laboratories) consisting of avidin D
and biotinylated horseradish peroxidase. Excess avidin-biotin complex was
washed away with 3 rinses of TBS and 3 rinses of 0.175M sodium acetate 10
min each. Fos protein was localized with a chromogen solution containing 0.01%
hydrogen peroxide, 0.05% DAB, and nickel sulfate in 0.175M sodium acetate.
Sections incubated in the chromogen for 3 min, and were washed 3 times in
0.175M sodium acetate and 3 times in TBS for 10 min each . Sections were then
incubated in 3% N68 in TBS/TX for 40 min to prevent non-specific binding with
the secondary antibody. Cells positive for Fos protein contained a black
insoluble precipitate localized within the nucleus.

Sections processed for Fos protein were incubated in primary rabbit anti-
TH (Chemicon International Inc.) for 40—64 h diluted 1220,000 in TBS/T X
containing 1.5% normal goat serum. Excess primary antibody is washed away
with TBS 6 times 10 min each. Sections were then incubated with secondary
antibody biotinylated goat anti-rabbit lgG (Jackson lmmunoresearch
Laboratories) diluted 1:500 in TBS/T X for 2 h at room temperature and rinsed
with TBS 6 times for 10 min each. Secondary antibody was labeled with an
avidin-biotin complex (ABC-AP Kit; Vector Laboratories) consisting of avidin DH
and biotinylated alkaline phosphatase H. Sections were then rinsed 3 with TBS
and 3 times with 0.1M TBS (pH 8.5) 10 min each. The TH protein was lowlized
using the chromogen solutions in the alkaline phosphatase substrate kit (Vector

Laboratories) and the endogenous alkaline phosphatase inhibitor levamisole in

62

0.1M TBS (pH 8.5) for 30 min in the dark. Sections were washed 3 times in 0.1 M
TBS (pH 8.5) and 3 times in TBS. A pinkish-red precipitate formed in the
cytoplasm of cells positive for TH. Sections were stored in 24-well plates with

TBS at 4°C until mounted onto gelatin—coated glass slides.

5. Tissue Mounting

Sections were free-floated in a petri dish of TBS, were mounted onto
gelatin—coated slides and allowed to air dry overnight. The sections were then
dehydrated in a series of ethanols (70%, 95%, 100%) and delipidated in 2 xylene
baths for 10 min each. Slides were coverslipped using DPX mounting media and

air dried overnight.

6. Quantification and Analyses

Each slide was coded to eliminate bias during counting. Sections were
viewed using a Leitz Laborlux S microscope under bright field microscopy (Leica;
Wetzlar, Germany). Neurons containing Fos protein had black stained nuclei and
neurons with TH immunoreactivity had a pinkish-red cytoplasm (Figure 2.4).

The total numbers of TH-lR neurons and TH-IR neurons containing Fos-IR
nuclei were counted bilaterally in the DM and VL subregions of the rostral,
middle, and caudal ARC. Mean values for each animal were calculated, and the
numbers of TH-IR neurons with Fos-IR nuclei were expressed as a percentage of

the total number of TH-lR neurons in each region.

63

 

Figure 2.4 High power (400x) computer-captured image of a section through the
arcuate nucleus (ARC) immunohistochemically processed using alkaline
phosphatase for the detection of TH and nickel intensified DAB for Fos under
bright field microscopy. TH-IR is identified by cells containing pinkish-red
cytoplasm and Fos-IR is identified by black stained nuclei. Arrows represent TH-
IR neurons colocalized with Fos-IR nuclei. 3V, third ventricle.

I. IN SITU HYBRIDIZATION HISTOCHEMICAL DETECTION OF TH mRNA

1. Slide Preparation

Prior to the experiment, the slides were cleaned and gelatin coated
(subbed). Slides were soaked in soapy distilled water for 1 h and rinsed
thoroughly with ddH2O. Slides were soaked overnight in filtered 80% EtOH and
rinsed with ddH2O 5 times. Slides were dipped once in subbing solution
consisting of 1.889 gelatin and 0.1889 chromium potassium sulfate dissolved in
750 ml sterile, 0.1% diethylpyrocarbonate (DEPC)-treated ddH2O at 60°C. Slides
were allowed to dry for approximately 1 h and dipped again into the subbing
solution. The slides were air dried overnight in a dust-free environment before

use.

2. Tissue preparation

Following appropriate treatments, rats were decapitated and brains were
quickly removed and frozen over dry ice. Fresh frozen brains were sectioned at
a thickness of 30 um in the frontal plane with a Hacker-Bright microtome cryostat
(model OTF/AS/O; Hacker Instruments Inc., Fairfield, NJ) under RNase-free
conditions. ARC sections were thaw-mounted onto sterile, RNase—free gelatin-
coated subbed slides. Slides were stored in a slide box containing desiccant at
-80°C until in situ hybridization processing. On the day of processing, one slide
per animal containing 6 sections through the middle ARC (beginning
approximately -Z.8 mm posterior to bregma) was chosen macroscopically and

placed in an autoclaved slide rack to thaw and airdry. In addition, 18 slides were

65

randomly chosen (approximately 20 mm posterior to bregma) as test slides (3
slides each for 6 weeks). Sections were fixed in 4% PF in sterile, DEPC-treated
1X PBS for 5 min, and then washed twice in sterile, DEPC-treated 1X PBS.
Sections were then treated with sterile DEPC-treated 0.1M TEA/0.9% saline (pH
8.0) containing 0.25% acetic anhydride for 10 min. The acetic anhydride
acetylates tissue to block positive charges on tissue and reduce nonspecific
binding. Sections were dehydrated with gradient ethanols (70%, 80%, 95%,
100%) for 1 min each, delipidated in chloroform for 5 min and rehydrated in

100% and 95% ethanol for 1 min each. Slides were allowed to air dry.

3. Labeling the TH mRNA probe

A 48 mer TH mRNA probe complimentary to bases 1441-1488 with the
sequence [5’-CGT GGG CCA GGG TGT GCA GCT CAT CCT GGA CCC CCT
CCA AGG AGC GGT-3’] (Grima et al., 1985; Young et al., 1987) was
synthesized and purified by the Macromolecular Facilities in the Department of
Biochemistry at Michigan State University. A 5 uM stock of the probe was
incubated with 35S-dATP (New England Nuclear Life Science Products; Boston.
MA), terminal transferase enzyme (Boehringer Mannheim Biochemicals;
Indianapolis, IN), and cacoadylate buffer for 20 min at 37°C to 3’ end label the
probe. The tailing reaction was stopped with the addition of sterile, DEPC-

treated Tris Base/EDTA buffer and yeast tRNA (Gibco BRL; Grand Island, NY).

66

4. Extraction of the Labeled Probe

For the first extraction, a phenol/chloroform/isoamyl alcohol (2522421)
solution was added to the probe and the mixture was vortexed for 30 s and
microfuged for 5 min. The aqueous layer was drawn off and transferred to a new
sterile microfuge tube while the organic layer was discarded. For the second
extraction, a chloroform/isoamyl alcohol (24:1) solution was added to the
aqueous layer, and the solution was briefly vortexed and microfuged for 2 min.
The aqueous layer was removed and transferred to a new sterile microfuge tube.
Cold absolute ethanol and 3M sodium acetate were added to this aqueous phase
and tubes were placed on ice for 45 min to initiate precipitation of the labeled TH
mRNA probe. The precipitant was then spun in a Sorvall RC-SB Superspeed
centrifuge (Du Pont Instruments; Hoffman Estates, IL) at 4000 rpm for 50 min at
0°C. The supernatant was poured off, and the pellet was allowed to dry for 1 h at
room temperature. The pellet was resuspended with Tris base/EDTA buffer and

1M dithiothreitol (DTT; Sigma) and the amount of radioactivity was counted.

5. Counting Radioactivity

One ul of probe was added to 5 ml of Safety Solve scintillation fluid and
the number of radioactive counts was determined using the Packard Tri-Carb
2100TR Liquid Scintillation Counter (Packard Instrument Co, Downers Grove,
IL). Each tailing reaction was counted in duplicate and the counts were
averaged. Calculations were carried out to determine the proper volume of

hybridization buffer and 5M DTT to dilute the labeled TH mRNA probe to 20,000

67

cpm/ul. The hybridization buffer consisted of 50% formamide (Fluka), 500 uglml
sheared single stranded DNA from salmon testes (Sigma), 250 ug/ml yeast tRNA
(Gibco), 1X Denhardt’s solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02%
bovine serum albumin; Sigma), 10% Dextran sulfate (Sigma), and 4X
saline/sodium citrate buffer (0.6M sodium chloride and 0.06M sodium citrate).
Formamide lowers the effective melting temperature of nucleic acid duplex
chains so that hybridization can occur at temperatures that preserve tissue
morphology. The sheared single stranded DNA and yeast tRNA were used as
non-homologous carriers. The Denhardt’s solution was a mixture of proteins that
stabilizes the TH mRNA probe and reduces nonspecific binding. Dextran sulfate
was a large, non-reactive polymer that increased effective probe concentration at
the tissue surface and helps reduce non-specific binding. DTT is an antioxidant
that stabilizes 35$ attachment to the probe by maintaining the sulfur molecule in a
reduced state. It also reduces the possibility of 35$ forming disulfide bonds with
sulfur molecules in the tissue, thereby eliminating another source of non-specific

probe binding.

6. Hybridization and Postwash

Sections previously fixed and dehydrated were incubated with
hybridization solution. Twenty-five ul of hybridization solution per brain section
was pipetted onto each slide and coverslipped with a sterile Hybri-Slip (Sigma).
All slides were incubated in a humidity chamber in a Labline Imperial Il incubator

(Labline Instruments Inc, Melrose Park, IL) at 37°C for approximately 18 h.

68

 

Slides were removed from the incubator and soaked in 1X saline/sodium
citrate buffer to remove the coverslips. The slides were transferred to a slide
rack and soaked in 2X saline/sodium citrate buffer/50% formamide at 40°C 4
times 15 min each. The slides were rinsed in 1X saline/sodium citrate buffer at
room temperature twice for 30 min each. The sections were then dehydrated in
a series of ethanols containing 0.3M ammonium acetate (50%, 70%, 90%, 95%)

and 100% EtOH, and allowed to air dry.

7. Emulsion Dip

In the darkroom under a No. 2 safelight filter, NTBZ photographic emulsion
(Eastman Kodak Co., Rochester, NY) was liquefied in a warm water bath and
diluted 1:2 with ddHZO. Dried slides were dipped into the emulsion and the
excess was wiped from the back of the slide. The slides were placed in a vertical
slide holder and allowed to dry for 1-2 h in a light-tight cardboard box. Dried
slides were placed in small slide boxes containing a desicwnt capsule, sealed
with black electrical tape, and wrapped in aluminum foil. The boxes were then

placed in a —20°C freezer to incubate.

8. Developing the Slides

Each week, a box of three test slides was removed from the freezer and
developed to determine the extent of labeling of the TH mRNA. Once adequate
labeling of TH mRNA was achieved (typically after a 4 — 6 week incubation), the

experimental slides were removed from the freezer and allowed to warm to room

69

tern

temperature for about 1 h. During this period, 300 ml each of Kodak D19
Developer and Kodak Fixer were prepared and cooled on ice to 17°C. In the
darkroom under a No. 2 safelight filter, slides were removed from the black slide
boxes and placed in a slide rack. The slides were developed in Kodak D19 for 2
min, washed in ddH2O for 15 sec, and placed in Kodak Fixer for 3 min. The
slides were then washed under slow running cool water for 30 min,
counterstained with thionin, dehydrated in a series of ethanols (70%, 95%,
100%) and dilipidated in 2 baths of xylene for 10 min each. Slides were

coverslipped using DPX mounting media and allowed to air dry overnight.

9. Quantification and Analysis

TH mRNA was visualized in sections using high-resolution darkfield optics
(Figure 2.5; Darklite Illuminator, Micro Video Instruments Inc, Avon, MA).
Images of labeled cells in the ARC are captured with the Spot 2 Digital Camera
(Diagnostic Instruments, Inc.) on a Leitz Laborlux S microscope (Leica; Wetzlar,
Germany). The number of silver grains over a chosen cell was counted using
NIH Image program (version 1.62). The number of silver grains over the
adjacent background of the same area size was also counted. The density of
mRNA per cell was reported as a labeling ratio, which was calculated by dividing
the number of silver grains over the cell by the average number of silver grains
over the adjacent background. This ratio corrects for subtle differences in grain
density due to differential thickness of the emulsion. A cell was considered

labeled if the labeling density ratio was 3 or greater. Data included the total

70

 

 

Figure 2.5 High power (400x) computer-captured images of a section through
the arcuate nucleus (ARC) processed for TH mRNA using in situ hybridization.
Top Panel: Localization of silver grains over the soma of cells containing TH
mRNA under dark-field microscopy. Bottom Panel: Same image under bright—
field microscopy demonstrating cells that are counterstained with thionin. Arrows
represent thionin stained cells with silver grains localized over the soma. 3V,
third ventricle.

71

number of labeled neurons and the average labeling ratio of those neurons

resulting from bilateral quantification of DM-ARC and VL-ARC.

J. Statistical Analyses

Results from all experiments were expressed as the mean i 1 SEM with
the n equal to the number of experimental animals. Data that were
nonparametric (i.e. did not follow a normal distribution) was transformed by
taking the square root before analysis was carried out. Statistical analyses were
conducted by one way analyses of variance followed by Student Newman Keuls
test for least significant difference. Kruskal Wallis one way analysis of variance
on ranks was carried out when data failed the equal variance test followed by
Dunn’s method to determine least significant difference. Differences were

considered significant if the probability of error was less than 5%.

K. Images

Images in this dissertation are presented in color.

72

 

CHAPTER THREE

SEXUAL DIFFERENCES IN THE DISTRIBUTION OF TH-IR NEURONS AND
EXPRESSION OF FOS AND RELATED ANTIGENS IN SUBDIVISIONS OF
THE ARCUATE NUCLEUS

Introduction

The perikarya of the TIDA neurons are located in the ARC and their axons
project ventrally and terminate in the ME. DA released at the ME diffuses into
the hypophysial portal blood system, and is carried to the anterior pituitary. DA
acts at DZ receptors located on pituitary Iactotrophs to inhibit the secretion of
PRL.

Early studies (conducted primarily in male rats) used histofluorescent
(Lc'ifstrém et al., 1976), electron microscopic (Ajika and Hokfelt, 1973), and
biochemical techniques (Selmanoff, 1981) to demonstrate that the distribution of
DA in the ME is not uniform. Higher levels of DA are located in the medial
portion of the ME compared to the lateral portions (Selmanoff, 1981; Palkovits et
al., 1982), and DA concentrations in both medial and lateral ME increase in a
rostrocaudal gradient (Palkovits et al., 1982).

The ARC is a relatively long structure in the rat that can be sectioned into
rostral, middle and caudal regions. In addition to dividing the ARC
rostrocaudally, the TH-IR neurons are distributed in such a way that they can
also be located in either the DM- or VL-ARC. These divisions are based on
differences in size (Van den Pol et al., 1984), projection (Selmanoff, 1981; Van
den Pol et al., 1984; Everitt et al., 1986) histochemistry, and biochemistry

(Meister et al., 1988; Komori et al., 1991; Balan et al., 2000).

73

den Pol et al., 1984; Everitt et al., 1986) histochemistry, and biochemistry
(Meister et al., 1988; Komori et al., 1991; Balan et al., 2000).

Basal activity of TIDA neurons is determined by the positive feedback
effects of circulating levels of PRL. Changes in PRL secretion from the anterior
pituitary lead to corresponding changes in the activity of TIDA neurons
(Demarest et al., 1981 ). Neurochemical techniques have demonstrated no
difference in the concentration of DA in the ME between male and female rats
(Demarest et al., 1981; Gunnet et al., 1986) indicating there is no sexual
difference in the density of DA innervation. However, there is greater tumover,
synthesis, and metabolism of DA in the ME in females compared to males
(Demarest et al., 1981; Gunnet et al., 1986; Lookingland et al., 1987), which
corresponds to greater neuronal activity in females. This difference is due to the
direct stimulatory effects of estrogen on PRL secretion in female rats (Moore et
al., 1987), and the ability of testosterone to inhibit the basal activity of TIDA
neurons, independent of PRL, in male rats (Toney et al., 1991).

One hypothesis for this sexual difference is that there are greater numbers
of active neurons in females versus males. A second line of thought is that there
are equal numbers of active neurons; however, each neuron is more active in
females due to increased DA synthesis, which is the result of increases of TH
mRNA (Arbogast and Voogt, 1990) and protein cycling (Porter, 1986b; Arbogast
and Voogt, 1990) or a greater amount of phosphorylation of TH protein (Porter,

1986a)

74

 

Because most of what is known concerning the regulation of TIDA
neurons is based on neurochemical estimates of DA synthesis and metabolism in
axon terminals converging in the ME, it is not known if anatomically distinct
subpopulations of these neurons respond differently to specific stimuli.
Neurochemical techniques are useful tools for studying the effects of
pharmacological and physiological manipulations on neuronal activity at axon
terminals, yet there is a limitation. While this technique can sample the area of
interest as a whole or even subpopulations of it (Selmanoff, 1981; Palkovits et
al., 1982), it is not possible to discern changes in individual neurons.

2-DG can also be used to study neuronal activity in axon terminals. This
technique permits quantitative and regionally localized analysis of glucose
utilization, an index of metabolic requirements of actively firing neurons and
neuronal terminals (Brown et al., 1996; Eells et al., 2000). Advantages of this
technique are that most types of physiological stimuli increase Z-DG
accumulation in the brain and there is the potential for demonstrating all of the
brain regions involved with a specific stimulus (Sharp et al., 1993). However,
increases in Z-DG uptake produced by synaptic stimulation occur primarily in
presynaptic neuronal and possibly glial elements (Sharp et al., 1993). While the
use of this technique can identify active brain regions, it does not allow for
chemical differentiation between types of neurons, such as TH neurons from
vasopressin neurons. Thus, as with neurochemistry, Z-DG provides an index of
activity of nerve terminals and does not allow for cellular resolution (Komisaruk et

al., 2000; Eells et al., 2000).

75

Electrophysiology studies allow for the study of cell bodies focusing on the
electrical activities of individual neuroendocrine neurons. Earlier in vivo studies
were laborious to carry out because of difficulty accessing these neurons located
deep within the brain. In addition, it was not possible to verify probe location until
after the experiment and the required use of anesthetic may have altered
neuronal response (Pan et al., 1986). In vitro studies using brain slices remove
these obstacles of electrophysiology and can provide information concerning
individual neurons in specific brain regions. While a few studies have
successfully labeled the neurons of interest (Wagner et al., 1998), most neurons
remain unidentified (Lin et al., 1993; Pan et al., 1996).

Immunohistochemistry has become a useful technique to visualize and
identify neurons and their projections, but which cells were active under specific
stimuli was unknown. Through studies with the immediate early gene FRA, it
was demonstrated that FRA expression provides a metabolic map of individual
neuronal responses. FRA are immediate early genes that are rapidly and
transiently expressed in a variety of tissues including the brain following such
stimuli as growth factors, electrical stimulation and various stressors (Sagar et
al., 1988; Hoffman et al., 1993; Hoffman et al., 1994). Thus, it has been used as
a tool for mapping neuronal responses, localizing subpopulations of chemically
similar neurons, and determining if there are changes in the numbers of active
neurons in that subpopulation.

The goals of the studies in this chapter are two fold. First is to compare

the numbers and the distribution of TH-IR neurons throughout the ARC in male

76

and female rats. Innervation of the ME by TIDA neurons, as estimated by DA
concentrations, is equivalent in male and female rats. Thus there should be
similar numbers of TH-IR neurons in males and females. Second is to calculate
the percentage of those neurons expressing FRA. Previous studies have
demonstrated that TIDA neuronal activity, as estimated by DOPAC
concentrations in the ME, are two to three times greater in females compared to
males. The working hypothesis is that if the expression of FRA correlates with
neuronal activity, then female rats will have a greater number of TIDA neurons
containing FRA in the ARC compared to male rats. For comparison lHDA
neurons of the MZI were examined. Unlike TIDA neurons, there is no sexual
difference in DA concentration or activity in axon terminals of lHDA neurons.
These neurons are not responsive to gonadal steroids or PRL (Gunnet et al.,
1986), but instead are regulated by DA receptor-mediated mechanisms
(Lookingland and Moore, 1984). The numbers of TH-IR neurons in the MZI were
determined and the percentage of those neurons expressing FRA calculated in

male and female rats.

Materials and Methods

Gonadally intact female and male rats were used in these experiments.
Care and handling of the rats was carried out as described in the Material and
Methods in Chapter 2. The estrous cycles of female rats were monitored by
daily vaginal lavage, and only diestrous females exhibiting two or more

consecutive 4 to 5 day cycles were used in these experiments.

77

Untreated rats were anesthetized with Equithesin and perfused with 4%
paraformaldehyde. Brains were removed and sectioned as described in
Materials and Methods in Chapter 2. The identification of TH-IR neurons using
fluorescent rhodamine and FRA—IR nuclei using nickel intensified DAB in the
ARC and MZI was carried out using immunohistochemical procedures described
in the Materials and Methods in Chapter 2. An indepth analysis of the entire
length of the ARC was initially carried out by dividing the ARC into 10 regions
spanning the area 1.8mm posterior to 3.96 mm posterior to bregma.
Observations during initial analysis suggested that subsequent trends may exist
in more defined subpopulations of TH-IR neurons within the ARC, thus the
original slides were re-evaluated. For this analysis, the ARC was regrouped into
rostral, middle, and caudal regions (2.3 mm, 2.8 mm and 3.3 mm posterior to
bregma, respectively) and then further partitioned into DM-ARC and VL-ARC.
FRA were localized with anti-FRA antiserum (Genosys Biotechnologies)
generated against a synthetic peptide derived from a conserved region of the c-
fos gene common to all FRA genes. The dilution used was 122500 in TBS/T X
containing 1.5% normal donkey serum. TH was detected using a monoclonal
anti-TH antiserum (Incstar Corp) generated in mouse against a midportion
fragment of purified portion of TH isolated from PC-12 cells. The final dilution

was 121000 in TBS/T X containing 1.5% normal horse serum.

78

Results

Figure 3.1 depicts the distribution of TH-IR neurons through
representative sections of the ARC of male rats. The numbers of TH-IR neurons
were significantly higher in the middle regions and continued through the caudal
regions of the ARC. The percentage of TH-lR neurons expressing FRA in males
were low in the rostral and more rostral middle regions of the ARC ranging from
3.3% to 6.1%, and significantly increased in the more caudal middle and caudal
regions to a maximum level of 11.7% (Figure 3.2). In females, higher numbers
of TH-IR neurons occurred in the rostral regions and declined in the caudal
regions of the ARC (Figure 3.3). The percentage of TH-IR neurons expressing
FRA remained relatively constant throughout the ARC ranging from 8.9-13.8%
(Figure 3.4).

In addition to the sexual differences in the distribution of TH-IR neurons
and FRA-IR nuclei within those neurons, there were visual clues that differences
may be present within the dorsoventral regions of the ARC. The ARC was
further analyzed by partitioning it into DM-ARC and VL-ARC.

In the DM-ARC, there was a significantly decreasing gradient in the
numbers of TH-IR neurons that extended caudally in both males and females
(Figure 3.5). The actual numbers of neurons within each rostrocaudal region
was similar between male (64.6 rostral, 50.2 middle, and 41.0 caudal) and
female (65 rostral, 54.9 middle, 33.6 caudal) indicating no sexual differences.
The number of TH-IR neurons containing FRA-IR nuclei, however, was almost 3

times greater in the rostral DM-ARC of females (14.2%) compared to males

79

Lateral -0.10 mm

 

 

 

 

m
c
2
8 60 *
2
0.!
E 4ol
t.—
0
fit:
20 ~
0 M l
1.80 2.04 2.28 2.52 2.76 3.00 3.24 3.48 3.72 3.96
Rostral— — Middle— —Caudal
ARC ARC ARC

Figure 3.1 Distribution of TH-IR neurons throughout the rostro-caudal extent of
the ARC in male rats. Coordinates for these regions extend from 1.80—3.96 mm
posterior to bregma (Paxinos and Watson, 1997). Columns represent means
and vertical lines + 1 SEM from 5 animals. *, Values that are significantly different
(p < 0.05) from 1.80 mm posterior to bregma. 3V, third ventricle; APit, anterior
lobe of pituitary; Arc, arcuate nucleus; DMH, dorsomedial hypothalamic nucleus;
InfS, infundibular stalk; IPit, intermediate lobe of pituitary; ME, median eminence;
ox, optic chiasm; PPit, posterior lobe of pituitary; sox, supraoptic decussation;
VMH, ventromedial hypothalamic nucleus

80

Lateral -0.10 mm

 

 

 

 

 

100 —
*
*

80 2
m
c
g

o 60 —
2
E
2':

l— 40 '1
Q—
0
1|:

20 l

0 _

1.80 2.04 2.28 2.52 2.76 3.00 3.24 3.48 3.72 3.96
Rostral— ——Middle — —— Caudal
ARC ARC ARC

Figure 3.2 Distribution of TH-IR neurons throughout the rostro-caudal extent of
the ARC in female rats. Coordinates for these regions extend from 1.80—3.96 mm
posterior to bregma (Paxinos and Watson, 1997). Columns represent means and
vertical lines + 1 SEM from 5 animals. *, Values that are significantly different (p
< 0.05) from 1.80 mm posterior to bregma. Abbreviations are as defined in
Figure 3.1.

81

Lateral -0.10 mm

% of TH-IR Neurons Expressing F RA

 

 

1.80 2.04 2.28 2.52 2.76 3.00 3.24 3.48 3.72 3.96

 

 

Rostral— — Middle— -—Caudal
ARC ARC ARC

Figure 3.3 Percentages of TH-IR neurons expressing FRA throughout the
rostro—caudal extent of the ARC in male rats. Coordinates for these regions
extend from 1.80—3.96 mm posterior to bregma (Paxinos and Watson, 1997).
Columns represent means and vertical lines + 1 SEM from 5 animals. *, Values
that are significantly different (p < 0.05) from 1.80 mm posterior to bregma.
Abbreviations are as defined in Figure 3.1.

