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DOPAMINE RECEPTOR-MEDIATED REGULATION OF
TUBEROINFUNDIBULAR DOPAMINERGIC NEURONS

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Robert Alan Durham

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DOPAMINE RECEPTOR-MEDIATED REGULATION OF
TUBEROINFUNDIBULAR DOPAMINERGIC NEURONS

By

Robert Alan Durham

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
Neuroscience Program

1999

ABSTRACT

DOPAMINE RECEPTOR-MEDIATED REGULATION OF
TUBEROINFUNDIBULAR DOPAMINERGIC NEURONS

By

Robert Alan Durham

Although it has long been recognized that neurotransmission in most dopaminergic
(DA) neurons in the brain is subject to DA autoreceptor-mediated negative feedback,
tuberoinfundibular (TDDA neurons in the mediobasal hypothalamus are not responsive to this
mode of DA receptor-mediated regulation. This assertion is supported by numerous studies in
which DA agonists and antagonists that affect autoreceptor-mediated processes in other DA
neurons have no effect on TIDA neurons. The development of DA drugs with greater
selectivity for the various DA receptors has revealed that TIDA neurons are subject to DA
receptor-mediated regulation, but that this is different in many important ways from that
mediated by autoreceptors in other DA neurons. In contrast to autoreceptor-mediated
inhibition of neuronal activity, the selective DA D2/3 agonist quinelorane stimulates TIDA
neurons. Studies described in this dissertation have utilized a combination of molecular,
neurochemical, neuroanatomical and pharmacological experimental approaches to characterize
DA receptor-mediated regulation of TIDA neurons.

The results. reveal that quinelorane-induced activation of TIDA neurons is mimicked
by the D2 receptor agonist PNU95666, but not by the D3 agonist PD128907. Unlike the
activation of TIDA neurons by prolactin, this D2 receptor-mediated activation is dependent

upon afferent neuronal input to the mediobasal hypothalamus. Although neurotensin and the

delta opioid or opioid-1 (delta/0P1) receptor agonist DPDPE can acutely activate these
neurons, quinelorane-induced activation of TIDA neurons is not dependent on either
neurotensin- or delta/OPl-receptor activation. A role for kappa opioid or opioid-2
(kappa/0P2) receptors was demonstrated by experiments that revealed that quinelorane-
induced activation of TIDA neuron is prevented by pre-treatment with the kappa/0P2
antagonist norbinaltorphimine (nor-BN1) and reversed by administration of the kappa/0P2
receptor agonist U-50488. In addition, in Jim mRNA hybridization studies demonstrated that
quinelorane reduces the expression of prodynorphin mRN A in the arcuate nucleus. This
reduction in dynorphin messenger RNA taken together with the pharmacological data suggests
a role for inhibition of dynorphinergic neurons in the DA receptor-mediated activation of
TIDA neurons.

Experiments utilizing selective D, receptor agonists and antagonists have revealed that
activation of D, receptors exerts an inhibitory influence on TIDA neuronal activity. This
inhibitory response has also been demonstrated to oppose the stimulatory action of D2
receptors. This finding provides an explanation for the lack of effect of non-selective agonists
and antagonists on TIDA neuronal activity. Thus, it is apparent that the activity of TIDA
neurons depends on a balance between these opposing D,/D2 receptor influences.
Accordingly, TIDA neuronal activity may serve as an important marker for the relative activity

of various pharmacological agents at D, and D2 receptors.

This work is lovingly dedicated to my wife Cathy.

"Res multae filiae congregaverunt divitias tu
supergressa es universas."

Proverbs 31 :29

iv

ACKNOWLEDGMENTS

This dissertation represents the combined efforts of many that have provided a
fertile environment in which I could develop as a scientist. First and foremost I wish to
thank Dr. Keith Lookingland who as my thesis advisor endured much and still had the
stamina to help me see this project through to completion. I deeply appreciate all the time
and energy he devoted to help me grow as a scientist. A special thanks goes to Dr. Ken
Moore who was instrumental in my having the opportunity to study pharmacology at
Michigan State University. In addition, I appreciate the time, talent and support of Drs.
Peter Cobbett and Cheryl Sisk who faithfully served on my guidance committee. I wish also
to pay tribute to Dr. Monty Piercey who served as a member of my committee prior to his
untimely death. I will always have fond memories of his enthusiasm for science, great
intellect, and the kindness he always extended to me.

Along with my professors there were several post-doctoral fellows, and students who
often lent their expertise and support. These have included Drs. Jorge Manzanares and
Misty Eaton who often gave me much of their time and patience in teaching me the ropes in
“the lab of the most loveable & agreeable investigators”. There were also the close
supportive relationships with senior students in the lab, Drs. Ed Wagner, John Goudreau,
and Annette Fleckenstein as well as my contemporaries Ken Hentschel and Yvonne Will.
Thanks to them the memories of long hard hours spent at the bench have faded and all that
remains is the fun we had and the laughter shared. A special thank-you to Erika Bronz for
providing technical assistance in many of my endeavors. Thanks also to John Johnson who
put himself at my disposal for a summer that turned into two years. He exceeded my

grandest expectations and readily became a valuable collaborator and friend.

I also want to extend my appreciation to the folks in the front office, Dianne
Hummel, Mickey Vanderlip and Nelda Carpenter who steered me through the many
administrative obstacles so I could concentrate on science.

Last but certainly not least, I need to thank my home team. Eric Wing, Dick
Latham, and their families have given me the love, support and many prayers needed to see
this project through to completion. For my wife Cathy words don’t adequately express how
thankful I am to have woman at my side who never fails to inspire. These are the people
who were cheering loudest when there didn’t seem to be anyone else in the stadium. Their
faithfulness helped me to know that even on the darkest of days the love of Jesus was always

at hand.

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

LIST OF TABLES ......................................................................................................................... X1
LIST OF FIGURES ..................................................................................................................... XII
CHAPTER ONE ........................................................................................................... 1
NEUROBIOLOGY OF DOPAMINE .................................................................................................... 1
Historical Permective 2
Neumpycbiatric Drug Diccooegy: Serendipiy to Rational Dengn 4
Dopamine receptor familie: (DI and D2) 10
Development of Selective Drug: for Subgpe: of DA Receptor: 13
Anatony and Function of DA Neuronal .S'jrtem: in the Mammalian Brain - - 18
Mesotelencephalic DA Systems ............................................................................................ 18
Hypothalamic DA Systems ................................................................................................... 23
Tubemin/undibular DA Neuron: and tbe Regulation of Prolactin Secretion ...... 28
Dopamine: Tbe Prolactin I n/n'bitog' Factor 30
Prolactin Feedback Regulation of NBA N eumnr ....... . . 32
Aflerent Modulation of'IYDA Neuronal Acting: ..... . 37
Dopamine Receptor Mediated Regulation of TIDA Neurons ........................................ 37
SPECIFIC AIMS ................................................................................................................................. 42
CHAPTER TWO ......................................................................................................... 44
GENERAL METHODS ...................................................................................................................... 44
Animalr 44
Drugs 44
Surgical Procedures 44
Intracerebroventricular Cannulation .................................................................................... 44
Orchidectomy .......................................................................................................................... 45
Neumcbemical ertimation tyneumnal acting! 46
Tissue and Blood Processing ................................................................................................. 48
High Performance Liquid Chromatography ....................................................................... 53
Radioimmunoassay for Prolactin .......................................................................................... 54

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Measurement oijnorpbin mRNA bi in ritu Hybridization Hirtocbemirtpl 56
Labeling The mRNA Probe ........................................................ 57
Extraction of the Labeled Probe .......................................................................................... 57
Preparation of Tissue ............................................................................................................. 58
In Situ Hybridization Procedure ........................................................................................... 59
Image Analysis ......................................................................................................................... 61

Dual Immunobtlrtocbemical Determination of For/ Pm and TH. ....... .. 64
Tissue Preparation .................................................................................................................. 65
Dual Immunohistochemistry ................................................................................................ 65

StatirticalAnabue: 71

CHAPTER THREE .................................................................................................... 72
D2 RECEPTORS MEDIATE THE STIMULATORY EFFECTS OF QUINELORANE ON
TUBEROINFUNDIBULAR DA NEURONS ...................................................................................... 72

Met/Jodr 74
Drugs ........................................................................................................................................ 74

Result: 75

Discum'on 86

CHAPTER FOUR -- - - ........ --92
D2 RECEPTOR-MEDIATED STIMULATION OF TIDA NEURONS IS DEPENDENT ON
AFFERENT NEURONAL INPUT TO THE MEDIOBASAL HYPOTHALAMUS ................................ 92

Metbodr 95
Deafferentation of the Mediobasal Hypothalamus ............................................................ 9S
Verification of the Deafferentation ...................................................................................... 96

Rerult: 97

Dtlramion 101

CHAPTER FIVE -- ................................................................................. 107
EVIDENCE THAT ACTIVATION OF DELTA / CPI-RECEPTORS STIMULATES TIDA
NEURONS ....................................................................................................................................... 107

Metbodr 108
Drugs ...................................................................................................................................... 108

Result: 109

Dircum'on ..... 1 20

 

 

CHAPTER SIX _ -- - - - -- .... ............ 123
ACTIVATION OF D2 RECEPTORS FAILS TO INDUCE THE RELEASE OF A

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NEUROTRANSMI’I’I‘ER THAT STIMULATES TIDA NEURONS .................................................. 123
Delta/0P1 opioid receptors ............................................................................................... 124
Neurotensin receptors .......................................................................................................... 125
Metbodr . - 127
Drugs - . - -_ . ........................................................................................................... 127
Rerult: ........... 127
Dircum'on 131
CHAPTER SEVEN ................................................................................................... 135

EVIDENCE THAT D2 RECEPTOR-MEDIATED ACTIVATION OF TIDA NEURONS OCCURS

VIA DISINHIBITION ...................................................................................................................... 13S

Met/)odr ................. 1 38
Drugs ...................................................................................................................................... 1 38
Rem/tr 1 39
Dircum'on 147
CHAPTER EIGHT ................................................................................................... 152
INHIBITION OF TUBEROINFUNDIBULAR DOPAMINERGIC NEURONAL ACTIVITY BY
SELECTIVE ACTIVATION OF D1 RECEPTORS ........................................................................... 152
Metbodr 154
Drugs ...................................................................................................................................... 1 54
Rein/t: 1 54
Diram‘ion .- 1 60
CHAPTER NINE ...................................................................................................... 163
OPPOSING ROLES FOR D1 AND D2 DOPAMINERGIC RECEPTORS IN THE REGULATION OF
TUBEROINFUNDIBULAR DOPAMINERGIC NEURONAL ACTIVITY .......................................... 163
Met/Md: .. 1 66
Drugs ...................................................................................................................................... 167
Rerult: 1 67
Diramion 1 72

 

CHAPTER TEN- -- ........ .................................................................... 176

 

 

 

 

GENERAL SUMMARY AND DISCUSSION ..................................................................................... 176
D2 Rec¢tor—mediated activation of TIDA neuron: - ..... 176
D1 recq)tor-med1'atedinbibition of TIDA neuron: and prolactin :ecretion ....... 181
Interaction: between DI and D2 receptor: in TIDA neuronal regulation ....... -- 185

APPENDIX .................................................................................................................................. 1 89

BIBLIOGRAPHY ....................................................................................................................... 1 92

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

LIST OF TABLES

A comparison of three generations of DA agonists and antagonists by affinity for
DA receptors .................................................................................................................... 17

Effects of quinelorane on dopamine and DOPAC/ DA ratios in the median
eminence and nucleus accumbens ................................................................................ 78

Effect of PNU-95,666 on dopamine and the DOPAC to dopamine ratio in the
median eminence and nucleus accumbens .................................................................. 81

Lack of effect of PD128907 on dopamine and the DOPAC to dopamine ratio in
the median eminence and nucleus accumbens ............................................................ 82

Effects of surgical deafferentation on the concentration of dopamine and
norepinephrine in the median eminence, and dopamine in the intermediate lobe
of the pituitary of male rats one week after surgery ................................................... 98

Lack of effect of DPDPE on DOPAC concentrations in the striatum and
intermediate pituitary lobe. .......................................................................................... 11 1

Lack of effect of DPDPE on DOPA concentrations in striatum and intermediate
pituitary lobe ..................................................... - ...................................... 1 13

 

Lack of effect of DPDPE on DOPAC concentrations in striatum and
intermediate pituitary lobe ............................................................................................ 1 15

Lack of effect of naltrindole on DOPAC concentrations in the striatum and
intermediate pituitary lobe. .......................................................................................... 1 18

Table 10 List of drugs used by generic name chemical name (source) and vehicle solvent

used. ............................................................... 1 90

 

 

LIST OF FIGURES

Figure 1 The structure of dopamine drawn in two conformational extremes designated:

alpha rotomer and beta rotomer adapted from Cannon, (1975). ........................... 15
Figure 2 Drawing of a sagittal View of the rat brain modified from Fuxe et a/., (1985)
depicting the nigrostriatal DA neuronal pathway. .................................................... 20
Figure 3 Drawing of a sagittal View of the rat brain modified from Fuxe et al., (1985)
depicting the mesolimbic DA neuronal pathway. ..................................................... 21
Figure 4 Drawing of a sagittal View of the rat brain modified from Fuxe et al., (1985)
depicting the mesocortical DA neuronal pathway .................................................... 22
Figure 5 Drawing of a sagittal View of the rat brain modified from Fuxe et al., (1985)
depicting the hypothalamic dopaminergic DA neuronal cell bodies ..................... 27
Figure 6 Prolactin feedback regulation of TIDA neurons .......................................................... 36
Figure 7 Neurochemical events in axon terminals of central dopaminergic neurons ........... 47

Figure 8 The anatomical locations of the nucleus accumbens (shaded), and striatum (cross
hatched) tissue samples dissected for neurochemical analysis. ............................... 50

Figure 9 The anatomical location of the median eminence (cross-hatched) tissue sample
dissected for neurochemical analysis ........................................................................... 51

Figure 10 A sagittal section through the pituitary gland showing the anatomical location of
the neural lobe of the pituitary (shaded) that was removed in order to allow
dissection of the intermediate pituitary lobe (cross hatched) for neurochemical

 

 

 

 

analysis ................ - ............................................................................... 52
Figure 11 Scheme for in :itu hybridization with synthetic oligonucleotides. ............................ 60
Figure 12 Computer captured micrograph depicting specific labeling of a cell containing
dynorphin mRNA in the arcuate nucleus. ................................................................. 63
Figure 13 Computer-captured image of the ME and ARC in a frontal brain section. ........... 67
Figure 14 Computer-captured micrograph depicting a frontal brain section
irnmunocytochemically labeled for both TH and lFos/ F RA. .................................. 69
Figure 15 Dose response effects of quinelorane on DOPAC concentrations in the median
eminence and nucleus accumbens of male rats ......................................................... 77
Figure 16 Time course of the effects of PNU-95,666 and PD—128907 on prolactin
concentrations in plasma. ................................... 79
Figure 17 Effects of PNU-95,666 and PD-128907 on DOPAC concentrations in the
median eminence and nucleus accumbens. ................................................................ 80
Figure 18 Time course of the effects of PNU-95,666 and PD-128907 on DOPAC
concentrations in the median eminence. ....... 84

Figure 19 Effects of PD-128907 on DOPA concentrations in the median eminence and
nucleus accumbens. ...... . . - ............................................................ 85

 

Figure 20 Schematic diagram depicting a Halasz knife and the region of the mediobasal
hypothalamus circumscribed by the knife—cut procedure which includes median
eminence (ME), arcuate nucleus (ARC) and the pituitary. ...................................... 94

Figure 21 Effects of quinelorane on DOPAC concentrations in the median eminence of rats
receiving either sham surgery (Sham Control) or surgical deafferentation of the
mediobasal hypothalamus (MBH Knife-cut). ............................................................ 99

Figure 22 Effects of quinelorane on DOPAC concentrations in the nucleus accumbens rats
receiving either sham surgery (Sham Control) or surgical deafferentation of the

mediobasal hypothalamus (MBH Knife-cut). .......................................................... 100
Figure 23 Dose-response of the effects of DPDPE on DOPAC concentrations in the

median eminence and nucleus accumbens. .............................................................. 110
Figure 24 Dose-response of the effects of DPDPE on the accumulation of DOPA in the

median eminence and nucleus accumbens ............................................................... 112

Figure 25 Time course of the effects of DPDPE on DOPAC concentrations in the median
eminence and nucleus accumbens. ............................................................................ 114

Figure 26 Lack of effect of naltrindole on DOPAC concentrations in the median eminence
and nucleus accumbens ............................................................................................... 117

Figure 27 Effects of naltrindole on DPDPE-induced increases in DOPAC concentrations
in median eminence and nucleus accumbens. ......................................................... 119

Figure 28 Lack of effect of naltrindole on quinelorane-induced increases in DOPAC

concentrations in the median eminence and nucleus accumbens. ....................... 129
Figure 29 Lack of effect of SR-48692 on quinelorane-induced Changes in DOPAC
concentrations in the median eminence and nucleus accumbens. ....................... 130

Figure 30 Effects of quinelorane on DOPA concentrations in the median eminence and
nucleus accumbens of vehicle- and U-50,488-treated rats ..................................... 141

Figure 31 Effects of quinelorane on DOPA concentrations in the median eminence and
nucleus accumbens of vehicle-, nor-BNI- and prolactin-treated rats .................. 142

Figure 32 Effects of quinelorane on DOPA concentrations in the median eminence of
intact and orchidectomized rats. ................................................................................ 144

Figure 33 Effects of nor-BN1 on DOPA concentrations in the median eminence of intact
and orchidectomized rats ............................................................................................ 145

Figure 34 Effects of quinelorane on the expression of dynorphin mRNA in the arcuate

(ARC) and ventromedial hypothalamic (VMN) nuclei of the rat. ........................ 146

Figure 35 Dose-response effects of SCH39166 on the concentrations of DOPAC in the
median eminence. ........................................................................................................ 156

Figure 36 Time-course of effects of SCH39166 on the concentrations of DOPAC in the
median eminence. ........................................................................................................ 157
Figure 37. Effects of SKF38393 on the concentrations of DOPAC in the median eminence
and prolactin in plasma. .............................................................................................. 158

Figure 38 SCH391663 blocks the SKF38393-induced decrease in the median eminence

 

DOPAC concentrations. ............................................................................................ 159
Figure 39 Dose—response effect of SKF38393 on DOPAC concentrations in the median
eminence quinelorane-treated rats. - ........................................... 169

Figure 40 Effects of apomorphine on DOPAC concentrations in the median eminence of
vehicle- and quinelorane-treated rats. ....................................................................... 170

Figure 41 Effect of SKF38393 on quinelorane-induced changes in the number of perikarya
in the ARC co-expressing TH-IR and Fos/FRA-IR. ............................................. 171

Figure 42 A schematic representation of the hypothetical relationship between inhibitory
D1 and stimulatory D2 receptors on the regulation of TIDA neuronal activity..178

xiv

Chapter One

Neurobiology of Dopamine

Interest in dopaminergic (DA) neurotransmission in the brain over the
past forty years has been driven primarily by two seminal Clinical discoveries in
neuropharmacology. Firstly, that L-3,4—dihydroxyphenylalanine (L-DOPA)
treatment could transiently alleviate the symptoms of Parkinson’s disease by

enhancing DA neurotransmission in the basal ganglia, and secondly that
blockade of dopamine receptors in limbic centers was found to be associated
With the antipsychotic efficacy of neuroleptic drugs. These important discoveries
along with advances in molecular biology identifying multiple DA receptors have
encouraged the development of drugs with greater selectivity for the various
SubtyPes of these receptors. Although it has long been recognized that
nellretransmission of most DA neurons in the brain is subject to DA
(autoreceptor) mediated negative feedback, tuberoinfundibular DA neurons in
the mediobasal hypothalamus are not subject to this mode of DA receptor-
Illecliated regulation. This assertion is supported by numerous studies in which
DA agonists and antagonists that affect autoreceptor—mediated processes in
Qfllét DA neurons have no effect on TIDA neurons. The development of DA
(It-“gs with greater selectivity for the various DA receptors has uncovered the

possibility that TIDA neurons are subject to DA receptor-mediated regulation.

 

The DA regulation of TIDA neurons is different in many important ways from
that mediated by autoreceptors in other DA neurons. Thus, studies described in

this dissertation have made use of newer pharmacological tools to better

characterize the mechanism of this regulation.

Historical Perspective

The first reports Of dopamine synthesis were published in 1910 (Mannich
and Jacobsohn; Barger and Ewins). However, discovery of a potential
physiological role for this compound was overshadowed by comparison to the
C10de related biogenic amines epinephrine and norepinephrine. Experiments
COil’lparing a series of biogenic amines demonstrated that dopamine had
relatively mild vasoconstrictive properties when compared with epinephrine and

norepinephrine (Barger and Dale 1910).

The eventual discovery of dopamine as a full-fledged neurotransmitter in
its OVm right is inexorably linked to the discovery of its immediate precursor
DOPA (Funk et al., 1911). During this period the pathway by which these
airlines were fonned in vivo received much attention. DOPA was discovered as a
byPthuct of this effort It was nearly thirty years later when Holtz et al., (1938)
demonstrated the existence of L-DOPA decarboxylase in mammalian kidney.
This discovery led Blaschko (1939) to propose that L-DOPA and dopamine

were intermediates in the biosynthesis of norepinephrine and epinephrine from

L-tyrosine. These studies, in conjunction with the lack of an observable
response when tested in various animal models Of adrenergic activity served to
strengthen the notion that both L—DOPA and dopamine were merely
intermediates in biosynthesis of adrenergic amines.
Elevation of dopamine from mere biosynthetic intermediate to
neurotransmitter status took an additional 20 years. Blaschko (1957) asserted that
dopamine may have some physiological function following studies that examined
the content of dopamine in various tissues. The role of dopamine as a
biosynthetic intermediate seemed most plausible in Chromaffin tissue where it
makes up only 2-3 °/o of the total catecholamine content. However, in
adrenergic neurons dopamine levels were comparable to those of norepinephrine
(Schfimann 1956) and this suggested a potential, yet undiscovered physiological
role for dopamine in these neurons. Why store large quantities of dopamine if it
Serves only as a biosynthetic precursor? The experiment demonstrating a unique
physiological role for dopamine had been done fifteen years earlier by Holtz and
Ctel'lcler (1942) who demonstrated that dopamine administered to guinea pigs
Cans ed a vasodilation rather than the expected vasoconstriction (Barger and Dale
1 9 1 0). However, this unique finding went unrecognized because Holtz and
Ctetlcier (1942) had suggested that this puzzling effect was the result of
dopatnine being metabolized to 3,4-dihydroxyphenylacetaldehyde via

ITlotfloamine oxidase. Thus, they dismissed this observation by suggesting that it

was the result of a non-specific effect of this metabolite. However, when
monoamine oxidase inhibitors became available Hornykiewicz (1958) was able to
ConfimI that dopamine and not the phenylacetaldehyde metabolite was the active
principle responsible for the vasodilatory effects observed in the guinea pig.
This finally confirmed Blaschko’s hypothesis that dopamine has a physiological

role that is independent from that of norepinephrine and epinephrine.