82

Lateral -0. 10 mm

 

% of TH-IR Neurons Expressing FRA
3

 

 

8

6

4

2

0

1.80 2.04 2.28 2.52 2.76 3.00 3.24 3.48 3.72 3.96

Rostral— — Middle— —Caudal
ARC ARC ARC

Figure 3.4 Percentages of TH-IR neurons expressing FRA throughout the
rostro-caudal extent of the ARC in female rats. Coordinates for these regions
extend from 1.80—3.96 mm posterior to bregma (Paxinos and Watson, 1997).
Columns represent means and vertical lines + 1 SEM from 5 animals.
Abbreviations are as defined in Figure 3.1.

83

DM-ARC

7O _ T - Male

[:l Female

 

60-

 

50-

40‘

 

30-

# of TH-IR Neurons

20-

10-

 

 

 

 

 

 

 

 

 

 

 

 

 

l l

Rostral Middle Caudal

Figure 3.5 Comparison of the numbers of TH-IR neurons in the rostral, middle,
and caudal regions of the DM-ARC in male (filled columns) and female rats
(open columns). Coordinates for these regions of the ARC were approximately
2.3mm (rostral), 2.8 mm (middle), and 3.3 mm (caudal) posterior to bregma
(Paxinos and Watson, 1997). Columns represent means and vertical lines + 1
SEM from 5 animals.

84

(5.2%). In the middle and caudal regions, the percentage of FRA-IR nuclei was
similar between females (8.6% and 6.5%) and males (6.2% and 4.8%) (Figure
3.6).

Unlike the DM—ARC, there were differences in the number of TH-IR
neurons between sexes in two of the subregions of the VL-ARC. The numbers of
neurons were almost two times greater in male versus female rats in the middle
and caudal VL-ARC (Figure 3.7). In the rostral VL-ARC, the numbers of neurons
were relatively low in males (16.5 cells/section) and females (10.0 cells/section).
Figure 3.8 represents the percentage of TH-IR neurons expressing FRA-IR
nuclei in the VL-ARC of males and females. The percentages were relatively
constant throughout the rostrocaudal VL-ARC in both sexes (13-17%), but there
was high variability in this area especially in the rostral VL-ARC.

In the MZI, the numbers of TH-IR neurons in males were comparable to
the numbers in females (260 vs 235 cells/section) (Figure 3.9, Bottom Panel).
However, this was a stark contrast to the numbers of TH-IR neurons within the
DM-ARC or VL-ARC; the numbers of TH-IR neurons in the MZI ranged 4 to 20
times higher. As with the numbers of neurons, there was no difference between
males and females in the percentage of TH-IR neurons containing FRA-IR nuclei
(Figure 3.9, Top Panel). Unlike the ARC, however, the percent of FRA-IR nuclei

in the MZI was very low, less than 1%.

85

DM-ARC

.s
G
J

T - Male
* [:1 Female

 

..L
A
J

.5
N
1

..5
O
1

 

 

 

% of TH-IR Expressing FRA
———I

 

 

 

 

 

 

 

     

 

 

 

I I

Rostral Middle Caudal

Figure 3.6 Comparison of the percent of TH-IR neurons expressing FRA in the
rostral, middle, and caudal regions of the DM-ARC in male (filled columns) and
female rats (open columns). Coordinates for these regions of the ARC were
approximately 2.3mm (rostral), 2.8 mm (middle), and 3.3 mm (caudal) posterior
to bregma (Paxinos and Watson, 1997). Columns represent means and vertical
lines + 1 SEM from 5 animals. *, Values for females that are significantly higher
(p < 0.05) than males.

86

VL-ARC

70 w
- Male

60 1 [:1 Female

50—
40—

sol

 

 

204

# of TH-IR Neurons

 

10*

 

 

 

 

 

 

 

 

 

 

   

0 ‘ r T

Rostral Middle Caudal

Figure 3.7 Comparison of the numbers of TH-IR neurons in the rostral, middle,
and caudal regions of the VL-ARC in male (filled columns) and female rats (open
columns). Coordinates for these regions of the ARC were approximately 2.3mm
(rostral), 2.8 mm (middle), and 3.3 mm (caudal) posterior to bregma (Paxinos
and Watson, 1997). Columns represent means and vertical lines + 1 SEM from 5
animals. *, Values for females that are significantly lower (p < 0.05) than males.

87

VL-ARC

(.10
O
4

— Male
l:l Female

25*

N
O
l

 

 

154 j

 

 

 

 

A
O
l

01
4

  

 

% of TH-IR Neurons Expressing FRA

 

 

 

 

 

 

 

 

 

 

 

l

O
I

l

Rostral Middle Caudal

Figure 3.8 Comparison of the percent of TH-IR neurons expressing FRA in the
rostral, middle, and caudal regions of the VL-ARC in male (filled columns) and
female rats (open columns). Coordinates for these regions of the ARC were
approximately 2.3mm (rostral), 2.8 mm (middle), and 3.3 mm (caudal) posterior
to bregma (Paxinos and Watson, 1997). Columns represent means and vertical
lines + 1 SEM from 5 animals.

88

MZI

01
r

_ Male
l:l Female

03 «h»
1 .4

N

_\
————I

 

 

% of TH-IR Neurons Expressing FRA

 

 

 

 

O

 

I I

Male Female

300 -

250 4

 

200 -

150 ‘

100 J

# of TH-IR Neurons

 

 

 

 

 

 

Male Female

Figure 3.9 Comparison of the numbers of TH-IR neurons (Bottom Panel) and
the percent of these neurons expressing FRA (Top Panel) in the MZI of male
(filled columns) and female rats (open columns. Coordinates for this region was
approximately 2.8 mm posterior to bregma (Paxinos and Watson, 1997).
Columns represent means and vertical lines + 1 SEM from 5 animals.

89

Discussion

The goals of these studies were to determine the distribution of TH-IR
neurons as well as the basal level of FRA expression in these neurons in the
DM-ARC and VL ARC in male and diestrous female rats.

The overall numbers and distribution of TIDA neurons throughout the ARC
correlate with previous studies describing the distribution of TH-IR neurons
throughout the hypothalamus in male rats (Chan-Palay et al., 1984; Van den Pol
et al., 1984). The results from initial studies presented here demonstrate an
increase in numbers of TIDA neurons in a rostrocaudal gradient in the ARC of
male rats. This correlates with DA concentrations, an indirect estimate of
innervation, of the ME. Distribution studies of the ME divided the region into five
rostrocaudal segments and compared DA concentrations (Palkovits et al., 1982).
The ME has an increasing rostrocaudal gradient of DA with the maximum level
located at the site of attachment of the pituitary stalk. TIDA neurons project
ventrally and connect each portion of the ARC to a corresponding part of the ME
(Lindvall and Bjorkland, 1978). Taken together these data indicate that there is
an increase in innervation in the caudal portion of the ME in male rats. In
females, there is a decrease in the numbers of TIDA neurons from the rostral to
caudal portions of the ARC. While there are no published data describing the
distribution of innervation in subdivisions of the ME in females, it would be logical
that, like with male rats, the numbers of TIDA neurons would correlate with the
innervation of the ME. Thus in females, one would anticipate that there would be

greater DA concentrations and innervation in the rostral portion of the ME.

90

Expression of immediate early genes such as FRA have been shown to
reflect activity of TIDA neurons of the ARC (Hoffman et al., 1994; Yang et al.,
1999). F RA are stimulated through depolarization or ligand-mediated activation
of membrane receptors located on neuronal soma or dendrites. This triggers a
second messenger system (e.g. cyclic AMP and calcium) and phosphorylation
cascade involving protein kinases A or C to activate constitutive transcription
factors like CREB (Sheng and Greenberg, 1990; Sim et al., 1994). CREB
induces the transcription and translation of immediate early genes like FRA.
FRA dimerize with Jun proteins and bind to the AP-1 site (Curran and Franza,
1988) to regulate transcription and translation of long-term genes such as TH
(Gizang-Ginsberg and Ziff, 1990; Icard-Liepkalns et al., 1992).

In males, there is an increase in the expression of FRA in the middle and
caudal regions of the ARC. In contrast, the expression of FRA in female rats
remains relatively constant throughout the ARC. These data suggest the
presence of subpopulations of active TIDA neurons present in males that may
subserve some gender-specific function which is absent or quiescent in females.

In the DM-ARC, there was a difference in distribution of TH-IR neurons in
both males and females. There were a greater number of neurons in the rostral
DM-ARC compared to the middle and caudal regions. Thus, the results
presented here correlate with neurochemical studies demonstrating the density
of innervation to the ME is the same for male and female rats (Gunnett et al.,

1986).

91

Several studies have demonstrated that axon terminal activity, using
neurochemical techniques (Lookingland et al., 1987), as well as glucose
utilization, using Z-DG autoradiography (Brown et al., 1996), is 2-3 times greater
in diestrous female rats compared to male rats under basal conditions. One
explanation for the higher activity in females could simply be that an identical
number of neurons are firing in males and females, but the rate is 2—3 times
greater in females. A second possible explanation is that while the firing rate is
constant, there are 2-3 times more neurons firing in females compared to males.
By using F RA as an index of activity, it may be possible to identify which scenario
accounts for the difference in female rats. It was the goal of these studies to
determine if there were sexual differences in the expression of FRA that
correlated with neurochemistry. If the percentage of TIDA neurons expressing
FRA does correlate with neuronal activity, it should be 2-3 times greater in
female rats compared to male rats. These results in Figure 3.6 demonstrate that
females have almost three times greater F RA expression in the rostral DM-ARC
compared to males. This indicates that there are a greater number of neurons
that are active in a subpopulation of TIDA neurons in female rats.

This difference is due to the effects of gonadal steroids as well as a
difference in responsiveness of these neurons to PRL. Estrogen has a
stimulatory effect on TIDA neuronal activity (Gunnet et al., 1986) as well as FRA
expression (Cheung et al., 1997) via a mechanism mediated by PRL. Blockade
of PRL secretion from the anterior pituitary prevents the estrogen-induced

increases in TIDA neuronal activity (Toney et al., 1992). Furthermore, estrogen

92

increases synthesis of PRL by regulating gene transcription (Shull and Gorski,
1989) and disrupts the inhibitory actions of DA on PRL release from the anterior
pituitary (Ferland et al., 1979). On the other hand, castration and hormone
replacement studies demonstrated that testosterone has an inhibitory effect on
both TIDA neuronal activity (Toney et al., 1991) and the expression of FRA
(Cheung et al., 1997). Testosterone also attenuates the responsiveness of TIDA
neurons to tonic stimulatory actions of PRL, but not the delayed activation by
PRL (Toney, et al., 1991). Females are more sensitive to the stimulatory actions
of PRL compared to males, and increases in PRL secretion stimulate DA release
(Demarest and Moore, 1981) as well as FRA expression (Hentschel, 2000) in
TIDA neurons of females to a greater extent than males. In addition,
hypoprolactinemia decreases the activity of TIDA neurons in females, but
decreases in PRL have almost no effect in males (Demarest and Moore, 1981 ).
In the VL-ARC, there is a sexual difference in the numbers of TH-IR
neurons. The numbers of TH-IR neurons are greater in the middle and caudal
aspects of the VL-ARC in males, but not in females. The neurons of the VL—ARC
project to the lateral ME (Everitt et al., 1986). The TH-IR neurons of the VL-ARC
are unique from those of the DM-ARC in that they lack the DD enzyme to convert
DOPA to DA. Instead they only synthesize TH and are limited to producing only
DOPA (Meister et al., 1988). This difference may indicate a physiological
difference in the function of these “DOPA—ergic” neurons. Their exact function is
not known; however, DOPA nerve terminals have been detected in vesicle-like

aggregates in the external layer of the ME (Misu et al., 1996), and DD activity is

93

present in the vascular wall of the brain (Meister et al., 1988). This suggests that
DOPA released from these neurons may be converted to DA at the level of the
ME or in the portal blood system (Meister et al., 1988).

The expression of FRA in the VL-ARC is equal in males and females but
highly variable. In the rostral portion of the VL-ARC this is most likely due to the
low numbers of TIDA neurons. Nonetheless, this suggests that there is no
sexual difference in the activity of these neurons.

For comparison, the numbers lHDA neurons of the MZI and the
percentage of those neurons expressing FRA were examined in males and
females. These neurons project to various areas of the brain including the
paraventricular nucleus, horizontal limb of the diagonal band of Broca and central
nucleus of the amygdala (Wagner et al., 1995; Eaton et al., 1994) and may play a
role in Iuteinizing hormone secretion in female rats (MacKenzie et al., 1984).
Compared to the ARC, the MZI has a significantly greater number of DA
neurons, but significantly lower expression of FRA. The finding that there was no
sexual difference in the number of lHDA neurons or in F RA expression correlates
with previous neurochemical studies demonstrating that these neurons are not
regulated by PRL or gonadal steroids and there is no sexual difference in basal
activity of lHDA neurons (Gunnett et al., 1986b)

The results confirm the presence of subpopulations of TH-IR neurons that
can be grouped into rostral, middle, and caudal DM-ARC and VL-ARC. In
addition, there are sexual differences in FRA expression in TH-IR neurons in the

rostral DM-ARC that coincide with differences in neurochemical activity of TIDA

94

neurons that terminate in the ME. These studies indicate that there are 2 to 3
times more TIDA neurons in the rostral DM-ARC that are active in female
compared male rats. It is these TH-IR neurons in the DM-ARC that should be
considered true TIDA neurons. In contrast the lHDA neurons of the MZI, which
are regulated by DA—receptor mediated mechanisms (and not PRL or gonadal
steroids as are TIDA neurons) demonstrate no sexual differences in the numbers

or in the percentage of neurons expressing FRA.

95

CHAPTER FOUR
D2 RECEPTOR REGULATION OF IMMEDIATE EARLY GENE EXPRESSION
IN TH-IR NEURONS IN SUBDIVISIONS OF THE ARCUATE NUCLEUS
Introduction

As discussed in Chapter Three, TIDA neurons are primarily regulated by
prolactin, which is influenced by estrogen. This is in contrast to the
mesotelencephalic DA neurons, which are regulated by DA receptor-mediated
mechanisms consisting of autoreceptors and long loop neuronal feedback
circuits. Several lines of evidence suggest TIDA neurons do not have
autoreceptors, although there is controversy as to whether or not this is the case.
TIDA neurons are unresponsive to nonselective DA agonists suggesting that
TIDA neurons do not have DA autoreceptors (Moore, 1987). y-Butyrolactone
(GBL) disrupts impulse flow in DA neurons and thereby reduces the release of
DA (Moore, 1987). In the mesotelencephalic and lHDA systems, less DA in the
synapse leads to decreased activation of pre-synaptic autoreceptors and a
disinhibition of TH (Demarest and Moore, 1979; Lookingland and Moore, 1984).
The result is an increase in the rate of synthesis and concentration of DA in axon
terminals. The increases of DA can be reversed with DA agonists (Moore, 1987).
However, the concentration and rate of synthesis of DA does not increase in
nerve terminals of the ME following GBL administration (Demarest and Moore,
1979; Lookingland and Moore, 1984; Gunnet et al., 1987).

Autoradiography and in situ hybridization studies have demonstrated D2

heteronuclear RNA and mRNA in many of the dopaminergic cell groups of the

96

hypothalamus including the MZI, but not the ARC (Weiner et al., 1991; Fox et al.,
1993). Local infusion of selective DZ agonists and antagonists through
microdialysis had no effect on DA and DOPAC levels in the medial basal
hypothalamus (Timmerman et al., 1995). However, application of DA on rat
hypothalamic brain slices inhibits single-unit activity of 70% of DM-ARC neurons
suggesting that these neurons may contain autoreceptors (Lin and Pan, 1999).
These neurons were not identified as being dopaminergic, however.

Following the discovery of receptor subtypes, in hopes of minimizing side
effects often encountered, (i.e. dyskinesias) more specific DA receptor agonists
and antagonists have been developed for the treatment of Parkinson’s disease
and schizophrenia. The receptor subtypes have been divided into two families:
D1-Iike and DZ-like. D1-like receptors are stimulatory and found post-
synaptically, while DZ-like receptors are inhibitory and found pre- and post-
synaptically (Spano et al., 1978; Kebabian and Calne, 1979; Vallone et al.,
2000). Autoreceptors fall into the DZ-like category. Systemic administration of
selective D2 agonists decreases neuronal activity in DA receptor-regulated
mesotelencephalic systems neuronal systems (Foreman et al., 1989; Eaton et
al., 1993; Durham et al., 1996). Surprisingly, DZ agonist effects on TIDA neurons
result in an increase in activity (Eaton et al., 1993). This occurs independently of
PRL and is mediated through tonically active afferent dynorphin neurons and
kappa opioid receptors (Durham et al., 1996).

In addition to causing DA release from the axon terminal, stimulation of

TIDA neurons can lead to changes in cellular events initiated by expression of

97

immediate early gene transcription factors. One set of immediate early genes
that has been correlated with neuronal activity is FRA (Hoffman et al., 1993;
Hoffman et al., 1994). FRA expression is increased throughout the brain
following various pharmacological and physiological manipulations.

There are several lines of evidence demonstrating regulation of FRA
expression via DA receptors in various brain regions. Haloperidol increases c-
fos mRNA in the dorsolateral regions of the rat neostriatum via an action on post-
synaptic DZ receptors (Merchant, et al., 1994). Quinelorane can reverse the
stimulatory effects of haloperidol on c-fos (and c-jun) mRNA in the striatum
(Rogue and Vincendon, 1992). Changes in FRA expression have also been

demonstrated in the hypothalamus. In the paraventricular nucleus, quinelorane
sti mulates FRA expression in corticotropin-releasing hormone (CRH) neurons
(Eaton, et al., 1996), while quinelorane inhibits FRA expression in somatostatin
r‘lelJrons of the periventricular nucleus (Cheung, et al., 1997). Fos expression
3'80 been studied in neonatal TH immunoreactive neurons cultured from the MZI
(S im, et al., 1994). These studies demonstrated that D2 agonists can inhibit Fos
e><pression and that a D2 antagonist can block this inhibition. Thus, the D2
receptor system regulates Fos levels in lHDA neurons. However, no information
is available regarding DA receptor-mediated regulation of gene expression in
T I DA neurons.
The goal of these studies was to determine if DZ receptors regulate
immediate early gene expression in TIDA neurons in male rats. The working

hypothesis is that activation of D2 receptors stimulates Fos and Jun expression

98

in subpopulations of these neurons. Experiments in Chapter 3 used a
nonspecific antibody that identified Fos, FosB, Fra-1 and Fra-Z. By measuring
FRA it is possible to identify increases and decreases in neuronal
responsiveness (Hoffman et al., 1994); however, it is not possible to determine
which transcription factor(s) is (are) being expressed, for each gene is induced
by different types of stimuli and has a different half-life (Hoffman et al., 1992;
Hughes and Dragunow, 1995; Kovacs, 1998). Activation of D2 receptors rapidly
increases TIDA neuronal activity. If a Fos family member is induced by acute
stimulation of these neurons, it is logical that it would be a protein that is induced
fol lowing acute challenges. c-fos is the most rapidly induced by acute challenges
(Hughes and Dragunow, 1995; Kovacs, 1998), thus it was chosen as the
lm mediate early gene to be studied. To this end, the time course effects of
Clu inelorane on Fos and c—Jun expression were determined. For comparison, the

effects on Fos and c-Jun in the lHDA neurons of the MZI were also studied.

Materials and Methods

Gonadally intact male rats were used in these experiments. Care and
handling of the rats was carried out as described in the Materials and Methods
in Chapter 2. Following appropriate treatments, rats were anesthetized with
Equithesin and perfused with 4% paraformaldehyde. Brains were removed and

sectioned as described in Materials and Methods in Chapter 2. The
identification of TH-IR neurons using diaminobenzidine or alkaline phosphatase

and Fos-IR using nickel-intensified diaminobenzidine in the ARC and MZI was

99

carried out using immunohistochemical procedures in Materials and Methods in
Chapter 2. The ARC sections were sorted into rostral, middle and caudal regions
and then visually partitioned into DM-ARC and VL-ARC. Fos was localized with
anti-Fos antiserum generated against the amino terminus of human c-Fos p62
( Santa Cruz) or against residues 4 to 17 of human fos (Oncogene). The dilution
used was 1:5000 (Santa Cruz) or 1210000 (Oncogene) in TBS/T X containing
1 .5% normal donkey serum. TH was detected using anti-TH serum (Chemicon)
generated against rat adrenal medullary tumor tissue. The final dilution was

1 220,000 in TBS/l“ X containing 1.5% normal goat serum.

Results
Photomicrographs in Figure 4.1 illustrate Fos expression in TH—IR
I“leaurons of middle DM-ARC 2 h after injection of either vehicle (Top Panel) or
cilL-linelorane-treated (Bottom Panel) male rats. These images show that Fos
expression in TH-IR neurons is increased after quinelorane administration.
Fi Qure 4.2 illustrates the time course effects of quinelorane on the percentage of
TFI—IR neurons expressing Fos in the DM—ARC. Quinelorane significantly
increased Fos expression in a time dependent manner, reaching maximum levels
at 2 h and returning to basal levels by 4 h in the rostral, middle and caudal
I"egions of the DM-ARC. While the number of TH-IR neurons declines through
the rostral-caudal extent of the DM-ARC, there were no changes in the numbers
of TH-IR neurons at any time point after quinelorane within each region of the

lDM-ARC (Figure 4.3). In contrast, quinelorane caused significant decreases of

100

 

 

Figure 4.1 High power (400x) computer-captured images demonstrating
‘hcreased Fos expression in TH-IR neurons of the DM-ARC 2 h following the
administration of quinelorane. Rats were injected with either vehicle (Top Panel;
0-9% saline; 1 mllkg; ip) or quinelorane (Bottom Panel; 100 uglkg; ip) and
sacrificed 2 h later. Arrowheads represent colocalization of brown TH-IR
Neurons containing black Fos-IR nuclei. 3V, third ventricle.

101

DM-ARC

 

 

 

20

g 1 —o— Rostral

I... * —D— Middle

2 * —A— Caudal

'_ \

g 15 ~ .

2

a.

><
LI...

2 10 -«

2

:3

a:
z
E ‘
:l": 5 “ -
|_.
9.—

o o \
°\°
0 I I I I I
O 1 2 4 8

Hours After Quinelorane

I=igure 4.2 Time course effects of quinelorane on the percentage of TH-IR
neurons expressing Fos in the rostral (circles), middle (squares), and caudal
(triangles) regions of the DM-ARC. Rats were injected with quinelorane (filled
8symbols; 100 jig/kg; ip) and sacrificed 1, Z, 4 or 8 h later. Zero time controls
(Open symbols) were injected with 0.9% saline vehicle (1 mllkg; ip) and sacrificed
2 h later. Symbols represent the means and vertical lines + 1 SEM from 6-8
animals. Where vertical lines are not depicted, 1 SEM is less than the radius of
the symbol. *, Values for quinelorane-treated rats that are significantly different
(D<0.05) from vehicle-treated controls.

102

DM-ARC

80 "
[1:] Vehicle
70 2 - Quinelorane

60— I1

50~
40* II

30—

#IH-IR Neurons

204

10‘

 

 

 

 

 

 

 

 

 

 

0 1 ‘ ‘
01248 01248 01248

Rostral Middle Caudal
Hours After Quinelorane

Figure 4.3 Time course effects of quinelorane on the numbers of TH-IR neurons
rt the rostral, middle, and caudal regions of the DM-ARC. Rats were injected
With quinelorane (filled columns; 100 ng/kg; ip) and sacrificed 1, Z, 4 or 8 h later
ero time controls (open columns) were injected with 0.9% saline vehicle (1.

rY‘lI/kg' ip) and sacrificed 2 h later Columns re resent th '
2 . . e means and
"hes + 1 SEM from 6-8 animals. p vertical

103

 

   

 

 

Fos expression at 4 and 8 h throughout the rostral-caudal extent of the VL-ARC
(Figure 4.4). As shown in Figure 4.5, there is an increasing gradient in the
numbers of TH-IR neurons in vehicle-treated rats that extends caudally in the VL-
ARC. As in the DM-ARC, there were no changes in the numbers of TH-IR
neurons at any time point within each region of the VL-ARC. In the MZI, there
was a gradual decrease in Fos expression following quinelorane such that there
was a significant decrease in the percentage of TH-IR neurons with Fos-IR nuclei
at the 8 h time point (Figure 4.6). Quinelorane had no effect on the numbers of
TH-IR neurons in the MZI at any time point (Figure 4.6; Inset).
To determine if the effects of quinelorane on Fos were mediated through
D2 receptors a study was carried out using the D2 receptor antagonist raclopride.
B l ockade of D2 receptors with raclopride had no effect per se, but reversed the
stimulatory effects of quinelorane on Fos expression throughout the rostral—
C>audal extent of the DM-ARC (Figure 4.7). There were no changes in the
numbers of TH-IR neurons following either raclopride or quinelorane (Figure
4-8). In the VL-ARC, there was a decreasing trend in the percent of TH-IR
heurons expressing Fos-IR nuclei following raclopride and quinelorane
administration, but there were no significant differences when compared to their
rSspective control group (Figure 4.9). There were no changes in the numbers of
TH-IR neurons in the VL-ARC following the administration of either raclopride or
cIllinelorane (Figure 4.10). In the MZI raclopride increased Fos expression and
the coadministration of quinelorane reversed this effect (Figure 4.11; Top

lDamel). Quinelorane alone had no effect on the percentage of TH-IR neurons

104

VL-ARC

N
U1
l

-—o— Rostral
—l:l— Middle
—£>r— Caudal

N
O
I

 

..1.
0'1
1

(J1
4

 

 

% of TH-IR Neurons Expressing Fos

 

-l
-I
—1
—4
_

 

Hours After Quinelorane

l'tigure 4.4 Time course effects of quinelorane on the percentage of TH-IR
neurons expressing Fos in the rostral (circles), middle (squares), and caudal
(triangles) regions of the VL-ARC. Rats were injected with quinelorane (filled
Symbols; 100 uglkg; ip) and sacrificed 1, 2, 4 or 8 h later. Zero time controls
(Open symbols) were injected with 0.9% saline vehicle (1 mllkg; ip) and
sacrificed 2 h later. Symbols represent the means and vertical lines + 1 SEM-for
S~8 animals. *, Values for quinelorane-treated rats that are significantly different
(p<0.05) form vehicle-treated controls.

105

VL-ARC

 

 

 

 

 

 

 

 

 

 

 

60 -
[:1 Vehicle
— Quinelorane
501 l
.. TH
r:
e 40 -
:3
a:
2
m 30 4
:r':
I'-
“5 20 .
1::
10 J
0 I I I
01248 01248 01248
Rostral Middle Caudal

Hours After Quinelorane

Figure 4.5 Time course effects of quinelorane on the numbers of TH-IR neurons
in the rostral, middle, and caudal regions of the VL-ARC. Rats were injected with
quinelorane (filled columns; 100 ug/kg; ip) and sacrificed 1, 2, 4 or 8 h later. Zero
time controls (open columns) were injected with 0.9% saline vehicle (1 mllkg; ip)
and sacrificed 2 h later. Columns represent the means and vertiml lines + 1
SEM from 6-8 animals.

106

MZI

    

u, 10 _ 250 .
O 11:9 Vehicle
u. — Quinelorane 200 ~
a: g ,
.E 8 1 e
In = 150
w 2
95,. E 00
' 1 .
X 6 _ E , ,
I“ "6
m at 50 ’
= l
a: 4 I o 1 2 4 8
2 Hours After Quinelorane
E
I
I _
P 2
a—
O
o\°

 

 

Hours After Quinelorane

Figure 4.6 Time course effects of quinelorane on the numbers of TH-IR neurons
(Inset) and the percentage of these neurons expressing Fos in the MZI. Rats
were injected with quinelorane (filled symbols and columns; 100 jig/kg; ip) and
sacrificed 1, 2, 4 or 8 h later. Zero time controls (open symbols and columns)
were injected with 0.9% saline vehicle (1 mllkg; ip) and sacrificed 2 h later.
Symbols and columns represent the means and vertical lines + 1 SEM from 6-8
animals. *, Values for quinelorane-treated rats that are significantly different
(p<0.05) from vehicle-treated controls.

107

DM-ARC

:1 Vehicle

50 7 - Quinelorane

N (O b
O O O
l l l

% of TH-IR Neurons Expressing Fos
8

 

ill .

Vehicle Raclopride Vehicle Raclopride Vehicle Raclopride
Rostral Middle Caudal

 

O
L

Figure 4.7 Effects of quinelorane on the percentage of TH-IR neurons
expressing Fos in the rostral, middle and caudal regions of the DM-ARC of
vehicle- or raclopride-treated rats. Rats were injected with either raclopride (3
mglkg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 2 h prior to sacrifice, and with
either quinelorane (100 jig/kg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 2 h prior
to sacrifice. Columns represent the means and vertical lines + 1 SEM form 6-8
vehicle- (open columns) and quinelorane- (solid columns) treated rats. *, Values
that are significantly different (p<0.05) from vehicle-treated rats.

108

DM-ARC

80 -
l I: Vehicle
70 ~ g — Quinelorane

t

40« I?

30—

# of TH-IR Neurons

204

101

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0 l .
Vehicle Raclopride Vehicle Raclopride Vehicle Raclopride

Rostral Middle Caudal

Figure 4.8 Effects of quinelorane on the numbers of TH-IR neurons in the
rostral, middle and caudal regions of the DM-ARC of vehicle- or raclopride-
treated rats. Rats were injected with either raclopride (3 mglkg; ip) or its 0.9%
saline vehicle (1 mllkg; ip) 2 h prior to sacrifice, and with either quinelorane (100
uglkg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 2 h prior to sacrifice. Columns
represent the means and vertical lines + 1 SEM from 6—8 vehicle- (open columns)
and quinelorane- (solid columns) treated rats.

109

VL-ARC

Q)
0"
r

l::l Vehicle
- Quinelorane

j I

N (A)
01 O
r I

N
O
1

_L
O
1

% of TH-IR neurons expressing Fos
01 6‘1
l——l

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I I

Vehicle Raclopride Vehicle Raclopride Vehicle Raclopride
Rostral Middle Caudal

0

Figure 4.9 Effects of quinelorane on the percentage of TH-IR neurons
expressing Fos in the rostral, middle and caudal regions of the VL-ARC of
vehicle- or raclopride-treated rats. Rats were injected with either raclopride (3
mglkg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 2 h prior to sacrifice, and with
either quinelorane (100 jig/kg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 2 h prior
to sacrifice. Columns represent means and vertical lines + 1 SEM form 6-8
vehicle- (open columns) and quinelorane- (solid columns) treated rats.

110

VL-ARC

451

:l Vehicle
_ Quinelorane I

351 l
301 l

257

404

20-

15~ %
10- I:

# of TH-IR neurons

 

 

 

 

 

 

 

 

 

 

 

 

 

0 1
Vehicle Raclopride Vehicle Raclopride Vehicle Raclopride

Rostral Middle ‘ Caudal

 

 

 

 

Figure 4.10 Effects of quinelorane on the numbers of TH-IR neurons in the
rostral, middle and caudal regions of the VL-ARC of vehicle- or raclopride-treated
rats. Rats were injected with either raclopride (3 mglkg; ip) or its 0.9% saline
vehicle (1 mllkg; ip) 2 h prior to sacrifice, and with either quinelorane (100 jig/kg;
ip) or its 0.9% saline vehicle (1 mllkg; ip) 2 h prior to sacrifice. Columns
represent the means and vertical lines + 1 SEM from 6-8 vehicle- (open columns)
and quinelorane- (solid columns) treated rats.

111

MZI

.3
O

:3 Vehicle
— Quinelorane

00
X-

 

 

% of TH-IR Neurons Expressing Fos
-1

 

 

 

 

 

 

 

 

 

Vehicle Raclopride

250 'l I

 

 

200 1

150 -

100 1

# of TH-IR Neurons

501

 

 

 

 

 

 

 

 

 

Vehicle Raclopride

Figure 4.11 Effects of quinelorane on the numbers of TH-IR neurons (Bottom
Panel) and the percentage of these neurons expressing Fos (Top Panel) in the
MZI of vehicle- or raclopride-treated rats. Rats were injected with either
raclopride (3 mglkg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 2 h prior to
sacrifice, and with either quinelorane (100 jig/kg; ip) or its 0.9% saline vehicle (1
mllkg; ip) 2 h prior to sacrifice. Columns represent the means and vertical lines +
1 SEM from 6-8 vehicle- (open columns) and quinelorane- (solid columns)
treated rats. *, Values that are significantly different (p<0.05) from vehicle-
treated rats. #, Values that are significantly different (p<0.05) from raclopride-
treated rats.

112

expressing Fos. There were no effects on the numbers of TH-IR neurons
following the administration of either raclopride or quinelorane (Figure 4.11;
Bottom Panel).

The TH-IR neurons of the VL-ARC did not respond to quinelorane and
raclopride thus expression of Fos in these neurons is not regulated by D2
receptors. While the 'TH-IR neurons of the VL-ARC and the MZI were not
responsive at 2 h, there was a gradual and late decrease in Fos expression.
This suggests other factors such as the stress of receiving an intraperitoneal
injection or circadian rhythm may account for the decrease in Fos expression.
Restraint stress decreases DOPAC levels in the ME as demonstrated by
neurochemical studies (Lookingland and Moore, 1988; Lookingland et al., 1990).
Circadian rhythms also induce changes in TIDA neuronal activity in female rats
as shown in neurochemical indices (Mai et al., 1994) and this is associated with a
corresponding decrease in FRA expression (Lerant and Freeman, 1997). The
circadian rhythm is regulated by the suprachiasmatic nucleus (SCN) and
amplified by estrogen for the induction of the PRL surge. There is a decrease in
TIDA activity over the afternoon corresponding to the PRL surge.

To determine if stress or time of day regulated Fos expression in TH-IR
neurons in the VL-ARC, the time course effects of an intraperitoneal saline
injection on Fos expression were examined. Uninjected animals served as
controls in this experiment. Throughout the rostral-caudal extent of VL-ARC,
there was no significant change at any time point after saline as compared with

uninjected vehicle controls (Figure 4.12). There were no changes in the

113

40-

35~

30-

251

201

15-

104

 

% of TH-IR Neurons Expressing Fos

VL-ARC

—o— Rostral
—l:l— Middle
—A— Caudal

 

 

Figure 4.12 Time course effects of saline on the percentage of TH-IR neurons
expressing Fos in the rostral (circles), middle (squares), and caudal (triangles)
regions of the VL-ARC. Rats were injected with 0.9% saline (filled symbols; 1
mllkg; ip) and sacrificed 1, 2, 4 or 8 h later. Zero time controls (open symbols)
received no injection prior to sacrifice. Symbols represent the means and vertical

I I I

1 2 4
Hours After Saline

lines : 1 SEM from 6-8 animals.

114

VL-ARC

 

 

 

 

 

 

 

 

 

 

 

50 —
[:1 No Injection
_ Saline
40 -
2 g i
323
m 30 7
2
E
:i': 20 -
'—
.,_ i
O
11:
10 ~
0 l .
01248 01248 01248
Rostral Middle Caudal

Hours After Saline

Figure 4.13 Time course effects of saline on the numbers of TH-IR neurons in
the rostral, middle, and caudal regions of the VL-ARC. Rats were injected with
0.9% saline (filled columns; 1 mllkg; ip) and sacrificed 1, 2, 4 or 8 h later. Zero
time controls (open columns) received no injection prior to sacrifice. Columns
represent the means and vertical lines + 1 SEM from 6-8 animals.

115

numbers of TH-IR neurons in the VL-ARC at any time point after saline
administration (Figure 4.13). Time of day linear regression analysis revealed r
values of -0.355 (rostral), -0.371 (middle), and -0.316 (caudal) in the VL-ARC.
The r value is the correlation coefficient which represents the measure of the
relationship between two variables. This number varies between -1 and +1. An r
value of -1 indicates there is a perfect negative relationship between the two
variables, with one always decreasing as the other increases. An r value of +1
indicates a perfect positive relationship between variables, with both always
increasing together. When there is no relationship between variables, the rvalue
is 0.

The correlation analysis demonstrated a negative correlation between
time of day and Fos expression in the rostral and middle the VL-ARC, but not the
caudal VL-ARC (Figures 4.14-4.16). Fos expression decreased as the day
progressed. There was a gradual decrease in Fos expression in the middle and
caudal DM-ARC following saline administration such that there was a significant
decrease in the percentage of TH-IR neurons with Fos-IR nuclei at 8 h (Figure
4.17). There were no changes in the numbers of TH-IR neurons in the DM-ARC
at any time point after saline administration (Figure 4.18). Time of day
regression analysis revealed r values of 0.121 (rostral), -0.498 (middle) and -
0.437 (caudal) in the DM-ARC. There was a negative correlation between time
of day and Fos expression in the middle and caudal DM-ARC (Figures 4.20 and
4.21), but not in the rostral DM-ARC (Figure 4.19). Saline had no effect on the

number of TH-IR neurons (Figure 4.22; Inset) or on the percentage of those

116

Rostral VL-ARC

40

 

r = - 0.355
35 — .

 

 

% of TH-IR Neurons Expressing Fos

 

 

O ' I ' I T l ' I ' I ' I ' I ' I

07:00 08:00 09:00 10:00 11:00 12:00 13:00 14:00 15:00

Time of Day

Figure 4.14 Effects of time of day on the percentage of TH-IR neurons
expressing Fos of the rostral VL-ARC. Rats were injected with 0.9% saline (1
mllkg; ip) and controls received no injection. Each circle represents the % of TH-
IR neurons expressing Fos from a single animal. Analysis of the data by linear
regression indicates that a significant decline in Fos expression occurred
(p<0.05).

117

Middle VL-ARC

 

r = - 0.371

N
01
1

.1.
01
1

 

..s

O
L
I

% of TH-IR Neurons Expressing Fos
N
0" o

 

 

 

O ' I ' I I I I I I t r I I

07:00 08:00 09:00 10:00 11:00 12:00 13:00 14:00 15:00
TimeofDay

Figure 4.15 Effects of time of day on the percentage of TH-IR neurons
expressing Fos of the middle VL-ARC. Rats were injected with 0.9% saline (1
mllkg; ip) and controls received no injection. Each sqaure represents the % of
TH-IR neurons expressing Fos from a single animal. Analysis of the data by
linear regression indicates that a significant decline in Fos expression occurred
(p<0.05).

118

Caudal VL-ARC

40

 

r = - 0.316

35— 2

 

% of TH-IR Neurons Expressing Fos

 

 

 

5 -
O s . . , . . . . . . . fl . r .
07:00 09:00 11:00 13:00 15:00

Time of Day

Figure 4.16 Effects of time of day on the percentage of TH-IR neurons
expressing Fos of the caudal VL-ARC. Rats were injected with 0.9% saline (1
mllkg; ip) and controls received no injection. Each triangle represents the % of
TH-IR neurons expressing Fos from a single animal.

119

DM-ARC

 

 

 

 

 

20 —
fl)
0 —O— Rostral
u. .
a, —Cl— Middle
.5 1 —A- Caudal
g 15
93
G
x
I.I.I
U)
c 10 ..
9
3
d)
2
I!
‘1 5 4
I \L\
I- are
U—
s\°

0 l l I I l

Hours After Saline

Figure 4.17 Time course effects of saline on the percentage of TH-IR neurons
expressing Fos in the rostral (circles), middle (squares), and caudal (triangles)
regions of the DM-ARC. Rats were injected with 0.9% saline (filled symbols; 1
mllkg; ip) and sacrificed 1, 2, 4 or 8 h later. Zero time controls (open symbols)
received no injection prior to sacrifice. Symbols represent the means and vertical
lines at 1 SEM from 6-8 animals.

120

DM-ARC

 

 

 

 

 

 

 

 

 

 

601
L E:l No Injection
- Saline
50 4
8 j I
e 40
3
d}
2
m 30 ‘1 El:
:r':
1..
“5 20 -
=I=I=
10 ~
0 1 r 1
01248 01248 01248
Rostral Middle Caudal

Hours After Saline

Figure 4.18 Time course effects of saline on the numbers of TH-IR neurons in
the rostral, middle, and caudal regions of the DM-ARC. Rats were injected with
0.9% saline (filled columns; 1 mllkg; ip) and sacrificed 1, 2, 4 or 8 h later. Zero
time controls (open columns) received no injection prior to sacrifice. Columns
represent the means and vertical lines + 1 SEM from 6—8 animals.

121

Rostral DM-ARC

 

 

 

 

 

 

14
8 r=0.121
"-124
a)
.5
$101
2
‘x‘ 84
“J o

o

5 .0 o . . .OOI
2 4‘ . . . I . 0
g 2« " e o 0 o .
E . . oo o
It- . 0
0 0'1 . C C .
e\°

 

07:00 08:00 09:00 10:00 11:00 12:00 13:00 14:00 15:00
TimeofDay

Figure 4.19 Effects of time of day on the percentage of TH-IR neurons
expressing Fos of the rostral DM-ARC. Rats were injected with 0.9% saline (1
mllkg; ip) and controls received no injection. Each circle represents the % of TH-
lR neurons expressing Fos from a single animal.

122

Middle DM-ARC

_L
A

 

r = - 0.498

 

% of TH-IR Neurons Expressing Fos

 

 

 

07:00 09:00 1 1 :00 13:00 15:00
Time of Day

Figure 4.20 Effects of time of day on the percentage of TH-IR neurons
expressing Fos of the middle DM-ARC. Rats were injected with 0.9% saline (1
mllkg; ip) and controls received no injection. Each square represents the % of
TH-IR neurons expressing Fos from a single animal. Analysis of the data by
linear regression indicates that a significant decline in Fos expression occurred
(p<0.05).

123

Caudal DM-ARC

 

.3
A

A n r = - 0.437

.3

N

L
D

..x
O
4

 

 

 

% of TH-IR Neurons Expressing Fos

 

T I T Y I I T I l

07:00 09:00 1 1 :00 13:00 15:00
Time of Day

Figure 4.21 Effects of time of day on the percentage of TH-IR neurons
expressing Fos of the caudal DM-ARC. Rats were injected with 0.9% saline (1
mllkg; ip) and controls received no injection. Each triangle represents the % of
TH-IR neurons expressing Fos from a single animal. Analysis of the data by
linear regression indicates that a significant decline in Fos expression occurred
(p<0.05).

124

MZI

10 w
—<>— No Injection 200
+ Saline

150

l
100 l ,
6 - l I
50 ‘ ‘
j . l
4 — 0 I E ‘ ..-1 . .2
0 1 2 4 8

Hours After Saline

ft of TH-IR Neurons

    

% of TH-IR Neurons Expressing Fos

 

 

Hours After Saline

Figure 4.22 Time course effects of saline on the numbers of TH-IR neurons
(Inset) and the percentage of these neurons expressing Fos in the MZI. Rats
were injected with 0.9% saline (filled symbols and columns; 1 mllkg; ip) and
sacrificed 1, 2, 4 or 8 h later. Zero time controls (open symbols and columns)
received no injection prior to sacrifice. Symbols and columns represent the
means and vertical lines 1' 1 SEM from 6-8 animals.

125

MZI

 

 

 

 

 

5
8 r=0.170
LL
.8 4*
II)
8
'a 3 2
X
11.1
In
8 2 ‘ e .
5 0 o
m o o . o . 2 . o
z 1 J u e
“-3 0’ ”
:1: o
t 0 - 9 ° e e o o
o
e\°
07:00 09:00 1 1 :00 13:00 15:00
Time of Day

Figure 4.23 Effects of time of day on the percentage of TH-IR neurons
expressing Fos of the MZI. Rats were injected with 0.9% saline (1 mllkg; ip) and
controls received no injection. Each diamond represents the % of TH-IR neurons
expressing Fos from a single animal.

126

neurons expressing Fos in the MZI (Figure 4.22). The r value from the time of
day regression analysis in the MZI was 0.170. There was no correlation between
Fos expression and time of day (Figure 4.23).

Fos protein dimerizes with a Jun protein before binding to the AP-1 site. A
common combination of heterodimers is Fos and c-Jun, so if there is an increase
in Fos, there could also be an increase in c-Jun. To determine if this is the case,
the effects of quinelorane on c—Jun expression in the ARC were examined.
Figure 4.24 illustrates the immunohistochemical labeling of c-Jun-IR nuclei and
TH-IR neurons in the ARC. This photomicrograph demonstrates the extremely
low numbers of co-Iocalized c—Jun-IR nuclei in TH-IR neurons in the middle ARC
2 h after quinelorane administration. While c-Jun-IR was virtually lacking in the
TH-IR neurons, it was present in areas of the VL-ARC devoid of TH-IR neurons.
Figure 4.25 illustrates the time course effects of quinelorane on the percentage
of TH-IR neurons expressing c-Jun in the DM-ARC. The percentage of TH-IR
neurons containing c-Jun-IR nuclei in the DM-ARC was less than 2% and
remain ed unchanged throughout the time course. There were no changes in the
numbe rs of TH-IR neurons in the DM-ARC (Figure 4.25; Inset). In the VL-ARC,
quinelorane had no effect on the percentage of TH-IR neurons containing c—Jun-
IR nuclei at any timepoint (Figure 4.26). The percentage of co-localization
remained low as seen in the DM-ARC, below 3%. There were no changes in the
numbers of TH-IR neurons in the VL-ARC (Figure 4.26; Inset). Quinelorane had
no effect on the number of TH-IR neurons (Figure 4.27; Inset) or the percentage

of those neurons expressing c—Jun (Figure 4.27) in the MZI.

127

 

Figure 4.24 Medium power (200x) computer-captured image demonstrating c-
Jun-IR nuclei (blue arrows), TH-IR neurons (red arrows) and colocalization of
Jun—IR nuclei in TH-IR neurons (yellow arrows) of the middle DM—ARC following
administration of quinelorane. Rats were injected with quinelorane (100 jig/kg;
ip) and sacrificed 2 h later.

128

DM-ARC

 

 

 

: 1O ‘ - 60
a —0— Vehicle
0 + Quinelorane g 50 , ,
O V l
.E’ 8 I 4o
2 .
a n_: 30 1
2 :t ,
Q 6 ‘ I- 20 1 ‘
0’3 '5
3k
in 10 .
s , ,
4 _ ._ . ,
5 o 1 2 4 3
g Hours After Quinelorane
°—.‘ 2 ~
I
I‘-
h
0
.\° 0 . . . j
0 1 2 4 8

Hours After Quinelorane

Figure 4.25 Time course effects of quinelorane on the numbers of TH-IR
neurons (Inset) and the percentage of those neurons expressing c-Jun in the
middle region of the DM-ARC. Rats were injected with quinelorane (filled
symbols and columns; 100 jig/kg; ip) and sacrificed 1, 2, 4 or 8 h later. Zero time
controls (open symbols and columns) were injected with 0.9% saline vehicle (1
mllkg; ip) and sacrificed 2 h later. Symbols represent the means and vertical
lines :I: 1 SEM from 6-8 animals.

129

VL-ARC

       

 

 

C 10 ‘ 50 1,

g —0— Vehicle

6 + Quinelorane m 40 j

a) 8 J 5 .

r: 5 ' l

'5 9’ 30 i . I

tn 2 l « .

2 °-.= *

Q 6 _ I 20 ‘ ‘

x ':

I.I.I O ‘

a, it 10 l l

c ‘ .

g 0 1 2 4 8

Hours After Quinelorane

I 2 _

T

I

I-

l.—

O

°\o 0
l l l I fil
0 1 2 4 8

Hours After Quinelorane

Figure 4.26 Time course effects of quinelorane on the numbers of TH-IR
neurons (Inset) and the percentage of those neurons expressing c-Jun in the
middle region of the VL-ARC. Rats were injected with quinelorane (filled symbols
and columns; 100 119/kg; ip) and sacrificed 1, 2, 4 or 8 h later. Zero times
controls (open symbols and columns) were injected with 0.9% saline vehicle (1
mllkg; ip) and sacrificed 2 h later. Symbols and columns represent the means
and vertical lines 3: 1 SEM from 6-8 animals.

130

MZI

—o— Quinelorane

I L
150 l j l ‘ ‘ ‘
l l
100 1
so l
4" J
o L s
o 1 2 4 8

Hours After Quinelorane

0)
# of TH-IR Neurons

    

 

% of TH-IR Neurons Expressing c-Jun

 

Hours After Quinelorane

Figure 4.27 Time course effects of quinelorane on the numbers of TH-IR
neurons (Inset) and the percentage of those neurons expressing c-Jun in the
MZI. Rats were injected with quinelorane (filled symbols and columns; 100
uglkg; ip) and sacrificed 1, 2, 4, or 8 h later. Zero time controls (open symbols
and columns) were injected with 0.9% saline vehicle (1 mllkg; ip) and sacrificed 2
h later. Symbols and columns represent the means and vertical lines 1: 1 SEM
from 6—8 animals.

131

Discussion

Quinelorane is a DZ receptor agonist that acutely decreases neuronal
activity in the DA neurons of the mesotelencephalic system via action on
autoreceptors and long loop neuronal feedback circuits. Interestingly, this
agonist has the opposite action on TIDA neurons by increasing their activity via a
mechanism that is independent of PRL (Eaton et al., 1993; Durham et al., 1996).
The previous chapter demonstrated that the expression of FRA in the DM-ARC
correlates with neuronal activity of TIDA nerve terminals in the ME. The aim of
the present study was to determine if DZ receptors regulate Fos and c-Jun
expression in TIDA neurons located in subdivisions of the rostral, middle, and
caudal ARC.

The results from these studies reveal that activation of DZ receptors
increases Fos expression in TIDA neurons throughout the rostral-caudal extent
of the DM-ARC in a time dependent manner in a fixed population of TH-IR
neurons. This increase in Fos is delayed and transitory (peaking at 2 h)
compared to the rapid (occurring at 30 min) and prolonged effects (persisting for
at least 8 h) of quinelorane on neuronal activity as estimated from neurochemical
indices (Eaton et al., 1993). This delay of expression in the DM-ARC is
characteristic of the induction of Fos and other immediate early genes. Fos
protein has a half life of 90 to 120 min, with maximum levels appearing between
1 and 3 h, gradually disappearing from the nucleus by 4 to 6 h (Sonnenberg et
al., 1989; Chan et al., 1993; Kovacs and Sawchenko, 1996). This time course of

F as expression is similar to that seen following PRL stimulation of TIDA neurons.

132

Exogenous PRL as well as haloperidol administration increases F RA expression
in the DM-ARC with a peak response at 3 h (Hentschel et al., 2000).

Raclopride is able to block the stimulatory effect of quinelorane on Fos
expression indicating that the actions of quinelorane are mediated through DZ
receptors. These results correlate with the findings from neuronal activity studies
demonstrating that DZ agonists induce an acute stimulatory response in TIDA
neurons and this can be reversed with a DZ receptor antagonist (Eaton et al.,
1993; Durham et al., 1997). The lack of effect of DZ receptor blockade on Fos
expression in the DM-ARC suggests that DZ receptors do not tonically inhibit Fos
expression in these neurons. If TIDA neurons were indeed regulated by
presynaptic autoreceptors, one would expect a DZ receptor antagonist to have a
stimulatory affect on Fos expression and a DZ agonist to be able to reverse the
affects as has been demonstrated in the striatum (Merchant et al., 1994; Rogue
and Vincendon, 1992).

The lHDA neurons of the MZI are regulated by DA receptors as are the
DA neurons of the mesotelencephalic systems (Lookingland and Moore, 1984).
DA agonists and locally applied DA decrease, while DA antagonists increase the
activity of lHDA neurons (Lookingland and Moore, 1995; Eaton et al., 1992). If
Fos correlates with neuronal activity, then blockade of DZ receptors on lHDA
neurons should increase Fos expression. These studies demonstrate that this
does occur. While quinelorane has no effect on Fos expression, it reverses the
stimulatory effect of raclopride. These results demonstrate that Fos expression

is regulated through DZ receptors. This is consistent with in vitm studies

133

performed in cultured TH-lR hypothalamic neurons demonstrating that DZ
agonists can reverse stimulated Fos expression (Sim et al., 1994). These
receptors are most likely located on lHDA cell bodies and/or dendrites as
opposed to axon terminals. Antidromic stimulation of hypothalamic neurons does
not induce immediate early gene expression for it bypasses the second
messenger system necessary to induce them (lcard-Liepkalns et al., 1992;
Luckman et al., 1994; Hoffman and Murphy, 2000). Thus, since Fos can be
induced through DZ receptors, the receptors must be somatodentritic.

In the VL-ARC, the percentage of Fos levels in TH-IR neurons in the
control group is higher than the DM-ARC (20% vs 5% respectively) suggesting
that greater numbers of these neurons are tonically active. While quinelorane
had no stimulatory effect on Fos expression in the VL-ARC at 2 h as in the DM-
ARC, there was a decrease after 4 h. These results in the VL-ARC from the
raclopride study demonstrated no effect at 2 h at a time when DM-ARC TH-IR
neurons are activated suggesting that these neurons are not regulated through
DZ receptors. This is in contrast to the specific effects of PRL on the TH-lR
neurons of the VL-ARC.