Neuropsychiatric Drug Discovery: Serendipity to Rational Design

Dopamine was defined as a neurotransmitter in the periphery at about the

same time the potent antipsychotic chlorpromazine was developed for
SChizophrenia, and LDOPA was discovered for the treatment of Parkinson’s
disease. The recognition of dopamine as a common denominator in both
diseases shifted the focus of this research away from peripheral effects to the
fill'1C1:ion DA neurotransmission in the central nervous system.

Traditionally drug discovery has been an empirical process that has
depended on serendipity coupled with astute clinical observations that have lead
to a. greater understanding of biology and the development of new treatments for
disease. The discovery of chloropromazine as a tranquilizer and subsequent
utility as an antipsychotic agent is a classic example of the traditional drug
disc()very process. Henri Laborit discovered the progenitor to chlorpromazine,

promethazine after searching for a drug treatment for the prophylaxis of surgical

 

Shock (1949). Promethazine was selected for its histamine blocking activity in
hopes that this would be effective in preventing the cardiovascular response to

the autonomic nervous system instability. However, some unique secondary

Central nervous system actions (hypnotic, analgesic and hypothermic)

differentiated this compound from other similar antihistaminergic agents and
made it a useful adjuvant to anesthesia. These beneficial secondary properties
Were exploited when a series of phenothiazide drugs was evaluated by RhOne-
Poulenc pharmacologist Simone Courvoisier in 1950 (Swazey 1977a). The results
Of these studies led to the selection of promazine as a lead compound for
“eminent of surgical shock (Swazey 1977a). Further experiments demonstrated
that the addition of a chlorine group to the phenothiazine ring of promazine
resulted in chlorpromazine, a compound that had exceptional activity with low
to3(icity (Swazey 1977a). Subsequent Clinical use confirmed that it did indeed
have excellent anti-shock activity. However, Laborit also noted its ability to calm
at1)<i<)us or agitated patients in the pre-operative period and recommended its use
by his psychiatric colleagues. This latter observation led to chlorpromazine’s trial

“(1 ultimate success in treating agitated or psychotic patients (Deniker et al.,

1 952; Swazey 1977b).

The dramatic impact of chlorpromazine and related pharmacological

approaches in psychiatric therapy can be appreciated by examining the number

0f iIlpatients in United States mental hospitals before and after the introduction

and widespread use of chlorpromazine. United States Public Health Service
Census figures show that the number of mental hospital inpatients reached a
peak of 559,000 in 1955 and began to decline after the introduction of
Chlorpromazine by 1956. Subsequently the number of inpatients continued to
decline to 426,000 by 1967 according to United States Public Health Service
Statistics (Kline 1968). Thus, in only a decade pharmacological therapy of
psychiatric disease reduced the need for mental hospital beds by more than
130,000. This decrease was all the more impressive given that the rate of mental
hospital admissions doubled during this period. These early successes spurred
the development of many other compounds with what became known as
neuroleptic “tranquilizing” actions. However, the dependence of these actions
on blockade of dopamine receptors would remain unrecognized for nearly 20
Years (Matthysee 1974).

At the time chlorpromazine was being introduced to psychiatry the
tre‘glttnent of Parkinson’s disease had been wholly empirical since the ailment was
described in 1817. The mainstay of therapy had been the finding that
atltiCliolinergic compounds isolated from the root of the belladona plant
ptc)‘7ided at least some palliative relief for some of the motor dysfunction in
lDittlitinson’s patients. The recognition that a chemically induced Parkinson-like

Sytl(itome occurred with chlorpromazine proved to be key to understanding

both the neurobiology of dopamine and the biochemical basis for Parkinson’s

disease.
An independent neurotransmitter function for dopamine was originally
Suggested by the observation that this compound is a normal constituent of the

mammalian brain and occurs in nearly equal abundance with norepinephrine

(Montague, 1957; Carlsson, 1958). Two additional pieces of evidence presented

by Carlsson (1958) lent support to this view. Firstly, dopamine, like

norepinephrine and 5-hydroxytryptamine, was depleted in response to reserpine,
and secondly, the behavioral response to reserpine was catalepsy and a
Parkinson’s disease-like syndrome. These data provided strong evidence for a

dOPaInine storage and release mechanism analogous to that of norepinephrine

211d S-hydroxytryptamine, and thus added dopamine to the list of

neurotransmitters that could mediate the behavioral and motor effects observed
after reserpine treatment. Based on similar findings in humans (Bertler and
I{C’sengren 1959; Sano et al., 1959), it was concluded that dopamine in the basal
gal'lgiia was linked with central motor function. These investigators were the
first to suggest that dopamine was the key neurotransmitter in the Parkinson’s
dis @use-like syndrome induced by reserpine.
These initial findings were extended by Eringer and Hornykiewicz (1960)
Who demonstrated an almost complete lack of dopamine in the corpus striatum

thaimed from postmortem patients who had suffered from Parkinson’s disease.

This study unequivocally linked dopamine depletion in the reserpine-induced
Parkinson’s disease-like syndrome to genuine Parkinsonism observed in humans.
This was the first instance where the lack of a simple neurotransmitter in a well-
defined brain region led to Clinical manifestations of a chronic degenerative brain
disease. Replacement of dopamine by administration of the precursor L—DOPA
led to the development of drugs that could substitute for dopamine in this
disease. Among the early drugs with efficacy in this regard were the ergot
derivative bromocriptine and pergolide (Lieberman and Goldstein 1985;
Lieberman et al, 1981).

At about the same time the connection between the loss of dopamine in
basal ganglia and Parkinson’s disease was being made, a parallel link was being
drawn between excessive dopaminergic activity in limbic areas, and psychotic or
scl'lizophrenic disorders. This notion became known as the DA hypothesis of

S(:llizophrenia. At its core, the hypothesis simply states that excess brain DA
fill'ltttion is the cause of the positive psychotic symptoms e.g. delusions
hall1:1Cinations, incoherence and loose associations of the disorder (Matthysee
1 974). Primarily two lines of evidence have supported this hypothesis. Firstly,
die antipsychotic efficacy of neuroleptic drugs is closely correlated with their
ability to block dopamine receptors (Creese et al., 1976, Seeman et al., 1976;
Johnaone et al., 1978). Secondly, amphetamine use or chronic treatment with

DA agonists induces a schizophrenia-like paranoid state, including visual

hallucinations in non-psychotic individuals, and can aggravate these symptoms in
individuals with pre-existing schizophrenia (Connell 1958; Angrist et al., 1974).

In order to further investigate the DA hypothesis, studies were
undertaken to determine if schizophrenic individuals exhibited symptoms that
Would suggest that intrinsic DA activity was elevated compared to control

Subjects. There is very little evidence to suggest that dopamine turnover is
Significantly altered in these patients as studies measuring dopamine metabolites
in cerebrospinal fluid found these to be unchanged (Bowers 1974, Farley et al.,
1977) - This lack of alteration in dopamine output led investigators to refine the

Original DA hypothesis of schizophrenia by suggesting that the disturbance in

DA transmission may lie in the basal ganglia input structures. That is, DA
hyperactivity may result from altered sensitivity in or numbers of the
Postsynaptic-receptors (Bowers 1974; Crow et al., 1976). However, in spite of
Vet), powerful molecular biological information about the genetics and
distm’bution of dopamine receptors (Baldessarini 1997), to date these efforts have
failed to identify a simple relationship between dopamine and schizophrenia.

Perhaps this whole line of investigation has suffered from too close an

ass(>ciation with the earlier work on Parkinson’s disease where there is a relatively
Simple association between the disappearance of dopamine and the symptoms of
tl'le disease. It has long been a tempting analogy to view Parkinson’s disease and

Selli20phrenia as opposite sides of the same coin. Even though dopamine is one

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common denominator in both diseases the study of etiological factors in
schizophrenia is complicated by the lack of an Objective measure to differentiate
it from a family of related psychiatric disorders. These difficulties not
withstanding, it is clear that the neurobiology of dopamine owes much to the
nearly four decades of intensive work that have attempted to illuminate the root

causes of these devastating neuropsychiatric diseases.

Dopamine receptor families (Di and DZ)

Once a distinct neurotransmitter function had been established for
dopamine, a significant effort was directed at the study of specific DA binding
sites in order to elucidate the various functions of this neurotransmitter. The
emerging role for dopamine depletion in the pathogenesis of Parkinson’s disease
and the efficacy of DA antagonists in the treatment of affective disorders have
provided the fuel that has propelled the basic science of DA receptor
pharmacology for nearly 30 years. In addition, these discoveries have moved the
search for therapies from a wholly empirical approach to a more rational
hypothesis-driven endeavor. Based on their review of a decade of dopamine
receptor research, Cools and van Rossum (1976) forwarded the concept that
there are two populations of DA receptors in the mammalian brain: excitation-
mediating (DAC) and inhibition-mediating (DAi). This notion of a homeostatic

balance between DAe - DA, activity accounted for many discrepancies observed

10

in various animal studies and provided a new way of interpreting data from
various animal models.

A number of investigators accepted and built upon this notion of two
distinct receptors for dopamine (Katzman et al., 1977; Spano et al., 1978). This
classification was based largely on the finding that dopamine or various DA
agonists could stimulate or inhibit the activity of adenylyl cyclase thus altering the
levels of cyclic adenosine monophosphate (CAMP) within target cells. In
general, there was a good correlation between antipsychotic efficacy and the
ability of compounds to block dopamine stimulated increases in CAMP. There
were some conspicuous exceptions to this rule since neither butyrophenones
such as haloperidol nor substituted benzamides such as sulpiride could block
dopamine dependent CAMP production, in spite of their high antipsychotic
efficacy (Roufogalis et al., 1976; Spano et al., 1978). However, the advent of
radioligand-binding assays Clearly identified a subpopulation of receptors that
were targeted by haloperidol (Burt et al., 1976; Seeman et al., 1975). Kebabian and
Calne (1979) further extended this idea and named the excitation-coupled
receptors D1 and the inhibitory-coupled receptors D2. Thus, identification of
two distinct DA receptors explained the earlier conflicting reports by
demonstrating that dopamine stimulated CAMP production was mediated by D1
receptors, whereas neuroleptic responses depended upon blockade of D2

receptors.

11

Advances in molecular biological techniques that used the complimentary
DNA predicted from known protein sequence and structural information
allowed for the discovery of gene sequences and products associated with the
super-family of G—protein coupled receptors. These gene-cloning discoveries
eventually provided the insight necessary to identify dopamine receptor genes
and gene-products as a subset of D1 and D2 families of receptors. Based on the
structural similarity of the seven transmembrane spanning sequences for the
beta-adrenergic receptor the rat D2 receptor was discovered. Using a similar
strategy with the known D2 sequence the D, receptor followed by the D3, D4 and
D5 were identified in rapid sequence (Civelli et al., 1993). This information
provided a way to clone and then express these dopamine receptors in various
model systems. Subsequently, these model systems have allowed for a rigorous
examination of DA receptor biochemistry, pharmacology and associated
physiological functions. The results of these studies altered the simple concept
of two dopamine receptors and replaced it with at least five distinct receptor sub-
types. However, these receptors could still be grouped into sub-families based
upon identified second messenger systems and gene sequence similarity. Thus,
D1 receptors were divided into D, and D5 subtypes, while D2 receptors were
comprised of D2, D3, and D4 subtypes. Note that whenever reference is being

made to specific subtypes of DA receptors the subscript will be used (e.g. D,).

12

On the other hand when referring to a family of DA receptors no subscript will

be used (e.g.Dl).

Development of Selective Drugs for Subtypes of DA Receptors

One of the limiting factors in the treatment of Parkinson’s disease using
DA agonists is the development of psychotic side effects such as disturbing
Visual hallucinations (Calne 1978). Similarly, during the treatment of
schizophrenia with DA antagonists the emergence of a Parkinson-like syndrome
occurs that presents acutely as akinesia and motor slowing. With longer-term
treatment these drugs induce the more severe motor impairment of tardive
dyskinesia. These extrapyramidal side effects are correlated with greater D2
receptor blockade in the basal ganglia (Nordstrém et al., 1993). The discovery of
multiple dopamine receptors has provided hope that development of drugs that
interact with a specific subset of these receptors would lead to more effective
clinical management of both Parkinson’s disease and schizophrenia with minimal
or no unwanted side effects.

The process of in developing specific drugs for these diseases has
progressed in a stepwise fashion whereby generations of drugs have been
developed based on the biological and pharmacological tools of the time. Thus,
the first generation of drugs was developed prior to understanding the role of

dopamine as a neurotransmitter. Early dopamine agonists (e.g. apomorphine

13

and bromocn'ptine) had been selected empirically because of their utility as
adjunctive therapies with L—DOPA in the treatment of Parkinson’s disease.
Likewise, the development of first generation DA receptor antagonist
chlorpromazine was the result of serendipity as discussed above. The utility of
chlorpromazine encouraged the mass screening of chemical libraries of
compounds to discover other drugs with similar properties such as haloperidol.
In general the first generation of DA agonists and antagonists tend to interact
with a variety of other receptors and have no clear preference for a single DA
receptor subtype (Table 1).

The discovery of multiple dopamine receptors has fostered the
development a second generation of DA compounds. The availability this
biological information and the technology to readily express and study DA
receptors in vitro has allowed for the development of detailed structure activity
relationships for large libraries of compounds. It is from these libraries that these
partial ergoline compounds such as quinelorane (Foremann et al., 1989) and
raclopride (Ogren et al., 1988) were developed because of their preference for the
D2 family of receptors. Similarly, the substituted benzazepines SKF38393 (Sibley
et al., 1982) and SCH39166 (Chipkin et al., 1988) were developed for their D1
receptor preference (Table 1).

Recently, the availability of powerful computers to rigorously compare

these structure activity relationships have allowed for the development of a

14

molecular drug design approach. This strategy has been developed to achieve
the goal of discovering more selective drugs for the treatment of these
neuropsychiatric disorders. Initially a hypothetical model of the target receptor is
developed based on the structural Characteristics of known ligands for the
receptor. This model is referred to as the “pharmacophore” and describes the
structural features of the ideal ligand with theoretical affinity and functional
efficacy at a particular receptor. One particular advantage to using this approach
to discover new DA ligands has been the observation that many DA ligands
incorporate elements of dopamine in their structure. Although dopamine has a
somewhat lower affinity for D2 receptors the flexibility of dopamine allows for
full efficacy and reasonable affinity at all DA receptors. Dopamine can exist in

one of two chemical forms i.e. the alpha rotomer and the beta rotomer (Cannon,

1975; Figure 1).
Axis of rotation

   

Alpha Rotomer Beta Rotomer

Figure 1 The structure of dopamine drawn in two conformational extremes designated:
alpha rotomer and beta rotomer adapted from Cannon, (1975). These conformations are
possible due to the free rotation of the aliphatic amine in relation to the catechol moiety.

15

The finding that the alpha rotomer corresponds best with the
pharrnacophore of D2-like receptors whereas the beta rotomer corresponds best
with Dl-like receptor subtypes provides an explanation for the selectivity of
various chemical classes to either of these receptor families. Although this
principle cannot account for the selectivity of all chemical substances at
dopamine receptors it has provided an organizing framework by which the DA
receptor selectivity of various chemical substances may be predicted. A
productive approach to discovering compounds with greater selectivity for one
receptor subtype has been the use of rigid dopamine analogs that represent
dopamine locked in either the alpha- or beta-rotomeric form. Intentionally
designing drugs that are locked into or favor the beta-rotomeric conformation
has led to the D1 selective agonists SKF38393 (Setler et al, 1978) and
dihedrexadine (Mottola et al., 1992). Those that are rigid analogs of the alpha-
rotomer seem to be relatively more selective for the D2 family, the partial
ergoline quinelorane is a prominent example that has activity at both D2 and D3
receptors (Gackenheirner et al, 1995). The 2—arninotetralins (7 OH-DPAT) and
benzoquinolines (PD128907) are ridgid alpha-rotomer analogs that show a

preference for D3 receptors.

16

Table 1 A comparison of three generations of DA agonists and antagonists by affinity for
DA receptors

 

 

 

 

D1 Family D2 Family
D1 D5 D2 D3 D4
First Generation
Agonist
Apomorphine +/- + +++ ++ +++
Bromocriptine + + +++ +++ +
Antagonists
Chloropromazine + + +++ ++ ++
Haloperidol + + ++++ ++ +++
SCH23390 ++++ ++++ +/- +/- +/-
Second Generation
Agonists
SKF38393 +++ ++++ + +/- +/-
Dihydrexidine ++ ND + + ND
Quinperole - ND + /- ++ ++
Quinelorane - ND +++ +++ ND
Antagonists
SCH39166 + ++ ND + /_ ND ND
Raclopride - ND + + + + + + + /-
Third Generation
Agonists
PNU95666 - ND + + + /- -
PD128907 - ND + + + +
7OH-DPAT + /- ND + + ++ + + /-

 

++++, Inhibition constant (K) < 0.5 nM; +++, 0.5 nM < K, < 5 nM; ++, 5 nM < K,< 50
nM; +, 50 nM < K,< 500 nM; +/-, 500 nM < K< 5 11M; -, K>5 11M; ND, not determined;
(Modified from Missale et al., 1998)

17

Anatomy and Function of DA Neuronal Systems in the Mammalian Brain

A technical advance allowing the visualization of monoarnines in tissue
sections via fluorescence microscopy (Falck et al, 1962) allowed for rapid
advances in our understanding of the distribution and function of dopamine in
the mammalian brain. Soon after this technique was developed, a system for
mapping the distribution of DA and noradrenergic cell bodies in the brain was
described by DahlstrOm and Fuxe (1964). One of the intriguing features of
central DA neurons that distinguishes them from other arninergic systems in the
brain is the discrete nature of DA cell body origins and terminal field projections.
This differs markedly from the widely diffuse patterns of innervation observed in
Cholinergic, 5-hydroxytryptaminergic, and noradrenergic neuronal systems. The
discrete nature of its neuronal network has enabled neurobiologists to more
readily appreciate the correlation between anatomy, biochemistry and function in
these systems. The DA neuronal systems are loosely categorized as being either
mesotelencephalic or hypothalamic according to the distribution of their cell

bodies and axon terminals.

Mesotelencephalic DA Systems

The mesotelencephalic DA neuronal systems originate predominantly in

the mesencephalon with cell bodies clustered in the par: corrpacta of the

18

substantia nigra and in the ventral tegmental area (Anden et al, 1964; Lindvall
and Bjérklund 1974). These brain regions correspond with areas A8, A9 and A,O
according to the alpha-numeric system developed by DahlstrOm and Fuxe
(1964). These mesotelencephalic DA projections have been sub-divided into
three systems based on their terminal field projections: nigrostriatal, mesolimbic
and mesocortical.

As shown in figure 2, nigrostriatal DA neurons originate in the substantia
nigra and project to the caudate-putamen; designated as striatum in the rat.
Mesolimbic DA neurons originate in the ventral tegmental area and project to
the nucleus accumbens and other subcortical structures such as the lateral
septum and olfactory tubercle (Figure 3). Mesocortical DA neurons project to
several cortical regions including the cingulate, entorhinal, prefrontal and
pyriforrn cerebral cortices (Figure 4). These mesotelencephalic pathways have
projections that are highly divergent and subserve different functions within the

brain (Bloom 1983).

19

 

 

 

 

 

Figure 2 Drawing of a sagittal View of the rat brain modified from Fuxe et al, (1985)
depicting the nigrostriatal DA neuronal pathway. The locations of cell bodies are indicated
by the triangles, axons are represented as solid lines and dots represent axon terminals and
synaptic boutons. Abbreviations: accumbens nucleus (Acb), caudate pummen (CPu), globus
pallidus (GP), medial forebrain bundle (mfb), substantia nigra pars compacta (SNC),
substantia nigra pars reticulata (SNR), subthalamic nucleus (STh), and olfactory tubercle
(TU).

20

 

 

 

 

Figure 3 Drawing of a sagittal view of the rat brain modified from Fuxe et al, (1985)
depicting the mesolimbic DA neuronal pathway. The locations of neuronal cell bodies are
indicated by the triangles, axons are represented as solid lines, and dots represent axon
terminals and synaptic boutons. Abbreviations: accumbens nucleus (Acb), caudate putamen
(CPu), medial forebrain bundle (mfb), olfactory tubercle (I' u).

21

 

 

 

 

Phi. .

EN

154‘

is
‘5

 

 

 

 

Figure 4 Drawing of a sagittal View of the rat brain modified from Fuxe et al, (1985)
depicting the mesocortical DA neuronal pathway. The locations of neuronal cell bodies are
indicated by the triangles, axons are represented as solid lines, and dots represent axon
terminals and synaptic boutons. Abbreviations: accumbens nucleus (Acb), anterior olfactory
nucleus posterior (AOP), caudate putamen (Cpu), lateral habenular nucleus (LHb), lateral
septal nucleus (LS), olfactory tubercle (Tu).

Electrophysiological evidence suggests that mesotelencephalic DA
projections are inhibitory (Bannon et al, 1986; Bunney et al, 1987) and, on the
basis of behavioral evidence, subserve a permissive function with regard to
emotional responsiveness and initiation of movements (BjOrklund and Lindvall
1983). Perturbations in these systems are implicated in the pathogenesis of
Parkinson’s disease (DA hypofunction in nigrostriatal DA system) and
schizophrenia (DA hyperfunction in mesolimbic DA system) as discussed in the
previous section. In addition these DA neurons have a role in learning
acquisition and positive reinforcement, and may play an important role in the

mediating the positive reinforcing effects of drugs of abuse (Callahan et al., 1997).