PRL increases FRA levels in the VL-ARC in a transient pattern at 3 h
(Hentschel et al., 2000) similar to that seen in the DM-ARC following quinelorane
administration. The VL-ARC contains PRL receptors (Bakowska and Morrell,
1997) and thus Fos (and perhaps long term gene) expression may be regulated
by PRL as opposed to DA receptors. However, unlike the neurons of the DM-

ARC, those of the VL-ARC do not produce DD, thus only DOPA is synthesized

134

and released (Komori et al., 1991; Meister et al., 1988; Balan et al., 2000). Their
function has not been completely elucidated, but evidence indicates that DOPA
may be released and taken up and converted to DA in the ME or at the anterior
pituitary (Meister et al., 1988; Misu et al., 1996). DD has been demonstrated in
the walls of blood vessels, so it is possible that DOPA released into the portal
blood system is converted to DA and can act at the anterior pituitary (Meister et
al., 1988). These “DOPA-ergic” neurons co-express TH and neuropeptides
(Chronwall 1985; Ershov et al., 2002) and DOPA may contribute to the regulation
of peptide turnover (Everitt et al., 1996).

The decrease in Fos expression in the VL-ARC may be due to other
factors such as stress (Lookingland et al., 1990; Campeau et al 1991; Persico et
al., 1993) or circadian rhythms (Lerant and Freeman, 1997; Yang et al., 1999).
Restraint stress has been demonstrated to decrease TIDA neuronal activity in
female rats (Lookingland et al 1990) while handling of rats for the first time
(Campeau et al., 1991) or acute intraperitoneal injection of saline results in a
strong induction of Fos and FRA in the hypothalamus after 2 h (Ryabinin et al.,
1999). There was no effect on Fos expression in the VL-ARC at any timepoint
following the injection of saline suggesting that stress of injection or handling has
no effect on Fos expression in TH-IR “DOPA-ergic” neurons in the VL-ARC.

A time of day analysis was also carried out to determine if there is a
circadian rhythm influence on the expression of Fos in the ARC. There is
evidence of circadian rhythm in TIDA neuronal activity in female rats. An

endogenous rhythm exists in all adult female rats and is estrogen dependent.

135

 

There is a decrease in TIDA neuronal activity (Mai et al., 1994) as well as in FRA
expression (Lerant and Freeman, 1997) in the late afternoon of proestrus which
corresponds with an afternoon PRL surge. There are no similar studies
examining activity of TIDA neurons in males. Interestingly, time of day analysis
revealed a correlation with the percent of Fos expression and the time the rats
were sacrificed in the middle and caudal portions of the DM-ARC as well as the
rostral and middle portions of the VL-ARC. As the day progresses, Fos
expression decreases in TIDA neurons, hinting at a circadian rhythm; however, a
24 h time course would need to be performed to make any further conclusions.
This would be unique for male rats for previous studies have not demonstrated a
circadian rhythm in the ARC (Pan, 1996).

In addition to Fos, Jun proteins can be induced following specific
stimulation (Herdegen and Leah, 1998). Jun proteins are a family of immediate
early genes consisting of c-Jun, JunB and JunD. Studying Jun proteins in
conjunction with Fos appears to be a logical choice, for these two immediate
early genes form heterodimers and bind to the AP-1 site on the TH gene
promoter. Fos can regulate the transcriptional activity and increase
transactivating potential of c-Jun. It has a similar half-life to c—Fos (90-1ZO min).
c-jun mRNA and Jun proteins have been detected throughout the brain (Rogue
and Vincendon, 1992; Hughes and Dragunow, 1995). In the hypothalamus, c-
Jun—IR nuclei have been located in LHRH and magnocellular neurons and are
co-expressed with c—Fos following such stimuli as proestrous LH surge and

hypertonic saline (Hoffman et al., 1993).

136

 

Interestingly, however, the activation of DZ receptors did not result in an
increase of c-Jun at any time point. The first possibility is that c-Jun is not the
Jun protein that is co-expressed with Fos in TIDA neurons following DZ receptor
activation. Indeed, c-Jun—lR nuclei were identified in the ARC, but the nuclei
were located mostly in the parvocellular region of the ARC and in the medial
portion of the VL-ARC where TH-lR neurons are the fewest, if not absent. Fos
may form a heterodimer with JunB in TIDA neurons. Though thought to be less
stable than c-Jun, JunB can be rapidly induced and has a half-life similar to c-
Jun. Another possible explanation is that not all stimuli have the same effect on
Fos and Jun family members. c-Jun is induced by serum, phorbol ester (which
stimulates the protein kinase C pathway), growth factors and cytokines (such as
platelet derived growth factor, epithelial growth factor, nerve growth factor,
fibroblast growth factor), but not by increases of protein kinase A via membrane
depolarization or stimulation of nicotinic receptors in PC12 cells. All of these
stimuli, however, increase c—Fos and JunB expression (Hughes and Dragunow,
1995; Herdegen 2000). DA receptors are linked to CAMP and thus to protein
kinase A. Keeping this in mind, it is conceivable that c—Jun would not be induced
following quinelorane administration, and the Jun protein that may dimerize with
Fos could be JunB.

Taken together the results presented in this chapter reveal that DZ
receptors regulate Fos, but not c-Jun, expression in TIDA neurons throughout the
rostral-caudal extent of the DM-ARC. These results correspond with the

neurochemical studies examining the nerve terminal activity of the ME following

137

DZ receptor activation. Unlike the DM-ARC, however, Fos expression in TH-IR

neurons in the VL-ARC is not regulated by DZ receptors.

138

CHAPTER FIVE
THE ROLE OF KAPPA OPIOID RECEPTORS IN D2 RECEPTOR
REGULATION OF FOS EXPRESSION IN TH-IR NEURONS IN SUBDIVISIONS
OF THE ARCUATE NUCLEUS
Introduction

As described in Chapter Four, immediate early gene expression in TIDA
neurons can be modulated by 3 DZ receptor-mediated mechanism. This is
evidenced by the finding that activation of D2 receptors increases the expression

of Fos protein in the cell bodies of TIDA neurons in the DM-ARC. It is unlikely

that this effect is due to a direct action on TIDA neurons since DZ receptors are

inhibitory, leading to decreases in CAMP and Ca+2 conductance as well as

increases in K+ conductance within target neurons (Vallone et al., 2000). A

stimulatory response of TIDA neurons following activation of OZ receptors is not
consistent with the response of neurons regulated by inhibitory autoreceptors
located on DA terminals, cell bodies and dendrites.

Instead, DZ receptor-induced stimulation of TIDA neuronal activity is
mediated through a neuronal system originating outside the mediobasal
hypothalamus (Durham et al., 1996). This was demonstrated through studies
involving mediobasal hypothalamic deafferentation surgeries. Those animals
receiving the deafferentation surgery failed to demonstrate an increase in TIDA
neuronal activity following the activation of DZ receptors, indicating that the DZ
receptors must be located outside the mediobasal hypothalamus. There were
two possibilities to explain this; 1) activation of a quiescent stimulatory neuronal

system, or Z) suppression of a tonically active inhibitory neuronal system. Due to

139

their inhibitory effects on target cells, it is more likely that DZ receptors would
“turn off” an already active system as opposed to triggering a stimulatory system
to “turn on”.

Dynorphin is an endogenous opioid neuropeptide highly selective for
kappa opioid receptors (Chavkin et al., 1982) which exerts an inhibitory action on
hypothalamic neurons (MacMiIlin and Clark, 1983; Lin and Pan, 1995). Several
lines of pharmacological evidence suggest that the stimulatory effects of DZ
receptors are mediated through the inhibition of tonically active dynorphin
neurons that inhibit TIDA neurons via kappa opioid receptors. Blockade of kappa
receptors with the antagonist nor-BNI, or immunoneutralization of endogenous
dynorphin, both increase TIDA neuronal activity like the DZ agonist quinelorane
(Manzanares et al., 19923; Manzanares et al., 1992b; Durham et al., 1996).
When co—administered, nor-BNI and quinelorane have no additive effect,
suggesting they share a common pathway (Durham et al., 1996). In agreement,
U50-488, a kappa receptor agonist, reverses the stimulatory effect of quinelorane
on TIDA neurons (Durham et al., 1996). Taken together, these studies imply that
tonically active inhibitory dynorphin neurons participate in DA receptor regulation
of TIDA neurons.

While these studies do not prove that these dynorphin neurons are located
within the mediobasal hypothalamus, microscopic analysis and immunoassay
studies lead one to believe that this is the case. Dynorphin-IR and mRNA are
located in the ARC and outside the mediobasal hypothalamus in the

ventromedial hypothalamic nucleus (Zamir et al., 1983; Zamir et al., 1984;

140

Durham, 1999). When DZ receptors are stimulated with quinelorane, expression
of dynorphin mRNA in the ARC is decreased, whereas the mRNA in the
ventromedial hypothalamic nucleus is not (Durham, 1999). The downstream
effect of this would be a decrease in synthesis and possibly release of dynorphin,
which is consistent with the view that dynorphin neurons located in the ARC are
selectively inhibited by DZ receptors via an unknown upstream neuron. Kappa
receptor mRNA as well as binding sites are located in the ARC, indicating that
not only do neurons produce the receptor mRNA, but they also express these
receptors (Mansour et al., 1988; Mansour et al., 1995). Though these studies did
not chemically identify the neurons with kappa receptors, other studies examined
TIDA neurons specifically. Dynorphin terminals interact with and are
concentrated around TIDA neurons, and electron microscopy has revealed
synaptic vesicles and postsynaptic thickening at these terminals (Fitzsimmons et
al., 1992). Figure 5.1 illustrates this proposed neuronal pathway under basal

conditions.

141

  
    
   

3rd
Ventricle

Figure 5.1 Frontal section schematic depicting the proposed neuronal pathway
that mediates the stimulatory effects of D2 receptors on TIDA neuronal activity.
Under basal conditions, a chemically unidentified neuron (depicted in green)
located outside the mediobasal hypothalamus tonically stimulates dynorphin
neurons located in the ARC. Dynorphin neurons in turn release dynorphin that
acts at the kappa opioid receptor located on TIDA neurons to tonically inhibit DA
release. Abbreviations: ?, unknown neuron/neurotransmitterlreceptor; ARC,
arcuate nucleus; DA, dopamine; DYN, dynorphin; ME, median eminence.

142

While there are several studies on the role of dynorphin and kappa opioid
receptors in regulating TIDA neurochemical activity associated with DA release,
no information is available regarding kappa opioid receptor regulation of gene
expression in TIDA neurons. The aims of the studies described in this chapter
are two fold. The first aim was to determine if kappa opioid receptors regulate
Fos expression in TIDA neurons. Secondly, if this is the case, then what role do
they play in D2 receptor regulation of Fos expression in TIDA neurons. The
working hypothesis of these studies is that kappa receptors tonically inhibit Fos
expression in TIDA neurons and that activation of DZ receptors inhibits dynorphin
neurons and stimulates Fos expression in TIDA neurons. For comparison, the
effects on Fos expression in the “DOPA-ergic” neurons in the VL-ARC and the

lHDA neurons of the MZI were also studied.

Materials and Methods

Gonadally intact male rats were used in these experiments. Care and
handling of the rats was carried out as described in the Materials and Methods
in Chapter 2. Following appropriate treatments, rats were anesthetized with
Equithesin and perfused with 4% paraformaldehyde. Brains were removed and
sectioned as described in Materials and Methods in Chapter 2. The
identification of TH-IR neurons using alkaline phosphatase and Fos-IR using
nickel-intensified diaminobenzidine in the ARC and MZI was carried out using
immunohistochemical procedures in Materials and Methods in Chapter 2. The

ARC sections were sorted into rostral, middle and caudal regions and then

143

visually partitioned into DM—ARC and VL-ARC. Fos was localized with anti-Fos
antiserum generated against residues 4 to 17 of human fos (Oncogene). The
dilution used was 1:10,000 in TBS” X containing 1.5% normal donkey serum.
TH was detected using anti-TH serum (Chemicon) generated against rat adrenal
medullary tumor tissue. The final dilution was 120,000 in TBS/T X containing

1.5% normal goat serum.

Results

Photomicrographs in Figure 5.2 illustrate Fos expression in TH-IR
neurons of middle DM-ARC 2 h after injection of either vehicle (Top Panel) or
nor-BNI (Bottom Panel) to male rats. These images show an increase in
colocalization of Fos-IR nuclei in TH-IR neurons following nor-BNI administration.
Figure 5.3 illustrates the time course effects of nor-BNI in the percentage of TH-
IR neurons expressing Fos-IR nuclei in the DM-ARC. Nor-BNI significantly
increased Fos expression in a time dependent manner reaching maximum levels
at 2 h and returning to basal levels by 8 h in the rostral, middle, and caudal
regions of the DM-ARC. There was a decrease in the numbers of TH-IR neurons
throughout the rostrocaudal extent of the DM-ARC; however, there were no
changes in the numbers of neurons within each region of the DM-ARC at any
time point following nor-BNI (Figure 5.4). In contrast, in the VL—ARC nor-BNI
had no effect on the percentage of TH-IR neurons containing Fos-IR nuclei
(Figure 5.5). There was an increasing gradient in the numbers of TH-IR neurons

in vehicle-treated rats that extended caudally in the VL-ARC. As in the DM-ARC,

144

 

 

Figure 5.2 High power (400x) computer-captured images demonstrating
increased Fos expression in TH-IR neurons of the DM-ARC 2 h following
administration of nor-BNI. Rats were injected with either vehicle (Top Panel;
ddeo; 3 til/rat; icv) or nor-BNI (Bottom Panel; 12.5 jig/rat; icv) and sacrificed 2
h later. Arrows represent colocalization of pink TH-IR neurons containing black
Fos-IR nuclei. 3V, third ventricle.

145

DM-ARC

(D
O
1

—o— Rostral

—I:I— Middle
/\\\ + Caudaj

M N
O 0'1
L 4

% of TH-IR Neurons Expressing Fos
a

 

 

10 4
5 J ‘
O I I l 1 fl
0 1 2 4 8
Hours After Nor-BNI

Figure 5.3 Time course effects of nor-BNI on the percentage of TH-IR neurons
expressing Fos in the rostral (circles), middle (squares), and caudal (triangles)
regions of the DM-ARC. Rats were injected with nor-BNI (filled symbols; 12.5
jig/rat; icv) and sacrificed 1, 2, 4, or 8 h later. Zero time controls (open symbols)
were injected with dngO vehicle (3 ul/rat; icv) and sacrificed 2 h later. Symbols
represent the means and vertical lines + 1 SEM from 6-8 animals. *, Values for
nor-BNI-treated rats that are significantly different (p<0.05) from vehicle-treated
controls.

146

70-
so. 9
50—
40-

30-

# of TH-IR Neurons

204

10-

 

 

 

0 .

 

0

Figure 5.4 Time course effects of nor-BNI on the numbers of TH-IR neurons in
the rostral, middle, and caudal regions of the DM-ARC. Rats were injected with
nor-BNI (filled columns; 12.5 uglrat; icv) and sacrificed 1, Z, 4, or 8 h later. Zero
time controls (open columns) were injected with ddH20 vehicle (3 ullrat; icv) and
sacrificed 2 h later. Columns represent the means and vertical lines + 1 SEM
from 6-8 animals.

1

 

2 4 8
Rostral

DM-ARC

 

 

 

 

 

 

o 1 2 4 a
Middle
Hours After Nor-BNI

147

l

0

l:l Vehicle
- Nor-BNI

1 2 4 8
Caudal

VL-ARC

 

 

 

25
8 l —-O— Rostral
"é, —o— Middle
.5 20 - —A— Caudal
t0
tn
2
it
I“ 15 " 3
2 i
e 2
3
d) 10 1
z
E —
:i': -
,_ 5
q—
o
°\°
0 I l fl j l
0 1 2 4 8
Hours After Nor-BNI

Figure 5.5 Time course effects of nor-BNI on the percentage of TH-IR neurons
expressing Fos in the rostral (circles), middle (squares), and caudal (triangles)
regions of the VL-ARC. Rats were injected with nor-BNI (filled symbols; 12.5
uglrat; icv) and sacrificed 1, Z, 4, or 8 h later. Zero time controls (open symbols)
were injected with ddH20 vehicle (3 ul/rat; icv) and sacrificed 2 h later. Symbols
represent the means and vertical lines + 1 SEM from 6-8 animals.

148

there were no changes in the numbers of TH-IR neurons at any time point within
each region of the VL-ARC following nor-BNI administration (Figure 5.6). In the
MZI, nor-BNI had no effect on the numbers of TH-IR neurons (Figure 5.7;
Bottom Panel) or the percentage of those containing Fos-IR nuclei (Figure 5.7;
Top Panel).

To determine if the effects of nor-BNI on Fos expression were mediated
through kappa opioid receptors, a study was carried out using the kappa receptor
agonist USO-488. Activation of kappa opioid receptors had no effect per se, but
reversed the stimulatory effects of nor-BNI on Fos-IR nuclei throughout the
rostrocaudal extent of the DM-ARC (Figure 5.8). There were no changes in the
numbers of TH-IR neurons following USO-488 or nor-BNI administration (Figure
5.9). In the VL-ARC, U50-488 significantly decreased the percentage of TH-IR
neurons containing Fos-IR nuclei and this effect was blocked by nor-BNI (Figure
5.10). Nor-BNI alone had no effect on the percentage of the TH-IR neurons
containing Fos-IR nuclei in the VL-ARC. There were no changes in the numbers
of TH-IR neurons in the VL-ARC following administration of either U50—488 or
nor-BNI (Figure 5.11). In the MZI, neither nor-BNI nor USO-488 had any effect
on the numbers of TH-lR neurons (Figure 5.12; Bottom Panel) or the

percentage of those neurons containing Fos-IR nuclei (Figure 5.12; Top Panel).

149

VL-ARC

 

 

 

 

 

 

 

 

 

 

 

50 -
[:1 Vehicle
- Nor-BNI I
40 -1
(I)
= I
§
w 30 _
2
E
:r':
I" 20 ‘ F321
“5
=11:
10 -
0 j I
0 1 2 4 8 0 1 Z 4 8 0
Rostral Middle
Hours After Nor-BNI

Figure 5.6 Time course effects of nor-BNI on the numbers of TH-IR neurons in
the rostral, middle, and caudal regions of the VL-ARC. Rats were injected with
nor-BNI (filled columns; 12.5 jig/rat; icv) and sacrificed 1, 2, 4, or 8 h later. Zero
time controls (open columns) were injected with ddeo vehicle (3 ullrat; icv) and
sacrificed 2 h later. Columns represent the means and vertical lines + 1 SEM

from 6-8 animals.

150

1 2 4 8
Caudal

MZI

 

 

 

8 10
u. 1 —<>— Vehicle
2’ + Nor-BNI
._ 8 l
m
in
2
o.
a: 6 —
in
c
2
a 4;
z
E
2': 2 1
j.
“6
°\° O ‘17 r r I '
0 1 2 4 8
Hours After Nor-BNI

200 -
in 150 —
c:
o
h
z
o
2
n: 100 —
:E
I-
M-
o
=tt: 50 4

0 «F—fiw

 

 

 

0 1 2 4 8
Hours After Nor-BNI

Figure 5.7 Time course effects of nor-BNI on the numbers of TH-IR neurons
(Bottom Panel) and the percentage of these neurons expressing Fos (Top
Panel) in the MZI. Rats were injected with nor-BNI (filled symbols and columns;
12.5 uglrat; icv) and sacrificed 1, 2, 4, or 8 h later. Zero time controls (open
symbols and columns) were injected with ddHZO vehicle (3 ul/rat; icv) and
sacrificed 2 h later. Symbols and columns represent the means and vertical lines
1 1 SEM from 6—8 animals.

151

DM-ARC

l::l Vehicle
- Nor-BNI
* *

N (A)
01 O
1 1

x-

N
O
1

ES
i—l
H
H
H

% of TH-IR neurons expressing Fos
01 if:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

O

I I I

Vehicle USO-488 Vehicle U50-488 Vehicle USO-488
Rostral Middle Caudal

Figure 5.8 Effects of nor-BNI on the percentage of TH-IR neurons expressing
Fos in the rostral, middle and caudal regions of the DM-ARC of vehicle— or
U50488-treated rats. Rats were injected with either U50488 (10 mglkg; so) or its
ddH20 vehicle (1 mllkg; sc) 2 h prior to sacrifice, and with either nor-BNI (12.5
uglrat; icv) or its ddH20 vehicle (3 ullrat; icv) 2 h prior to sacrifice. Columns
represent the means and vertical lines + 1 SEM from 6—8 vehicle- (open
columns), and nor-BNI- (solid columns) treated rats. *, Values that are
significantly different (p < 0.05) from vehicle-treated rats.

152

DM-ARC

80 1
[:3 Vehicle
70 4 - Nor-BNI

60-
_'L‘

50-

404

30‘ .:L1
204 a

10‘

# of TH-IR Neurons
l—l
J—1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

O l I I I I I
Vehicle USO-488 Vehicle USO-488 Vehicle USO-488

Rostral Middle Caudal

Figure 5.9 Effects of nor-BNI on the numbers of TH-IR neurons in the rostral,
middle and caudal regions of the DM-ARC of vehicle- or U50488-treated rats.
Rats were injected with either USO-488 (10 mglkg; so) or its ddH20 vehicle (1
mllkg; sc) 2 h prior to sacrifice, and with either nor-BNI (12.5 uglrat; icv) or its
ddH20 vehicle (3 ullrat; icv) 2 h prior to sacrifice. Columns represent the means
and vertical lines + 1 SEM from 6-8 vehicle- (open columns), and nor-BNI- (solid
columns) treated rats.

153

VL-ARC

0)
O
1

l:l Vehicle
- Nor-BNI

1 i l

N
01
u

N
Q
g

A
O
l

x.

4l—4
J—l

% of TH-IR neurons expressing Fos
a

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

O 1 r
Vehicle USO-488 Vehicle U50-488 Vehicle USO-488

Rostral Middle Caudal

Figure 5.10 Effects of nor-BNI on the percentage of TH-IR neurons expressing
Fos in the rostral, middle and caudal regions of the VL-ARC of vehicle- or
U50488-treated rats. Rats were injected with either U50488 (10 mglkg; sc) or its
ddH20 vehicle (1 mllkg; sc) 2 h prior to sacrifice, and with either nor-BNI (12.5
uglrat; icv) or its ddH20 vehicle (3 ullrat; icv) 2 h prior to sacrifice. Columns
represent the means and vertical lines + 1 SEM from 6-8 vehicle- (open
columns), and nor-BNI- (solid columns) treated rats. *, Values that are
significantly different (p < 0.05) from vehicle-treated rats.

154

VL-ARC

40 1
I:l Vehicle .
35 J - Nor-BNI l

30‘ l l

25~

204 1‘ l

15J

# of TH-IR neurons

104

51

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0 I I l
Vehicle USO-488 Vehicle U50-488 Vehicle U50488
Rostral Middle Caudal

 

I I I

Figure 5.11 Effects of nor-BNI on the numbers of TH-IR neurons in the rostral,
middle and caudal regions of the VL-ARC of vehicle- or U50488-treated rats.
Rats were injected with either U50488 (10 mglkg; so) or its ddH20 vehicle (1
mllkg; sc) 2 h prior to sacrifice, and with either nor-BNI (12.5 jig/rat; icv) or its
ddH20 vehicle (3 ullrat; icv) 2 h prior to sacrifice. Columns represent the means
and vertical lines + 1 SEM from 6-8 vehicle- (open columns), and nor-BNI- (solid
columns) treated rats.

155

MZI

1O -
[:1 Vehicle
— Nor-BNI
8 .l
6 -

 

 

  

 

% of TH-IR Neurons Expressing Fos

 

 

 

 

 

 

 

Vehicle U50488

200 1

175 1

 

150 i

 

125i
100 1

751

# of TH-IR Neurons

50-

 

25i

 

 

 

 

 

 

 

Vehicle U50488

Figure 5.12 Effects of nor-BNI on the numbers of TH-IR neurons (Bottom
Panel) and the percentage of these neurons expressing Fos (Top Panel) in the
MZI of vehicle- or U50488-treated rats. Rats were injected with either U50488
(10 mglkg; sc) or it ddHZO vehicle (1 mllkg; sc) 2 h prior to sacrifice, and with
either nor-BNI (12.5 jig/rat; icv) or its ddH20 vehicle (3 ullrat; icv) 2 h prior to
sacrifice. Columns represent the means and vertical lines + 1 SEM from 6-8
vehicle- (open columns) and nor-BNI- (solid columns) treated rats.

156

If quinelorane blocks the inhibitory effects of dynorphin at kappa receptors
thereby increasing Fos expression in TIDA neurons, then the addition of a kappa
receptor agonist should restore activity at kappa receptors and block the effects
of quinelorane on Fos expression. U50-488 had no effect per se, but was able to
reverse the stimulatory effects of quinelorane on the percentage of TH-IR
neurons containing Fos-IR nuclei in the DM-ARC (Figure 5.13). There were no
changes in the numbers of neurons after quinelorane or USO-488 (Figure 5.14).
As shown in Figure 5.15, quinelorane significantly decreased the percentage of
Fos-IR nuclei in TH-IR neurons throughout the rostrocaudal extent of the VL-
ARC at an earlier time point than in Chapter 4. USO-488 alone also decreased
the Fos expression, but there was no additive effect of quinelorane and USO-488.
Neither quinelorane nor USO-488 had an effect on the numbers of TH-IR neurons
within each region of the VL-ARC (Figure 5.16). In the MZI, quinelorane
significantly decreased the expression of Fos in TH-IR neurons (Figure 5.17;
Top Panel). USO-488 had no effect on the percentage of TH-IR neurons
expression Fos or on the numbers of TH-IR neurons (Figure 5.17; Bottom

Panel) in vehicle or quinelorane-treated animals.

157

DM-ARC

l:l Vehicle
- Quinelorane

N

O
A

x.

*

.3
(J1
1

01
j

1 a f i

% of TH-IR neurons expressing Fos
8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I I

O

I

Vehicle USO-488 Vehicle USO-488 Vehicle USO-488
Rostral Middle Caudal

Figure 5.13 Effects of quinelorane on the percentage of TH-IR neurons
expressing Fos in the rostral, middle and caudal regions of the DM-ARC of
vehicle- or U50488-treated rats. Rats were injected with either U50488 (10
mglkg; sc) or its ddHZO vehicle (1 mllkg; sc) 2 h prior to sacrifice, and with either
quinelorane (100 pg/kg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 2 h prior to
sacrifice. Columns represent the means and vertical lines + 1 SEM from 6-8
vehicle— (open columns), and quinelorane- (solid columns) treated rats. *, Values
that are significantly different (p < 0.05) from vehicle-treated rats.

158

DM-ARC

80 1
I:l Vehicle
70 4 - Quinelorane

60i
501 % L
401

30~

# of TH-IR Neurons

204

101

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0 I I I
Vehicle USO-488 Vehicle USO-488 Vehicle USO-488

Rostral Middle Caudal

I

Figure 5.14 Effects of quinelorane on the numbers of TH-IR neurons in the
rostral, middle and caudal regions of the DM-ARC of vehicle- or U50488-treated
rats. Rats were injected with either U50488 (10 mglkg; sc) or its ddH20 vehicle
(1 mllkg; sc) 2 h prior to sacrifice, and with either quinelorane (100 jig/kg; ip) or
its 0.9% saline vehicle (1 mllkg; ip) 2 h prior to sacrifice. Columns represent the
means and vertical lines + 1 SEM from 6-8 vehicle- (open columns), and
quinelorane- (solid columns) treated rats.

159

VL-ARC

40 ~
[:1 VehiCIe
35 - - Quinelorane

1 i

I.
25~

20‘
15-
* *
10‘ * *
*
51 H I
0 I I I I I

Vehicle U50-488 Vehicle 050-488 Vehicle USO-488
Rostral Middle Caudal

% of TH-IR neurons expressing Fos

 

 

 

 

 

 

 

 

 

 

Figure 5.15 Effects of quinelorane on the percentage of TH-IR neurons
expressing Fos in the rostral, middle and caudal regions of the VL-ARC of
vehicle- or U50488-treated rats. Rats were injected with either U50488 (10
mglkg; so) or its ddH20 vehicle (1 mllkg; sc) 2 h prior to sacrifice, and with either
quinelorane (100 jig/kg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 2 h prior to
sacrifice. Columns represent the means and vertical lines + 1 SEM from 6-8
vehicle- (open columns), and quinelorane- (solid columns) treated rats. *, Values
that are significantly different (p < 0.05) from vehicle-treated rats.

160

VL-ARC

501

45 1 CI] Vehicle ¥ I1
_ Quinelorane

40- L L

35~
30-
251
201

15- 1 IT

101

# of TH-IR neurons

54

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

O I l I I I
Vehicle USO-488 Vehicle USO—488 Vehicle USO-488
Rostral Middle Caudal

Figure 5.16 Effects of quinelorane on the numbers of TH-IR neurons in the
rostral, middle and caudal regions of the VL-ARC of vehicle- or U50488-treated
rats. Rats were injected with either U50488 (10 mglkg; sc) or its dngO vehicle
(1 mllkg; sc) 2 h prior to sacrifice, and with either quinelorane (100 uglkg; ip) or
its 0.9% saline vehicle (1 mllkg; ip) 2 h prior to sacrifice. Columns represent the
means and vertical lines + 1 SEM from 6-8 vehicle- (open columns), and
quinelorane- (solid columns) treated rats.

161

MZI

A
O
J

[:3 Vehicle
— Quinelorane

G)

 

 

21

 

 

 

 

 

  

 

 

% of TH-IR Neurons Expressing Fos
a.
a.

 

Vehicle

200 -

175 ~

 

150 -

 

125 1

100 1

751

# of TH-IR Neurons

501

251

 

 

 

 

 

 

 

 

Vehicle U50488

Figure 5.17 Effects of quinelorane on the numbers of TH-IR neurons (Bottom
Panel) and the percentage of these neurons expressing Fos (Top Panel) in the
MZI of vehicle- or U50488-treated rats. Rats were injected with either U50488
(10 mglkg; so) or it ddHZO vehicle (1 mllkg; sc) 2 h prior to sacrifice, and with
either quinelorane (100 uglkg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 2 h prior
to sacrifice. Columns represent the means and vertical lines + 1 SEM from 6-8
vehicle- (open columns) and quinelorane- (solid columns) treated rats. *, Values
that are significantly different (p < 0.05) from vehicle-treated rats.

162

 

 

DISCUSSION

The major finding of these studies is that opioid receptors regulate Fos
expression in TIDA neurons of the DM-ARC. Blockade of kappa opioid receptors
increases Fos expression in TIDA neurons throughout the rostrocaudal extent of
the DM-ARC in a time dependent manner. This time frame is characteristic of
the induction of Fos and other immediate early genes in a variety of tissues.
Once induced, c—fos mRNA increases as early as 15 min and is subsequently
translated into Fos protein (Herdegen and Leah, 1998). The half life of Fos
protein is 90-120 min, with maximum levels reached between 1 and 3 h, and
gradually disappearing from the nucleus by 4-6 h (Sonnenberg et al., 1989; Chan
et al., 1993; Kovacs and Sawchenko, 1996). This rapid down-regulation of Fos is
not fully understood, but there are three main processes that could account for
this. First is down-regulation of transcription by Fos protein cis-repressing its
own promoter (Sassone-Corsi et al., 1988). Secondly, the mRNA of c-fos and
other oncogenes are not stable and contain elements that make them targets for
exonucleases and thus rapid degradation (Veyrune et al., 1995). Thirdly, in
addition to mRNA degradation, the protein itself is also rapidly broken down by
lysosomes and proteasomes (Stancovski et al., 1995; Aniento et al., 1996).

This time course of Fos expression shown here is similar to that seen by
others following the blockade of mu and kappa opioid receptors. For example,
naloxone and nor-BNI both increase Fos expression in unidentified neurons in
various areas of the brain 90 to 120 min following administration (Gestreau et al.,

2000; Carr et al., 1999). Kappa opioid receptors are linked to G1 and Go proteins,

163

 

 

which inhibit CAMP and hyperpolarize cell membranes (Childers, 1991; Childers
et al., 1998; Takekoshi et al., 2000). These data suggest that kappa receptors
tonically suppress Fos expression in TIDA neurons. Precedent for induction of
immediate early genes through the loss of tonic opioid activity has been
demonstrated in the central amygdala (Carr et al., 1999) and nucleus tractus
solitarii (Gestreau et al., 2000). In this case, the blockade of dynorphin induces a
disinhibition of TIDA neurons by a postsynaptic action which results in an
increase in neuronal discharge and metabolic activities strong enough to induce
Fos expression (Morgan and Curran, 1991).

In the DM-ARC, the time course of Fos expression in TH-IR neurons
following blockade of kappa receptors is also identical to the time course
following activation of DZ receptors shown in Chapter 4. In this respect blockade
of kappa receptors mimics the effects of D2 receptor activation on Fos
expression in TIDA neurons of the DM-ARC. These results, along with the
neurochemical experiments using nor-BNI and U50-488, (Manzanares et al.,
1992a and 1992b; Durham et al., 1996) further support a role of kappa receptors
in mediating the effects of DZ receptor stimulation of Fos expression.

If expression of Fos is regulated by kappa opioid receptors, then a kappa
receptor agonist should reverse the effects of a kappa opioid receptor antagonist.
Indeed, U50-488 blocks the stimulatory effect of nor-BNI on Fos expression
confirming that the actions of nor-BNI are mediated through kappa opioid
receptors. These results correlated with findings from neuronal activity studies

demonstrating that kappa antagonists induce an acute stimulatory response in

164

TIDA neurons and this can be reversed by a kappa receptor agonist
(Manzanares et al., 1992). The lack of effect of a kappa agonist suggests that
dynorphin neurons are tonically active and maximally inhibit Fos expression in
TIDA neurons of the DM-ARC under basal conditions. In addition to TIDA
neurons, the neurons of the periventricular DA system are also tonically inhibited
by dynorphin as revealed by neurochemical studies (Manzanares et al., 1991).
This is in contrast to the effects on the nigrostriatal and mesolimbic DA systems
in neurochemical studies where there was no effect following the blockade or
activation of kappa receptors on neuronal activity in these two systems
(Manzanares et al., 1991). This suggests that nigrostriatal and mesolimbic
neurons are not tonically inhibited by dynorphin.

Having established that Fos expression in TIDA neurons is regulated by
kappa opioid receptors, the next aim was to determine if these receptors share a
common pathway that mediates the stimulatory effects of DZ receptors on Fos
expression. If quinelorane increases Fos expression in TIDA neurons by
preventing dynorphin release and activation of kappa opioid receptors, then the
administration of a kappa receptor agonist should restore activity at these
receptors and prevent stimulation of Fos expression. The results from this study
reveal that this is indeed the case, and correlate with previous neurochemical
studies showing that USO-488 reverses the stimulatory effects of quinelorane on
TIDA neuronal activity (Durham et al., 1996). These studies demonstrate that

the D2 receptor-mediated neuronal regulation of Fos expression in the DM-ARC

165

 

of TIDA neurons occurs through the loss of tonic inhibition of dynorphin neurons
via kappa opioid receptors.

This is the first time this type of regulation of Fos expression has been
demonstrated in DA neurons. Kappa receptors are involved DA receptor-
mediated regulation of immediate early genes, but in non-DA striatonigral
neurons (striatal neurons which project to the substantia nigra). Dynorphin
released in the ST from striatonigral neurons following D1-receptor stimulation
acts locally on pre-synaptic kappa receptors on DA terminals or postsynaptic
kappa receptors on the striatonigral neurons themselves to inhibit Fos and zif268
induction (Steiner and Gerfen, 1995, 1996 and 1998). In the substantia nigra
and ventral tegmental areas, kappa receptor mRNA (Mansour et al., 1994) and
receptor binding (Speciale et al., 1993) has been demonstrated, as well as
dynorphin-IR terminals forming synapses with TH-labeled dendrites (Pickel et al.,
1993). Dynorphin released in the substantia nigra from striatonigral terminals
could act to inhibit nigrostriatal neurons; however, none of these studies
examined Fos immunoreactivity in these DA neurons. Only one study has been
reported to examine the effects of kappa receptor blockade on Fos expression in
the ventral tegmental area (Carr et al., 1999). Unlike TIDA neurons in the DM-
ARC, nor-BNI had no effect on neurons in the ventral tegmental area suggesting
that mesolimbic DA neurons are not tonically suppressed by dynorphin.

Figure 5.18 illustrates the proposed pathway following D2 receptor
activation with quinelorane. From the available evidence it is likely that the DZ

receptors are located outside the mediobasal hypothalamus, whereas dynorphin

166

  

DA

     

D, Receptor

 

 

- 7
Medlobasal - Receptor

Hypothalamus

ARC Kappa Opioid Receptor

ADA

Figure 5.18 Schematic depicting the proposed neuronal pathway following DZ
receptor activation. DA acts at a DZ receptor on the chemically unidentified
neurons (depicted in green) located outside the mediobasal hypothalamus. In
turn, there is a decrease in the release of stimulatory neurotransmitter which
leads to inhibition of dynorphin neurons. The decrease in dynorphin at the kappa
opioid receptor leads to a disinhibition of TIDA neurons and subsequent increase
in DA release. Abbreviations: ?, unknown neuron/neurotransmitter/receptor;
ARC, arcuate nucleus; DA, dopamine; DYN, dynorphin; ME, median eminence.

167

neurons are located in the ARC and terminate at kappa opioid receptors on TIDA
neurons. There are still several unknowns in this pathway; 1) the origin of DA, 2)
location of the DZ receptor-expressing target cells, 3) the neurochemical identity
of the target cell and its receptors on dynorphin neurons.

Although no thorough studies have been carried to answer these
questions, it is tempting to speculate about what these unknowns might be. One
area of the hypothalamus that may be involved in this neuronal pathway is the
dorsomedial hypothalamic nucleus. Electrical stimulation of this nucleus
stimulates DA release from TIDA neurons (Gunnet and Moore, 1988). Since
current spread to the ARC was ruled out in these studies it is likely that the
stimulation causes DA release from an unknown source that, in turn, activates
DZ receptors and mimics the stimulatory effect of quinelorane. The DMN also
contains extensive dendritic processes from the lHDA neurons of the MZI (Chan-
Palay et al., 1984), therefore a possible origin of DA is the MZI. The lHDA
neurons project to the paraventricular nucleus, the horizontal diagonal band, and
the central amygdala (Eaton et al., 1994; Wagner et al., 1995), hence it is

possible that one of these areas contains the DZ receptor expressing target cell.

168

If the proposed pathway is correct, then the neuron targeted by DA should
originate outside the mediobasal hypothalamus and be stimulatory in nature,
tonically activating dynorphin neurons. One clue to the identity of this stimulatory
neuron is that activation of its target receptor should mimic the effects of
dynorphin neurons and tonically inhibit TIDA neurons. Though it is not known
what the neuron might be, there are certain neurotransmitter and receptor
systems that can certainly be ruled out. For example, several lines of evidence
demonstrate that the neuropeptide neurotensin is not involved. First, neurotensin
activates TIDA neurons (Berry and Gudelsky, 1990) and mediates the stimulatory
effects of PRL on the activation of these neurons (Hentschel et al., 1998). In
addition, co—administration of a neurotensin receptor antagonist has no effect on
the quinelorane-induced increases in TIDA neuronal activity (Durham, 1999).

Two neurotransmitters that tonically inhibit TIDA neurons are GABA
(acting at GABAA receptors; Wagner et al., 19943) and glutamate (acting at

AMPA receptors; Wagner et al., 1994b). However, GABA is an inhibitory
neurotransmitter so if the unidentified neuron were GABAergic it would inhibit
(rather that stimulate) dynorphin neurons thus leading to activation of TIDA
neurons. Interestingly, blockade of AMPA receptors increases the activity of
TIDA neurons (Wagner et al., 1994) thus mimicking the effect of quinelorane.

However, this increase is not reversed by activating kappa opioid receptors as it
is by a GABAA receptor agonist (Wagner et al., 1994c). Therefore, the AMPA

receptor-mediated tonic inhibition of TIDA neurons occurs by a mechanism

involving GABAA receptors and not kappa receptors.

169

The results of the VL-ARC reveal that “DOPA-ergic” neurons are not
regulated by kappa opioid receptors under basal conditions. In these TH-IR
neurons, activation of kappa opioid receptors decreased Fos expression while
blockade of the receptors had no effect. Thus, kappa opioid receptors do not
tonically inhibit these neurons. There is evidence that there are dynorphin nerve
terminals in the ARC (Fitzsimmons, et al., 1992); however, those studies do not
differentiate between the VL-ARC vs. DM-ARC. There are also dynorphin
neurons in the ventromedial nucleus (VMN) of the hypothalamus (Zamir et
al.,1983), and like “DOPA-ergic” neurons, these neurons are not regulated by DZ
receptors (Durham, 1999). Though no studies are available concerning the
projection of dynorphin neurons of the VMN, it is possible they regulate “DOPA-
ergic” neurons. While it is possible that these neurons have kappa receptors, it
cannot be concluded from these studies if or whether or not the effect of
dynorphin on “DOPA-ergic” neurons is direct or indirect.

In these experiments, quinelorane decreased Fos expression in the
“DOPA-ergic” cells of the VL-ARC at an earlier time point compared to results
reported in Chapter 4. One possibility for this difference in response is time of
day influences. However, the animals of the quinelorane groups in these studies
and in Chapter 4 followed the same dosing schedule and were sacrificed at the
same time of day. Thus, time of day influences do not account for this difference.
The response of “DOPA-ergic” neurons to the D2 receptor agonist has been

variable throughout, reiterating that, unlike the TIDA neurons in the DM-ARC, D2

170

receptors do not regulate these neurons. The significance of this finding remains
to be elucidated.

The results for the lHDA neurons of the MZI demonstrate that there is no
tonic kappa opioid receptor regulation of Fos expression, nor is there any
response to the kappa agonist. This is in agreement with neurochemical studies
demonstrating a lack of kappa opioid receptor regulation of lHDA neuronal
activity (Tian et al., 1992). Thus, it is concluded that there is no kappa opioid
receptor regulation of gene expression in the lHDA neurons of the MZI.

It is interesting to note that quinelorane significantly decreases Fos
expression in these neurons. Recall in Chapter 4 quinelorane alone had no effect
on Fos expression in the lHDA neurons at 2 h. One possibility for this difference
in response is time of day influences. However, the animals receiving
quinelorane in these studies and in Chapter 4 were sacrificed at the same time of
day. Thus, time of day influences can not account for this difference.

The percentage of neurons expressing Fos in the vehicle groups is higher
in this experiment (approximately 6%) compared to previous studies
(approximately 4% or less). In the raclopride/quinelorane study in Chapter 4
(Figure 4.11), blockade of D2 receptors with raclopride increased the expression
of Fos in the MZI to about 6% and this was reversed with quinelorane. It is
possible that, for a reason not explained or due to some natural variation, the
lHDA neurons were slightly more active and thus expressing more Fos at the
time of this experiment. If the D2 receptors were not fully occupied, quinelorane

may have been able to bind and decrease Fos expression.

171

In conclusion, dynorphin neurons projecting to TIDA neurons are tonically
active, and they regulate Fos expression via kappa opioid receptors in TIDA
neurons in the DM-ARC. The expression of Fos in “DOPA-ergic” neurons of the
VL—ARC is regulated by kappa receptors as well; however, this regulation is not
present under basal conditions. Finally, DZ receptor mediated regulation of Fos
in TIDA neurons in DM-ARC occurs via a mechanism involving inhibition of
endogenous dynorphin release and loss of tonic inhibition of gene expression by

kappa opioid receptors.

172

CHAPTER SIX
D2 RECEPTOR REGULATION OF TH GENE EXPRESSION IN SUBDIVISIONS
OF THE ARCUATE NUCLEUS
Introduction

In Chapter 4, immunohistochemical techniques were utilized to
characterize DZ receptor-mediated changes in Fos expression in TIDA neurons.
These studies revealed that following D2 receptor activation, Fos increases
throughout the rostral-caudal extent of the DM-ARC in a time dependent manner,
but not in the VL-ARC. These results correlated with previous neurochemical
studies examining DZ receptor-mediated stimulation of TIDA nerve terminal
activity in the ME (Eaton et al., 1993). Activation of D2 receptors increases DA
synthesis and turnover in these neurons within 30 min and persists for at least 8
h (Eaton et al., 1993; Durham et al., 1996).

TH is the rate limiting enzyme in the catecholamine biosynthesis pathway.
The catalytic activity of this molecule determines the synthetic rate of DA in the
TIDA system (Wang et al., 1993). TH can be regulated by activation of the
existing enzyme, such as through phosphorylation (Porter, 1986a) and by
increased synthesis of the enzyme beginning at the transcription level (Biguet et
al., 1991; Kumer and Vrana, 1996).

There are several lines of evidence demonstrating a correlation between
TH neuronal activity and TH mRNA expression in TIDA neurons regulated by
PRL. Chronic administration of pharmacological agents that cause changes in

PRL levels results in corresponding changes in TIDA neuronal activity and TH

173

mRNA expression (Arbogast and Voogt, 1991). Also, afferent neuronally-
mediated physiological stimuli originating from suckling rat pups decreases TIDA
neuronal activity and TH mRNA expression in dams, which can be reversed with
the removal of the pups (Wang et al., 1993; Berghom et al., 2001 ). However, the
acute effects of DA receptor activation on TH gene expression in TIDA neurons
have not been studied.

The specific aim of these studies was to determine if an acute increase in
neuronal activity caused by activation of DZ receptors was associated with
changes in the expression of the long term gene TH in TIDA neurons. The
working hypothesis is that acute activation of DA release from TIDA neurons is
accompanied by an increase of TH mRNA expression in these neurons. To this
end, the time course of effects of quinelorane on TH mRNA expression were
determined in the DM-ARC and for comparison, “DOPA-ergic” neurons in the VL-

ARC and lHDA neurons in the MZI.

Materials and Methods

Gonadally intact male rats were used in these experiments. Care and
handling of the rats was carried out as described in Materials and Methods in
Chapter 2. Following appropriate treatments, rats were decapitated and brains
were quickly frozen. Brains were sectioned using a cryostat as described in
Materials and Methods in Chapter 2. The identification of neurons containing
TH mRNA in the ARC and MZI was carried out using in situ hybridization

procedures in Materials and Methods in Chapter 2. Results from studies

174

presented earlier demonstrated that the stimulation of Fos expression was
consistent throughout the rostrocaudal extent of the DM-ARC. Because there
were no differences in this response, the in situ studies focused on the rostral
and middle ARC. The rostral and middle ARC sections were chosen and then
visually partitioned into DM-ARC and VL-ARC. TH mRNA was localized with a

TH oligonucleotide probe labeled with 3‘58.

Results

Photomicrographs in Figure 6.1 illustrate TH mRNA expression in neurons in the
middle DM-ARC 4 h after the injection of either vehicle (Left Panel) or
quinelorane (Right Panel). There is an increase in grain density of TH mRNA
following quinelorane administration. Figure 6.2 illustrates the time course
effects of quinelorane on the labeling density ratio of TH mRNA expression per
neuron in the DM-ARC. Quinelorane significantly increased TH mRNA
expression in a time dependent manner reaching maximum levels at 4 h and
remained elevated for at least 24 h in the DM-ARC. There were no changes in
the numbers of neurons containing TH mRNA within the DM-ARC at any
timepoint following quinelorane administration (Figure 6.3). In contrast, in the
VL-ARC quinelorane had no effect on the labeling density ratio of TH mRNA per
neuron (Figure 6.4). As in the DM-ARC, there were no changes in the numbers
of neurons containing TH mRNA at any timepoint within the VL-ARC following

quinelorane administration (Figure 6.5). In the MZI, quinelorane had no effect on

175

 

Figure 6.1 High power (400x) computer-captured images demonstrating
increased TH mRNA expression in neurons of the DM-ARC 4 h following
administration of quinelorane. Rats were injected with either vehicle (Left Panel;
0.9% saline; 1 mllkg; ip) or quinelorane (Right Panel; 100 uglkg; ip) and
sacrificed 4 h later. Arrows represent silver grains localized over the cell bodies
of neurons containing TH mRNA. 3V, third ventricle.

176

 

DM-ARC

 

7 “ 0 Vehicle
0 Quinelorane

 

O I I I I I 7
O 2 4 8 16 24

 

Labeling Density Ratio of TH mRNA per Neuron
0)

Hours After Quinerlorane

Figure 6.2 Time course effects of quinelorane on the labeling density ratio of TH
mRNA expression per neuron in the DM-ARC. Rats were injected with
quinelorane (filled symbols; 100 jig/kg; ip) and sacrificed 2, 4, 8, 16, or 24 h later.
Zero time controls (open symbols) were injected with 0.9% saline vehicle (1
mllkg; ip) and sacrificed 2 h later. Symbols represent the means and vertical
lines :I: 1 SEM from 6-8 animals. *, Values for quinelorane-treated rats that are
significantly different (p<0.05) from vehicle-treated controls.

177

DM-ARC

l:l Vehicle
80 I - Quinelorane

701
601

501

201

101

# of Neurons Containing TH mRNA
A
O

 

 

 

 

 

0 2 4 8 16 24
Hours After Quinelorane

Figure 6.3 Time course effects of quinelorane on the number of neurons
containing TH mRNA in the DM-ARC. Rats were injected with quinelorane (filled
columns; 100 jig/kg; ip) and sacrificed 2, 4, 8, 16, or 24 h later. Zero time
controls (open columns) were injected with 0.9% saline vehicle (1 mllkg; ip) and
sacrificed 2 h later. Columns represent the means and vertical lines + 1 SEM
from 6-8 animals.

178

VL-ARC

 

 

:

O

'5 12 -

g 0 Vehicle

5 11 1 o Quinelorane
CD

Q 10 1

E 9‘

E 8 ‘

I 4

,_ 7

..5 61

O

“s 51

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3"

ca 3 ..

5 2 _

D

0'1 1 1

E

E 0 I I I I I I
'8 0 2 4 8 15 24
.1

Hours After Quinelorane

Figure 6.4 Time course effects of quinelorane on the labeling density ratio of TH
mRNA per neuron in the VL-ARC. Rats were injected with quinelorane (filled
symbols; 100 uglkg; ip) and sacrificed 2, 4, 8, 16, or 24 h later. Zero time
controls (open symbols) were injected with 0.9% saline vehicle (1 mllkg; ip) and
sacrificed 2 h later. Symbols represent the means and vertical lines :1: 1 SEM
from 6-8 animals.

179

 

VL-ARC

35 1
:l Vehicle

30 q - Quinelorane

25- I

201

 

15-

101

# of Neurons Containing TH mRNA

 

 

 

 

 

 

0 2 4 8 15
Hours After Quinelorane

Figure 6.5 Time course effects of quinelorane on the numbers of neurons
containing TH mRNA in the VL-ARC. Rats were injected with quinelorane (filled
columns; 100 uglkg; ip) and sacrificed 2, 4, 8, 16 or 24 h later. Zero time controls
(open columns) were injected with 0.9% saline vehicle (1 mllkg; ip) and sacrificed
2 h later. Columns represent the means and vertical lines + 1 SEM from 6-8
animals.

180

the numbers of neurons containing TH mRNA (Figure 6.6; Bottom Panel) or the
labeling density ratio of TH mRNA (Figure 6.6; Top Panel).

To determine if the effects of quinelorane on TH mRNA were mediated by
DZ receptors, a study was carried out using the D2 receptor antagonist
raclopride. Blockade of D2 receptors had no effect per se, but reversed the
stimulatory effects of quinelorane on TH mRNA expression in the DM-ARC
(Figure 6.7). There were no changes in the numbers of neurons containing TH
mRNA following raclopride or quinelorane administration (Figure 6.8). In the VL-
ARC, neither quinelorane nor raclopride had any effect on the numbers of
neurons containing TH mRNA (Figure 6.10) or on the labeling density ratio of TH
mRNA of those neurons (Figure 6.9). In the MZI, neither quinelorane nor
raclopride had any effect on the numbers of neurons containing TH mRNA
(Figure 6.11; Bottom Panel) or the labeling density ratio of TH mRNA of those

neurons (Figure 6.11; Top Panel).

181

 

 

MZI

64 0 Vehicle
0 Quinelorane

Labeling Density Ratio of
TH mRNA per Neuron
A

 

orl I I l j
024 8 16 24

Hours After Quinelorane

 

N

O

O
J

_|
01
O

01
O

 

# of Neurons Containing TH mRNA
8
O

 

 

 

o
i
1

O 2 4 8 16 24
Hours After Quinelorane

Figure 6.6 Time course effects of quinelorane on the numbers of neurons
containing TH mRNA (Bottom Panel) and the labeling density ratio of TH mRNA
per neuron (Top Panel) in the MZI. Rats were injected with quinelorane (filled
symbols and columns; 100 jig/kg; ip) and sacrificed 2, 4, 8, 16, or 24 h later.
Zero time controls (open symbols and columns) were injected with 0.9% saline
vehicle (1 mllkg; ip) and sacrificed 2 h later. Symbols and columns represent the
means and vertical lines + 1 SEM from 6-8 animals.