Hypothalamic DA Systems
Hypothalamic DA neurons are organized into distinct systems with

clusters of cell bodies having discrete axonal projections. The terminal fields of
these local circuits remain mostly within the hypothalamus but are less well
defined than those of the extrahypothalarnic systems. Four DA systems have
been characterized in this region: incertohypothalamic, periventricular,
periventricular—hypophysial and tuberoinfundibular (TI) DA neurons.

The cell bodies of incertohypothalamic DA neurons designated A, 3 are
located in the medial zona incerta (DahlstrOm and Fuxe 1964; BjOrklund, et al,

1975). Short fibers from the medial zona incerta project ventromedially to the

23

dorsomedial hypothalamic nucleus, and these fibers have been identified as
dendrites (Wagner et al., 1993; Chan-Palay et al., 1984). Projections of the
incertohypothalamic DA neurons to the central nucleus of the amygdala,
diagonal band of Broca and paraventricular nucleus have been identified by
anterograde tracing and neurochemical experiments (Wagner et al, 1995; Eaton et
al., 1993a; 1994). In support of these observations, retrograde tracing studies
have demonstrated tyrosine hydroxylase positive neuronal projections to the
central amygdala , horizontal limb of the diagonal band of Broca and
paraventricular nucleus (PVN) originating from the medial zona incerta (Cheung
et al, 1998).

These tracing studies have provided important clues to the function of
incertohypothalamic DA neurons. The horizontal diagonal band contains cell-
bodies of gonadotropin-releasing hormone neurons (Merchenthaler et al, 1989)
and thus may be the target of these incertohypothalamic DA projections. This
hypothesis is strengthened by studies that have implicated the medial zona
incerta in the regulation of luteinizing hormone surges and ovulation (Mackenzie
et al, 1984; Sanghera et al, 1991). In contrast to either the central amygdala or
horizontal diagonal band which receive DA projections from multiple sites, the
DA projections to the paraventricular nucleus appear to originate from medial
zona incerta. In addition, other studies have demonstrated a close proximity of

DA and corticotrophin—releasing hormone neurons in this region (Liposits and

24

Paul] 1989). Taken together with the demonstration DA agonists increase
corticotropin—releasing hormone mRNA levels in the PVN, suggests IHDA
neuronal involvement in stress-induced activation of the hypothalamic-pituitary-
adrenal axis (Cheung et al., 1998).

A,4 Periventricular DA cell bodies are distfibuted in the periventricular
nucleus along the entire rostrocaudal extent of the third ventricle (van den Pol,
Herbst and Powell 1984). Although not well characterized, fibers of
periventricular DA neurons in the rostral periventricular nucleus (see Figure 5)
are believed to project laterally into adjacent medial preoptic nucleus and anterior
hypothalamic area (BjOrklund and Lindvall 1984). The function of
periventricular DA neurons is not well characterized, but medial preoptic lesions
(Bitran et al., 1988) or injection of DA antagonists into this region (Pehek et al.,
1988) inhibits copulatory behavior in male rats suggesting a role for these DA
neurons in the regulation of sexual behavior.

Periventricular-hypophysial IDA neuronal system consists of a
subpopulation of A, 4 DA cells in the periventricular nucleus located ventral to
the paraventricular nucleus; these DA neurons project to the pituitary
intermediate lobe (Luppi et al., 1986; Goudreau et al., 1992). Dopamine released
from these neurons tonically inhibits the release of pro-opiomelanocortin-
derived hormones such as alpha—melanocyte stimulating from the intermediate

lobe of the pituitary (Millington et al. 1988).

25

Of all the intrahypothalamic DA neurons TIDA neurons are the best
studied. Cell bodies designated A,2 (DahlstrOm and Fuxe 1964) for these
neurons are located in the arcuate nucleus and short axons project ventrolaterally
to the external layer of the median eminence (Bjérklund and Nobin, 1973).
These DA neurons function in the regulation of prolactin secretion as discussed

in the next section.

26

 

 

 

 

 

 

 

 

 

Flgtlte 5 Drawing of a sagittal view of the rat brain modified from Fuxe et al., (1985)
:ePipu'ng the hypothalamic dopaminergic DA neuronal cell bodies. The location of DA cell
vod-les is indicated by the filled triangles. Abbreviations: 3V third ventricle, 4V fourth
- etltticle, arcuate nucleus (ARC), anterior pituitary lobe (AP), dorsomedial nucleus (DM),
rte-mediate pituitary lobe (IL), mamillothalamic tract (mt), median eminence (ME), medial
Qua incerta (MZI), neural pituitary lobe (NL), optic chiasm (ox), periventricular nucleus
e\fbl), and ventromedial nucleus (V M).

27

Tuberoinfundibular DA Neurons and the Regulation of Prolactin Secretion
Unlike the stimulatory effect of hypothalamic releasing factors on the
other anterior pituitary hormones the primary action of the hypothalamus upon
prolactin secretion is inhibitory. It has long been known that isolated pituitaries
grafted into other animals hypersecrete prolactin. Everett (1954) transplanted
pituitary glands beneath the kidney capsule and reported on the biological effects
of prolactin hypersecretion. Likewise, in studies where the portal blood flow is
interrupted by pituitary stalk transection plasma prolactin levels are rapidly
elevated (within 30 min) and remain elevated up to 6 months later (N ikitovich-
Winer, 1965; Welsch et al., 1971; Kanematsu and Sawyer, 1973; Murai et al.,
1 989). Conversely, grafting isolated pituitaries to an intact portal blood supply do
not hypersecrete prolactin as they did if grafted elsewhere (Lu and Meites, 1972).
That the hypothalamus is the source of the prolactin inhibitory factor
carried in portal blood was suggested by the finding that hypothalamic lesions
l.Ilduce lactation in rats (Halasz et al., 1962). This possibility was demonstrated
more directly by showing that bilateral lesions in the median eminence increase
Plasma prolactin levels, whereas lesions in the central nucleus of the amygdala
have no effect (Chen et al., 1970). These studies suggested that the mediobasal

lJYI’thalamus was the source of or stimulated the release of a prolactin

28

inhibitory factor that controls the secretion of prolactin from the anterior
pituitary gland.

The notion that the prolactin inhibitory factor originates from the
hypothalamus is also supported by a number of in vitro studies that have shown
that prolactin secretion in isolated anterior pituitaries is inhibited when exposed
to homogenates or acid extracts of hypothalami. These extracts were known to
contain releasing factors for follicle-stimulating hormone, luteinizing hormone,
thyroid stimulating hormone and adrenocorticotropin in addition to inhibiting
factor for prolactin (Pasteels, 1961; Talwaker et al., 1963). Similar in vivo

experiments utilizing hypothalamic extracts also reduced plasma prolactin
concentrations 1 and 4 hours after injection into non-lactating female rats
(Amenmori and Meites, 1970). The possibility that these effects were direct on
the pituitary was suggested by Kamberi et al., (1971) who found that infusion of
hypothalamic extracts into a portal vessel inhibited prolactin secretion. Talwalker
0” al., (1963) noted that many neurotransmitters found in the hypothalamus
(acetylcholine, epinephrine, norepinephrine, serotonin, histamine, oxytocin, and
Vas Opressin) had no effect on in vitro prolactin release.
A role for biogenic amines in the hypothalamic control of prolactin
SeCretion was first suggested by studies in catecholamine-depleted rabbits
CI<a~l’lematsu et al., 1963). In these experiments reserpine injections given to

Ovatectomized rabbits induced lactation and increased the pituitary prolactin

29

m

content (presumably via hormone synthesis) compared to controls. However,
electrolytic lesions placed in the mediobasal hypothalamus mimicked the effect
of reserpine and thereafter administration of reserpine had no further effect.
Thus, it was concluded that reserpine exerted its action via the hypothalamus.
Taken together with the description of TIDA axon terminals in the external layer
of the median eminence, adjacent to the portal blood supply, these data
presented an alternate hypothesis to the dogmatic view that prolactin inhibiting

effects of catecholamines occurred via stimulation of a prolactin inhibiting factor

(Van Maanen and Smelik 1968).

Dopamine: The Prolactin Inhibitory Factor

In View of the mounting evidence that catecholamines could act directly
on the anterior pituitary as a prolactin inhibitory factor, there was significant
concern centered on the effects of L-DOPA treatment on serum prolactin
secretion in patients with Parkinson’s disease. It is clear from a review by Meites

at al., (1972) that although catecholamines were known to be involved in the
regulation of prolactin secretion this involvement was supposed to be indirect by
controlling the release of a prolactin inhibitory factor. The reason for this View
1:)t Obably stems from a bias that like all other hormones of the anterior pituitary,
I)1~'()1actin secretion is likely regulated via a peptide hypothalamic factor. This

hyPOthesis was bolstered by experiments in which i.c.v. administration of

30

(223

a-
r1.

mu-

5".

FAI‘

(J

-'1

t-

catecholamines decreased plasma prolactin levels whereas, direct infusion into

the portal blood vessel was without effect (Kamberi at al., 1971). However, this
finding was contradicted by Takahara et al., (1974) who demonstrated that
purified porcine hypothalamic extracts or dopamine infused into a portal blood
vessel were sufficient to inhibit prolactin secretion. Thus, the failure of Kamberi

et al., (1971) to show an effect was thought possibly to be due to degradation of
dopamine in their preparation. Shaar and Clemens (1974) studied the
catecholamine content of hypothalamic extracts that had been shown to inhibit
prolactin secretion and reported that the dopamine and norepinephrine content
in the hypothalamus could account for the prolactin inhibitory activity in these
extracts. In addition, treatment of these extracts with proteases did not alter
their prolactin inhibitory factor activity whereas, treatment with monoamine
oxidase rendered them inactive. By 1978 Gibbs et al. had further demonstrated
that dopamine levels in the hypothalamic pituitary stalk were sufficient to inhibit

Prolactin secretion in viva.

Support for the hypothesis that dopamine was the prolactin inhibitory

faCtor was provided by Macleod (1969); Birge et al., (1970) and Koch et al., (1970)
who used an in vitm system to demonstrate direct effects of dopamine on
13"5(>lactin release from anterior pituitaries. Their system made use of 3H-leucine
Whith when incubated with isolated pituitaries is incorporated into newly

S3’1'1t11esized prolactin. Application of dopamine to these pituitary cultures

31

significantly reduced the level of radiolabeled prolactin in the medium and
increased 3‘H-prolactin in the anterior pituitary. Electron micrographic
examination of these pituitary cells demonstrated that following dopamine
treatment there was a build-up of secretory granules containing prolactin in the
rough endoplasmic reticulum (MacLeod and Lehmeyer, 1974).

Once dopamine was established as having a direct prolactin inhibiting
action, TIDA neurons emerged as an important link in the regulation of
prolactin secretion. Anatomical studies performed by Hokfelt and Fuxe, (1972)
demonstrated that TIDA neurons projected short axons that coursed ventrally
from the arcuate nucleus to terminate in the external layer of the median
eminence. Thus, with terminals immediately adjacent to the portal vasculature
these DA neurons would be a likely source for the “prolactin-inhibiting”

dopamine in the found in the portal circulation.

Prolactin Feedback Regulation of TIDA Neurons
A number of studies suggested that TIDA neurons participated in the
Prolactin-mediated negative feedback of its release from the pituitary (for a
review see Moore and Lookingland 1995). An elevation in plasma prolactin via
exogenous administration or grafted anterior pituitaries leads to an increase in
file synthesis of dopamine in TIDA neurons (Demarest and Moore, 1981;

Morgan and Herbert, 1980; Selmanoff 1981). These findings lent support to the

32

hypothesis that feedback regulation of prolactin secretion occurs via stimulation
of TIDA neurons. According to this scenario, elevations in plasma prolactin
would activate TIDA neurons causing the release of dopamine, which enters
hypophysial portal blood. Dopamine is then carried to the anterior pituitary
where it activates D2 receptors (Creese, et al., 1977; Caron at al., 1978) and
inhibits prolactin secretion from lactotrophs (Ben-Jonathan, 1977; 1985).

One of the central questions remaining to be addressed is by what
pathway does prolactin act upon TIDA neurons to accomplish this feedback
regulation? One possibility is retrograde transport via the long hypophysial
portal vessels (Olivier at al., 1977) whereby high levels of prolactin may be
delivered to the median eminence and terminals of TIDA neurons. The
presence of prolactin receptors in the median eminence is consistent with this
hypothesis (Barton at al., 1989). Alternatively, prolactin can be taken up from the
general circulation into the brain via transport through the choroid plexus
(Clemens and Sawyer 1974; Walsh et al., 1978).

Although the precise mechanism by which prolactin activates TIDA
neurons is unknown, there is evidence to suggest that this stimulatory effect
takes place within the mediobasal hypothalamus. Deafferentation of the
Itlediobasal hypothalamus (Hilasz and Pup 1965) has been used as a means to

Smdy hypothalamo-pituitary function independent from afferent neuronal

1Ilflllences from the rest of the brain. Indeed, this knife-cut deafferentation

33

procedure has been used successfully to determine the site of prolactin mediated
activation of TIDA neurons (Gudelsky et al., 1978). Exogenous administration
of prolactin is able to increase TIDA neuronal activity in rats with surgical
deafferentation of the mediobasal hypothalamus suggesting that this response is
not dependent upon afferent neuronal input to the mediobasal hypothalamm.
Rather, prolactin acts within the area circumscribed by the knife-cut to stimulate
TIDA neurons. Unlike hypophysectomy or surgical ablation of the mediobasal
hypothalamus, this deafferentation procedure has no effect on basal prolactin
levels. Thus, tonic inhibitory regulation of prolactin secretion is not dependent
upon afferent input to the TIDA neurons from outside the mediobasal
hypothalamus (Krulich et al., 1975, Turpen and Dunn 1976, Gudelsky et al.,
1 978).

Prolactin feedback activation of TIDA neurons is composed of “rapid
tonic” and “delayed inductive” components (Demarest et al., 1984; 1986). The
“rapid tonic” component regulates the set point for basal circulating prolactin

levels by acutely altering TIDA neuronal activity in response to acute changes in
Cil’culating prolactin. The “delayed inductive” component of prolactin feedback
inVOlves a change in the capacity of TIDA neurons to respond to more
Prolonged periods of elevated prolactin. Induced activation does not appear

uEltil 12 hr following systemic or central administration of exogenous prolactin

34

(Hokfelt and Fuxe, 1972, Selmanoff at al., 1981; Demarest et al., 1984, 1986) and

is neurotensin dependent (I-Ientschel et al., 1998).

Thus, as summarized in Figure 6, TIDA neurons provide a key link in the
inhibitory feedback regulation of prolactin secretion from the anterior pituitary.
When serum prolactin levels are high TIDA neurons are activated and release
more dopamine into the hypophysial portal circulation. Dopamine is then
carried to the anterior pituitary where it reduces further prolactin secretion via an

action at D2 dopamine receptors located on lactotrophic cells.

35

 

 

 

 

 

 

 

u ' 2 Receptor

 
 
  
 

  

Anterior
Pituitary

  

Figure 6 Prolactin feedback regulation of TIDA neurons. Dopamine (DA) released from
terminals of TIDA neurons in the median eminence (ME) is carried via hypophysial portal
vasculature to the anterior pituitary. There it binds to D2 receptors on lactotrophs and
inhibits prolactin secretion. Prolactin, in turn, feeds back to activate TIDA neurons via a
direct action on cell bodies or axon terminals and/ or an indirect action mediated by afferent
interneurons. Abbreviations: 3V third ventricle.

36

Afferent Modulation of TIDA Neuronal Activity

In addition to prolactin, pharmacological evidence suggests that a number
of neurotransmitters are involved in the acute afferent modulation of TIDA
neuronal activity. These effects can be either stimulatory (e.g. enkephalin;
neurotensin) or inhibitory (e.g. dynorphin) modulators of TIDA activity. Finally,
there is accumulating evidence that the activity of these neurons may be subject
to DA-receptor—mediated stimulation or inhibition. The goal of the studies
contained herein is to examine in detail the DA receptor mediated mechanisms
whereby the activity of TIDA neurons is altered and the relationship, if any, to
the aforementioned stimulatory and inhibitory modulators. Details will be

discussed in the introductions of the respective chapters.

Dopamine Receptor Mediated Regulation of TIDA Neurons

The responses of DA neuronal systems to a variety of DA agonists and
antagonists have been intensively studied. First generation DA agonists such as
apomorphine and bromocriptine, and antagonists such as haloperidol acutely
modulated the activity of mesotelencephalic and hypothalamic DA neurons
except TIDA neurons. DA agonists decrease the firing rate (Bunney et al., 1973),
and the synthesis, release and metabolism of dopamine in these neurons
(Carlsson, 1975; Roth et al., 1976; Lookingland and Moore, 1984; Imperato and

DiChiara, 1985; Imperato et al., 1988) whereas DA antagonists increase these

37

paramaters of neuronal activity. These responses to DA agonists and
antagonists are thought to be due to an action at presynaptic DA inhibitory
autoreceptors and at postsynaptic DA receptors involved in inhibitory neuronal
feedback loops.

The regulation of TIDA neurons represents a departure from other
mesotelencephalic or hypothalamic DA neurons in that these neurons are not
subject to DA receptor-mediated mechanisms. This was established by
observations that acute administration of first generation DA agonists
(apomorphine, bromocriptine) and antagonists (haloperidol) had no acute effect
on the activity of TIDA neurons (Gudelsky and Moore, 1976; 1978; Demarest
and Moore, 1979; Lookingland and Moore 1984). Although these neurons are
responsive to high doses of these compounds, these responses are delayed and
are mediated by prolactin (Moore and Lookingland 1995).

Given the numerous observations that DA agonists have no acute effect
on the activity of TIDA neurons it was surprising when subsequent studies using
the partial ergoline-derived agonists quinpirole and quinelorane (i.e., second
generation DA agonists) were shown to activate TIDA neurons (Berry and
Gudelsky, 1991; Eaton et al. 1993). This stimulatory effect is present even in
female rats where circulating levels of prolactin provide tonic stimulatory
feedback onto TIDA neurons (Demarest et al. 1985). In females, the stimulatory

effect of quinelorane is masked by decreases in plasma prolactin, which in turn

38

reduces tonic stimulation of TIDA neurons. The net result is no change in the
activity of these neurons (Eaton et al., 1993). The ability of quinelorane to induce
increases in the activity of these neurons is only apparent when circulating
prolactin is immmoneutralized by systemic administration of a prolactin
antibody. In these prolactin—neutralized female rats basal TIDA neuronal activity
is significantly lower than in normal rabbit serum-treated controls. Under these
conditions the stimulatory effect of quinelorane can be observed and is
qualitatively similar to that seen in male rats (Eaton et al. 1993). In the studies
presented in this dissertation the male rat has been used, in part, to avoid the
complications of the tonic stimulatory effect of circulating prolactin on TIDA
neurons in female rats.

The mechanism by which the D2 agonists such as quinelorane (Foreman
at al 1989) activate TIDA neurons appears to be DA receptor mediated, as this
stimulatory effect is blocked by second-generation DA receptor antagonists such
as raclopride (Eaton et al. 1992; 1993). However, it is not known if the
quinelorane-induced activation of TIDA neurons results from its ability to
activate D2 or D3 receptors since in vitro studies have confirmed that this agonist
binds to both of these receptor subtypes (Gackenheimer et al. 1995). The
development of PNU-95,666 a selective D2 agonist (Smith et al. 1996) and
PD128907 a selective D3 agonist (Pugsley at al. 1995), i.e., third generation DA

agonists, have made it possible to determine the extent to which either the D2 or

39

D3 receptors contribute to the stimulatory effects of quinelorane on TIDA
neurons.

Stimulatory DA receptor-mediated afferent neuronal regulation of TIDA
neurons could occur either by increasing the release of an endogenous stimulator
of these neurons or by suppression of a tonically active inhibitory system
(disinhibition). This hypothesis is attractive due to the abundant evidence that
activation of D2 receptors is coupled to inhibitory cellular events: reduction of
CAMP production, or calcium conductance, or increases in potassium
conductance (jackson and Westlind-Danielsson 1994). Thus, a mechanism
involving disinhibition is invoked to explain how inhibitory D2 receptor
activation would induce a net increase in TIDA neuronal activity. For this
disinhibition hypothesis to be correct two basic criteria must be met: (1) some
neuronal circuit must tonically inhibit TIDA neurons, and (2) exogenous
administration of the inhibitory neurotransmitter or its analog should block the
Dz-mediated increase in TIDA neuronal activity. At least two inhibitory
neurotransmitters; dynorphin and ‘Y-aminobutryic acid (GABA) via actions at
kappa/0P2 and GABAA receptors respectively meet this first criterion.

The discovery of multiple DA receptor subtypes (Civelli et al., 1993) has
spawned the development of more selective DA receptor ligands in order to

better assess the functional role of each of the various DA receptor subtypes,

m

(I

EEC-

and to identify new drugs that may have potential for treating neuropsychiatrtric
disorders. Among these compounds are the substituted benzazepines
SKF38393 (Sibley et al., 1982) and SCH39166 (Chipkin et al., 1988) with
selectivity for the D1-like receptors. In contrast to the stimulatory effects on
TIDA neurons observed after activation of D2-like receptors, the D1 receptor
agonist SKF38393 can inhibit activated TIDA neurons (Berry and Gudelsky,
1990). Indeed, SKF38393 is able to inhibit TIDA neurons that had been
activated following a variety of treatments: neurotensin, reserpine or haloperidol.
Thus, the lack of effect following administration of non-selective agonists and
antagonists may be due to opposite effects at D1 (inhibitory) and D2

(stimulatory) receptors.

41

Specific Aims

The overall goal of studies described in this dissertation is to characterize

the mechanisms underlying DA receptor-mediated regulation of TIDA neurons,

with special emphasis on the roles of inhibitory D1 and stimulatory D2 receptors

in this process. For comparison, the responses of DA neurons comprising the

nigrostriatal and mesolimbic systems will also be examined in key experiments.

An underlying theme in these studies is that anatomically distinct populations of

DA neurons are differentially regulated by DA receptors. The development of

DA agonists and antagonists that are highly selective for D1 and D2 receptor

families has made it possible to distinguish these differences. A combination of

molecular, neurochemical, neuroanatomical and pharmacological experimental

approaches will be used to address the following specific aim$:

1.