182

DM-ARC

:1 Vehicle
- Quinelorane

 

 

 

 

 

 

 

 

 

 

0 . .
Vehicle Raclopride

Labellng Density Ratio of TH mRNA per Neuron
A

Figure 6.7 Effects of quinelorane on the labeling density ratio of TH mRNA per
neuron in the DM-ARC of vehicle- or raclopride-treated rats. Rats were injected
with either raclopride (3 mglkg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 4 h prior
to sacrifice, and with either quinelorane (100 pig/kg; ip) or its 0.9% saline vehicle
(1 mllkg; ip) 4 h prior to sacrifice. Columns represent the means and vertical
lines + 1 SEM from 68 vehicle— (open columns) and quinelorane- (solid columns)
treated rats. *, Values that are significantly different (p<0.05) from vehicle-
treated rats.

183

DM-ARC

60-

 

 

 

 

 

 

 

 

 

 

 

< [:1 Vehicle
2 - Quinelorane
E -
E 50 T
..I. L
a) 40 1
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.E
s so.
0
0
8 20 «
2
:3
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2 10
h
O
43:
0 l r
Vehicle Raclopride

Figure 6.8 Effects of quinelorane on the numbers of neurons containing TH
mRNA in the DM-ARC of vehicle- or raclopride-treated rats. Rats were injected
with either raclopride (3 mglkg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 4 h prior
to sacrifice, and with either quinelorane (100 pig/kg; ip) or its 0.9% saline vehicle
(1 mllkg; ip) 4 h prior to sacrifice. Columns represent the means and vertical
lines + 1 SEM from 6-8 vehicle- (open columns) and quinelorane- (solid columns)
treated rats.

184

"kg

VL-ARC

 

 

 

 

 

 

 

 

 

 

 

:
O
5 5 ‘ :3 Vehicle
% - Quinelorane
h
a:
a.
< 4 1 T T
Z
or
E
E 31
Q—
0
.2
...-a
1
g 2
9?.
2
0 _
D 1
a)
.E
E 0
a I T
-‘ Vehicle Raclopride

Figure 6.9 Effects of quinelorane on the labeling density ratio of TH mRNA per
neuron in the VL-ARC of vehicle- or raclopride-treated rats. Rats were injected
with either raclopride (3 mglkg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 4 h prior
to sacrifice, and with either quinelorane (100 pglkg; ip) or its 0.9% saline vehicle
(1 mllkg; ip) 4 h prior to sacrifice. Columns represent the means and vertical
lines + 1 SEM from 6-8 vehicle- (open columns) and quinelorane- (solid columns)
treated rats.

185

 

VL-ARC

50 1
[:2] Vehicle
45 1 - Quinelorane

401
T

 

 

351
30—
251
201
151
10-

54

0

# of Neurons Containing TH mRNA

 

 

 

 

 

 

 

 

 

I

Vehicle Raclopride

Figure 6.10 Effects of quinelorane on the numbers of neurons containing TH
mRNA in the VL-ARC of vehicle- or raclopride-treated rats. Rats were injected
with either raclopride (3 mglkg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 4 h prior
to sacrifice, and with either quinelorane (100 jig/kg; ip) or its 0.9% saline vehicle
(1 mllkg; ip) 4 h prior to sacrifice. Columns represent the means and vertical
lines + 1 SEM from 6-8 vehicle- (open columns) and quinelorane- (solid columns)
treated rats.

186

MZI

E Vehicle
- Quinelorane

 

 

Labeling Density Ratio of
TH mRNA per Neuron

 

 

 

 

 

 

0 e .
Vehicle Raclopride

 

 

250 1

200 1

 

 

150 1

100 1

# of Neurons Containing TH mRNA
0|
0

 

 

 

 

 

 

0 . .
Vehicle Raclopride

Figure 6.11 Effects of quinelorane on the numbers of neurons containing TH
mRNA (Bottom Panel) and the labeling density ratio of TH mRNA per neuron
(Top Panel) in the MZI of vehicle- or raclopride-treated rats. Rats were injected
with either raclopride (3 mglkg; ip) or its 0.9% saline vehicle (1 mllkg; ip) 4 h prior
to sacrifice, and with either quinelorane (100 uglkg; ip) or its 0.9% saline vehicle
(1 mllkg; ip) 4 h prior to sacrifice. Columns represent the means and vertical
lines + 1 SEM from 6-8 vehicle- (open columns) and quinelorane- (solid columns)
treated rats. *, Values that are significantly different (p<0.05) from vehicle-
treated rats.

 

 

187

DISCUSSION

The aim of the present study was to determine if DZ receptors regulate TH
gene expression in TIDA neurons located in DM-ARC. The results from these
studies reveal that activation of DZ receptors increases TH mRNA expression in
a time dependent manner in a fixed population of TIDA neurons.

There were no changes in the numbers of neurons containing TH mRNA
at any time point. This is in agreement with other in situ hybridization studies of
TIDA neurons demonstrating a constant number of neurons expressing TH
mRNA following exposure to bromocriptine, haloperidol, and PRL (Arbogast and
Voogt, 1991) and during the estrous cycle (Arbogast and Voogt, 1994). The
numbers of neurons containing TH mRNA is consistent with the numbers of TH-
IR neurons counted in the immunohistochemical studies in Chapter 4. These
results indicate that the amount of TH mRNA is increasing per neuron, as
opposed to an increase in the numbers of neurons expressing TH mRNA.

This is different from Fos expression. There are increases in the actual
numbers of TH-IR neurons containing Fos protein. This implies that there is a
subset of TIDA neurons that are responsive to D2 receptor activation. These
results can also be explained in part by the technique used for analyzing TH
mRNA. All the neurons that contained TH mRNA were counted (which is
equivalent to the numbers of TH-IR neurons found in immunohistochemical
studies presented in Chapters 3, 4, and 5). However, for the analysis of the
amount of TH mRNA present, not all neurons labeled with TH mRNA were

evaluated. Only 10 of the “brightest” neurons were chosen (i.e. those neurons

188

 

that visually had the greatest amount of silver grains). While it is possible to
determine the amount of TH mRNA expressed in each neuron (though time-
prohibitive), it is not known if those neurons that were considered the brightest
were also the neurons expressing Fos. Fos binds to the AP-1 site on the TH
gene promoter, which can regulate the synthesis of TH. Therefore, it’s very
plausible that those neurons with increased Fos expression also have an
increase in expression of TH mRNA.

DZ receptor-mediated activation of TH mRNA expression is delayed (4 h)
and persistent (24 h) compared to D2 receptor-mediated activation of TIDA
neuronal activity which increases rapidly (30 min) and is less prolonged (at least
8 h). The time course for the induction of TH mRNA varies depending on the
experimental model and design. Many studies have used cell cultures for
examining TH mRNA expression. In vitro studies using fetal hypothalamic cell
cultures stimulated with forskolin (which induces the PKA pathway) also
increases TH mRNA expression at 3 and 6 h (Tohei et al., 2000). However,
studies using PC12 cells find TH mRNA expression as early as 1 h following
stimulation with nerve growth factor and epidermal growth factor (Gizang-
Ginsberg and Ziff, 1990 and 1994).

The results of the studies presented here are in contrast to Arbogast and
Voogt (1995) who examined the effects of bromocriptine, a DA receptor agonist,
on TH mRNA expression in TIDA neurons. Exposure to bromocriptine for 4 h
decreases PRL and TH catalytic activity, but not TH mRNA expression. Other

studies examining the induction of TH mRNA looked at chronic manipulations. In

189

the ARC, chronic administration of DA antagonist haloperidol increases PRL and
TIDA neuronal activity as well as TH mRNA (Arbogast and Voogt, 1991), while
chronic administration of bromocriptine results in decreases in these parameters
(Arbogast and Voogt, 1991 and 1995). While bromocriptine is a DA receptor
agonist and haloperidol a DA receptor antagonist, they are not selective for D2
receptors as are quinelorane and raclopride, respectively. Their effects on TIDA
neurons are through their actions on PRL and not a direct effect on TIDA
neurons.

DZ receptor induced activation of TH mRNA expression in TIDA neurons
is unique in that a single acute stimulus induces a long term response, which has
not been demonstrated in other dopaminergic systems (Arbogast and Voogt,
1991; Sirinathsinghji et al., 1994; Iwata et al., 2000). D2 receptor-mediated
activation of TIDA neuronal activity last 8 h, but TH mRNA levels remained
elevated for at least 16 more hours. Indeed, removal of suckling pups induced
changes in TH mRNA expression in a similar time course as quinelorane
administration (Wang et al., 1993; Berghom et al., 2001); however, what should
be noted is that the suckling pups were absent for the full 48 h of experiment.
The response of TH mRNA expression following the return of suckling was not
studied. Therefore, this study has a chronic stimulus, unlike the acute stimulation
of D2 receptors.

Expression of TH mRNA is very prolonged after the acute activation of D2
receptors. However, under basal conditions, the half life of TH mRNA in the ARC

is 6-7 h as estimated by transcription inhibition studies in slice explant cultures

190

(Maurer and Wray, 1997). TH can be regulated through a variety of mechanisms
such as transcriptional regulation of mRNA levels, alternative RNA processing,
regulation of RNA stability, translational regulation, feedback inhibition, allosteric
regulation by polyanions, enzyme phosphorylation and enzyme stability (Kumer
and Vrana, 1996). Though it is not possible to determine exactly what specific
regulation of TH can account for the sustained TH mRNA expression, it is most
likely regulation of transcription or mRNA stability. Transcription regulation can
be mediated through changes in the transcription rate (due to the physiological
state of the animal or various pharmacological agents) and/or through cell type-
specific expression (Kumer and Vrana, 1996). Indeed other mechanisms of
regulation of TH may be occurring under these experimental conditions;
however, these mechanisms would most likely not be reflected as increases in
mRNA.

The D2 receptor antagonist raclopride is able to reverse the stimulatory
effect of quinelorane on TH mRNA expression indicating that the actions of
quinelorane are mediated through D2 receptors. The lack of effect following
blockade of D2 receptors also indicates that these neurons are not tonically
inhibiting TH expression. These results correlate with the finding from neuronal
activity studies demonstrating that D2 agonists induce an acute stimulatory
response in TIDA neurons and this can be reversed with a D2 receptor
antagonist (Eaton et al., 1993; Durham et al., 1997). This also correlates with the

immunohistochemical findings in Chapter 4 showing that D2 receptors mediate

191

Fos expression in the DM-ARC in a time-dependent manner, and this increase
precedes the increase in TH mRNA.

Taken together, these results suggest a causal relationship between DZ
receptor-mediated stimulation of DA release from TIDA neurons and Fos—induced
activation of TH synthesis. The increase in TH mRNA following activation of D2
receptors is delayed and prolonged compared to Fos expression as illustrated in
Chapter 4. Neuronal activity increases 30 min following D2 receptor activation
followed by immediate early gene expression at 2 h. This time course is similar
to the effects of lactation and subsequent removal of suckling pups. Lactation
inhibits TIDA neuronal activity, and FRA and TH mRNA expression. When
suckling pups are removed from the mother, there is an increase in maternal
TIDA neuronal activity (Selmanoff and Wise, 1981; de-Greef et al., 1981),
followed by increases in FRA expression at 3 h (Hoffman et al., 1984) and TH
mRNA at 6 h (Wang et al., 1993; Berghom et al., 2001). These studies
demonstrate a temporal effect of a stimulus: the induction of neuronal activity
followed by an increase in immediate early gene expression and then an
increase in long term gene expression. Figure 6.12 summarizes the seqdences

of neuronal activity and gene expression following the activation of DZ receptors.

192

 

r 70C

160 -
- ~ 500
150 -
+ 500
E 140 - \/ :0.
a u
c r 400 c
8 130 ' Long Tel’li‘l Gene 8
'6 120 - _ 300 “5
5° «1"
110 - h 200
100 - i ‘ ‘00
fii ' 0

 

 

90

 

 

16 24

Hours after quinelorane
Figure 6.12 Sequence of TIDA neuronal activity and gene expression following
activation of D2 receptors. D2 receptor-mediated DA release (red) precedes the

stimulation of immediate early (green) and long term gene expression (blue) in
the DM-ARC of TIDA neurons. Abbreviations: IEG, immediate early gene.

First there is a rapid increase in neuronal activity; associated with this is
an increase in immediate early gene (Fos) expression. This is transitory and
decreases in the face of sustained activity of the neurons. Following the
induction and synthesis of Fos protein, it dimerizes with a Jun protein and binds
to the AP-1 site (Curran and Franza, 1988; Angel and Karin, 1991) located within
the TH gene promoter of DA neurons (Biguet et al., 1991; Icard—Liepkalns et al.,
1992; Kumer and Vrana, 1996). Binding of the AP-1 site regulates the
transcription and translation of TH. The increase in Fos is then followed by an
increase in expression of the long term gene product, TH mRNA. This D2

receptor-mediated DA release precedes the stimulation of immediate early and

193

long term gene expression in the DM-ARC of TIDA neurons. This temporal
sequence of events also supports a role for Fos in the regulation of TH mRNA in
TIDA neurons.

In the VL-ARC, activation of D2 receptors has no effect on TH mRNA
gene expression which coincides with the lack of stimulation of F 03 expression in
"DOPA-ergic” neurons in this region. Furthermore, the levels of TH mRNA
remain relatively constant in the VL-ARC throughout the time course of the
experiment, which is in contrast with the expression of Fos. As demonstrated in
Chapter 4, there is a significant decrease in Fos expression at the 4 and 8 h time
points. This decrease in Fos does not appear to have an effect on the level of
TH mRNA. The results presented here demonstrate that “DOPA-ergic” neurons
of the VL-ARC are regulated differently than the TIDA neurons of the DM-ARC.
Studies pertaining to gene expression in the VL-ARC are very limited, thus no
definitive conclusions can be made concerning the function or regulation of these
neurons.

The lHDA neurons of the MZI are regulated by DA receptors as are the
DA neurons of the mesotelencephalic systems (Lookingland and Moore, 1984).
DA agonists and locally applied DA all decrease, while DA antagonists increase
the activity of lHDA neurons (Moore and Lookingland, 1995; Eaton et al, 1992).
Interestingly, the results from the present studies reveal that TH gene expression
is not mediated through DZ receptors in lHDA neurons. Similar findings were
found following administration of DA agonists (apomorphine and bromocriptine)

and DA antagonists (eticlopride or haloperidol) (Arbogast and Voogt, 1991;

194

Sirinathsinghji et al., 1994; Iwata et al., 2000). There were no changes in the
expression of TH mRNA in the lHDA neurons in the MZI following treatment with
either bromocriptine or haloperidol (Arbogast and Voogt, 1991).

In the mesolimbic and nigrostriatal DA systems, time course studies
examining the effects of the DA antagonist eticlopride on TH mRNA revealed no
changes in expression at any time point (5 min to 24 h) in the ventral tegmental
area or substantia nigra (Sirinathsinghji et al., 1994). Other studies determined
that TH protein and TH mRNA are inhibited by chronic continuous infusion, but
not intermittent injection of DA agonist apomorphine (Iwata et al., 2000). Chronic
infusion of a DA agonist works at post-synaptic receptors via a long-loop
feedback circuit to exert negative feedback on the DA neurons (Roth and
Elsworth, 1995). This continuous negative feedback can lead to decreases in TH
mRNA. A single injection of DA agonist or antagonist is capable to altering DA
neuronal activity in the lHDA neurons of the MZI through pre-synaptic
autoreceptors (Lookingland and Moore, 1984); however, TH mRNA is not
affected by acute actions because antidromic stimulation (i.e. stimulation at pre-
synaptic autoreceptors) does not effect gene expression (Icard-Liepkalns et al.,
1992)

Taken together the results presented in this chapter reveal that D2
receptors regulate TH mRNA expression and support a role for Fos in this
regulation in TIDA neurons in the DM-ARC. In addition, TH gene expression in
the “DOPA-ergic” neurons in the VL-ARC and in lHDA neurons in the MZI is not

regulated by 02 receptors.

195

CHAPTER SEVEN

THE ROLE OF FOS IN THE REGULATION OF TH GENE EXPRESSION IN
TIDA NEURONS IN SUB DIVISIONS OF THE ARCUATE NUCLEUS

Introduction

In Chapter Four, it was demonstrated that stimulation of DZ receptors can
increase expression of Fos in a time dependent manner in TIDA neurons located
in the DM-ARC. These same neurons, as illustrated in Chapter Six, also have
increases in TH mRNA following the activation of DZ receptors. The TH gene
promoter contains an AP—1 site (Figure 7.1), and it has been well documented
through in vitro studies that the Fos family of proteins dimerize with the Jun
family of proteins and bind to the AP-1 site (Bohmann et al., 1987; Franza et al.,
1988; Rauscher et al., 1988; Gizang-Ginsberg and Ziff, 1994) and thereby
regulate TH transcription (Gizang-Ginsberg and Ziff, 1990; Icard-Liepkalns et al.,
1992; Nagamoto-Combs et al., 1997; Sun and Tank, 2002). Indirect evidence
has shown in vivo that increases in TH mRNA in TIDA neurons occur after the
induction of FRA expression, suggesting that FRA may play a role in the
regulation of TH in these neurons (Wang et al., 1993; Hoffman et al., 1994).
There have not, however, been any in vivo studies examining a direct link

between expression of Fos and TH.

196

 

TH Gene Promoter

 

Figure 7.1 A schematic representation of the rat TH proximal gene promoter.
The putative and functional elements involved in regulation of TH transcription
are shown. HIF: Binding site for hypoxia-induced protein. AP-Z: Binding site for
AP-Z transcription factor found in adrenergic and noradrenergic neurons. AP-1:
Highlighted in red; functional binding site for Fos/Jun; involved in stimulated
expression. E box: Overlaps with AP-1 site and is involved with cell specificity.
Oct/Hept: Binding site for Oct-Z; represses expression. Sp1/Egr1: Binding sites
for Sp1 and Egr1; involved in basal and stimulated expression. CRE-2: Binding
site for CREB, involved in stimulated expression, works as a modulator possibly
with CRE. CRE/CaRE: Binding site for cyclic AMP response element binding
protein (CREB), regulates basal and cyclic AMP induced expression. TATA
box: Conserved AoT-rich core promoter site. Abbreviations: AP, activated
protein; CaRE, calcium response element; CRE, CAMP response element; Egr,
early growth response; Hept, heptamer; HIF, hypoxia-inducible factor; Oct,
octamer; Sp, specificity protein. Modified from Papanikolaou and Sabban, 2000.

197

The specific aim of these studies was to assess the role of Fos in the
regulation of TH gene expression in TIDA neurons. The working hypothesis is
that Fos mediates quinelorane-induced activation of TH mRNA expression in
TIDA neurons. To determine a role for Fos, an experimental technique needed
to be developed that could remove the influence of Fos. The use of antisense
oligonucleotide probes provides a way to transiently “knock out” proteins, such as
Fos by preventing the translation of specific mRNA’s (Chiasson et al., 1992;
Merchant, 1994; Hooper et al., 1994).

A gene is comprised of a specific sequence of bases. In order for a gene
to be expressed a strand of mRNA must be copied from DNA that encodes the
gene of interest. In double stranded DNA, one strand is the coding strand while
the other is the template from which mRNA is transcribed. The mRNA sequence
is thus complimentary to the DNA template strand and identical to the coding
strand. Normally after the mRNA is transcribed, a ribosome can attach and carry
out translation of proteins (Pilowsky and Suzuki, 1994). The objective of
antisense oligonucleotides is to interfere with gene expression by preventing the
translation of proteins from mRNA. Two of the primary mechanisms of
antisense, translational arrest and activation of RNase H, are depicted in Figure

7.2.

198

 

 

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Figure 7.2 A schematic representation of the process of normal gene
expression and two of the possible mechanisms by which antisense can
attenuate translation of protein from mRNA. DNA consists of two complimentary
strands: a coding strand and a template strand. mRNA is transcribed from the
template strand so that its sequence is identical to the coding strand. Under
normal conditions a ribosome attaches to the mRNA and carries out translation
of the protein (A). Antisense oligonucleotides bind to the mRNA and block the
ribosome causing translational arrest (B) or induce RNase H which degrades
RNA/DNA hybrids (C). Modified from Pilowsky and Suzuki, 1994 and Zen, 1995.

199

An antisense oligonucleotide has a sequence that is complimentary to a
portion of the mRNA of interest. The antisense and mRNA hybridize to form a
complex that inhibits the ribosome and halts the translation of the desired protein
(Pilowsky and Suzuki, 1994). RNase H is an endonuclease that recognizes
RNA-DNA complexes and selectively cleaves the RNA strand (Zon, 1995). Once
the RNA is cleaved, the antisense oligonucleotide is thought to dissociate from
the duplex and become available to bind to a second target mRNA (Myers and
Dean, 2000). In addition, it is possible for the antisense to enter the nucleus and
interact directly with the gene of interest by forming a triple helix which would
prevent transcription from taking place (Miller and Das, 1998). Intron-exon
junction sites are also possible targets of antisense leading to inhibition of proper
splicing and consequently the maturation of the transcript (Phillips and Gyurko,
1995; Keller et al., 2000).

Certain aspects need to be considered when employing antisense
oligonucleotides including the route of administration, dose, and time course of
action. The route of administration can either be directly into the target brain
tissue (intracerebral) or into the cerebrospinal fluid of the ventricle
(intracerebroventricular; icv). Several studies have examined the effects of c-fos
antisense in the striatum using a direct intracerebral route of administration
(Chiasson et al., 1992; Merchant, 1994; Dragunow et al., 1993). The injection of
antisense intracerebrally causes tissue damage and necrosis, and consequently
this route is best used in a brain region that is relatively large (like the striatum)

where adequate viable tissue remains to detect a response. Icv administration is

200

 

 

more appropriate when multiple brain regions are to be studied or the target
region of interest is small and difficult to reach without causing extensive tissue
damage.

The proper dose of antisense is in accordance with the route of
administration and certain characteristics of the target gene. Small doses of only
1 or 2 nmol are sufficient for intracerebral injections (Chiasson et al., 1992) since
the oligonucleotide is injected directly into the tissue and concentrated in a small
area. Larger doses ranging from 10-50 nmol are necessary for icv administration
due to dilution in the CSF (Chiu et al., 1994). If the gene of interest has low
basal expression and turns over rapidly (such as Fos) one injection may be
sufficient (Merchant, 1994). However, if the basal expression of the gene is high
and the turnover is low (such as a receptor), then multiple dosing is usually
necessary before effective protein depletion is observed (Qin et al., 1995). The
timing of administration of c-fos antisense has been examined from 4—72 hours
with 10-12 hours being the most successful at consistently preventing the
translation of Fos protein (Hooper et al., 1994; Morrow et al., 1999).

Preliminary studies for this thesis were carried out to determine the most
appropriate route of administration that would assure delivery of Fos antisense
oligonucleotide to the ARC. lntracerebral injections resulted in extensive tissue
damage from the cannula and fluid pressure from the injection of antisense. It
was concluded that the ARC is too small to be suited for intracerebral injections.
Accordingly, one aim of the studies included in this chapter was to establish the

effective dose of antisense oligonucleotide probe when administered via icv route

201

 

of administration. Initial experiments established the time course effect of c-fos
antisense and its specificity in preventing Fos expression in quinelorane-
stimulated TIDA neurons. The last experiment used these parameters to
determine the effect of c—fos antisense on TH mRNA in TIDA neurons under
basal conditions and following activation of DZ receptors. For comparison, the
effects of icv c-fos antisense administration on Fos and TH mRNA expression in
the “DOPA-ergic” neurons of the VL-ARC and the lHDA neurons of the MZI were

also studied.

Materials and Methods

Gonadally intact male rats were used in these experiments. Care and
handling of the rats was carried out as described in Materials and Methods in
Chapter 2. Following appropriate treatments in the first set of experiments, rats
anesthetized with Equithesin and perfused with 4% paraformaldehyde. Brains
were removed and sectioned as described in Materials and Methods in Chapter
2. The identification of TH-lR neurons and Fos-IR nuclei in the ARC and MZI
was carried out using immunohistochemical procedures in Materials and
Methods in Chapter 2. The ARC sections were sorted into rostral, middle, and
caudal regions and then visually partitioned into DM-ARC and VL-ARC.

For the last experiment, rats were decapitated and brains were quickly
frozen and sectioned. The identification of neurons containing TH mRNA in the
ARC and MZI was carried out using in situ hybridization procedures as described

in Materials and Methods in Chapter 2. Results from studies presented earlier

202

 

 

demonstrated that the stimulation of Fos expression was consistent throughout
the rostrocaudal extent of the DM-ARC. Because there were no differences in
this response, the in situ studies focused on the middle ARC. The middle ARC
sections were chosen and then visually partitioned into DM-ARC and VL-ARC.
c-fos antisense, c-fos “sense”, and c-fos "nonsense” probes were
prepared as described in Material and Methods in Chapter 2. The antisense
probe sequence was complimentary to the translation start site of c-fos mRNA.
The “sense” probe sequence consisted of the same sequence as the translation
start site, and the “nonsense” probe had the same G:C:T:A content of the

antisense probe but had a mismatched sequence of bases.

Results

Figure 7.3 depicts the time course effects of c-fos antisense on the
percentage of quinelorane-stimulated TH-lR neurons containing Fos-IR nuclei in
the DM-ARC. Quinelorane significantly increased Fos expression in the rostral,
middle and caudal regions of the DM-ARC as previously demonstrated in
Chapters 4 and 5. c-fos antisense significantly blocked quinelorane-induced Fos
expression in a time dependent manner, effective by 6 h in the rostral and middle
regions, and by 12 h in the caudal region of the DM-ARC. As seen in previous
studies, the distribution of TH-IR neurons declines through the rostral-caudal
extent of the DM-ARC, but there were no changes in the numbers of TH-IR

neurons at any time point following c-fos antisense administration within each

203

 

DM-ARC

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 7.3 Time course effects of c-fos antisense oligonucleotide on the
percentage of TH-IR neurons expressing Fos in the rostral, middle, and caudal
regions of the DM-ARC in quinelorane-treated rats. Rats were injected with c-fos
antisense (gray columns; 40 nmol/rat; icv) 3, 6 or 12 h prior to sacrifice and with
quinelorane (100 jig/kg; ip) 2 h prior to sacrifice. Zero time controls (black
columns) were injected with 0.9% saline vehicle (3 ullrat; icv) 12 h prior to
sacrifice and with quinelorane (100 ug/kg; ip) 2 h prior to sacrifice. Vehicle
controls (open columns) were injected with 0.9% saline vehicle (3 ul/rat; icv) 12 h
prior to sacrifice and again with 09% saline (1 mllkg; ip) 2 h prior to sacrifice.
Columns represent the means and vertical lines + 1 SEM from 6—8 animals. *,
Values for quinelorane-treated rats that are significantly different (p<0.05) from
vehicle-treated controls. #, Values for antisense-treated rats that are significantly
different (p<0.05) from quinelorane-treated rats.