Determine the D2 receptor subtype that mediates the quinelorane-

mediated activation of TIDA neurons

Determine if quinelorane-induced activation of TIDA neurons is mediated

by stimulatory delta/0P1 or neurotensin receptors

Determine if quinelorane—induced activation of TIDA neurons is mediated

by the release of an endogenous stimulator of TIDA neurons

Determine if quinelorane-induced activation of TIDA neurons is mediated

by inhibitory kappa/0P2 receptors

Characterize D1 receptor-mediated regulation of TIDA neurons

42

6. Determine if the activation of inhibitory D1 receptors opposes the

stimulatory effects of quinelorane on TIDA neurons.

43

Chapter Two

General Methods

Animals

All experiments were performed in male Long-Evans rats purchased from
Harlan Breeding Laboratories (Indianapolis, IN) and maintained in a
temperature- (22i1°C) and light-controlled room (lights on between 06:00 h and

20:00 h) with food (T eklad Rodent Diet) and tap water provided ad libitum.

Drugs

Drugs are referred to by their generic names throughout the text. A listing
of chemical names, vehicle solvents and sources for these compounds can also
be found in Table I of the APPENDIX. Dosages of all drugs are calculated
based on either the salt or free base. Routes and times of drug administration

are indicated in the legends of the appropriate figures.
Surgical Procedures

Intracerebroventricular Cannulation

Rats receiving intracerebroventricular (i.c.v.) injections were anesthetized
via intraperitoneal (imp) administration of 3 ml Equithesin/ kg body wt. ' Once
anesthesia was evident the surgical site was shaved and cleaned with betadine

scrub and 75% ethyl alcohol. Rats were then assessed for surgical anesthesia by

44

 

 

 

pinching a toe; a lack of reflex movement was judged as sufficient anesthesia to
begin the procedure. Rats were then placed in a stereotaxic frame (David Kopf
Instruments, Tujunga, CA) and stainless steel guide cannulae were implanted
into right lateral cerebral ventricle 5 days prior to the experiment. For this
procedure the incisor bar on the stereotaxic frame was set 2.4 mm below the
intraaural line (Konig and Klippel, 1963). A 23-gauge stainless steel guide
cannula was implanted 1.4 mm lateral to the midline at bregma and lowered 3.2
mm below the dura matter surface. This guide cannula was then anchored to the
skull with stainless-steel screws and denture acrylic. On the day of the
experiment, i.e.v. injections were carried out by connecting a 10 pl Hamilton
microsyringe to a 30-gauge stainless steel injector which protruded 1 mm below
the tip of the guide cannula and into the right lateral ventricle. Drugs or their
vehicles were manually injected at an approximate rate of 3-5 pl per minute (total

volume 3-5 Ill). The placement of each cannula was verified histologically and

only those animals with a cannula track in the right lateral ventricle were included

in the data analysis.

Orchidectomy

Orchidectomy was performed in some experiments to assess the role, if
any, of gonadal hormones on the effects of pharmacological manipulations on

TIDA neuronal activity. Rats were anesthetized with diethylether seven days

45

prior to the experiment and the surgical site was shaved, and scrubbed with
betadine and 75% ethyl alcohol. Testicles were removed by making a single
incision along the midline of the lower abdomen through the skin and muscle
layers. The testicles were exteriorized, ligated with silk suture material and then
excised. The muscle layer was closed with sutures and the skin was closed with

wound clips.

Neurochemical estimation of neuronal activity

Within axon terminals of DA neurons there is tight coupling between
synthesis, release and metabolism to insure that adequate levels of releasable
neurotransmitter are maintained (Figure 7). Dopamine is synthesized from
dietary tyrosine, which is then converted to DOPA by the rate limiting enzyme
tyrosine hydroxylase. DOPA is then rapidly decarboxylated by the action of L-
aromatic amino acid decarboxylase thereby forming a pool of cytoplasmic
dopamine which is either packaged into storage vesicles for future release or
metabolized by monoamine oxidase to form 3,4-dihydroxyphenylacetic acid
(DOPAC). This coupling between dopamine release, and de novo synthesis and
metabolism affords 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. Increased neuronal

activity (synthesis and release) is reflected as increases in free intra-neuronal

46

 

 

 

dopamine. Monoamine oxidase has access to the dopamine in these unprotected
non-vesicular pools and converts it to DOPAC. Procedures which increase or
decrease DA neuronal activity will produce a concomitant change in the

concentration of this metabolite (Lookingland et al., 1987).

 

Post-Synaptic
Tyrosine-TH->DOPA-DO>DA ® Cell Body or
Dendrite

 

 

Tyrosine

I
1 If. -

Tyrosine-TH->DOPA-Do; W—> DA

w

Figure 7 Neurochemical events in axon terminals of central dopaminergic neurons. Dashed
lines represent inhibitory feedback loops. Abbreviations: DA (dopamine), DC (DOPA
decarboxylase), DOPA (3,4~dihydroxyphenylalanine), DOPAC (3,4~dihydroxyphenylacetic
acid), MAO (monoamine oxidase), and TH (tyrosine hydroxylase).

Median Eminence

Anterior
Pituitary

 

 

The second method of indexing neuronal activity involves estimating the
synthesis of dopamine. Synthesis of new dopamine can be estimated by
measuring DOPA accumulation following administration of an L-aromatic
amino acid decarboxylase inhibitor (Carlsson et al., 1972). This estimate is based
on the tight coupling between tyrosine hydroxylase activity and its end-product

inhibition by intra-neuronal free dopamine. Thus, following DOPA

47

 

decarboxylase inhibition procedures which increase or decrease neuronal activity
produce a corresponding change in the rate of DOPA accumulation (Demarest
et al, 1981). In experiments described in this dissertation the concentration of

DOPAC or accumulation of DOPA 30 min after decarboxylase inhibition will

be measured as indices of DA neuronal activity.

Tissue and Blood Processing

Following in viva manipulations rats were killed by decapitation. Brains and
pituitaries were rapidly removed from the skull, frozen on aluminum foil placed
over dry ice. Frontal brain sections (600 um thick) were prepared in a cryostat at
-9°C. Ten adjacent serial sections were collected beginning at approximately 2.2

mm anterior to bregma and ending at approximately 3.8 mm posterior to bregma
(Pardnos and Watson 1986). These sections were placed on glass slides and re-
frozen. The slides were then transferred to the stage of a stereo microscope and
the nucleus accumbens, striatum and median eminence were dissected using a
modification (Lookingland and Moore, 1984) of the method of Palkovits (1973).
The nucleus accumbens was dissected from sections #1 and #2 by means of a
21 gauge hypodermic needle, the striatum from section #2 using an 18 gauge
hypodermic needle (Figure 8), and the median eminence from sections #8 and
#9 using an oval shaped 18 gauge needle (Figure 9). The pituitaries were

dissected according to the method of Lookingland et al., (1985). An 18 gauge

48

needle was used to first remove the neural lobe and then the intermediate lobe
was collected by separating it from the underlying anterior pituitary (Figure 10).
Tissue samples were placed in 100 ill (nucleus accumbens and striatum) or 50 pl
(median eminence and intermediate lobe of the pituitary) of a 0.1 M phosphate-
citrate buffer (pH 2.5) containing 15% methanol and stored at -20°C until

assayed.

49

 

 

SE

CTION #2

Figure 8 The anatomical locations of the nucleus accumbens (shaded), and striatum (cross
hatched) tissue samples dissected for neurochemical analysis. Each frontal section was 600
11m thick and section #1 was from approximately 2.2 mm and section #2 was from

approximately 1.6 mm anterior to bregma respectively. Figures were modified from Paxinos
and Watson, 1986.

50

 

SECTION #9

Figure 9 The anatomical location of the median eminence (cross-hatched) tissue sample
dissected for neurochemical analysis. Each frontal section was 600 [1m thick and section #8
was taken from approximately 2.56 mm and section #9 from approximately 3.3 mm
posterior to bregma respectively. Figures were modified from Paxinos and Watson, 1986.

51

      
     
   

v o'o’o‘o » “vs 3 . ‘v's l nte I'm ed I ate
v o'o’o’o’o’t’o'o’o'o'o'o'o'o'o'o'o'o’o’o"9 - °
- satAt.tsVIN)”.fifololofofofofotozoto}, P IIU 'tary L0be
.. . A\,

Anterior Pituitary

 

Figure 10 A sagittal section through the pituitary gland showing the anatomical location of
the neural lobe of the pituitary (shaded) that was removed in order to allow dissection of the
intermediate pituitary lobe (cross hatched) for neurochemical analysis (Modified from
Paxinos and Watson, 1986).

52

High Performance Liquid Chromatography

On the day of the assay, tissue samples were thawed, sonicated for 3 s
(Sonicator Cell Disruptor, Heat Systems-Ultrasonic, Plainview, NY) and
centrifuged for 30 s in a Beckman 152 Microfuge. Contents of DOPAC,
DOPA, dopamine and norepinephrine in supernatants were determined by high
performance liquid chromatography coupled with electrochemical detection as
described previously (Lindley et al., 1990). The amounts of these catecholamines
in tissue samples were measured by comparing peak heights (determined by a
Hewlett Packard Integrator, Model 3395) with those obtained from external

standards run on the same day. The standards contained a mixture of the above
analytes each at a concentration of 0.5 or 1.0 ng/50 111. The lower limit of
sensitivity of this assay for these compounds was 1-5 pg/ sample.

Fifty microliters of each sample supernatant was injected onto a C18
reverse-phase analytical column (Slim spheres; 250m in length; Biophase ODS,
Bioanalytical systems, West Lafayette IN) preceded by a pre—column filter
cartridge (5 [1m spheres; 30mm). Following separation analyte signals from the
nucleus accumbens, striatum, or median eminence samples were determined
using a electrochemical detector (LC4A; Bioanalytical systems, West Lafayette
IN) equipped with a TL-5 glassy carbon electrode set at +0.75 V relative to an

Ag/AgCl reference electrode. Because samples from the intermediate lobe of

53

the pituitary contain lower levels of these catecholamines, they were analyzed
using a single coulometric electrode conditioning cell in series with dual electrode
analytical cells (ESA, Bradford MA). The conditioning cell electrode was set a +
0.4 V and the analytical electrodes were set at +0.12 V and — 0.4 V respectively,
relative to the Ag/AgCl reference electrode.

The high performance liquid chromatography mobile phase consisted of a
0.1 M phosphate/ citrate buffer (pH 2.65) containing 0.1 mM
ethylenediaminetetraacetic acid, 0.03-0.045°/o sodium octylsulfate and 15 - 25%
methanol. Depending on the condition of the column, components of the
mobile phase were adjusted slightly to maintain separation of the analytes of
interest and to minimize total retention times (Chapin et al., 1986). Tissue pellets

from each sample were dissolved in 100 or 200 ill of 1.0 N sodium hydroxide

depending on the size of the tissue punch and assayed for protein (Lowry et al.,
1951). All neurochemical data have been reported as concentrations (ng/ mg

protein) to correct for variability in tissue sample size.

Radioimmunoassa y for Prolactin

In some studies plasma prolactin measurements were made as an indirect
physiological index of dopamine release from TIDA neurons, or to determine

the effect of various pharmacological manipulations on D2 receptors in the

pituitary gland. Immediately after rats were decapitated, trunk blood was

54

toicc

310:6:

PAM

tbes

:I:
24

 

collected into glass tubes containing heparin and 150 mg of bacitracin, and
stored on ice. Following collection, tubes were centrifuged for 30 min at 4°C,
plasma was separated from red cells and stored in sodium citrate containing

tubes at -20°C until the day of the assay.

Plasma prolactin concentrations were determined by a double-antibody
radioirnmunoassay. This assay made use of reagents and procedures supplied by
Drs. A. F. Parlow and S. Raiti, NIADDK National Hormone and Pituitary
Program. NIDDK rat prolactin (RP—3) was use as the standard and rat prolactin
(rat PRL-L6; NIDDK) was labeled with 125Iodine (Amersham Arlington
Heights, IL) using the chloramine-T technique (Chard, 1990). Rat plasma
samples were incubated with anti-rat prolactin (anti-rat PRL S-9 NIDDK) and
125I-labeled prolactin at 4° C for 24 hours. On the next day the precipitating
secondary antibody (goat anti-rabbit gama globulin) was added to the samples
and incubated for an additional 24 hours at 4° C. The following day the mixture
was centrifuged, the supernatant decanted and the precipitate was counted in a

gamma counter (Micromedic model 4/ 600). Using a 100 pl aliquot of plasma the

lower limit of sensitivity for prolactin in these assays was 100—200 pg/ sample,

and the inter-assay coefficient of variability was approximately 10%.

55

Measurement of Dynorphin mRNA by in situ Hybridization Histochemistry
In catecholaminergic neurons the cellular machinery for synthesis, storage
and release of neurotransmitters is located in the axon terminals. Thus, in
neurochemical determinations of neuronal activity it is useful to make
measurements by sampling tissue from a brain region corresponding to the axon
terminal field projections of the neurons of interest. However, peptidergic
neurotransmitters are synthesized in neuronal perikayra. Using sensitive
immunohistochemical methods it is possible to qualitatively determine the
presence of such neuropeptides and determine their precise location. However,
it is exceedingly difficult to make quantitative assessments that reflect changes in
neuronal activity in a way that was described above for DA systems. In situ
hybridization histochemistry is a method whereby the mRNA transcripts for a
particular protein can be measured as a quantitative index of the activity of
specific peptidergic neurons. The underlying theory is that changes in
neuropeptide release from axon terminals are accompanied by corresponding
changes in do: nova synthesis of mRNA transcripts in the neuronal perikarya.
Thus, in situ hybridization provides anatomical and quantitative information

about the activity of peptidergic neurons.

56

COII

GG

Gil

tit

193:

i"):

5 .
fdcl

Labeling The mRNA Probe

The sequence of the 47 base synthetic oligomer probe was
complementary to bases 862-909 of the rat prodynorphin gene [5’GC TAT
GGG GGC TI’C CT G CGG CGC ATT CGC CCC AAG CTT AAG TGG
GAC3’] (Civelli at al., 1985). Purified oligodeoxynucleotides were 3’ end-labeled

with 3SS-dATP using terminal deoxynucleotidyl transferase (T (H) (Young at al.,
1986). Briefly, 1 111 of 5 11M probe was reacted with 4 ill of TdT enzyme
(25U/tll; Boehringer Manheim; Indianapolis, IN), 10 [ll of 5X DNA tailing
buffer containing 0.5 M potassium cacodylate, pH 7.2, 10 mM cobalt chloride
and 1 mM dithiothreitol, 5 ill of 35 S—dATP (1389 Ci/mmol; New England

Nuclear; Boston MA) and 30 [ll sterile diethyl pyrocarbonate (DEPC)-treated

water for 5 min at 37°C.

Extraction of the Labeled Probe

The labeling reaction was stopped by addition of 400 ill of Tris-EDTA

(TE) buffer containing 10 mM Tris HCL/EDTA, pH 7.5 and 2 [1.1 yeast tRNA

(25 mg/ ml) The labeled probe was extracted with the addition of 400 ml
phenol/chloroform/isoamyl alcohol (50:49:1). The mixture was vortexed then
centrifuged and the recovered aqueous phase was extracted with the addition of
400 ml of chloroform : isoamyl alcohol (24:1). The recovered aqueous phase

was then precipitated with the addition of cold absolute ethyl alcohol containing

57

0.3M sodium acetate on ice for 45 min and then centrifuged for 45 min at 4°C.
The supernatant was discarded and the pellet was allowed to air dry. The air

dried pellet was dissolved in 100 ml TE buffer containing 1 [ll of 1 M

dithiothreitol (DTT) and stored at 4°C until the hybridization procedure was

done.

Preparation of Tissue

Following in viva experimental manipulation rats were euthanized by
decapitation, brains were removed and rapidly frozen on aluminum foil covered
dry ice (see page 48). Frontal sections (25 m thick) through the arcuate nucleus
in these fresh frozen brains were prepared in a microtome cryostat (IEC
Minotome, International Equipment Corp.) and affixed directly to gel-coated

slides. These slides were then placed in a vacuum/ desiccator at 4°C to allow

them to dry. Slides were stored in boxes containing desiccant at -80°C until the
day of the hybridization procedure. All solutions used in pre-hybridization are
made using sterile DEPC-treated water. On the day of hybridization slides were
warmed to room temperature loaded into autoclaved racks and fixed for 5 min
using 4% formaldehyde in sterile phosphate buffered saline (PBS; pH 7.4).
Slides were then rinsed 2 times with PBS to re-hydrate the tissue. Once re-
hydration was complete tissues were treated with acetic anhydride 0.25% in 0.1

M triethanolamine (TEA) / 0.9% saline (pH 8.0) for 10 min. Acetylation of the

58

tissue via acetic anhydride reduces non-specific binding. Sections were then
dehydrated in serial ethanol solutions (70% 80% 90% and 100%), de-fatted for 5
min in absolute chloroform, re—hydrated in graded ethanol solutions (100% and
95%) and air dried.
In Situ Hybridization Procedure

Labeled probe was added to hybridization buffer containing: 50 %
formamide, 500 ug/ ml sheared ss DNA (Sigma Chemical St. Louis, MO), 250
mg/ ml yeast tRNA, 4X saline-sodium citrate (SSC), 1X Denhardt’s solution, and
10% dextran sulfate to reach a concentration of 20,000 cpm/ [.11. Approximately
25 ul of this hybridization solution was added to each section and incubated

under a sterile coverglass on humidified trays at 37°C for 18 hr. After
hybridization, coverslips were removed and slides rinsed in 1X SSC and then
washed in each of the following: 50% formamide in 2X SSC at 40°C (4 X 15

min), 1X SSC at room temperature (2 X 30 min). Tissues were then dehydrated
in serial ethanol solutions (50, 70, 90 and 95%) containing 300 mM arnoniurn
acetate then in 100% ethanol.

Air dried slides were then dipped in NTB-Z liquid photographic emulsion
(Eastman Kodak; Rochester, NY) diluted 1:1 with water for autoradiographic

analysis. Slides were exposed at -20°C for 7 days. After development slides were

rinsed, counter-stained with nissil stain, dehydrated and coverslipped to facilitate

59

regional localization of exposed silver grains in the photographic emulsion. A

summary of the in situ hybridization histochemistry procedure is depicted in

 

Figure 11.
Dynorphin (DYN) oligonucleotide probe 19:17
+ TDT + [358] *dATP* i .. = ‘ _
A*A*A*
Label DYN oligonucleotide probe with Cut sections and mount
terminal deoxynucleotidyl transferase onto gel-coated slides

 

Apply probe solution onto slides,
hybridize overnight at 37°C

Dissolve probe in
hybridization buffer /@ @/

Post hybridization

63 63 A/wash and dehydrate

Dip slides in photographic emulsion
(NTB—2), expose and develop

 

 

 

 

 

 

 

Figure 11 Scheme for in situ hybridization with synthetic oligonucleotides.

60

 

Image Analysis

Specific labeling of dynorphin mRNA was visualized using dark field
optics (Micro Video Instruments, Avon MA) connected with a Leitz LaborLux S
microscope (Leica, Germany). Quantitative analysis of autoradiographic grain
density was done using the microscope mounted video camera model CCD-725
(Dage-MTI, Michigan City, IN) coupled to a LG3 video card (Scion Corp.
Frederick MD) on a Macintosh 8800 PowerPCTM computer. NIH image (ver.
1.6) and an automated grain counting macro was used to quantify the number of
exposed silvergrains in the emulsion overlying brain sections. The labeling ratio
of a cell was defined as the ratio of exposed silver grains atop its perikarya to the
background number of exposed silver grains in an adjacent cell-sized area of the
neuropil (Popper at al., 1992a; 1992b). An individual cell was considered to be
specifically labeled when the labeling ratio was greater than or equal to three
(Figure 12).

Slides were coded such that the treatment groups were not known until
after the data analysis was complete. Brain sections at approximately the same
level in the hypothalamus -2.30 mm anterior/ posterior to bregma (Paxinos and
Watson 1986) were chosen macroscopically. Ten specifically labeled cells in each

section were counted and background determinations were made to obtain a

61

labeling ratio for each cell. These ten determinations were then averaged to

obtain a labeling ratio for each brain (11 = 6 / treatment group).

62

 

 

Figure 12 Computer captured micrograph depicting specific labeling of a cell containing
dynorphin mRNA in the arcuate nucleus. The arrow indicates a cell specifically labeled by
the dynorphin mRNA probe observed using both darkfield optics (left panel) and brightfield
optics right panel.

63

Dual lmmunohistochemical Determination of Folera and TH

The c-fos gene encodes the F 03 transcription factor which is involved
with the modulation of gene activity in response to cell surface signals (Curran
and Franza 1988). Although neuronal Fos is expressed at very low levels under
baseline conditions, the level of this protein peaks 60 to 90 min after stimulation
and the magnitude of its induction reflects the number of activated neurons
recruited by the stimulus (Lee at al., 1992; Verbalis at al, 1991). Fos/Fra
expression complements neurochemical estimates of TIDA activity since
changes in the synthesis or metabolism of dopamine in the median eminence do
not provide information about the responsiveness of distinct subpopulations of
TIDA neurons. The arcuate nucleus has been subdivided into two distinct
populations of tyrosine hydroxylase immmoreactive (PH-IR) neurons
(dorsomedial and ventrolateral). These distinctions are made based upon
phenotypic differences in size, location of their perikarya and neurochemistry
(Everitt at al., 1986). In studies described in chapter 7 of this dissertation, the
activity of these two groups of TH positive cells were compared by measuring
the expression of Fos/FRA in TH-immunoreactive cell bodies located in the

dorsomedial and ventrolateral arcuate nucleus (Figure 13).

64

it:
mi.
buff
pm

the:

m
. BID

biou

B.

Tissue Preparation

The numbers of TH-IR neurons containing Fos/FRA-IR nuclei were
determined using dual irnmunohistochemistry. After drug administration, rats
were anesthetized with Equithesin (3 ml/ kg; i.p.) and perfused through the aorta
with 0.9%saline (4°C), followed by 4% paraformaldehyde in 0.1 M phosphate
buffer. Brains were removed from the skull and post-fixed overnight in 4%
paraformaldehyde buffer. The brains were then cryoprotected by immasing
them in 0.1 M phosphate buffer containing 20% sucrose at 4°C for 2448 hours.
Frontal sections (36 um) through the medial arcuate nucleus (Cheung at al.,
1997a) were placed in 24 well cell culture plates for immunohistochemical

staining procedures.