204

region of the DM-ARC (Figure 7.4). In contrast, c-fos antisense had no effect on
the percentage of TH-IR neurons expressing Fos (Figure 7.5) or the numbers of
TH-IR neurons throughout the rostral-caudal extent of the VL-ARC in
quinelorane-treated animals (Figure 7.6). In the MZI, there were no changes in
the numbers of TH-IR neurons (Figure 7.7; Bottom panel) or in the percentage
of those neurons containing Fos-IR nuclei (Figure 7.7; Top panel) at any
timepoint following c-fos antisense administration.

To characterize the specificity of c-fos antisense in preventing Fos
expression, a study was carried out using c-fos “sense” and c-fos “nonsense”
oligonucleotides. While c-fos antisense was able to block the stimulatory effects
of quinelorane on the percentage of TH-IR neurons expressing Fos, c—fos
“sense” and c-fos “nonsense” probes had no effect (Figure 7.8). There were no
changes in the numbers of TH-IR neurons following the administration of
quinelorane or the three different c—fos oligonucleotides (Figure 7.9). In the VL-
ARC, quinelorane significantly decreased the percentage of TH-IR neurons
expressing Fos in the middle and caudal regions; however, c-fos antisense,
“sense” and “nonsense” had no effect in quinelorane-treated animals (Figure
7.10). There were no changes in the numbers of TH-IR neurons within each
rostrocaudal region of the VL-ARC following any of the treatments (Figure 7.11).
In the MZI, there were no changes in the numbers of TH-IR neurons (Figure
7.12; Bottom panel) or in the percentage of these neurons containing Fos-IR

nuclei (Figure 7.12; Top panel) following any of the treatments.

205

 

DM-ARC

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 7.4 Time course effects of c-fos antisense oligonucleotide on the
numbers of TH-IR neurons in the rostral, middle, and caudal regions of the DM-
ARC in quinelorane-treated rats. Rats were injected with c-fos antisense (gray
columns; 40 nmol/rat; icv) 3, 6 or 12 h prior to sacrifice and with quinelorane (100
jig/kg; ip) 2 h prior to sacrifice. Zero time controls (black columns) were injected
with 0.9% saline vehicle (3 ul/rat; icv) 12 h prior to sacrifice and with quinelorane
(100 jig/kg; ip) 2 h prior to sacrifice. Vehicle controls (open columns) were
injected with 0.9% saline vehicle (3 ullrat; icv) 12 h prior to sacrifice and again
with 09% saline (1 mllkg; ip) 2 h prior to sacrifice. Columns represent the means
and vertical lines + 1 SEM from 6-8 animals.

206

 

 

 

 

 

 

 

 

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Figure 7.5 Time course effects of c-fos antisense oligonucleotide on the
percentage of TH-IR neurons expressing Fos in the rostral, middle, and caudal
regions of the VL-ARC in quinelorane-treated rats. Rats were injected with c-fos
antisense (gray columns; 40 nmol/rat; icv) 3, 6 or 12 h prior to sacrifice and with
quinelorane (100 uglkg; ip) 2 h prior to sacrifice. Zero time controls (black
columns) were injected with 0.9% saline vehicle (3 ullrat; icv) 12 h prior to
sacrifice and with quinelorane (100 jig/kg; ip) 2 h prior to sacrifice. Vehicle
controls (open columns) were injected with 0.9% saline vehicle (3 ullrat; icv) 12 h
prior to sacrifice and again with 09% saline (1 mllkg; ip) 2 h prior to sacrifice.

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Columns represent the means and vertical lines + 1 SEM from 6-8 animals.

207

 

 

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Figure 7.6 Time course effects of c-fos antisense oligonucleotide on the
numbers of TH containing neurons in the rostral, middle, and caudal regions of
the VL-ARC in quinelorane-treated rats. Rats were injected with c-fos antisense
(gray columns; 40 nmol/rat; icv) 3, 6 or 12 h prior to sacrifice and with
quinelorane (100 jig/kg; ip) 2 h prior to sacrifice. Zero time controls (black
columns) were injected with 0.9% saline vehicle (3 ullrat; icv) 12 h prior to
sacrifice and with quinelorane (100 jig/kg; ip) 2 h prior to sacrifice. Vehicle
controls (open columns) were injected with 0.9% saline vehicle (3 uI/rat; icv) 12 h
prior to sacrifice and again with 09% saline (1 mllkg; ip) 2 h prior to sacrifice.
Columns represent the means and vertical lines + 1 SEM from 6-8 animals.

208

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Figure 7.7 Time course effects of c-fos antisense oligonucleotide on the
numbers of TH-IR neurons (Bottom Panel) and the percentage of these neurons
expressing Fos (Top Panel) in the MZI in quinelorane-treated rats. Rats were
injected with c-fos antisense (gray columns; 40 nmol/rat; icv) 3, 6 or 12 h prior to
sacrifice and with quinelorane (100 uglkg; ip) 2 h prior to sacrifice. Zero time
controls (black columns) were injected with 0.9% saline vehicle (3 til/rat; icv) 12 h
prior to sacrifice and with quinelorane (100 jig/kg; ip) 2 h prior to sacrifice.
Vehicle controls (open columns) were injected with 0.9% saline vehicle (3 ullrat;
icv) 12 h prior to sacrifice and again with 09% saline (1 mllkg; ip) 2 h prior to
sacrifice. Columns represent the means and vertical lines + 1 SEM from 6—8

animals.

209

 

DM-ARC

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 7.8 Effects of c-fos antisense, sense, and nonsense oligonucleotides on
the percentage of TH-IR neurons expressing Fos in the rostral, middle, and
caudal regions of the DM-ARC in quinelorane-treated rats. Rats were injected
with c-fos antisense (AS; 40 nmol/rat; icv), c-fos sense (8; 4O nmol/rat; icv), or c-
fos nonsense (NS; 40 nmol/rat; icv) 12 h prior to sacrifice and with quinelorane
(100 uglkg; ip) 2 h prior to sacrifice. Controls were injected with 0.9% saline
vehicle (3 pI/rat; icv) 12 h prior to sacrifice and with either quinelorane (V; 100
uglkg; ip) or 0.9% saline (V; 1 mllkg; ip) 2 h prior to sacrifice. Columns represent
the means and vertical lines + 1 SEM from 6-8 animals. *, Values that are
significantly different (p<0.05) from vehicle-treated controls. #, Values that are
significantly different (p<0.05) from quinelorane-treated controls. Abbreviations:
AS, antisense; NS, nonsense; S, sense; V, vehicle.

210

 

DM-ARC

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

      

 

 

 

 

 

 

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Figure 7.9 Effects of c-fos antisense, sense, and nonsense oligonucleotides on
the numbers of TH containing neurons in the rostral, middle, and caudal regions
of the DM-ARC in quinelorane-treated rats. Rats were injected with c-fos
antisense (AS; 40 nmol/rat; icv), c-fos sense (S; 40 nmol/rat; icv) or c-fos
nonsense (NS; 40 nmol/rat; icv) 12 h prior to sacrifice and with quinelorane (100
jig/kg; ip) 2 h prior to sacrifice. Controls were injected with 0.9% saline vehicle (3
til/rat; icv) 12 h prior to sacrifice and with either quinelorane (V; 100 jig/kg; ip) or
saline (V; 1 mllkg; ip) 2 h prior to sacrifice. Columns represent the means and
vertical lines + 1 SEM from 6-8 animals. Abbreviations: AS, antisense; NS,
nonsense; 8, sense; V, vehicle.

211

 

 

VL-ARC

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 7.10 Effects of c-fos antisense, sense, and nonsense oligonucleotides on
the percentage of TH-IR neurons expressing Fos in the rostral, middle, and
caudal regions of the VL-ARC in quinelorane-treated rats. Rats were injected
with c-fos antisense (AS; 40 nmol/rat; icv), c-fos sense (S; 40 nmol/rat; icv), or c-
fos nonsense (NS; 40 nmol/rat; icv) 12 h prior to sacrifice and with quinelorane
(100 jig/kg; ip) 2 h prior to sacrifice. Controls were injected with 0.9% saline
vehicle (3 ullrat; icv) 12 h prior to sacrifice and with either quinelorane (V; 100
ug/kg; ip) or 0.9% saline (V; 1 mllkg; ip) 2 h prior to sacrifice. Columns represent
the means and vertical lines + 1 SEM from 6-8 animals. *, Values that are
significantly different (p<0.05) from vehicle-treated controls. Abbreviations: AS,
antisense; NS, nonsense; S, sense; V, vehicle.

212

 

 

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V IVASSNSl V IVASSNSJ V lVASSNSJ

 

 

 

Quinelorane Quinelorane Quinelorane

Figure 7.1 1 Effects of c-fos antisense, sense, and nonsense oligonucleotides on
the numbers of TH-IR neurons in the rostral, middle, and caudal regions of the
VL-ARC in quinelorane-treated rats. Rats were injected with c-fos antisense (AS;
40 nmol/rat; icv), c-fos sense (S; 40 nmol/rat; icv) or c-fos nonsense (NS; 40
nmol/rat; icv) 12 h prior to sacrifice and with quinelorane (100 jig/kg; ip) 2 h prior
to sacrifice. Controls were injected with 0.9% saline vehicle (3 ul/rat; icv) 12 h
prior to sacrifice and with either quinelorane (V; 100 jig/kg; ip) or saline (V; 1
mllkg; ip) 2 h prior to sacrifice. Columns represent the means and vertical lines +
1 SEM from 6-8 animals. Abbreviations: AS, antisense; NS, nonsense; S, sense;
V, vehicle.

213

 

MZI

..3
O

 

 

 

 

" ~. .’ .....

.' .....

 

 

 

 

 

 

 

 

 

 

 

% of TH-IR Neurons Expressing Fos

V IV AS S Nsl

 

1757

150 .

 

 

125 -

100 4

75-

# of TH-IR Neurons

50*

25~

 

 

 

 

 

 

 

 

 

 

o- 1 I ‘7” r
V l V AS S NSJ

Quinelorane

 

 

 

Figure 7.12 Effects of c-fos antisense, sense, and nonsense oligonucleotides on
the numbers of TH-IR neurons (Bottom Panel) and the percentage of these
neurons expressing Fos (Top Panel) in the MZI in quinelorane-treated rats. Rats
were injected with c-fos antisense (AS; 40 nmol/rat; icv), c-fos sense (S; 40
nmol/rat; icv) or c-fos nonsense (NS; 40 nmol/rat; icv) 12 h prior to sacrifice and
with quinelorane (100 uglkg; ip) 2 h prior to sacrifice. Controls were injected with
0.9% saline vehicle (3 ul/rat; icv) 12 h prior to sacrifice and with either
quinelorane (V; 100 uglkg; ip) or saline (V; 1 mllkg; ip) 2 h prior to sacrifice.
Columns represent the means and vertical lines + 1 SEM from 6—8 animals.
Abbreviations: AS, antisense; NS, nonsense; S, sense; V, vehicle.

214

 

Once the c-fos antisense was determined to block Fos expression, a study
was carried out to determine if Fos mediates quinelorane-induced stimulation of
TH mRNA expression in TIDA neurons. Quinelorane significantly increased the
labeling density ratio of TH mRNA per cell in the DM-ARC (Figure 7.13). c-fos
antisense in the absence of quinelorane had no effect on the labeling density
ratio of TH mRNA in the DM-ARC; however, it was able to block quinelorane-
induced increases in the labeling density ratio. The “sense” and “nonsense”
oligonucleotide probes had no effect. There were no changes in the numbers of
neurons that contain TH mRNA in the DM-ARC following any of the treatments
(Figure 7.14). In the VL-ARC, there were no changes in the labeling density
ratio (Figure 7.15) or in the numbers of neurons containing TH mRNA following
any of the treatments (Figure 7.16). Similarly, there were no changes in the
labeling density ratio (Figure 7.17; Top Panel) or in the number of neurons
containing TH mRNA (Figure 7.17; Bottom Panel) in the lHDA neurons of the

MZI.

215

DM-ARC

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

S
5 101
g *
L
a 8 , T
<
2
m
E
E 6*
h
0
.2
fl
.3: 41
E‘
2
8 2 1
O)
.E
g o r s
Vehicle Quinelorane

Figure 7.13 Effects of c-fos antisense, sense, and nonsense oligonucleotides on
the labeling density ratio of TH mRNA per neuron in the DM-ARC in quinelorane-
treated rats. Rats were injected with c-fos antisense (AS; 40 nmol/rat; icv), c-fos
sense (8; 40 nmol/rat; icv), or c-fos nonsense (NS; 40 nmol/rat; icv) 14 h prior to
sacrifice and with quinelorane (100 uglkg; ip) 4 h prior to sacrifice. Controls were
injected with 0.9% saline vehicle (3 til/rat; icv) 14 h prior to sacrifice and with
either quinelorane (V; 100 ug/kg; ip) or 0.9% saline (V; 1 mllkg; ip) 4 h prior to
sacrifice. Columns represent the means and vertical lines + 1 SEM from 6—8
animals. *, Values that are significantly different (p<0.05) from vehicle-treated
controls. #, Values that are significantly different (p<0.05) from quinelorane-
treated controls. Abbreviations: AS, antisense; NS, nonsense; S, sense; V,
vehicle.

216

 

 

 

DM-ARC

80-
70*

60~

TH mRNA

 

 

 

50~

mrng

40-

 

30~

 

20*

10-

 

# of Neurons Conta

 

 

 

 

 

 

 

 

0 T .
j V AS I j V
Vehicle Quinelorane

 

 

 

Figure 7.14 Effects of c-fos antisense, sense, and nonsense oligonucleotides on
the numbers of neurons expressing TH mRNA in the rostral, middle, and caudal
regions of the DM-ARC in quinelorane-treated rats. Rats were injected with 0403
antisense (AS; 40 nmol/rat; icv), c-fos sense (S; 40 nmol/rat; icv) or c-fos
nonsense (NS; 40 nmol/rat; icv) 14 h prior to sacrifice and with quinelorane (100
uglkg; ip) 4 h prior to sacrifice. Controls were injected with 0.9% saline vehicle (3
ul/rat; icv) 14 h prior to sacrifice and with either quinelorane (V; 100 jig/kg; ip) or
saline (V; 1 mllkg; ip) 4 h prior to sacrifice. Columns represent the means and
vertical lines + 1 SEM from 6-8 animals. Abbreviations: AS, antisense; NS,
nonsense; S, sense; V, vehicle.

217

 

 

 

 

VL-ARC

 

 

 

 

 

 

 

 

 

 

 

     

 

 

8

5 1o-

0

z

8-

a

< 8‘

z

a:

E

E 6:

“—

O

.9 T

"’ - T

if 4

E‘

2

a 2:

O)

.E

g o . .

-‘ I V AS I l V AS
Vehicle Quinelorane

Figure 7.15 Effects of c-fos antisense, sense, and nonsense oligonucleotides on
the labeling density ratio of TH mRNA per neuron in the rostral, middle, and
caudal regions of the VL-ARC in quinelorane-treated rats. Rats were injected
with c-fos antisense (AS; 40 nmol/rat; icv), c-fos sense (S; 40 nmol/rat; icv), or c-
fos nonsense (NS; 40 nmol/rat; icv) 14 h prior to sacrifice and with quinelorane
(100 uglkg; ip) 4 h prior to sacrifice. Controls were injected with 0.9% saline
vehicle (3 ul/rat; icv) 14 h prior to sacrifice and with either quinelorane (V; 100
uglkg; ip) or 0.9% saline (V; 1 mllkg; ip) 4 h prior to sacrifice. Columns represent
the means and vertical lines + 1 SEM from 6—8 animals. Abbreviations: AS,
antisense; NS, nonsense; S, sense; V, vehicle.

218

 

 

VL-ARC

805
70~
60—

50*

 

 

 

 

40-

30-

20-

 

10-

# of Neurons Containing TH mRNA
H

 

 

 

 

 

 

 

 

 

 

O r .
lvrxsJ Lv Assnsl

Vehicle Quinelorane

 

 

 

 

 

Figure 7.16 Effects of c-fos antisense, sense, and nonsense oligonucleotides on
the numbers of neurons expressing TH mRNA in the rostral, middle, and caudal
regions of the VL-ARC in quinelorane—treated rats. Rats were injected with c-fos
antisense (AS; 40 nmol/rat; icv), c-fos sense (dark gray; 40 nmol/rat; icv) or c-fos
nonsense (light gray; 40 nmol/rat; icv) 14 h prior to sacrifice and with quinelorane
(100 ug/kg; ip) 4 h prior to sacrifice. Controls were injected with 0.9% saline
vehicle (3 ul/rat; icv) 14 h prior to sacrifice and with either quinelorane (V; 100
jig/kg; ip) or saline (V; 1 mllkg; ip) 4 h prior to sacrifice. Columns represent the
means and vertical lines + 1 SEM from 6-8 animals. Abbreviations: AS,
antisense; NS, nonsense; S, sense; V, vehicle.

219

 

 

MZI

10*

oo
1

 

 

 

61

 

Labeling Density Ratio of
TH mRNA per Neuron

 

 

 

 

 

 

 

 

 

 

0 f I fl
j V AS I I V AS S NS 1

Vehicle Quinelorane

 

 

 

 

 

1751

150*

 

 

 

 

1 25 _ _ w
100*

75*

 

5° ‘ J

 

 

25*

 

 

 

 

 

 

 

 

 

 

 

 

 

# of Neurons Containing TH mRNA

1 V AS I l V AS 3 NS
Vehicle Quinelorane

 

Figure 7.17 Effects of c-fos antisense, sense, and nonsense oligonucleotides on
the numbers of neurons expressing TH mRNA (Bottom Panel) and the labeling
density ratio of TH mRNA of these neurons (Top Panel) in the MZI in
quinelorane-treated rats. Rats were injected with c—fos antisense (AS; 40
nmol/rat; icv), c-fos sense (dark gray; 40 nmol/rat; icv) or c—fos nonsense (light
gray; 40 nmol/rat; icv) 14 h prior to sacrifice and with quinelorane (100 uglkg; ip)
4 h prior to sacrifice. Controls were injected with 0.9% saline vehicle (3 ullrat; icv)
14 h prior to sacrifice and with either quinelorane (V; 100 uglkg; ip) or saline (V; 1
mllkg; ip) 4 h prior to sacrifice. Columns represent the means and vertical lines +
1 SEM from 6-8 animals. Abbreviations: AS, antisense; NS, nonsense; S, sense;
V, vehicle.

220

 

 

DISCUSSION

These studies reveal that Fos mediates, at least in part, D2 receptor
activation of TH expression in TIDA neurons. Indeed, a single icv injection of c-
fos antisense is capable of blocking Fos protein expression in quinelorane-
stimulated TIDA neurons of the DM-ARC in a time-dependent manner. These
results establish that a single injection of 40 nmol of antisense oligonucleotide
into the lateral ventricle is concentrated enough to travel in the cerebrospinal fluid
to the third ventricle and penetrate into the adjacent DM-ARC. This is consistent
with other studies demonstrating that single injection of phosphorothioated
antisense (identical to that employed here) follows bulk flow from the lateral
ventricle through to the third and fourth ventricle finally to the subarachnoid
spaces (Yaida and Nowak, 1995).

These results are consistent with a previous report showing that
oligonucleotides can penetrate 1 to 2 mm into the tissue surrounding the lateral
ventricles and approximately 1 mm into the tissue surrounding the third and
fourth ventricle (Grzanna et al., 1998). This is in contrast to studies utilizing
phosphodiester (or unmodified) oligonucleotides that are subject to rapid
degradation by exonucleases and do not remain in the tissue long enough to
exert anti-translational effects (Szklarczyk and Kaczmarek, 1995; Yaida and
Nowak, 1995; Broberger et al., 2000). The phosphorothioated (or fully modified)
antisense has a modified backbone; the oxygen atoms in the phosphodiester
bond between nucleotides are replaced with sulfur atoms which impart a greater

stability in biological fluids (Phillips and Gyurko, 1995). The half life of a 15—mer

221

 

m

 

 

phosphorothioated oligonucleotide probe can be up to 19 h in vitro in CSF
(Campbell et al., 1990).

The results of the present study demonstrate that for c-fos antisense to
have its anti-translational effects it must be present for at least six hours. This is
consistent with previous reports demonstrating that c-fos antisense is capable of
blocking Fos protein expression between 6 to 18 h after its administration
(Chiasson et al., 1992; Dragunow et al., 1993; Hooper et al., 1994; Liu et al.,
1994; Grzanna et al., 1998; Morrow et al., 1999). This delayed action of
antisense suggests that it takes at least 6 h for the antisense oligonucleotide to
diffuse through tissue, internalize within neurons, and hybridize to the target
mRNA (Hooper et al., 1994; Liu et al., 1994; Phillips and Gyurko, 1995; Yaida
and Nowak, 1995; Grzanna et al., 1998; Cui et al., 1999). Although the precise
mechanism of how antisense gets into the cell is not known, it is believed to enter
through fluid phase pinocytosis and/or absorptive endocytosis (Loke et al., 1989;
Phillips and Gyurko, 1995; Myers and Dean, 2000). There is also evidence that
a membrane-bound receptor may internalize antisense when present in low
concentrations (Yakubov et al., 1989). This has been supported by the
identification of 30 kDA, 66 kDA and 80 kDA proteins that mediate the uptake of
oligonucleotides (Miller and Das, 1998; de Diesbach et al., 2000).

The antisense oligonucleotide probe used in these experiments was
phosphorothioated to increase its stability. Some studies have reported side
effects in animals following the use of modified oligonucleotides including

lethargy, piloerection and frequent urination (Cirelli et al., 1995; Schobitz et al.,

222

 

1997; Ho et al., 1998). In addition, this modification confers a charge to the
molecule that may cause it to interact with proteins in a nonselective fashion
(Perez et al., 1994; Szklarczyk and Kaczmarek, 1999; Eckstein, 2000).
Accordingly, an experiment was carried out to determine if the anti-translational
effects of c-fos antisense were indeed specific, and this was tested by using c-fos
“sense” and “nonsense” oligonucleotides. The c-fos “sense” oligonucleotide has
the same sequence of nucleotides found in the segment of targeted c-fos mRNA.
There are no base pairs that it can bind with so this oligonucleotide does not
prevent Fos expression (Phillip and Gyurko, 1995; Pilowsky and Suzuki, 1994).
The 0405 “nonsense” oligonucleotide is a jumbled sequence that has the same
base composition as the antisense oligonucleotide, but has no homology to any
known mRNA and likewise has no effect on Fos expression (Pilowsky and
Suzuki, 1994). The three oligonucleotide sequences were also run through
GenBank,® an NIH genetic sequence database, to determine if the sequence of
c—fos antisense was specific for the c-fos gene (and not fosb, fra1 or fraZ) of the
rat, and that the c—fos “sense” and “nonsense” sequences were not found on any
other mRNA.

The results from this experiment demonstrate that the effect of c-fos
antisense is specific. Antisense was able to attenuate quinelorane-induced Fos
expression in TH-IR neurons in the DM-ARC, whereas “sense” and “nonsense”
oligonucleotides were ineffective. These results also confirm and coincide with
other studies (Cirelli et al., 1995) that the behavioral effects (lethargy, piloerection

and urination) are specific to the antisense and not due to the phosphorothioation

223

 

or nonspecific protein binding, for the animals receiving the “sense” and
“nonsense” probes do not exhibit these behavioral effects.

It is important to note that throughout the antisense experiments the
numbers of neurons in each region of the DM-ARC remained constant which is
consistent with the conclusion that the effects of phosphorothioated c-fos
antisense is not toxic or damaging to these neurons. Thus, the decrease in the
expression of Fos caused by this oligonucleotide is not due to destruction of
neurons expressing Fos, but rather to a true blockade of protein synthesis.

Many studies have been carried out using antisense probes to block
immediate early genes, neuropeptides, as well as receptors. The link between
immediate early genes and long term genes has been insinuated, but studies
demonstrating this are few. With the route of administration and the time course
ascertained for employing c-fos antisense to block D2 receptor stimulation of Fos
protein synthesis, the next step was to establish a link between Fos and TH gene
expression. lf Fos mediates the stimulatory effects of D2 receptor activation on
TH mRNA, then c-fos antisense should prevent this increase in TH mRNA.

The results of this study reveal this is the case. Fos antisense blocks
quinelorane-induced activation of TH mRNA expression in neurons in the DM-
ARC. Evidently, antisense blocks the translation of Fos protein, and there is no
Fos to dimerize with Jun protein, resulting in decreased binding of the dimer at
the AP-1 site on the TH promoter. These results also suggest that other proteins
of the Fos family such as FosB, FRA1 or FRA2 are not part of the dimerization.

The c-fos antisense is specific for the translation start site of c-fos mRNA and

224

 

 

does not bind to fosB, fra1 or fra2 mRNA (GenBank,®). lf FosB, FRA1 or FRA2
were involved in DZ receptor activation of TH mRNA expression, then
accelerated translation of these proteins and dimerization with Jun would activate
TH mRNA expression even in the presence of c—fos antisense. By eliminating
Fos expression, TH mRNA does not increase following quinelorane
administration. This is a specific action of the antisense since c—fos “sense” and
“nonsense” probes do not alter the expression of TH mRNA. The
oligonucleotides and phosphorothioation are not toxic or destructive to the cells
containing TH mRNA, for there are no changes in their numbers.