Dual Immunohistochemistry

These sections were incubated in a primary sheep anti-Fos oncoprotein
antiserum (1 :2500; Cambridge Research Biochemicals) for 40 hours at 4°C. The
primary antiserum was localized using donkey anti-sheep antibody, the avidin-
biotin complex (Vector Laboratories) and diaminobenzadine subtrate with
nickel-intensification. The sections were then incubated in primary mouse anti-
TH antiserum (1:1000; Incstar Co.) for 40 hours at 4°C. The primary antiserum

was localized with horse anti-mouse antibody and rhodamine—tagged avidin

65

(Vector Laboratories). Thus, double-labeled neurons appear as red cells (T H)

with dark nuclei (F 05 / F RA) depicted in Figure 14).

66

Figure 13 Computer—captured image of the ME and ARC in a frontal brain section. Panel A.:
Depicts the distribution of TH-IR neurons in the ARC located at 2.6 mm posterior to
bregma (Paxinos and Watson, 1986). 3V (third ventricle). The inset is shown as panel B.
Panel B: Computer captured image showing the subdivision of the ARC into dorsomedial
(DM) and ventrolateral (VL) regions.

67

 

68

Figure 14 Computer-captured micrograph depicting a frontal brain section
immunocytochemically labeled for both TH and Fos/FRA. Panel A: Bright-field depiction
of a computer captured image showing Fos/FRA-IR neuclei Panel B: The same computer-
captured image using fluroescence to depict TH-IR neurons. Arrows correspond to cell
bodies where TH- and Fos/FRA-IR are co-localized in the DM-ARC.. Arrows correspond
to perikaryia in which Fos/FRA-IR and TH-IR are co-localized in the DM-ARC. 3V, third

ventricle.

69

 

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Statistical Analyses

In neurochemistry experiments data were tested to determine if they met
the basic requirements of an analysis of variance (AN OVA). That is, that these
data fell into a normal distribution and demonstrated equality of variance
between groups. Data not normally distributed were analyzed by a non-
parametric analysis of variance using Kruskal—Wallace and Dunn’s test for
multiple compansons. Data that violated the assumption of equal variance were
transformed using a logarithmic transformation to reduce the between groups
variance.

Data that were inherently non-parametric (i.e., exposed siver grain counts
for in situ hybridization procedures or cell counts in immunohistochemical
procedures) a square root transformation was performed to convert the data to a
normal distribution and / or analyzed by non-parametric procedures. When only
two treatment groups were compared significant differences were determined by
a Students t—test or if there were more than two groups an AN OVA was used to
determine significant differences among treatment groups. Differences were
considered significant if the probability that the null hypothesis was rejected due

to error was less than 5%.

71

 

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Chapter Three
0;. Receptors Mediate the Stimulatory Effects of Ouinelorane on

Tuberoinfundibular DA Neurons

The lack of an inhibitory response to quinelorane in TIDA compared to
mesolimbic DA neurons is not surprising since it has been well established that
TIDA neurons lack the DA autoreceptor-mediated responses (Demarest and
Moore, 1979) characteristic of other mesotelencephalic and hypothalamic DA
neuronal systems (Lookingland and Moore, 1984). Due to the presence of these
autoreceptors, the activity of most DA neuronal systems is inhibited following
administration of the DA agonist apomorphine and activated when exposed to
the DA antagonist haloperidol. The novel stimulatory effect induced by
quinelorane (Eaton et al., 1993) is intriguing in that it suggests a heretofore-
unrecognized mechanism whereby the activity of TIDA neurons can be
regulated by DA receptors. Although quinelorane has an advantage over first
generation compounds of greater selectivity for D2 versus D1 receptor families,
it has been shown to bind avidly to both D2 and D3 receptor subtypes within the
D2 subfamily (Gackenheirner et a1 1995). Thus, it is not clear which receptor
subtype is mediating the response to quinelorane.

Newer, even more selective agonsts have been developed in an effort to

better distinguish the functional roles of D2 and D3 receptors. PNU-95,666

72

 

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mi

exhibits a high degree of selectivity for the D2 receptor compared to the D1, D3,
or D4 receptors (Smith et a1 1996) whereas, PD128907 has an approximately 18
fold greater affinity for the D3 versus D2 receptor (Pugsley et a1 1995), and at
dosages up to 300 pg/ kg this compound acts selectively at D, receptors (Pugsley
at al 1995; Bristow at al 1996). The availability of these more selective agonists
could be used to determine which DA receptor subtype is mediating the
quinelorane-induced stimulation of TIDA neurons. Administration of selective
D2 or D3 antagonists in conjunction with quinelorane could be an alternative
strategy to determine which DA receptor subtype mediates the stimulatory
action of quinelorane. Since these pharmacological tools were not available
selective agonists were used in experiments described in this chapter.

The purpose of these experiments was to establish the DA receptor
subtype that mediates the stimulatory response of TIDA neurons to quinelorane.
To this end, the effects of PNU-95,666 and PD128907 on the activity of TIDA
neurons were compared in order to determine which selective agonist most
closely mimicked the stimulatory effect of quinelorane. For comparative
purposes the effects of these compounds on the activity of mesolimbic DA
neurons and plasma prolactin concentrations were also assessed. The hypothesis

to be tested is that the stimulatory effect of quinelorane on TIDA neurons is

73

1111

DR

PX

mediated by D2 receptors. If this is the case then PNU-95,666 should generate a

similar response to quinelorane, whereas PD128907 should have no effect.

Methods

Care and handling of the rats, and neurochemistry procedures for
determining the dopamine synthesis and metabolism in median eminence and
nucleus accumbens were carried out as described in the General Methods. The

radioimrnunoassay for plasma prolactin was carried out as described above in the
General Methods. The lower limit of detection in this assay was 120 pg/ 100 pl

of plasma and the intraassay variability was approximately 6%.

Drugs
Quinelorane (Dr. M. Niedenthal The Eli Lilly Company; Indianapolis IN),

PNU-95,666 (Dr. P. VonVoigtlander, The Pharmacia-Upjohn Company,
Kalamazoo, MI), and 3—hydroxybenzylhydrazine dihydrochloride (NSD 1015,
Sigma Chemical Company, St Louis, MO, USA) were dissolved in 0.9 % saline.
PD128907, (Dr. M. D. Davis, Parke Davis Pharmaceutical Research, Ann Arbor,
MI) was dissolved in distilled water. Drugs were administered as indicated in the
legends of the appropriate figures; doses of drugs were calculated as the

respective salts.

74

 

R.

10

C0

Results

The differential effects of incremental doses of quinelorane on DOPAC
concentrations in the median eminence and nucleus accumbens of male rats are
depicted in Figure 15. Quinelorane elicited a dose-related increase in DOPAC
concentrations in the median eminence, with significant increases attained at
doses of 50 and 100 pg/ kg. On the other hand, quinelorane reduced DOPAC
concentrations in the nucleus accumbens, even at the lowest dose employed (10
ug/ kg) In contrast, quinelorane had no effect on dopamine concentrations so
that the ratio of DOPAC to dopamine increased in the median eminence and
decreased in the nucleus (T able 2).

The hallmark of drugs with D2 agonist properties is inhibition of prolactin
secretion from the anterior pituitary (Ben-Jonathan, 1985). Consistent with the
effect of other D2 agonists 10 mg PNU-95,666/ kg significantly reduced plasma
prolactin levels by 30 min and this effect lasted at least 120 min (Figure 16). The
D3 receptor selective dose of PD128907 (300 ug/ kg, Pugsley at al, 1995), failed
to alter plasma levels of prolactin. However, PNU-95,666 induced a dose-related
increase in DOPAC levels in the median eminence and reduced DOPAC
concentrations in the nucleus accumbens (Figure 17). PNU-95,666 failed to alter
dopamine concentrations in these brain regions (Table 3) so that DOPAC to

dopamine ratios increased in the median eminence and decreased in the nucleus

75

accumbens. In contrast, PD128907, the selective D3 agonist, failed to alter
DOPAC (Figure 17) or dopamine in the median eminence or nucleus
accumbens. Thus, DOPAC to dopamine ratios were unchanged in these regions

as well (Table 4).

76

MEDIAN EMINENCE

 

 

 

 

 

- 1:: Vehicle

’5 — Quinelorane
’63 *
§ 21 - *
Q.
E’
3, 14 - T
5
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49 ' NUCLEUS ACCUMBENS
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2 35 - * *
0' *
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B:
5 21 -
2
a. 14 -
O
o 7-
0

 

 

 

 

VEH 10 50 100

Figure 15 Dose response effects of quinelorane on DOPAC concentrations in the median
eminence and nucleus accumbens of male rats. Rats were injected with various doses of
quinelorane (10, 50 or 100 pg/ kg; i.p.) or its 0.9% saline vehicle (1 ml/ kg; i.p.) 60 min prior
to decapitation. Columns represent the means and vertical lines 1 SEM of 8 determinations
of DOPAC concentrations in the median eminence and nucleus accumbens of vehicle-
(open columns) or quinelorane-treated (solid columns) rats. *p < 0.05, values from
quinelorane-treated rats that are significantly different from vehicle-treated controls

77

Table 2 Effects of quinelorane on dopamine and DOPAC/ DA ratios in the median
eminence and nucleus accumbens. Rats were injected with various doses of quinelorane (10,
50 or 100 ug/ kg; i.p.) or its 0.9% saline vehicle (1 ml/ kg; i.p.) 60 min prior to decapitation.
Values in each cell represent the means i 1 SEM of 8 determinations. *p < 0.05, values
from quinelorane-treated rats that are significantly different from vehicle-treated controls.

 

 

 

Median Eminence Nucleus Accumbens
gfidgfk; Dopamine Doggon Dopamine 001122431321
Vehicle 124.7 i 7.7 0.11 r 0.02 80.2 :t 2.5 0.46 i 0.02
10 141.3 i 8.7 0.11 :1: 0.01 93.1 r 3.5 0.30 r 0.01*
50 128.1 i 5.8 0.14 i: 0.01 95.2 i: 5.0 0.33 :l: 001*
100 125.9 :1: 6.9 0.15 :t 001* 85.2 :1: 6.7 0.35 :t: 001*

 

 

 

 

 

 

 

78

 

 

 

10-
A PNU-95,666
o PD-128907

a 8'

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a

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.5 4-

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e

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0. I;
0 30 60 90 120 240 360 480

MIN AFTER INJECTiON

Figure 16 Time course of the effects of PNU-95,666 and PD128907 on prolactin
concentrations in plasma. Rats were decapitated at various times after being injected with
PNU-95,666 (10 mg/ kg so) or PD128907 (0.3 mg/ kg i.p.) 30 minutes after their respective
vehicles (time 0). Symbols represent means and vertical lines 1 SEM. of plasma prolactin
concentration in 5-7 rats; where no vertical line is depicted 1 SEM is less than the radius of
the symbol. Solid symbols represent those values that are significantly different (p < 0.05)

from time 0 controls.

79

 

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Median Eminence Nucleus Accumbens

 

 

   

 

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2 ‘ o
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:wehicle
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1:. °- 50
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E 15 E 40
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0°39 (“g/k9)

Figure 17 Effects of PNU-95,666 and PD128907 on DOPAC concentrations in the median
eminence and nucleus accumbens. Rats were decapitated 30 minutes after being injected so
with incremental doses of PNU-95,666 or its vehicle, or 60 minutes after being injected i.p.
with incremental doses of PD128907 or its vehicle. Columns (open-vehicle; solid-drug)
represent means and vertical lines 1 SEM. of DOPAC concentrations in median eminence
or nucleus accumbens from 7-9 rats. *, Values in drug—treated rats that are significantly
different (p < 0.05) from vehicle-treated controls.

80

Table 3 Effect of PNU-95,666 on dopamine and the DOPAC to dopamine ratio in the
median eminence and nucleus accumbens. Rats were decapitated 30 minutes after being
injected s.c. with incremental doses of PNU-95,666 or its vehicle. Values represent means i
1 SEM. of dopamine, or the ratio of DOPAC to dopamine concentrations in median
eminence or nucleus accumbens from 7-9 rats. *, Values in drug-treated rats that are
significantly different (p < 0.05) from vehicle-treated controls.

 

 

 

 

 

 

 

 

 

 

Median Eminence Nucleus Accumbens
PNU-95,666 Do . DOPAC/DA DO . c DOPAC/ DA
(mg/kg) Pwm‘e Ratio 1mm“ Ratio
Vehicle 196.9 i 11.7 0.09 :I: 0.01 110.8 i: 2.7 0.48 :I: 0.01
1 219.4 i 13.2 0.09 :I: 0.01 108.6 :I: 5.7 0.41 :1: 0.01*
3 204.1 :1: 14.9 0.12 i 0.01* 107.9 d: 5.9 0.37 :I: 0.01*
10 190.8 i 16.1 0.12 i 0.01* 121.7 i 4.5 0.34 :I: 0.01*

 

81

 

Table 4 Lack of effect of PD128907 on dopamine and the DOPAC to dopamine ratio in the
median eminence and nucleus accumbens. Rats were decapitated 60 minutes after being
injected i.p. with incremental doses of PD128907 or its vehicle. Values represent means i 1
SEM. of dopamine, or the ratio of DOPAC to dopamine concentrations in median
eminence or nucleus accumbens from 7-9 rats.

 

 

 

 

 

 

 

 

 

 

PD128907 Median Eminence Nucleus Accumbens
Vehicle 146.1 i 8.3 0.10 i: 0.01 112.9 1: 4.4 0.48 i 0.01
3 144.9 i 7.9 0.10 i 0.01 114.3 1' 2.9 0.51 :1: 0.01
30 151.4 :I: 10.5 0.09 i 0.01 122.6 1: 7.2 0.47 :i: 0.01
300 146.1 i 12.2 0.09 i 0.01 115.3 t 3.9 0.47 :I: 0.03

 

 

82

A similar pattern emerged in the time course study (Figure 18). PNU—
95,666 caused a prompt (within 30 min) and a sustained (more than 120 min)

increase in DOPAC concentrations in the median eminence, while at the highest
dose employed (300 ug/ kg) PD128907 failed to alter the DOPAC content in the

median eminence or nucleus accumbens at any of the times examined.

The lack of effect of PD128907 raised concerns that the drug sample had
lost activity or was injected inappropriately. Thus, an additional experiment was
designed based upon reports that PD128907 reduced the rate of DOPA
accumulation in the nucleus accumbens (Pugsley et al, 1995; Bristow at al, 1996).
In our hands, PD128907 selectively reduced the accumulation of DOPA in the
nucleus accumbens 30 minutes after the administration of NSD1015, but had no
effect on DOPA accumulation in the median eminence (Figure 19).

Although there were no objective measures of motor activity made in
these studies a characteristic pattern of increased motor activity and stereotyped
behavior was observed in rats receiving either quinelorane or PNU-95,666
compared to vehicle control injections. On the other hand, administration of
PD128907 induced a period of reduced motor activity compared to vehicle

controls.

83

 

I“! l"..'

 

rlrle C . n U I u '1‘

30-

A PNU-95,666
25 - O PD-128907

20-

10( MM

DOPAC (11me protein)

 

 

0 30 60 90 120 240 360 480
MIN AFI'EFI INJECTION

Figure 18 Time course of the effects of PNU-95,666 and PD128907 on DOPAC
concentrations in the median eminence. Rats were decapitated at various times after being
injected with PNU-95,666 (10 mg/ kg, s.c.) or PD128907 (0.3 mg/kg i.p.) 30 minutes after
their respective vehicles (Time 0). Symbols represent means and vertical lines 1 SEM of
DOPAC concentrations in the median eminence of 5-8 rats; where no vertical line is
depicted 1 S.E.M. is less than the radius of the symbol. Solid symbols represent those values
that are significantly different (p < 0.05) from time 0 controls.

84

 

 

Artsvuotn. 0E\0£v (0.00.

Median Eminence Nucleus Accumbens

 

 

 

 

 

 

 

 

 

 

 

 

15- 70.
E E
,9 12- .9 56- r
9 9
o.
g 9- T g) 42-
E 5. E 28-
< <
o. 3. CL 14.
8 8
0 0
VEH 3 30 300 VEH 3 30 300

Figure 19 Effects of PD128907 on DOPA concentrations in the median eminence and
nucleus accumbens. Rats were decapitated 30 min after administration of NSD1015 and 60
min after i.p. administration of incremental doses of PD128907 or its vehicle. Columns
(open-vehicle; solid-drug) represent means and vertical lines 1 S.E.M. of DOPAC
concentrations in median eminence or nucleus accumbens from 7-9 rats. *, Values in drug-
treated rats that are significantly different (p < 0.05) from vehicle-treated controls.

85

 

Dis:

Hill

N

Discussion

The results presented here reveal that quinelorane activates TIDA
neurons while inhibiting other ascending DA systems such as the mesolimbic
DA neurons that tenninate in the nucleus accumbens. These findings are
consistent with those of Berry and Gudelsky ( 1991) and Eaton at al, ( 1993)
who demonstrated that second generation DA agonists (e.g., quinpirole and
quinelorane) with increased selectivity for D2 receptors induce a novel
stimulatory response in TIDA neurons. On the other hand, these agonists
induce the typical inhibitory response of first generation agonists (e.g.,
bromocriptine, and apomorphine) observed in other DA neurons via an action at
autoreceptors. The present results comparing the effects of these more selective
compounds with those of quinelorane reveal that the D2 selective agonist PNU-
95,666 closely mimicked the effects of quinelorane on plasma prolactin, TIDA
and mesolimbic DA neuronal activity. Whereas the D3 selective agonist
PD128907 was without effect on plasma prolactin or TIDA neuronal activity,
but did inhibit the activity of mesolimbic DA neurons.

One of the difficulties in making extrapolations about in viva receptor
functionality from DA agonists that have been defined primarily by in vitm

receptor binding assays, is the influence that intrinsic efficacy and receptor

86

 

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Ititli'
100
these
were

prol

RCC

reserve for each of the target receptor subtypes (i.e. D2 and D,) has on functional
activity in viva (Kenakin, 1993). Therefore, it is important to verify the efficacy of
these selective agonists in viva if possible. To this end, plasma prolactin levels
were used as an in viva measure of functional activity at the D2 receptor, since
prolactin secretion is regulated by the action of dopamine on D2 receptors
located on pituitary lactotrophs (Ben-Jonathan at al 1989). Thus, DA agonists
with in viva activity at D2 receptors will lower plasma prolactin concentration by a
direct action on pituitary lactotrophs. In these studies quinelorane and PNU-
95,666 reduced plasma prolactin concentration, while PD128907 was without
effect. These results confirm that both quinelorane and PNU-95,666 but not
PD128907 had in viva efficacy at the D2 receptor.

The subjective behavioral observations made in these experiments are in
accord with studies that have objectively reported decreases in motor activity
following administration of a D3 selective dose of this drug (Pugsley at al 1995;
Bristow et al, 1996). In spite of the lack of a rigorous assessment of in viva D3
receptor function these results confirm that PD128907 exerted a behavioral
effect that has been attributed to the D3 receptor at a dose that had no
measurable in viva efficacy at Dzreceptors (i.e. plasma prolactin concentration).

The D2 selective agonist PNU-95,666 mimics the effects of quinelorane
on TIDA neurons as measured by either synthesis or metabolism of dopamine

in the median eminence, whereas the D3 selective dose of PD128907 (up to 300

87

 

mg/ kg) is without effect These findings support the hypothesis that TIDA
neurons are subject to a novel D2 receptor-mediated stimulatory influence even
though they lack autoreceptor—mediated inhibition characteristic of other DA
neuronal systems. (Roth and Elsworth, 1996). These results also indicate that
TIDA neurons lack a regulatory mechanism mediated by the D3 receptor.

The D2 agonist PNU-95,666 lowered the concentration of DOPAC in the
nucleus accumbens at all dosages tested and these data are consistent with an
action at D2 autoreceptors on mesolimbic DA neurons. Interpretation of the
neurochemical results following administration of the D3 agonist PD128907 is
complicated by the discrepancy between a lack of effect on DOPAC
concentration, and a significant reduction in DOPA accumulation in the nucleus
accumbens. One possible explanation for the lack of effect of PD128907 on
mesolimbic DA metabolism could be that the compound was injected
inappropriately or had lost activity. However, observations that rats given the
drug exhibited a behavioral change consistent with reported effect of the D3
agonist (i.e. decreased motor activity) argues against either of these possibilities.
It therefore seems unlikely that failure to inject the drug correctly would explain
the lack of effect on dopamine metabolism in these studies.

This discrepancy between D, receptor-mediated effects on synthesis and
metabolism of dopamine in these neurons may be explained by one or two

potentially related phenomena. Autoreceptors found on DA neurons can be

88

 

 

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located on cell bodies, dendrites or on axon terminals. These receptors function
to maintain neurotransmitter homeostasis by regulating synthesis and/ or release
of dopamine within the neurons upon which they are located (Elsworth and
Roth 1997). The relative contribution of D2 and D3 receptors in the regulation
of dopamine release and synthesis has yet to be demonstrated unequivocally in
mesolimbic DA neurons. Studies using the D3-prefer1ing agonist 7-OH-DPAT
have suggested that D3 receptors function to modulate the synthesis of
dopamine in these neurons (Aretha et a1 1995). However this assertion is
weakened by the observation that 7OH-DPAT may not be as selective for D3
versus D2 in vivo as determined by in vitm affinity studies (Burris at al, 1995). In
addition, Seabrook and colleagues (1995) have suggested that claims of D3
receptor functionality based on selective agonists should be demonstrated at
dosages that are without effect on plasma prolactin, and be reversible with
selective D3 antagonists. Aretha at al, (1995) met this first criterion by showing
that the selective D3 antagonist UH232 could block the dopamine synthesis
inhibiting effect of 7—OH-DPAT on mesolimbic DA neurons. Although they
didn’t rule out involvement of D2 receptors by measuring plasma prolactin levels,
this finding suggests that D3 receptors may serve as the synthesis modulating
autoreceptors in these neurons.

The density of D2 receptors in the ventral tegmental area is much greater

than that for D3 receptors (Richtand at al 1995). Thus, greater affinity for

89

 

receptors in vitm may not have any functional relevance in viva if the number of

D2 receptors far exceeds that of D3 receptors. This may be highly relevant for
mesolimbic DA neurons with cell bodies located in the ventral tegmental area.
Based on these observations, it could be postulated that the efficacy of
PDl 28907 to affect changes in dopamine homeostasis would necessarily be less
than that of PNU-95,666. Thus, the discrepancy between a lack of effect on
dopamine metabolism and an inhibitory effect on dopamine synthesis may
reflect a lower functional efficacy for the D3 receptor in overall DA receptor-
Inediated autoregulation. However, the inhibition of dopamine synthesis by
PDT 28907 in these studies agrees with the findings of others (Pugsley er a1 1995;
Bristow et a1 1996) that show similar changes in dopamine synthesis. Taken
together, these results suggest that dopamine synthesis may be a more sensitive
aSSfily of the D3 receptor-mediated effect on mesolimbic DA neurons. However,
deSpite this small discrepancy it is clear that these data are in accord with the

hYPOthesis that both D2 and D3 receptors can function as autoreceptors in

IneSOlimbic DA neurons.

Although quinelorane binds to both D2 and D3 receptors, these results
demonstrate that D2 and not D3 receptors mediate the increase in TIDA
ne“tonal activity following administration of this second-generation DA agonist.

DeVelopment of more selective DA agonists such as quinelorane have

90

 

uncovered a previously unrecognized role for dopamine in the regulation of

TIDA neurons.

91

 

Chapter Four
D, Receptor-Mediated Stimulation of TIDA Neurons is Dependent

on Afferent Neuronal Input to the Mediobasal Hypothalamus

Pharmacological evidence reviewed in Chapter Two suggests that the
activity of TIDA neurons can be modulated by a D2 receptor-mediated
mechanism. Activation of D2 receptors with agonists (quinelorane or PNU-
95,666) stimulates rather than inhibits TIDA neurons which is not consistent
with a known action mediated by DA autoreceptors located on axon terminals,
cell bodies or dendrites of these neurons. The inhibitory nature of cellular
responses typically coupled to D2 receptors, e.g. inhibition of adenylyl cyclase,
reduced calcium conductance, or increased potassium conductance (Liu et al,
1994), suggests that the direct response to D2 receptor activation would
necessarily be inhibitory. More likely the stimulatory effect of quinelorane
represents the effect of D2 receptor inhibition of an inhibitory neuronal pathway
(disinhibiton) one or more neurons removed from TIDA neurons. Thus, an
investigation of this neuronal circuit and at least a rudimentary understanding
about the location of D2 receptors involved will be an important first step
toward defining the mechanism of this response.

Deafferentation of the mediobasal hypothalamus (Halasz and Pup 1965)

has been used to isolate the hypothalamo-pituitary axis from the rest of the

92

bit
the
Sul

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me
101
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Ill

thj
life

the

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brain. This technique was developed to study the functional capacity and role of
the mediobasal hypothalamus in control of trophic hormone secretion.
Subsequently, this knife—cut deafferentation procedure was also used to
determine the site of prolactin mediated activation of TIDA neurons (Gudelsky
at al, 1978). In these experiments exogenous administration of prolactin is able
to increase TIDA neuronal activity in rats with surgical deafferentation of the
mediobasal hypothalamus suggesting that the stimulatory effects of prolactin are
not dependent upon afferent neuronal input to the mediobasal hypothalamus.
Rather, prolactin acts within the area circumscribed by the knife-cut to stimulate
TIDA neurons.

The purpose of these experiments of these was to determine the extent to
which the D2 receptor-mediated activation of TIDA neurons is dependent on
afferent neuronal input from outside the mediobasal hypothalamus. To this end,
the effects of quinelorane were tested in rats bearing a complete deafferentation
of the mediobasal hypothalamus. The knife-cut procedure was employed to
isolate the mediobasal hypothalamus and TIDA neurons from afferent neuronal

connections to the rest of the brain (Figure 20).

93

 

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,’I M21." ,

         
 

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I

  

 

 

 

 

r=1.5mm

Figure 20 Schematic diagram depicting a Hillasz knife and the region of the mediobasal
hypothalamus circumscribed by the knife—cut procedure which includes median eminence
(ME), arcuate nucleus (ARC) and the pituitary (denoted by -. Abbreviations: third
ventricle (3V), anterior pituitary (AP), dorsomedial nucleus (DM), intermediate pituitary lobe
(IL), medial zona incerta (MZI), neural pituitary lobe (NL), optic chiasm (ox), periventricular
nucleus (PeVN), and ventromedial nucleus (V M)

The hypothesis tested was that D2 receptor mediated activation of TIDA
neurons is dependent upon afferent neuronal input arising from outside the
mediobasal hypothalamus. If the D2 receptors that mediate the stimulatory
effect on TIDA neurons reside outside the area circumscribed by the knife-cut,

then the effects of quinelorane should be blocked in deafferentated rats.

94

 

Met

Dea'

met

and

5161

bit

W35

T0<

3m!

Methods

Deafferentation of the Mediobasal Hypothalamus

Seven days prior to the day of the experiment Long-Evans rats receiving
mediobasal hypothalamic knife—cut lesions were anesthetized with Equithesin (3
ml/ kg i.p.) Once general anesthesia was apparent, the surgical site was shaved
and scrubbed with betadine and 75% ethyl alcohol. Rats were then placed in a
stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) with the incisor
bar 8 mm below the intraaural line (Konig and Klippel 1963). A small incision
was made along the midline of the skull and the skin retracted so that a 4 mm x 4
mm opening in the skull could be made using a small cutting drill (Dremel Moto-
Tool model 380-5, Racine, WI). A modified Hilasz knife (height 1.5 mm, radius
1.5 mm; (Figure 20) was attached to a stereotaxic carrier and lowered in the
midsagittal plane to the base of the skull 1.3 mm posterior to bregma. The
anterior portion of the mediobasal hypothalamic deafferentation was
accomplished by rotating the knife twice; 90° to the left and then to the right.
The knife was then moved in a posterior direction 1.5mm, rotated 180° and
moved hack 1.5 mm in the anterior direction. The knife was then rotated back
to midline and removed from the brain at the point of entry. Sham surgery
consisted of lowering the knife 3.0 mm below the dura mater surface without

rotation. After surgery the bone was sealed with bone wax and the skin closed

95

 

tit

hi1

Ve

hi2
prt
the
deg
inc
me

Hi?!

HOS

with surgical wound clips. The position and efficacy of the mediobasal
hypothalamic lesions was verified in each rat by histological and neurochemical
criteria described below. Only those animals that met both criteria were included

in the study.

Verification of the Deafferentation

In neurochemistry experiments rats were killed by decapitation and the
brains were removed and rapidly frozen over dry ice. Frontal sections were
prepared from these frozen brains as detailed in the General Methods. During
the preparation of these frontal sections the mediobasal hypothalamic
deafferentation was visually evaluated and rats in which the knife-cut was
incomplete, inappropriately placed, or had evidence of necrosis of the
mediobasal hypothalamus were eliminated from the study. In addition several
neurochemical measures were taken to verify that deafferentation of the
mediobasal hypothalamus was complete. Dopamine and norepinephrine were
measured by high performance liquid chromatography coupled to
electrochemical detection from median eminence and intermediate pituitary lobe
samples. Dopamine and norepinephrine served as indirect indices of DA and

noradrenergic innervation to these regions.

96

Results

The effectiveness of the mediobasal hypothalamic deafferentation was
verified by several neurochemical measures. The result of deafferentation on
dopamine and norepinephrine in the median eminence is shown in Table 5.
After deafferentation median eminence dopamine decreased by 26% and
norepinephrine by 62%. In contrast deafferentation produced a much larger
decrease (45%) in dopamine from samples in the intermediate lobe of the
pituitary (Table 5). As shown in Figure 21, deafferentation of the mediobasal
hypothalamus had no effect on the concentration of DOPAC in the median
eminence per re, but blocked quinelorane-induced increases in this dopamine
metabolite. On the other hand, the knife-cut lesion had no effect on the ability

of quinelorane to decrease DOPAC concentrations in the nucleus accumbens

(Figure 22).

97

 

 

 

Table 5 Effects of surgical deafferentation on the concentration of dopamine and
norepinephrine in the median eminence, and dopamine in the intermediate lobe of the
pituitary of male rats one week after surgery. One week following sham or deafferentation
surgery, rats were sacrificed by decapitation. Values represent means i 1 SEM of the
concentrations of dopamine and norepinephrine from 17 sham surgery control or

mediobasal hypothalamic (MBH) knife-cut male rats.

 

 

 

 

 

 

 

 

Sham Control MBH Knife-Cut
(n=17) (11:17) % Change
Median Eminence
Dopamine 171.83 :I: 7.62 126.23 :I: 14.67 - 26 %
Norepinephrine 29.95 i 2.35 11.43 i 0.87 _ 62 0/0
Intermediate
Pituitary Lobe

Dopamine 15.01 i 1.18 8.31 :I: 1.29 - 45 °/o
Norcpinephrine 2.14 :t 0.22 1.54 :1: 0.22 —28 %

 

98

 

 

:2 Vehicle

 

 

 

 

 

 

 

 

 

 

 

 

2° ' 2° ' — Quinelorane
*

’E‘ E

E 15 - '§ 15 - l

9 9

D. O.

E.” T E’

a, 10 - a, 10 ~

5 5

0 0

E E

O 5 ‘ o 5 *

o o

0 0
Sham Control MBH Knife-cut

Figure 21 Effects of quinelorane on DOPAC concentrations in the median eminence of rats
receiving either sham surgery (Sham Control) or surgical deafferentation of the mediobasal
hypothalamus (MBH Knife-cut). One week following sham or deafferentation surgery, rats
were injected with either quinelorane (100 pg/ kg; i.p.) or its 0.9% saline vehicle (1 ml/kg;
i.p.) 60 min prior to decapitation. Columns represent means and vertical lines 1 SEM of 5-12
determinations of DOPAC concentrations in the median eminence of vehicle- (open
columns) and quinelorane-treated (solid columns) rats. *p < 0.05, values from quinelorane-
treated rats that are significantly different from vehicle-treated controls.

99

 

1:! Vehicle
— Quinelorane

 

 

 

 

 

 

 

 

 

 

 

35 - 35
T
A A T
5 28+ * ,g 28 ~ *
2 2
9 e
O. D.
g, 21 " CE» 21 '1
Bi 3»
5 14 5 14
o 1 o ‘
g a
o 7 1 8 7 -
O 0
Sham Control MBH Knife-cut

Figure 22 Effects of quinelorane on DOPAC concentrations in the nucleus accumbens rats
receiving either sham surgery (Sham Control) or surgical deafferentation of the mediobasal
hypothalamus (MBH Knife-cut). One week following sham or deafferentation surgery, rats
were injected with either quinelorane (100 pg/ kg; i.p.) or its 0.9% saline vehicle (1 ml/ kg,
ip.) 60 min prior to decapitation. Columns represent means and vertical lines 1 SEM of 5-12
determinations of DOPAC concentrations in the nucleus accumbens of vehicle- (open
columns) and quinelorane-treated (solid columns) rats. *p < 0.05, values from quinelorane- '
treated rats that are significantly different from vehicle-treated controls.

100

Discussion

Data presented in the previous chapter demonstrated that TIDA neurons
are activated following administration of quinelorane or the D2 selective agonist
PNU-95,666, but not after administration of the D3 selective agonist PD128907.
These pharmacological data indicate that the activity of TIDA neurons is subject
to modulation by an action at D2 receptors. Since these neurons lack
autoreceptors and the pattern of increased neuronal activity after a DA agonist is
inconsistent with autoreceptor-mediated phenomena, TIDA neurons are not
likely the source of dopamine that could mediate this response. This study was
undertaken to test the hypothesis that the D2 receptor-mediated effect arises
from outside the mediobasal hypothalamus. Deafferentation of the mediobasal
hypothalamus was verified by both neurochemical and histological methods, and
used to determine the dependence of quinelorane-induced activation of TIDA
neurons on neuronal inputs from outside the mediobasal hypothalamus.

Both objective and subjective measures demonstrated that deafferentation
of the mediobasal hypothalamus effectively isolated TIDA neurons from the rest
of the brain. Neurochemical measurements provided evidence that the knife-cut
lesion disrupted afferent neurons originating from outside and terminating
within or passing through the mediobasal hypothalamus. Periventricular

hypophysial DA neurons with cell bodies located in the periventricular nucleus

101

have axons that course thorough the mediobasal hypothalamus and tenninate
within the intermediate lobe of the pituitary (Goudreau 1992). Dopamine levels
were reduced in the intermediate pituitary lobe in lesioned rats suggesting that
periventricular hypophysial DA neurons had been disrupted.

Surgical deafferentation of the mediobasal hypothalamus disrupts afferent
noradrenergic neuronal inputs from the pons medulla to the median eminence
(Fuxe 1965) and results in a 50-60% depletion of norepinephrine concentrations
with in this region (Gudelsky at al, 1978). The 62% loss of NE in lesioned rats
compared to control is consistent with these earlier findings and is evidence that
the knife-cut was complete and disrupted these noradrenergic projections. While
the remaining 38% of norepinephrine left in the median eminence after
deafferentation could indicate that the lesion was not complete, it is possible that
norepinephrine in this instance may come from a peripheral source. Alper at al,
(1980) found that surgical ligation of the superior cervical ganglion reduces
norepinephrine levels in the median eminence by 22%. This finding suggests
that some of the remaining norepinephrine in the median eminence of
mediobasal hypothalamic deafferentated rats may come from these autonomic
noradrenergic neurons.

Neurochemical measurements were also used to insure that TIDA
neurons were not disrupted by surgical deafferentation of the mediobasal

hypothalamus. There was no change in the basal activity of TIDA neurons.

102

However, the 24% decrease in the median eminence dopamine content might
indicate that some TIDA neurons originating in more rostral portions of the
arcuate nucleus were disrupted by the deafferentation procedure. Dopamine is a
precursor of norepinephrine therefore the loss of noradrenergic neurons
following knife-cut lesions may account for some of the loss of median
eminence dopamine. However, the number of DA neurons innervating the
median eminence far outnumber the noradrenergic neurons in this region so this
loss would be very small It is important to note that the loss of dopamine in the
median eminence was substantially less than in the intermediate lobe where DA
neurons had clearly been disrupted.

Physiological changes were also evident after the deafferentation
procedure. Hilasz and Pup (1965) reported that rats displayed a syndrome of
diabetes insipidus after complete mediobasal hypothalrnic deafferentation.
Diabetes insipidus is characterized by excessive drinking and urinating due to the
disruption of antidiuretic hormone-secreting neurons that arise from the
supraoptic and paraventricular nuclei in route to the posterior pituitary.
Increased urinary output and compensatory drinking is due to the loss of
antidiuretic hormone-mediated water reabsorption in the renal collecting duct.
Although no objective measure of water balance was recorded in the present
studies, it was apparent from the need to refill the water bottles and change the

bedding more frequently that animals receiving knife-cut lesions were drinking

103

and urinating significantly more than the sham-treated control rats. Taken
together, these observations suggest that TIDA neurons are relatively spared
compared to noradrenergic afferent projections to the median eminence, or
periventricular hypophysial DA or antidiuretic hormone neurons that pass
through the mediobasal hypothalamus in route to the pituitary gland.

The purpose of this experiment was to determine if activation of TIDA
neurons by D2 receptor-mediated events is dependent upon afferent neuronal
inputs that arise from outside the mediobasal hypothalamus. This possibility was
tested by examining the effects of quinelorane in rats where the TIDA neurons
(and associated mediobasal hypothalamus) was isolated from the rest of the
brain. The results of this experiment revealed that the stimulatory effect of
quinelorane on TIDA neurons is dependent upon afferent neuronal input to the
mediobasal hypothalamus. The inability of quinelorane to increase median
eminence DOPAC concentration in rats with a knife-cut lesion is not likely due
to the disruption of TIDA neurons because the surgical procedure has no effect
on the basal activity of these neurons. In addition, Gudelsky at al, (1978)
demonstrated that TIDA neurons were still responsive the effects of prolactin
after this deafferentation procedure. Nor is the lack of effect due the surgical
stress-induced alterations in the pharmacokinetics of quinelorane, since
mediobasal hypothalamic deafferentation did not alter the inhibitory response of

mesolimbic DA neurons to this D2 agonist. Rather, these results indicate that

104

activation of D2 receptors on neurons residing outside the area circumscribed by
the mediobasal hypothalamic knife-cut provides stimulatory neuronal input to
TIDA neurons.

Diencephalic DA neurons are organized into distinct systems with clusters
of cell bodies that have discrete axonal projections (Dahlstrom and Fuxe 1964;
Bjorklund at al, 1975). The terminal fields of these local circuits remain mostly
within the hypothalamus but are less well defined than those of the ascending
mesotelencephalic DA systems. Any DA neurons outside the region of the
knife-cut including hypothalamic (A13 or A14) or mesotelencephalic DA neurons
(A8 - A10) could be the source of dopamine that mediates this stimulatory
response. The lack of definitive information about DA neuronal circuits that
may be involved in the regulation of TIDA neurons make it difficult to more
specifically define the mechanism whereby this regulation could occur.
Interestingly, electrical stimulation of the dorsomedial nucleus (the site of A13
dendrites) increases the concentration of DOPAC in the median eminence
(Gunnet and Moore 1985), suggesting a role for incertohypothalamic DA
neurons in this process.

An alternative to this interpretation of the results might suggest that
afferent input to the mediobasal hypothalamus is not mediating the stimulatory
Dz-mediated effect on TIDA neurons but rather plays a permissive role. That is,

the loss of afferent input to the mediobasal hypothalamus leads to the loss of the

105

neuronal circuit within this brain region that more directly mediates the
response. If this scenario were true, then D2 receptors that mediate the
stimulatory effect of quinelorane should be located within the mediobasal
hypothalamus. But, localization studies have failed to demonstrate the existence
of significant levels of D2 receptors in this brain region. Autoradiographs of D2
receptor binding or measurement of mRNA for the D2 receptor, have not
demonstrated significant levels of the D2 receptor within the mediobasal
hypothalamus (Bouthenet at al, 1987; 1991). The most straightforward
interpretation of the results presented here support the hypothesis that
quinelorane induces an increase in TIDA neuronal activity by an action at D2
receptors that is mediated indirectly by afferent neuronal inputs that arise from

outside the mediobasal hypothalamus.

106

Chapter Five
Evidence That Activation of Delta/OP1-Fleceptors Stimulates

TIDA Neurons

Three pharmacologically distinct subtypes of opioid receptors have been
identified in the central nervous system: delta, kappa and mu (Goldstein and
James 1984; Martin 1984; and Paterson at al., 1983) recently renamed OP1, OP2
and OP3 respectively (Dhawan at al, 1996). Studies of the regulation of DA
neurons by subtypes of opioid receptors have been dependent upon the
availability of specific agonists and antagonists for these receptors.

Much of what is known about the effects of opioids on DA neurons has
been based upon studies using compounds that act at kappa/OP2 or mu/OP3
receptors. Activation of mu/OP3 receptors stimulates mesolimbic, nigrostriatal
(Di Chiara and Imperato, 1988) and incertohypothalamic DA neurons
(Lookingland and Moore, 1985), while inhibiting TIDA neurons (Deyo et al,
1979; Gudelsky and Porter 1979; Alper at al, 1980). On the other hand,
activation of kappa/OP2 receptors inhibits the basal activity of periventricular
DA neurons and pharmacologically—stimulated activities of mesolimbic DA,
nigrostriatal DA and TIDA neurons (Manzanares at al, 1991).

Comparatively less is known about the role of delta/0P1 receptors in the

regulation of DA neuronal systems in the brain. Administration of the

107

delta/OP1-selective enkephalin analog [D-Pen2,D-Pen5]enkephalin (DPDPE)
increases the release of dopamine in the nucleus accumbens (Spanagel et al,
1990), but has no effect in the striatum (Mulder at al, 1989). No information is
available regarding the effects of delta/0P1 receptor activation or blockade on
the activities of hypothalamic DA neurons. The purpose of these studies was to
determine the dose-response and time course effects of the delta/0P1 selective
agonist DPDPE and antagonist naltrindole on neurochemical estimates of the
activity of TIDA neurons. For comparison purposes the effects of these
treatments on the activity of mesolimbic, nigrostriatal, and periventricular-

hypophysial DA neurons were also evaluated.

Methods

Surgical care and handling of rats for implantation of i.c.v. guide cannula,
and neurochemistry procedures for determining the synthesis and metabolism of
dopamine in the median eminence, nucleus accumbens, striatum and

intermediate pituitary lobe were carried out as described in the General Methods.
Dmgs

The delta/0P1 agonist DPDPE (Peninsula Laboratories, Belmont, CA,
USA) and antagonist naltrindole hydrochloride (Donated by Dr. James H.
Woods University of Michigan Medical School, Ann Arbor, MI, USA) were

dissolved in distilled water. NSD 1015 was dissolved in 0.9% saline. Drugs were

108

administered as indicated in the appropriate figure legends and dosages were

calculated as the respective salts.

Results

Dose response effects of DPDPE on DOPAC concentrations in the
median eminence and nucleus accumbens are depicted in Figure 23.
Intracerebroventricular administration of DPDPE effected a dose-related
increase in DOPAC concentrations in the median eminence and nucleus
accumbens, but was without effect in either the striatum or intermediate lobe of
the pituitary (Table 6). Similarly, DPDPE increased the accumulation of DOPA
in the median eminence and nucleus accumbens (Figure 24), but again was
without effect in either the striatum or intermediate lobe of the pituitary (Table
7). The ability of DPDPE to increase the concentrations of DOPAC in terminals
of TIDA and mesolimbic DA neurons was observed again when the time course
of this delta/0P1 receptor agonist was determined. Figure 25 shows that i.c.v.
administration of DPDPE caused a rapid increase in DOPAC concentrations in
the nucleus accumbens (by 15 min) and median eminence (by 30 min), but again

was without effect in either the striatum or intermediate pituitary lobe (Table 8).

109

Median Eminence

_a _A
0' m

_L
I0

DOPAC (ng/mg protein)
0) (O

(A)

 

O

0.0 0.3 1.0 3.0 10.0

Nucleus Accumbens

   

DOPAC (ng/mg protein)
a '8

O

0.0 0.3 1.0 3.0 10.0
Dose of DPDPE (pg/rat)

Figure 23 Dose-response of the effects of DPDPE on DOPAC concentrations in the
median eminence and nucleus accumbens. Rats were injected i.c.v. with either DPDPE (0.3,
1, 3, 10 ug/rat) or its water vehicle (5 til/rat) 30 min prior to decapitation. Columns
represent means and vertical lines 1 SEM of DOPAC concentrations in tissues obtained
from 6-8 rats. *, values from DPDPE—treated rats (solid columns) that are significantly
different (p < 0.05) from vehicle-treated (open columns) controls.