Antisense alone has no effect on the levels of TH mRNA being expressed
by neurons in the DM-ARC revealing that blockade of constitutively expressed
Fos has no impact on basal TH gene expression. This implies that other
transcription factors may be involved. It has been demonstrated that the CRE
site and SP-1 site on the proximal TH gene promoter are necessary for the basal
expression of TH mRNA in neuroblastoma cell lines (Kim et al., 1993; Yang et
al., 1998). Studies in PC-18 cells (rat pheochromocytoma cells derived from PC-
12 cells) have revealed that induction of Fos is sufficient to stimulate the TH gene
transcription rate and that this is in part dependent on the AP1 site within the TH
proximal promoter (Sun and Tank, 2002). Taken together, these results suggest
that the induction of Fos is necessary for the increase in TH mRNA following DZ
receptor activation of TIDA neurons, but the AP-1 may not play a prominent role

in promoting basal levels of TH mRNA synthesis. Thus, it can be hypothesized

225

 

 

that an antisense to CREB or SP-1 may very well decrease the basal expression
of TH mRNA in TIDA neurons.

While various studies have been carried out using c—fos antisense to study
the effects of Fos protein expression, only a few studies have examined
antisense effects on other markers in vivo. c-fos antisense is capable of blocking
behaviors associated with fear and anxiety (Moller et al., 1994) as well as
preventing alcohol tolerance (Szabo et al., 1996). One study demonstrated the
ability of c-fos antisense to block haloperidol-stimulated neurotensin mRNA
induction in the striatum and ischemia-induced nerve growth factor mRNA
stimulation in the dentate gyrus, indicating a role of Fos in the regulation of
neurotensin gene and nerve growth factor, respectively (Merchant, 1994; Cui et
al., 1999). However, this is the first time that a link has been established
between Fos and the TH gene in vivo.

c—fos antisense has no effect on Fos expression in “DOPA-ergic” neurons
of the VL-ARC. One possibility could be that the antisense does not diffuse into
the VL-ARC. However, oligonucleotides have been shown to diffuse
approximately 1 mm into tissue from the third ventricle (Yaida and Nowak, 1995;
Grzanna et al., 1998), so it seems unlikely that the oligonucleotide failed to
penetrate into the VL-ARC.

In the time course study, the percentage of neurons TH-IR neurons
expressing Fos in the control group is only 3%, much lower than in previous
experiments (which have ranged from 15-30%). The actual numbers of TH-IR

neurons in each rostrocaudal region is similar to what has been reported in

226

previous experiments, so the lower percentage of Fos-IR is not due to a lack of
neurons present. Quinelorane has no stimulatory effect on Fos expression in the
VL-ARC, which correlates with the results shown in Chapters 4 and 5.

In the specificity study, the expression of Fos in the VL-ARC in the control
group is consistent with previous studies. In this experiment, quinelorane
significantly decreases the percentage of TH-IR neurons expressing Fos in the
middle and caudal regions of the VL-ARC. ln rostral region there is a decrease
in the percentage of Fos expression, but it is not significant most likely due to
variability. Interestingly, antisense had no further effect on the expression of Fos
in these neurons. The percentage of TH-IR neurons expressing Fos following
the administration of c-fos antisense was still between 10 and 15%, similar to that
determined after quinelorane administration. Although it would seem logical to
anticipate that antisense would further decrease Fos expression, it is possible
that the inhibitory effect of quinelorane attenuated the transcription of c-fos
mRNA sufficiently that antisense had very little c-fos mRNA to react with. As in
the DM-ARC, there are no changes in the numbers of TH-IR neurons in the VL-
ARC indicating that antisense, “sense” and “nonsense” have no deleterious effect
on the viability of the “DOPA—ergic neurons” of the VL-ARC.

The levels of TH mRNA in VL-ARC “DOPA-ergic” neurons were
unchanged regardless of the treatment. These neurons are not regulated by DZ
receptors, thus the lack of a stimulatory effect of quinelorane on TH mRNA is
consistent with the results presented in Chapter 6. It is interesting to note that

when quinelorane decreases Fos expression in the VL-ARC (though this effect is

227

 

rims..."

 

variable), there is no corresponding change in TH mRNA. It is possible that if the
CRE and SP-1 sites on the TH gene promoter are involved in the basal levels of
TH mRNA expression, then decreased levels of Fos would not have an affect on
gene expression.

In the MZI, the overall percentage of Fos expression in TH—IR neurons in
the control groups is low. Moreover, antisense administration had no effect on
Fos expression in these neurons. One reason could be that the antisense failed
to diffuse to the MZI in adequate amounts. Icv injections can diffuse about 1-2
mm from the ventricles (Yaida and Nowak, 1995; Grzanna et al., 1998), but the
MZI is greater than 2 mm from the lateral and third ventricles. Also, similar to the
VL-ARC, basal Fos expression may be so low that the c-fos antisense has no or
very little c-fos mRNA to bind to, thus no appreciable difference could be
determined in Fos protein expression. The numbers of lHDA neurons in the MZI
remain the same indicating that if the oligonucleotide probe diffused to the MZI
then it has no effect on the viability of these DA neurons.

As in the VL-ARC, TH mRNA expression remained unaffected in the lHDA
neurons of the MZI following Fos antisense administration. The labeling density
ratio of TH mRNA per cell is greater in the MZI compared to the VL-ARC
indicating that the basal level of TH mRNA content is greater in the MZI. This
correlates with the staining of the neurons in the immunohistochemistry studies;
the greater the intensity of the staining, the greater the protein content (Hoffman
and Murphy, 2000). In these studies, the TH immunostaining in the MZI is darker

than in the VL-ARC. While the MZI has greater basal levels of TH mRNA,

228

 

 

antisense alone was without effect. This indicates that Fos does not play a role
in the basal expression of TH gene expression, implying that other transcription
factors are involved such as CREB or SP-1.

In conclusion, these experiments reveal that c-fos antisense specifically
prevents the DZ receptor-mediated stimulation of Fos and TH gene expression in
TIDA neurons of the DM-ARC, but not the VL-ARC. With the use of c—fos
antisense, a link has been established between the immediate early gene Fos
and the long term gene TH. These results also reveal that the AP-1 site is not
involved with the basal expression of TH mRNA in the ARC or in lHDA neurons

of the MZI.

229

CHAPTER EIGHT

GENERAL SUMMARY

This body of work was aimed at determining if an acute increase in
neuronal activity caused by stimulation of DZ receptors was associated with the
regulation of gene expression in TIDA neurons. To this end, studies herein
detailed the sexual differences in the distribution of TIDA neurons and basal level
of FRA expression in subdivisions of the ARC, the temporal effects of DZ
receptor stimulation on immediate early gene expression in TIDA neurons, the
role of kappa opioid receptors in D2 receptor regulation, the temporal effects of
DZ receptor stimulation on TH gene expression and the role of Fos in TH gene

expression. The major points from these studies are summarized below.

DM-ARC

There are no sexual differences in the numbers of TH-IR neurons located
within the rostral-caudal extent of the DM-ARC, but the number of TH-IR neurons
in both females and males is higher in the rostral than in either the middle or
caudal regions. The percentage of TH-IR neurons containing F RA-IR nuclei was
2-3 times higher in females versus males only in the rostral DM-ARC.

A single injection of DZ receptor agonist quinelorane causes a delayed (2
h) and transient (through 4 h) increase in the percentage of TH-IR neurons
expressing Fos throughout the rostral-caudal extent of the DM-ARC. The

blockade of D2 receptors with the antagonist raclopride has no effect per se, but

230

 

reverses the stimulatory effects of quinelorane. These results reveal that DZ
receptors regulate Fos expression in TIDA neurons in the DM-ARC.

Blockade of kappa opioid receptors with the antagonist nor-BNl increases
the percentage of TH-IR neurons expressing Fos in a delayed and transient
manner similar to quinelorane throughout the rostral-caudal extent of the DM-
ARC. Activation of kappa opioid receptors with the agonist USO-488 reverses the
stimulatory effects of nor-BNI, indicating that kappa opioid receptors regulate Fos
expression. U50—488 also blocks the stimulatory effects of quinelorane on
percentage of TH-IR neurons expressing Fos in the DM-ARC. The results
suggest that kappa opioid receptors mediate D2 receptor regulation of Fos
expression.

Quinelorane significantly increases the labeling density ratio of TH mRNA
per neuron in the DM-ARC at 4 h and remains elevated for at least 24 h, but has
no effect on the numbers of neurons containing TH mRNA at any timepoint.
Blockade of DZ receptors with raclopride has no effect per se, but reverses the
stimulatory effects of quinelorane on TH mRNA expression. c—fos antisense,
which specifically blocks Fos expression, prevents quinelorane-induced
increases in the labeling density ratio of TH mRNA per neuron in the DM-ARC.
The c-fos “sense” and “nonsense” oligonucleotide probes have no effect. There
is no change in the numbers of neurons that contain TH mRNA in the DM-ARC
following any of the treatments. These results reveal that DZ receptors regulate
TH gene expression in TIDA neurons in the DM-ARC and that Fos, at least in

part, mediates this activation of TH gene expression.

231

 

 

VL-ARC

There are no sexual differences in the numbers of TH-IR neurons in the
rostral VL-ARC, but the number of TH-IR neurons in the middle and caudal VL-
ARC were higher in males than in females. There were no sexual differences in
the percentage of TH-IR neurons containing F RA-lR nuclei.

While there is no effect 2 h following quinelorane administration, there is a
decrease at 4h in the percentage of TH-IR neurons expressing Fos throughout
the rostral-caudal extent of the VL—ARC. Blockade of DZ receptors with
raclopride has no significant effect on Fos expression. These results suggest
that “DOPA-ergic” neurons of the VL-ARC are not regulated by DZ receptors.

In contrast to the DM-ARC, activation of kappa opioid receptors with U50-
488 significantly decreases the percentage of TH-IR neurons containing Fos-IR
nuclei throughout the rostral-caudal extent of the VL-ARC. While nor-BNI has
no effect per se, it reverses the inhibitory effects of USO-488. These results
indicate that while kappa opioid receptors regulate Fos expression, it is not under
basal conditions as seen in the DM—ARC.

Neither quinelorane nor raclopride has an effect on the labeling density
ratio of TH mRNA per neuron in the VL-ARC, indicating that DZ receptors do not
regulate TH gene expression in “DOPA-ergic” neurons. c-fos antisense, “sense,”
and “nonsense“ oligonucleotide probes have no effect on the labeling density
ratio or on the numbers of neurons containing TH mRNA in the VL-ARC. These

results reveal that D2 receptors do not regulate TH gene expression in “DOPA-

232

ergic” neurons in the VL-ARC and Fos does not play a role in TH gene

expression.

MZI

There is no sexual difference in the numbers of TH-IR neurons or in the
percentage of those neurons expressing F RA in the MZI.

As in the VL-ARC, quinelorane has no effect at 2 h; however, it does
decrease the percentage of TH-lR neurons containing Fos-IR nuclei at 8 h.
Blockade of DZ receptors with raclopride increases the percentage of TH-IR
neurons containing Fos-IR nuclei in the MZI, and this is reversed by the
administration of quinelorane. These studies demonstrate that Fos expression in
lHDA neurons of the MZI are regulated by DZ autoreceptors.

In contrast to the ARC, neither activation nor blockade of kappa opioid
receptors has any effect on the percentage of TH-IR neurons containing Fos-IR
nuclei, indicating that kappa opioid receptors do not regulate Fos expression in
the lHDA neurons of the MZI.

DZ receptors do not regulate TH gene expression in lHDA neurons of the
MZI, as demonstrated by the lack of effect of raclopride and quinelorane on the
labeling density ratio of TH mRNA. c—fos antisense, “sense,” and “nonsense”
oligonucleotide probes have no effect on the labeling density ratio in the MZI
suggesting that Fos does not play a role in the basal expression of TH gene

expression.

233

 

CONCLUDING DISCUSSION

DM-ARC

To review, DZ receptors are inhibitory and systemic administration of
selective DZ agonists decreases neuronal activity, including DA neuronal
systems regulated by DA autoreceptors or feedback neuronal circuits (Foreman
et al., 1989; Eaton et al., 1994a). TIDA neurons are unique in that activation of
DZ receptors stimulates TI DA neuronal activity (Berry and Gudelsky, 1991; Eaton
et al., 1993). This response is independent of PRL and is instead mediated
through an afferent neuronal system involving the inhibition of tonically active
dynorphin via kappa opioid receptors (Durham et al., 1996). The goal of these
studies was to determine if this mechanism is involved in the regulation of gene
expression in TIDA neurons.

For well over a decade, inducible transcription factors have been studied
and utilized as a tool for mapping neuronal activity. Under basal conditions Fos
expression is low, but is induced following transsynaptic activation. Immediate
early genes are stimulated within minutes to hours through depolarization or
ligand-mediated activation of membrane receptors located on neuronal cell
bodies or dendrites. This triggers a second messenger system and
phosphorylation cascade involving protein kinases and constitutive transcription
factors (Sheng and Greenberg, 1990; Sim et al., 1994). The constitutive
transcription factors induce the transcription and translation of immediate early

genes. The immediate early genes then bind to their DNA binding elements to

234

 

 

regulate transcription and translation of long-term genes (Gizang-Ginsberg and
Ziff, 1990; Icard-Liepkalns et al., 1992).

The studies described in this dissertation demonstrated that activation of
D2 receptors increases Fos expression in TIDA neurons throughout the rostral-
caudal extent of the DM-ARC. This is mediated through the release of inhibition
from tonically active dynorphin neurons via kappa opioid receptors. The origin of
the DA and the location of the DZ receptor-expressing target cells have yet to be
determined. From the available evidence described earlier, it has been
suggested that the DZ receptors are located outside the mediobasal
hypothalamus (Durham et al., 1996). The dorsomedial hypothalamic nucleus
may be one area of the hypothalamus that may be involved in the afferent
neuronal pathway. Electrical stimulation in this area increases TIDA neuronal
activity, mimicking the effect of quinelorane (Gunnet and Moore, 1988). Within
the dorsomedial nucleus are extensive dendritic processes from lHDA neurons of
the MZI (Chan-Palay et al., 1984), thus the MZI may be the origin of the DA.
Areas of the brain to be considered for the location of the D2 receptor-expressing
target cell are the paraventricular nucleus, the horizontal diagonal band, and the
central amygdala, the three areas to which the lHDA neurons project (Eaton, et
al., 1994; Wagner et al., 1995).

The neurochemical identity of the target cell and its receptors on
dynorphin neurons has yet to be elucidated. One characteristic that is required
of this upstream neuron is the ability to mimic the effect of dynorphin on TIDA

neurons. In addition, it is most likely a stimulatory neuron given that DZ

235

 

—
Fm 'A .4"
.1

 

receptors are inhibitory and activation of these receptors would most likely “turn
off” a stimulatory neuronal system. Neurotensin is a neuropeptide that
stimulates, rather than inhibits, TIDA neurons and is involved in mediating the
effects of PRL on the activation of these neurons (Hentschel et al., 1998). GABA
(acting at GABAA receptors; Wagner et al., 1994a) and glutamate (acting at
AMPA receptors; Wagner et al., 1994b) tonically inhibit TIDA neurons. However,
GABA is an inhibitory neurotransmitter and would inhibit dynorphin neurons
under basal conditions resulting in increased activity of TIDA neurons. Blockade
of AMPA receptors increases the activity of TIDA neurons, mimicking the effect
of quinelorane; however, this mechanism involves GABAA receptors and not
kappa opioid receptors (Wagner et al., 1994c).

The blockade of DZ receptors had no effect on Fos expression suggesting
a lack of tonic activation of these receptors. This might suggest that the DA
pathway that serves as a source of DA for these receptors may be active only
during specific situations (e.g. during different endocrinological states, stress,
etc). Alternatively, these DZ receptors may not be activated by endogenous DA,
but respond only to exogenbusly administered DA agonists. There is precedent
for this; for example, the activation of DA receptors in the area postrema.

Dynorphin neurons are located in the hypothalamus including the
ventromedial nucleus and ARC as demonstrated by microscopic analysis and
immunoassay studies and are concentrated around TIDA neurons (Zamir et al.,
1983 and 1984; Fitzsimmons et al., 1992; Durham, 1999). To help determine the

possible location of the unknown projection neurons future studies could include

236

 

 

retrograde tract tracing studies in conjunction with immunohistochemistry or in
situ hybridization. Dynorphin neurons located in the DM-ARC could be identified
using immunohistochemical or in situ hybridization techniques. The tracer could
be injected into the DM-ARC to identify nerve terminals clustered around the
dynorphin neurons and then examine where these projections to the DM-ARC
originate. If a specific area or nucleus of the brain could be identified, this may
assist in determining the identity of the interneuron. Immunohistochemical
studies could then be carried out using appropriate agonists and antagonists to
determine if that neurotransmitter mimics the effect of dynorphin on Fos
expression in TIDA neurons.

Dynorphin acts at kappa opioid receptors, which have been identified in
the ARC. Kappa opioid receptors are linked to Gi/o protein, inhibiting CAMP
(Childers, 1991; Childers et al., 1998b, Takekoski et al., 2000). If the theoretical
pathway is correct, under basal conditions the interneuron stimulates dynorphin
release, which acting at kappa opioid receptors inhibits TIDA neuronal activity
and the expression of Fos. Activation of DZ receptors would inhibit the
interneuron, thus releasing the inhibitory action of dynorphin on TIDA neuronal
activity and on Fos expression. The lack of dynorphin at kappa opioid receptors
may lead to increases in the CAMP and begin the second messenger cascade
eventually leading to an increase in the transcription and translation of Fos.

In addition to a correlation between TIDA neuronal activity and immediate
early gene expression, there is also a correlation between neuronal activity and

TH mRNA expression (Arbogast and Voogt, 1991; Wang et al., 1993; Berghom

237

 

 

et al., 2001). Acute increases in neuronal activity caused by activation of DZ
receptors were associated with increases in the expression of the TH gene in
TIDA neurons. Follow up studies could determine if kappa opioid receptors
mediate D2 receptor regulation of TH gene expression using in situ hybridization.
If this is true, blockade of kappa opioid receptors should increase TH gene
expression mimicking the effects of quinelorane, and activation of kappa
receptors should reverse the stimulatory effects of quinelorane.

The expression of TH is delayed, occurring after the induction of TIDA
neuronal activity and the expression of Fos. Following the induction and
synthesis of Fos protein, it dimerizes with a Jun protein and binds to the AP-1
site (Curran and Franza, 1988; Angel and Karin, 1991) located within the TH
gene promoter of DA neurons (Biguet et al., 1991; Icard-Liepkalns, et al., 1992;
Kumer and Vrana, 1996). Thus, the AP-l site regulates the transcription and
translation of TH.

The last set of experiments for this dissertation utilizing c—fos antisense
determined that Fos, at least in part, mediates D2 receptor activation of TH
expression in TIDA neurons. c—fos antisense blocks the translation of Fos protein
resulting in a lack of Fos to dimerize with Jun and decreases binding of the dimer
at the AP-1 site on the TH promoter. c—fos antisense alone has no effect on TH
gene expression implying that the AP-1 site does not play a major role in the
basal expression of TH. While there is some expression of Fos under basal
conditions that presumably binds to the AP-1 site with a Jun protein, other factors

may be needed to induce TH gene transcription. The E box is adjacent to the

238

 

 

AP-1 site and is believed to be involved with cell specificity (Papanikolaou and
Sabban, 2000). It is possible this site and/or other modulators or enhancers
must be present in addition to the binding of Fos/Jun dimers for AP-1 to induce
transcription of TH in this system. The Hept and Oct sites on the TH gene
promoter are repressor sites specificity (Papanikolaou and Sabban, 2000).
Under basal conditions, these or other repressor sites may interfere with the
binding of Fos to the AP—1 site. Studies have demonstrated that the CRE and
SP-1 sites on the proximal TH gene promoter are necessary for the basal
expression of TH (Kim et al., 1993; Yang et al., 1998). Additional studies could
examine the effects of a CREB or SP-1 antisense on the expression of TH. If
these binding sites are involved in the basal regulation of TH expression, then an
antisense to their respective binding proteins should decrease the basal
expression of TH.

This pathway of DZ receptor-mediated activity of TIDA neurons is
separate from the PRL pathway. First, coadministration of exogenous PRL and
quinelorane has an additive effect on TIDA neuronal activity (Durham et al.,
1996). Indeed PRL increases FRA expression in a time dependent manner
similar to the time course following the activation of D2 receptors (Hentschel et
al., 2000b). However, TIDA neuronal activity increases 6 — 12 h following PRL
administration (Hentschel et al., 2000a), but only 30 min following the activation
of D2 receptors (Eaton et al., 1993). Thus FRA expression precedes the
increases in PRL—induced TIDA neuronal activity, while Fos expression occurs

after quinelorane-induced neuronal activity.

239

 

 

 

VL-ARC

The neurons of the VL-ARC do not contain DD and thus can not
synthesize DA, but only the precursor DOPA. These neurons are not true TIDA
neurons as in the DM-ARC, but can be considered “DOPA-ergic” neurons.
Unlike the DM-ARC, DZ receptors do not regulate Fos expression in “DOPA-
ergic” neurons in the VL-ARC. Fos expression may instead be regulated by
PRL. The VL-ARC contains PRL receptors (Bakowska and Morrell, 1997) and
administration of exogenous PRL increases FRA expression in a time dependent
manner (Hentschel et al., 2000). The function of “DOPA-ergic” neurons is not
completely understood, but DOPA may be released into the portal blood system
where it is converted to DA by DD found in the walls of blood vessels (Meister et
al., 1988; Misu et al., 1996).

Fos expression in “DOPA-ergic” neurons is regulated by kappa opioid
receptors but this is not present under basal conditions. While there is evidence
of dynorphin neurons and nerve terminals being present in the ARC, dynorphin
neurons are also present in the ventromedial hypothalamic nucleus.
Ventromedial nucleus dynorphin neurons are not regulated by DZ receptors
(Durham, 1999) so it is possible that the “DOPA-ergic” neurons of the VL-ARC
are regulated by them. Future studies could include tract tracing studies to
determine the hypothalamic nucleus of origin of the dynorphin nerve terminals
found in the ARC in conjunction with immunohistochemical labeling of the

dynorphin neurons and “DOPA-ergic” neurons.

240

 

As with Fos expression, TH gene expression in “DOPA-ergic” neurons
also is not regulated by DZ receptors. What is interesting to note is the levels of
TH mRNA remain relatively constant and are not greater than in the DM-ARC in
light of the fact that Fos expression is greater in the VL-ARC under basal
conditions and decreases following D2 receptor activation. Fos does not appear
to have an effect on the level of TH mRNA in these neurons. “DOPA-ergic”
neurons co—express TH and neuropeptides (Chronwall 1985; Ershov et al.,
2002). The AP-1 site is found on other proximal gene promoter in addition to the
TH gene. It is possible that Fos may bind to an AP—1 site located on a promoter
of a neuropeptide gene coexpressed in “DOPA-ergic” neurons.

c-fos antisense has no effect on Fos or TH gene expression in
quinelorane—treated “DOPA-ergic” neurons. In these studies, activation of D2
receptors resulted in decreases in Fos expression at 2 h. Although it would
seem logical that antisense would further decrease Fos expression, it is possible
that the inhibitory effect of quinelorane attenuated the transcription of c-fos

mRNA sufficiently that antisense had very little c—fos mRNA to hybridize to.

MZI

The lHDA neurons of the MZI are regulated by DA receptors as are the
DA neurons of the mesotelencephalic systems (Lookingland and Moore, 1984).
DZ receptor agonists decrease while antagonists increase DA neuronal activity
(Lookingland and Moore, 1995; Eaton et al., 1992). The studies in this

dissertation demonstrate that D2 receptors agonists are without effect, but D2

241

antagonists increase Fos expression in lHDA neurons. The D2 receptors are
most likely located on lHDA cell bodies and/or dendrites as opposed to axon
terminals, for antidromic stimulation of hypothalamic neurons does not induce
immediate early gene expression (Icard-Liepkalns et al., 1992; Luckman et al.,
1994; Hoffman and Murphy, 2000).

Although Fos expression is regulated by acute activation of D2 receptors,
TH gene expression is not. Various studies in the mesolimbic and nigrostriatal
DA systems have demonstrated that chronic continuous infusions, but not
intermittent injections of DA agonists inhibit TH mRNA expression (Sirinathsinghji
et al., 1994; Iwata et al., 2000). Chronic infusion of apomorphine a very short
acting DA agonist works at post-synaptic receptors via a long-loop feedback
circuit to exert negative feedback on the DA neurons (Roth and Elsworth, 1995).
This continuous negative feedback can lead to decreases in TH mRNA. While
these studies were carried out in the mesolimbic and nigrostriatal DA systems, it
is logical to anticipate the same is for the lHDA neurons. To test this hypothesis,
quinelorane, a long acting DA agonist, could be administered to assess the effect
on TH gene expression in lHDA neurons. If this hypothesis is correct, chronic
injections of quinelorane should decrease the expression of TH mRNA. A single
injection of DA agonist or antagonist is capable to altering DA neuronal activity in
the lHDA neurons of the MZI through pre-synaptic autoreceptors (Lookingland
and Moore, 1984); however, TH mRNA is not affected by acute actions because
antidromic stimulation (i.e. stimulation at pre-synaptic autoreceptors) does not

effect gene expression (Icard-Liepkalns et al., 1992).

242

The results of these final studies demonstrate that Fos does not play a
role in TH gene expression in lHDA neurons following the acute activation of DZ
receptors. There are several possible reasons for this. First acute activation or
blockade of D2 receptors does not alter TH gene expression. Secondly, the
expression of Fos is low in lHDA neurons under basal conditions and following
quinelorane administration thus no appreciable difference could be determined in
Fos expression following c-fos antisense administration. To determine if Fos
plays a role in TH gene expression, the lHDA neurons would first need to be
stimulated with a D2 receptor antagonist to stimulate Fos. To ensure changes in
the TH gene expression chronic injections of the antagonist may not increases
Fos expression but increases FRA-1 or FRA-2 expression (which are induced
following chronic stimulation). Thus even under the proper conditions, Fos may
not mediate the expression of the TH gene.

In conclusion, the results of studies described in this thesis demonstrate
that DZ receptors regulate the expression of the immediate early gene Fos in
TIDA neurons of the DM-ARC. This is mediated through the release of inhibition
of tonically active dynorphin neurons via kappa opioid receptors It is believed
that the removal of the inhibitory effect by dynorphin on adenylate cyclase and
cAMP is a potent enough stimulus to induce Fos expression. In addition to
regulation of Fos, stimulation of D2 receptors also leads to increases in the
expression of TH mRNA. Fos, at least in part, plays a role in the regulation of TH
gene expression, a gene that is essential to maintain DA synthesis and PRL

inhibitory function of these TIDA neurons.

243

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