110

Table 6 Lack of effect of DPDPE on DOPAC concentrations in the striatum and
intermediate pituitary lobe. Rats were injected i.c.v. with either DPDPE (0.3, 1, 3, 10 ug/ rat)
or its water vehicle (5 ul/ rat) 30 min pn'or to decapitation. Values represent the means :1: 1
SEM of DOPAC concentrations (ng/ mg protein) in tissues obtained from 6-8 rats.

 

 

DPDPE Striatum Intermediate Pituitary Lobe
_£E£./m)
Vehicle 41.1 :i: 1.9 1.70 i 0.14
0.3 42.0 :t 1.7 1.88 i- 0.10
1.0 41.9110 1.92i0.16
3.0 44.3 :1: 2.1 1.73 i 0.08
10 47.4 :t 2.1 1.64 :1: 0.11

 

 

 

 

 

111

Median Eminence

DOPA (ng/mg protein)

 

 

0.0 0.3 1.0 3.0 10.0

Nucleus Accumbens
32 ' * *

 

 

 

 

0.0 0.3 1.0 3.0 10.0
Dose of DPDPE (ug/rat)

Figure 24 Dose-response of the effects of DPDPE on the accumulation of DOPA in the
median eminence and nucleus accumbens. Rats were injected i.c.v. with either DPDPE (0.3,
1, 3, 10 ug/ rat) or its water vehicle (5 p.1/ rat) and killed by decapitation 30 min later. All rats
were injected with NSD 1015 30 min prior to decapitation. Columns represent means and
vertical lines 1 SEM of DOPA concentrations in tissues obtained from 6-8 rats. *, values
from DPDPE-treated rats (solid columns) that are significantly different (p < 0.05) from
vehicle-treated (open columns) controls.

112

Table 7 Lack of effect of DPDPE on DOPA concentrations in striatum and intermediate
pituitary lobe. Rats were injected i.c.v. with either DPDPE (0.3, 1, 3, 10 ug/ rat) or its water

vehicle (5 [.11/ rat) 30 min prior to decapitation. Values represent the means :1: 1 SEM of
DOPA concentrations (ng/ mg protein) in tissues obtained from 6-8 rats.

 

 

 

DPDPE Striatum Intermediate Lobe
Ara/rat)
Vehicle 23.7 i 0.6 1.88 i 0.11
0,3 22.7 :t 1.8 1.77 i‘ 0.12
1.0 21.0 :t 0.8 1.82: 0.19
3.0 22.3 :1: 0.7 1.81 i 0.07
10 22.7 i 0.8 1.90 i: 0.04

 

 

 

 

113

Median Eminence
24 -

20‘
16*

12-

DOPAC (ng/mg protein)

 

 

o 1530 so 120 240
Nucleus Accumbens
7s -

Sad/\%
45- 4

30‘

 

DOPAC (ng/mg protein)

15‘

 

 

0 1530 60 120 240
Min After DPDPE

Figure 25 Time course of the effects of DPDPE on DOPAC concentrations in the median
eminence and nucleus accumbens. Rats were injected i.c.v. with DPDPE (10 ug/ rat) and
killed by decapitation either 15, 30, 60, 120 or 240 min later. Control rats were injected i.c.v.
with water vehicle (5 ul/ rat) and killed by decapitation 30 min later. Symbols represent
means and vertical lines 1 SEM of DOPAC concentrations in tissues from 5-9 rats. When
vertical lines are not visible 1 SEM is smaller than the radius of the symbol. Solid symbols
represent values from DPDPE-treated rats that are significantly different (p < 0.05) from
vehicle-treated controls.

114

Table 8 Lack of effect of DPDPE on DOPAC concentrations in striatum and intermediate
pituitary lobe. Rats were injected i.c.v. with DPDPE (10 ug/ rat) and killed by decapitation
either 15, 30, 60, 120 or 240 min later. Control rats were injected i.c.v. with water vehicle (5
111/ rat) and killed by decapitation 30 min later. Values are reported as means :I: 1 SEM of
DOPAC concentrations (ng/ mg protein) in tissues from 5-9 rats.

 

 

 

 

 

 

its: Striatum IntermedlizgeePituitary
DPDPE
Vehicle 38.5 3:19 1.50 i014
15 39.9 i1.7 1.52 i010
30 39.1 :10 1.52 i016
60 41.7 i- 2.1 1.52 i 0.08
120 37.6 i 1.1 1.60 i008
240 35.5 i 2.1 1.67 i 0.11

 

 

115

Intracerebroventricular administration of the selective delta/0P1 receptor
antagonist naltrindole had no effect on DOPAC concentrations in either the
median eminence or nucleus accumbens (Figure 26), nor did it alter the
concentrations of DOPAC in the striatum or intermediate pituitary lobe (Table
9). On the other hand, naltrindole completely blocked DPDPE-induced
increases in DOPAC concentrations in both the median eminence and nucleus

accumbens (Figure 27).

116

Median Eminence

-‘N
.A

000030100100

1

EL

1

_A-L

DOPAC (11ng protein)

A

 

 

 

 

0.0 0.3 1.0 3.0

Nucleus Accumbens

N (A) A O)
k» 0) on O

DOPAC (ng/mg protein)
‘16

 

O

0.0 0.3 1.0 3.0
Dose of Naltrindole (pg/rat)

Figure 26 Lack of effect of naltrindole on DOPAC concentrations in the median eminence
and nucleus accumbens. Rats were injected i.c.v. with either naltrindole hydrochloride (0.3, 1
and 3 pg/ rat) or its water vehicle (5 ul/ rat) 45 min prior to decapitation. Columns represent
means and vertical lines 1 SEM of DOPAC concentrations in tissues from 8-9 rats

117

Table 9 Lack of effect of naltrindole on DOPAC concentrations in the striatum and
intermediate pituitary lobe. Rats were injected i.c.v. with either naltrindole (0.3, 1 and 3
ug/ rat) or its water vehicle (5 pl/ rat) 45 min prior to decapitation. Values represent the
means i 1 SEM of DOPAC concentrations (ng/ mg protein) in tissues from 8-9 rats.

 

 

 

 

 

 

DPDPE Striatum Intermediate Pituitary Lobe
Ali's/m)
Vehicle 237 i 0.6 1.88 i 0.11
0.3 22.7 i1.8 1.77 i 0.12
1.0 21.0 i 0.8 1.82 2'.”- 0.19
3.0 22.3 i 0.7 1.81 i 0.07
10 I 22.7 i 0.8 1.90 i 0.04

118

Median Eminence

 

 

 

 

 

 

 

 

 

13-
,E t 1: Vehicle
#3 ‘5‘ — DPDPE
D. 12 e
“e” * ’
a, 9
5
0 6j
E
O 3'
o

0

Vehicle Naltrindole
so Nucleus Accumbens

70'1 a
60*
501

 

 

zoj
10-

DOPAC (rig/mg protein)
8

 

 

 

 

 

 

 

Vehicle Naltrindole

Figure 27 Effects of naltrindole on DPDPE-induced increases in DOPAC concentrations in
median eminence and nucleus accumbens. Rats were injected i.c.v. with either a mixture of
DPDPE (1O |.l.g/ rat and naltrindole hydrochloride (3 ug/ rat), or DPDPE (10 ug/ rat) alone
and killed by decapitation 45 min later. Control rats were injected ic.v. with water vehicle (5
Lll/ rat) 45 min prior to decapitation. Columns represent means and vertical lines 1 SEM of
DOPAC concentrations in tissues of 6-8 rats. *, values form DPDPE-treated rats (solid
columns) that are significantly different (p < 0.05) from vehicle-treated (open columns)
controls.

119

Discussion

The results of these studies indicate that i.c.v. administration of the
delta/0P1 selective agonist DPDPE increases the activity of mesolimbic and
TIDA neurons, but fails to alter the activity of either nigrostriatal or
periventdcular—hypophysial DA neurons. The stimulatory effect of DPDPE on
mesolimbic DA neurons is in agreement with a previous report (Spanagel et al,
1990) demonstrating that the DPDPE-induced increases of mesolimbic DA
neuronal activity is blocked by the selective delta/0P1 antagonist naltrindole.
These results indicate that this action is mediated through delta/0P1 receptors.
Kappa/OP2 and mu/OP3 receptor agonists inhibit and stimulate respectively,
the activities of both mesolimbic and nigrostriatal DA neurons in concert
(Manzanares et al, 1991; Di Chiara and Imperato, 1988). The selective
stimulatory effect of DPDPE on mesolimbic but not nigrostriatal DA neurons
represents a departure from effects observed after administration of kappa/OP2
or mu/OP3 opioid agonists.

The present study represents the first in viva demonstration that the
activity of TIDA neurons is increased following activation of delta/0P1
receptors with DPDPE. These results reveal that delta/0P1 receptor-mediated
regulation of DA neurons is not limited to mesolimbic DA neurons. Indeed, the

efficacy for delta/OPl-mediated increases in neuronal activity appears to be

120

 

 

greater for TIDA compared to mesolimbic DA neurons, as evidenced by a
greater magnitude of increase in DOPAC concentrations in the median
eminence compared to the nucleus accumbens following DPDPE
administration. On the other hand, DPDPE has no effect on the activity of
periventricular-hypophysial DA neurons that terminate in the intermediate lobe
of the pituitary, and in this respect, these neurons resemble the DA neurons of
the nigrostriatal system.

A possible explanation for the lack of effect of DPDPE on nigrostriatal
and periventriwlar—hypophysial DA neurons is that delta / 0P1 receptors are
already maximally occupied by an endogenous ligand. But, blockade of
delta/0P1 receptors with naltrindole had no effect on the activities of
periventricular-hypophysial, nigrostriatal, mesolimbic or TIDA neurons
suggesting that there is no tonic regulation of these DA neurons by endogenous
delta/0P1 selective neuropeptides. Thus, the lack of effect of DPDPE on
nigrostriatal and periventricular-hypophysial DA neurons is not due to pre-
existing maximum stimulation by endogenous delta/ 0P1 ligands.

There is substantial evidence for interactions between enkephalinergic and
mesotelencephalic DA neuronal systems. Exogenous application of dopamine
to rat striatal slices in vitro induces an efflux of MET-enkephalin that is blocked
by pretreatment with D2 antagonist and mimicked by apomorphine but not D1

selective agonists (Pasinetti et al, 1984). Administration of quinpirole induces a

121

stimulatory effect on enkephalin gene expression and release of enkephalin in the
striatum in viva (Bannon et al., 1989). In addition Pickel et al, (1992) have
provided anatomical evidence for these DA / enkephalinergic interactions.
Delta/0P1 receptors have a more restricted distribution than other
subtypes of opioid receptors. Dense areas of delta/0P1 receptors have been
located in olfactory-related areas, neocortex, striatum, nucleus accumbens and
amygdala, whereas few receptors have been located in the hypothalamus
(Mansour at al, 1988; Desjardins et al, 1990). While the stimulatory action of
DPDPE on mesolimbic DA neurons may occur at the receptors located in
nucleus accumbens, the absence of delta/0P1 receptors in regions containing
cell bodies or terminals of TIDA neurons suggests that the stimulatory action of
DPDPE on these neurons may occur indirectly, possibly via afferent neurons.
The studies described in this chapter represent the first in viva
demonstration that the activity of TIDA neurons like that of mesolimbic DA
neurons can be acutely activated by DPDPE via an action at delta/0P1

receptors.

122

Chapter Six
Activation of 02 Receptors Fails to Induce the Release of A

Neurotransmitter That Stimulates T IDA Neurons

Results from studies presented in Chapters Two and Three demonstrated
that activity of TIDA neurons is regulated via a modulatory pathway that
involves activation of D2 receptors, and that Dz-mediated stimulation TIDA
neurons is indirect and mediated by afferent neuronal input to the mediobasal
hypothalamus. In order to explore the nature of these afferent neurons two
basic hypotheses were formulated. The first of these states that activation of D2
receptors induces the release of a neurotransmitter that stimulates TIDA
neurons. A second (but not mutually exclusive) hypothesis states that activation
of TIDA may be the result of inhibition of an endogenous substance that
tonically inhibits TIDA neurons. These possibilities are examined in this and the
subsequent chapter.

In order for the first hypothesis to be correct two basic criteria would
have to be met; 1) administration of the putative stimulatory neurotransmitter or
its receptor agonist should activate TIDA neurons, and 2) pharmacological
blockade of receptors for these neurotransmitters should prevent the D2

receptor-mediated activation of TIDA neurons. At least two classes of receptors

123

for endogenous neuropeptides have been identified which fullfill this first

criterion.

Delta/0P1 opioid receptors
Experiments described in chapter 4 demonstrated that DPDPE acting via

delta/0P1 receptors acutely stimulates TIDA neurons. This suggests the
possibility that endogenous enkephalins acting selectively at delta/0P1 receptors
may be involved in quinelorane-induced activation of these neurons. In support
of this theory there is substantial evidence for interactions between
enkephalinergic and mesotelencephalic DA neuronal systems. Indeed,
exogenous application of dopamine to rat striatal slices in vitra induces an efflux
of MET-enkephalin that is blocked by pretreatment with a D2 antagonist and
mimicked by nonselective agonist apomorphine but not D1 selective agonists
(Pasinetti at al, 1984). In viva the D2 receptor agonist quinpirole increases
preproenkephalin gene expression in the stn'atum, whereas D1 agonists are
ineffective (Bannon at al, 1989). In addition, Pickel at al, (1992) have provided
anatomical evidence for DA / enkephalinergic interactions in mesotelencephalic
DA neurons, but there are no studies describing similar DA / enkephalinergic

interactions in the mediobasal hypothalamus.

124

Neurotensin receptors

The tridecapeptide neurotensin was initially isolated from bovine
hypothalamus (Carraway et al, 1973). Subsequently, neurotensin has been found
to be widely distributed throughout the central nervous system (U hl et al., 1977)
but is found in highest concentration in nucleus accumbens, preoptic area and
mediobasal hypothalamus (Kobayashi et al, 1977). Of particular interest is the
wealth of pharmacological, anatomical and neurochemical evidence that
neurotensin interacts with central DA neurons (N emeroff 1986). The
neuroleptic-like effects attributed to exogenous administration of neurotensin
have consequently focused much of the attention upon these interactions in the
mesolimbic and nigrostriatal DA neurons. (Nemeroff 1980; Ervin at al, 1981;
Blaha at al, 1992). The dopamine inhibiting property of endogenous neurotensin
was directly tested by Wagstaff et al, (1994) who demonstrated that
methamphetamine-induced dopamine release was facilitated in rats pretreated
with either neurotensin antiserum or SR48692, the non-peptide antagonist of
neurotensin receptors. Quinpirole, a second-generation D2 agonist, can increase
neurotensin release in the anterior nucleus accumbens and lateral caudate
nucleus (Wagstaff at al, 1996). Thus, elevated DA neuronal activity would

induce neurotensin release that, in turn, inhibits DA neurons.

125

Neurotensin imrnunoreactive cell bodies have also been localized to
mediobasal hypothalamic regions; including the arcuate nucleus (Kahn at al,
1980) which contains the perikarya of TIDA neurons. Additional observations
have provided direct evidence that neurotensin activates TIDA neurons (Berry
and Gudelsky 1990; Pan at al, 1992), and that this increased activity was
coincident with a reduction in the release of prolactin from the anterior pituitary
(Pan at al, 1992). The discovery of SR48692, a non-peptide antagonist of
neurotensin receptors (Gully at al, 1993) has made it possible to further test the
physiological role of neurotensin in the regulation of TIDA neurons and
prolactin secretion.

The goal of experiments presented in this chapter is to test the hypothesis
that endogenous enkephalins via delta/0P1 receptors and/ or neurotensin via
neurotensin receptors are involved in D2 receptor-mediated increases in TIDA
neuronal activity. To this end, the effect of quinelorane on DOPAC
concentrations in the median eminence were determined in rats pretreated with
either the delta/0P1 receptor antagonist naltrindole, or the neurotensin receptor
antagonist SR48692. For comparative purposes the concentration of DOPAC in
the nucleus accumbens was also measured. If release of either enkephalins or
neurotensin mediate the stimulatory effects of quinelorane, then administration
of these selective antagonists should block quinelorane-induced increases in the

concentration of DOPAC in the median eminence.

126

IMnhods

Care and handling of rats and brain tissue used in these experiments
followed the procedures for surgical implantation of i.c.v. guide cannula, and
neurochemistry procedures for measuring the concentration of DOPAC in the

median eminence and nucleus accumbens, were performed as described in the

General Methods.

[Mugs
SR48692 (Provided by Dr. Danielle Gully Sanofi Recherche, Toulouse,

France) was dissolved in 10% dimethyl-sulfoxide (DMSO), naltrindole
hydrochloride (Donated by Dr. James H. Woods University of Michigan Medical
School, Ann Arbor, MI, USA) was dissolved in distilled water. Quinelorane
dihydrochloride was dissolved in 0.9% saline. Drugs were administered as
indicated in the appropriate figure legends and dosages were calculated as the

respective salts.

Resuns

Figure 28 depicts the effect of quinelorane alone or when co-administered
with naltrindole on the concentration of DOPAC in the median eminence and
nucleus accumbens. These results show that naltrindole failed to block the
quinelorane-induced increase in the concentration of DOPAC in the median

eminence. Similarly, quinelorane-induced decreases in the concentration of

127

DOPAC in the nucleus accumbens were not altered by co-administration of
naltrindole.

The effect of co-administration of the selective neurotensin antagonist
SR48692 on quinelorane-induced changes in the concentration of DOPAC in
the median eminence and nucleus accumbens are shown in Figure 29. Co-
administration of SR48692 had no effect on the quinelorane-induced increase in
the concentration of DOPAC in the median eminence or the decrease in the

concentration of DOPAC in the nucleus accumbens.

128

Median Eminence

C: Vehicle
. — Quinelorane

d”
m—t

*

d
U"

A
N

 

 

9-1

DOPAC (rig/mg protein)
.4

 

 

 

 

 

o —
Vehicle Naltrindole

 

Nucleus Accumbens

21- T

18" a T

 

 

15‘

121

61

DOPAC (rig/mg protein)

 

 

 

 

 

 

 

 

Vehicle Naltrindole

Figure 28 Lack of effect of naltrindole on quinelorane-induced increases in DOPAC
concentrations in the median eminence and nucleus accumbens. Rats were injected with
quinelorane (100 ug/ kg; i.p.) or its 0.9% saline vehicle (1 ml/ kg i.p.), and naltrindole
hydrochloride (3 ug/ rat; i.c.v.) or its water vehicle (3 )11/ rat) 60 min prior to decapitation.
Columns represent means and vertical lines 1 SEM of DOPAC concentrations in tissues
sampled from 7-8 rats. *, values from quinelorane-treated rats (solid columns) that are
significantly different (p < 0.05) from vehicle-treated (open columns) controls.

129

DOPAC (rig/mg protein)

DOPAC (ng/mg protein)

'0
d

.5
m

_L
0"

_tL
N

 

Median Eminence

:21 Vehicle
— Quinelorane

*

 

 

 

 

 

 

 

 

 

Vehicle SIR-48692

Nucleus Accumbens

T

 

T

 

 

 

 

 

 

 

Vehicle SR48692

Figure 29 Lack of effect of SR48692 on quinelorane-induced changes in DOPAC
concentrations in the median eminence and nucleus accumbens. Rats were injected with
quinelorane (100 ug/ kg, i.p.) or its 0.9% saline vehicle (1 ml/kg i.p.), and SR48692 (100
[lg/kg; i.p.) or its 10 °/o DMSO vehicle (1 ml/ kg, i.p.) 60 min prior to decapitation. Columns
represent means and vertical lines 1 SEM of DOPAC concentrations in tissues sampled from
*, Values from quinelorane-treated rats (solid columns) that are significantly
different (p < 0.05) from vehicle-treated (open columns) controls.

130

Discussion

Quinelorane-induced activation of TIDA neurons is mediated by D2
receptors and dependent upon afferent neuronal input arising from outside the
mediobasal hypothalamus. There are a number of stimulatory neuropeptides that
could serve as intermediaries in this pathway most prominently enkephalins and
neurotensin. As demonstrated in the previous chapter pharmacological
activation of delta/0P1 receptors with the selective agonist DPDPE activates
TIDA neurons. However, little is known about endogenous
DA / enkephalinergic interactions in the hypothalamus of male rats. In female
rats during lactation and suckling there is an increase in the expression of
enkephalin in tyrosine hydroxylase positive neurons in the arcuate nucleus
(Merchenthaler 1993; 1994; Merchenthaler at al, 1995). In this instance
enkephalins are thought to help maintain elevated prolactin during lactation and
suckling by suppressing the tendency of TIDA neurons to become activated
thereby reducing prolactin secretion in response to this hyperprolactinemic state.

Experiments in this chapter were performed to test the hypothesis that
activation of TIDA neurons is dependent upon endogenous activation of
delta/OP1-receptors. The observation that naltrindole does not alter the
stimulatory effect of quinelorane on TIDA neurons indicates that endogenous

release of enkephalins acting at delta/0P1 receptors do not mediate this

131

stimulatory response. Thus, there is no evidence to suggest that quinelorane
activates TIDA neurons via an action on enkephalinergic neurons.

Comparatively more is known about neurotensin and DA neuronal
interactions in the mediobasal hypothalamus. Administration of neurotensin
directly into the third cerebral ventricle reduces the concentration of prolactin in
plasma via a dopamine dependent process (Koenig at al, 1982). In addition,
Gudelsky at al, (1989) have established that exogenous administration of
neurotensin induces a dose-related increase in the activity of TIDA neurons in
male rats. Similar results have been obtained in female rats where the increase in
TIDA activity following neurotensin administration was correlated with
reductions in the concentration of plasma prolactin (Pan at al, 1992).

Hentschel and coworkers (1998) have shown that SR48692 can block the
stimulatory effects of prolactin on TIDA neuronal activity. In these studies
SR48692 had no effect per re but did block the haloperidol—induced prolactin-
mediated activation of these neurons. Taken together, these results suggest that
the stimulatory effect of prolactin on TIDA neurons may be mediated, in part,
by endogenous neurotensin. These observations coupled with the finding that
D2—like agonists induce the release of neurotensin in the telencephalon raised the
possibility that endogenous release of neurotensin mediates the Dz-induced

increase in TIDA neuronal activity.

132

The failure of the neurotensin antagonist SR48692 to modify the
stimulatory effect of quinelorane on TIDA neurons argues against the
involvement of neurotenisn in this stimulatory pathway. One possible
explanation for the ineffectiveness of SR48692 could be that the antagonist does
not interact with the neurotensin receptor that mediates the activation of TIDA
neurons. Studies utilizing a variety of in vitm and in viva assays have demonstrated
that although SR48692 can antagonize neurotensin-induced hypomotility in the
rat, it fails to block neurotensin-induced hypothermia and analgesia (Dubuc at al,
1994). These disparate effects are suggestive of at least two neurotensin receptor
subtypes, one of which is SR48692 insensitive. On the other hand, SR48692 can
block the haloperidol-induced prolactin-mediated activation of TIDA neurons,
suggesting that this effect is mediated, in part, by endogenous neurotensin
(Hentschel at al, 1998). This observation establishes that prolactin feedback
activation of TIDA neurons is mediated by a SR48692 sensitive neurotensin
receptor and by extension is independent of the stimulatory effects mediated by
D2 receptor agonists.

For comparison purposes the effects of these treatments on the activity of
mesolimbic DA neurons were also determined. The results reveal that blockade
of either delta/0P1 receptors or neurotensin receptors has no effect on

quinelorane induced inhibition of these neurons. These results indicate that

133

neither endogenous enkephalins nor neurotensin are involved in the D2
receptor— mediated inhibitory feed back of these neurons.

Taken together, the results of these studies demonstrate that neither
endogenous NT nor enkephalins acting at NT- or delta/OPl-receptors,
respectively, mediates the quinelorane-induced increase in TIDA neuronal

activity.

134

Chapter Seven
Evidence that Dz Receptor-Mediated Activation of TIDA Neurons

Occurs via Disinhibition

Results presented in the previous chapter demonstrated that quinelorane-
induced activation of TIDA neurons is not mediated by the release of
endogenous neurotensin or enkephalin. Alternatively, stimulatory D2 receptor-
mediated afferent neuronal regulation of TIDA neurons could occur via
suppression of a tonically active inhibitory system. This disinhibition hypothesis
is attractive due to the abundant evidence that activation of D2 receptors is
coupled to inhibitory cellular events such as inhibition of adenylyl cyclase and
reduced calcium conductance, or increases in potassium conductance (Jackson
and Westlind-Danielsson 1994). Thus, a mechanism involving disinhibition is
invoked to explain how inhibitory D2 receptor activation would induce a net
increase in TIDA neuronal activity. For this disinhibition hypothesis to be
correct two criteria must be met: 1) TIDA neurons must be tonically inhibited,
and 2) exogenous administration of an inhibitory neurotransmitter or a receptor
agonist should block D2 receptor-mediated regulation of TIDA neurons. The
inhibitory neurotransmitter dynorphin via an action at kappa/OP2 receptors is

known to meet the first criterion.

135

In the male rat, the activity of TIDA neurons is tonically inhibited by
dynorphinergic neurons; an action that is mediated by kappa/OP2 receptors.
Administration of the kappa/OP2 receptor-selective agonist U-50,488 has no
dose- or time-related effect on either the accumulation of DOPA or
concentrations of DOPAC in the median eminence (Manzanares at al, 1991;
1992a). Whereas, administration of the selective kappa/OP2 antagonist nor-
binaltorphirnine (nor-BNI) induces an increase in the accumulation of DOPA in
median eminence that is reversed by U-50,488 (Manzanares et al, 1992a).
Furthermore, immunoneutralization of either the active fragment dynorphin],8 or
full-length dynorphin].17 induces an increase in the concentration of DOPAC in
the median eminence that was reversed by administration of U-50,488
(Manzanares at al, 1992b) These data indicate that the activity of TIDA neurons
in male rats is tonically inhibited by endogenous dynorphin acting at kappa/OP2
receptors. Tonic inhibition of TIDA neurons in the male rat is also dependent
upon the presence of androgens (Gunnet at al, 1986; Toney at al, 1991), and
Manzanares at al, (1992a) have demonstrated that this inhibitory action of
androgens is mediated, in part, by endogenous dynorphin acting at kappa/OP2
receptors.

The purpose of the experiments described in this chapter is to test the
hypothesis that inhibition of tonically active dynorphinergic neurons is the

mechanism that accounts for Dz-receptor-mediated activation of TIDA neurons.

136

To this end, the effects of the kappa/OPZ-selective agonist U-50,488 and
antagonist norBNI were tested for their ability of modify quinelorane-induced
changes in the synthesis of dopamine in the median eminence. For comparison,
neurochemical measurements were also made in the nucleus accumbens. Since
gonadal androgens are known to be important for maintenance of the tonic
dynorphinergic inhibition of TIDA neurons, an additional series of experiments
was carried out in orchidectomized males. In situ hybridization studies were also
conducted to determine if quinelorane-induced increases in TIDA neuronal
activity were accompanied by a reduction in the activity of dynorphinergic
neurons in the hypothalamus. If a reduction in dynorphin release and
subsequent activation of inhibitory kappa/OP2 receptors is involved in this
change in TIDA activity then administration of the kappa/OP2 agonist U-
50,488 should re-instate this inhibitory tone and block the stimulatory effects of
quinelorane. On the other hand, the blockade of kappa/0P1 receptors with nor-
BNI should acutely stimulate TIDA neurons, but prevent further activation by
quinelorane.

There was concern that a lack of quinelorane effect in nor-BNI treated
rats could be attributed to reaching a ceiling in the maximal possible activation of
TIDA neurons. Therefore some rats in these studies were pre-treated with
prolactin. The prolactin mediated activation of TIDA neurons is known to

occur by a mechanism different from thst of quinelorane, and these treatments

137

should be additive. Since maintenance of inhibitory dynorphinergic tone is
gonadal steroid dependent, orchidectomy alone should increase basal TIDA
activity and block both nor-BNI- and quinelorane-induced changes in median
eminence DOPA accumulation. Finally, if quinelorane induced changes are the
result of inhibition of a population of dynorphinergic neurons then expression of

dynorphin mRNA should be reduced after quinelorane treatment.

Methods

Surgical care and handling of rats for implantation of i.c.v. guide cannula,
gonadectomies, and neurochemistry procedures were carried out as described in
the General Methods. The level of dynorphin mRNA expression in the arcuate

and ventromedial nuclei was measured by in .ritu hybridization procedures

described in General Methods.

Drugs
U-50,488 HCl (Dr. P. VonVoigtlander, The Pharmacia and Upjohn

Company, Kalamazoo, MI), nor-BNI HCl, (RBI, Natic, MA) and rat prolactin
(Dr. A.F. Parlow, NIDDK-rPRL-B—S-SIAFP Lot # APP-3697A) were dissolved
in distilled water. Quinelorane dihydrochloride and NSD1015 were dissolved in
0.9 % saline. Drugs were administered as indicated in the legends of the
appropriate figures; doses of drugs were calculated based on the weight of the

free base or salt form of each drug.

138

Results

As shown in Figure 30, activation of kappa/OP2 receptors with U-50,488
(10 mg/ kg, s.c.) had no effect per re, but prevented quinelorane-induced increases
in the concentration of DOPA in the median eminence. In contrast, U-50,488
did not alter quinelorane-induced decreases in the concentration of DOPA in the
nucleus accumbens. Blockade of kappa/OP2 receptors with nor-BNI increased
median eminence DOPA accumulation, and prevented the stimulatory effects of
quinelorane on dopamine synthesis in this region (Figure 31). Administration of
prolactin also increased median eminence DOPA accumulation, but did not alter
the ability of quinelorane to further stimulate dopamine synthesis in the median
eminence. On the other hand, neither nor-BNI nor prolactin pretreatment
altered quinelorane-induced decreases in DOPA concentration in the nucleus
accumbens.

Orchidectomy increased the basal concentration of DOPA in the median
eminence but had no effect on the quinelorane-induced increases in DOPA
concentration in this region (Figure 32). As shown in Figure 33, nor-BNI
increased DOPA concentrations in the median eminence of gonadally-intact and
orchidectomized rats. Orchidectomy alone increased the concentration of
DOPA compared to intact controls but did not prevent the nor—BNI induced

increase in DOPA accumulation.

139

Specific hybridization of 3’sS-labeled cells in the arcuate and ventromedial
nuclei is consistent with the presence of dynorphin containing neuronal cells
within these brain regions. Administration of quinelorane (100 lig/ kg, i.p.)
significantly reduced the dynorphin mRNA labeling ratio in the arcuate nucleus

but was without effect in the ventromedial hypothalamic nucleus (Figure 34).

140

 

Median Eminence

.A.
U"

* 1:1 Saline
— Quinelorane

—L
N

 

 

DOPA (rig/mg protein)
H

 

 

 

 

 

 

 

Vehicle U50488

15 . Nucleus Accumbens

121

 

 

DOPA (rig/mg protein)

 

 

 

 

 

 

 

Vehicle U50488

Figure 30 Effects of quinelorane on DOPA concentrations in the median eminence and
nucleus accumbens of vehicle— and U-50,488-treated rats. Animals were injected with either
U—50,488 (10 mg/ kg; s.c.) or its distilled water vehicle (1 ml/ kg, s.c.) 65 min prior to
decapitation and with either quinelorane (100 pg/ kg, i.p.) or its 0.9% saline vehicle (1 ml/ kg;
i.p.) 60 min prior to decapitation. All rats were injected with NSD 1015 (100 mg/ kg; i.p.) 30
min prior to decapitation. Columns represent means and vertical lines 1 SEM of 6-9
determinations of DOPA concentrations in the median eminence and nucleus accumbens of
vehicle- (open columns) or quinelorane- (solid columns) treated rats. * P < 0.05, values from
quinelorane-treated rats that are significantly different from vehicle-treated controls.

141

Figure 31 Effects of quinelorane on DOPA concentrations in the median eminence and
nucleus accumbens of vehicle-, nor-BNI- and prolactin-treated rats. Rats were injected with
either nor-BNI (12.5 ug/ rat; i.c.v) or its distilled water vehicle (3 pl/ rat; i.c.v.) 120 min prior
to decapitation, or with either prolactin (10 pg/ rat; i.c.v) or its distilled water vehicle 12 hours
prior to decapitation. Vehicle-, nor-BNI- and prolactin-treated rats were injected with either
quinelorane (100 ug/ kg; i.p.) or its 0.9% saline vehicle (1 ml/ kg; i.p.) 60 min prior to
decapitation. All rats were injected with NSD 1015 (100 mg/ kg; i.p.) 30 min prior to
decapitation. Columns represent means and vertical lines 1 SEM of 6-9 determinations of
DOPA concentrations in the median eminence and nucleus accumbens of vehicle- (open
columns) or quinelorane- (solid columns) treated rats. * P < 0.05, values from quinelorane
treated rats that are significantly different from vehicle-treated controls. # P < 0.05, values
from nor-BNI and prolactin- treated rats that are significantly different from vehicle-treated
controls.

142

Median Eminence

 

 

 

 

 

 

 

 

 

2‘ ‘ 1:23 Saline *
— Quinelorane
18 . #
r: l #
.9 15-
e . J5
O.
a: 12 1
ti
5 9 ‘
E
o 5 ‘
o
3 l
o __ _
Vehicle NOR-BNI Prolactin
Nucleus Accumbens
36
A 30 -
CD
‘9‘ 24 J ”I“
Q.
E’
E, 18
I * * *
o- 12 '
O
o
6 .
0

 

 

 

 

 

 

 

 

 

—

Vehicle NOR-BNI Prolactin

143

 

 

 

 

 

 

 

 

 

 

 

28 1 a
1:: Saline

A 24 ‘ — Quinelorane
:
E 20 - T
9 t
a
a) 16 -
it
5 12 ~
E I
o 3 '
D

4 4

0

Intact Orchidectomy

Figure 32 Effects of quinelorane on DOPA concentrations in the median eminence of intact
and orchidectomized rats. Rats were injected with either quinelorane (100 itg/ kg i.p.) or its
0.9 0/o saline vehicle (1 ml/ kg; i.p.) 60 min prior to decapitation. All rats were injected with
NSD 1015 (100 mg/ kg; i.p.) 30 min prior to decapitation. Columns represent means and
vertical lines 1 SEM of 7-11 determinations of DOPA concentrations in the median
eminence of vehicle- (open columns) or quinelorane- (solid columns) treated-rats. * P <
0.05, values from quinelorane treated rats that are significantly different from vehicle-treated
controls. ** P < 0.05, values from orchidectomized rats that are significantly different from
intact controls.

144

N
.a

 

 

 

 

 

 

 

 

 

 

l_—_l Saline *
18 J _ Nor-BNI
E
9 15 1
9
O. a
g: 12 -
I l
|
E 9-
< T
o.
o 5 ‘
o
3 ..
0
Intact Orchidectomy

Figure 33 Effects of nor-BNI on DOPA concentrations in the median eminence of intact
and orchidectomized rats. Rats were injected with either nor-BNI (1 2.5)1g/ rat; i.c.v.) or its
distilled water vehicle 5 |J.l/ rat; i.c.v.) 120 min prior to decapitation. All rats were injected
with NSD 1015 (100 mg/ kg; i.p.) 30 min prior to decapitation. Columns represent means
and vertical lines 1 SEM of 7-8 determinations of DOPA concentrations in the median
eminence of vehicle- (open columns) or nor-BNI- (solid columns) treated rats. * p < 0.05,
values from nor-BNI-treated rats that are significantly different from vehicle-treated
controls. ** p < 0.05, values from orchidectomized rats that are significantly different from
intact controls.

145

 

 

:3 Saline
- Quinelorane

 

4i

Labeling Ratio DYN mRNA/Cell

 

 

 

 

 

 

 

 

 

ARC VMN

Figure 34 Effects of quinelorane on the expression of dynorphin mRNA in the arcuate
(ARC) and ventromedial hypothalamic (VMN) nuclei of the rat. Quinelorane (100 pg/ kg;
i.p.) or its 0.9% saline vehicle (1 ml/ kg; i.p.) was administered to rats 90 min prior to
decapitation. Columns represent means and vertical lines 1 SEM. of the labeling ratio/ cell
determined in 6 rats. * P < 0.05, values of quinelorane-treated rats (solid column) that are
significantly different from saline-treated rats (open column).

146

 

Discussion

Although TIDA neurons lack the DA autoreceptor-mediated regulation
that has been well characterized in the mesolimbic DA system, these neurons are
subject to activation that is mediated by DA D2 receptors. D2 receptor mediated
regulation of mesolimbic DA neurons is effected by autoreceptors located on
neurons which, either directly or indirectly via interneurons, provide reciprocal
innervation back to these DA neurons (Oades and Halliday 1987).
Pharmacological activation of D2 receptors inhibits the activity of mesolimbic
DA neurons by autoreceptor mediated prevention of dopamine release and
afferent neuronal inhibition of impulse conduction. In agreement, the results of
the present study demonstrate that activation of D2 receptors following
administration of quinelorane inhibits mesolimbic DA neurons terminating in
the nucleus accumbens.

Unlike mesolimbic DA neurons, TIDA neurons are not inhibited by
quinelorane; this finding is consistent with the conclusion that these neurons lack
D2 receptor-mediated afferent neuronal feedback inhibition (Demarest and
Moore 1979). Rather, results of experiments described in this dissertation have
demonstrated that activation of D2 receptors on neurons residing outside the
mediobasal hypothalamus provides stimulatory neuronal input to TIDA

neurons. In addition, this afferent stimulatory input is not dependent upon

147

 

endogenous activation of delta / OP1 , or neurotensin receptors. Experiments in
this chapter were designed to test the hypothesis that D2 receptor-mediated
activation of TIDA neurons is the result of disinhibition. The results reveal that
D2 receptor—mediated activation of TIDA neurons occurs, at least in part, via a
mechanism involving kappa / OP2 receptors.

If quinelorane attenuates kappa / OP2 receptor-mediated tonic inhibition
of TIDA neurons by preventing dynorphin release (Manzanares at al, 1992a;
1992b) then: 1) administration of an kappa/OP2 agonist should block the
actions of quinelorane, and 2) administration of an kappa/OP2 antagonist
should mimic the effects of quinelorane by stimulating TIDA neurons, and
prevent any further stimulation by quinelorane. The results presented here
indicate that this is the case. The inability of quinelorane to further increase
median eminence DOPA accumulation in nor-BNI-treated rats was not due to
limitations in the maximal rate of dopamine synthesis (i.e., ceiling effect). Indeed,
administration of prolactin increased median eminence DOPA accumulation but
did not alter the ability of quinelorane to further stimulate dopamine synthesis in
this region. Quinelorane was active when co-administered with nor-BNI since
DOPA accumulation in the nucleus accumbens was reduced following
administration of this agonist. Therefore, these results are consistent with the

hypothesis that D2 receptor-mediated activation of TIDA neurons occurs via

148

 

disinhibition that is dependent upon inhibition of tonically active dynorphinergic
neurons.

Contrary to our hypothesis, the results of these expedments also reveal
that Dz-mediated increases in TIDA neuronal activity are independent of the
effects of gonadal steroids. This conclusion is based on the observation that
removal of gonadal androgens by orchidectomy increased basal TIDA neuronal
activity but had no effect on either quinelorane— or nor-BNI induced increases in
the accumulation of DOPA. Earlier studies have demonstrated that TIDA
neurons are subject to tonic inhibition by the action of dynorphin (Manzanares at
al, 1992b) and that this tonic inhibition is dependent, in part, upon the presence
of gonadal androgens (Manzanares at al, 1992a). In vitra experiments
demonstrating that release of dynorphin from hypothalamic slices is lower in
tissue from orchidectomized versus gonadally-intact or orchidectomized
testosterone-treated male rats further support these in viva findings (Almeida at
al, 1987). It is possible that two populations of tonically active dynorphinergic
neurons modulate the activity of TIDA neurons. One that is responsive to
circulating gonadal androgens and another that is insensitive. Taken together,
these results suggest that an androgen insensitive population of dynorphinergic
neurons is responsive to the inhibitory actions of the D2 receptor agonists.

The results of the present study also confirm that in male rats the basal

activity of mesolimbic DA neurons terminating in the nucleus accumbens is not

149

 

regulated by kappa/OP2 receptors (Manzanares at al, 1992a; Manzanares at al,
1992b). Indeed, neither activation (with U-50,488) nor blockade (with nor-BNI)
of kappa/OP2 receptors had any effect on nucleus accumbens DOPA
accumulation in control rats. The inability of either U-50,488 or nor-BNI to
block the inhibitory effects of quinelorane in the nucleus accumbens reveals that
neither kappa/OP2 receptors nor dynorphinergic neurons participate in D2
receptor-mediated regulation of mesolimbic DA neurons.

These experiments also demonstrate that the DA D2 receptor-mediated
activation of TIDA neurons is independent of that mediated by prolactin on
these neurons. Prolactin-induced activation is delayed (requires 12 hours), is not
dependent upon afferent neuronal inputs to the mediobasal hypothalamus and is
mediated by endogenous activation of neurotensin receptors (Hokfelt and Fuxe,
1972; Gudelsky at al, 1978; Hentschel et al, 1998). In contrast, the D2 receptor-
mediated activation is acute (occurs within an hour), dependent upon afferent
neuronal input to the mediobasal hypothalamus, and independent of
neurotensin.

Activation of D2 receptors specifically reduces the capacity of
dynorphinergic neurons in the arcuate nucleus to synthesize new dynorphin.
This conclusion is based on the observation that quinelorane selectively reduces
the expression of dynorphin mRNA in the arcuate nucleus, but is without effect

on the expression of dynorphin mRNA in ventromedial nucleus. These data

150

 

support the hypothesis that activation of TIDA neurons via D2 receptors is
mediated, in part, by the inhibition of tonically active dynorphinergic neurons.
The reduction in dynorphin output from this population of neurons, in turn,
reduces the activation of inhibitory kappa/OP2 receptors thereby disinhibiting

TIDA neurons.

151

Chapter Eight
Inhibition of Tuberoinfundibular Dopaminergic Neuronal Activity

by Selective Activation of D1 Receptors

Previous chapters in this dissertation have dealt with D2 receptor-
mediated modulation of TIDA neurons. In this chapter the focus of attention
will be shifted to consider the role that D1 receptors may play in regulating
TIDA neuronal activity.

The discovery that antipsychotic efficacy was highly correlated with
antagonism of D2 receptors (Creese at al, 1976; Seeman at al, 1976) stimulated
the development of many D2 selective compounds. In contrast, there were
relatively fewer D1 receptor selective compounds. When SKF 38393 was initially
described it had a unique profile that was attributed to D1 receptor selectivity
(Setler at al, 1978). Although this agonist failed to alter plasma prolactin in viva it
was shown to mimic dopamine-stimulated adenylyl cyclase in rat striatal
homogenates. Subsequently, Sibley at al (1982) used radioligand displacement
studies to demonstrate that SKF 38393 bound preferentially to D1 receptors.

The first Dl-selective antagonist was SCI-123390 (Iorio at al, 1983). This
compound has been widely used as a tool to study the anatomical distribution
and function of D1 receptors. Reported interactions with SHT2 receptors in vitra

and and its short half-life in viva were serious limitations to its clinical utility

152

 

(Billard at al., 1984; Barnett at al, 1986). These problems led to the development
of a second generation D1 antagonist SCH39166 which has a longer half-life and
greater selectivity for D1 receptors (Chipkin at al, 1988; McQuade at al, 1991).
Acute administration of the D1 receptor agonist SKF38393 has been
shown to inhibit “activated” TIDA neurons (Berry and Gudelsky 1991), but little
information is available regarding the effects of D1 receptors on the basal
activity of these neurons. Thus, the purpose of the present series of experiments
was to characterize the role of D1 receptors in the regulation of TIDA neurons.
To this end, the effects of the selective D1 receptor agonist SKF38393 (Sibley at
al, 1982) and D1 receptor antagonist SCH39166 (Chipkin at al, 1988) were
examined on the activity of TIDA neurons as determined by measuring the
concentrations of DOPAC in the median eminence. In addition, the effects of
these treatments on plasma prolactin were also determined. These experiments
were designed to test the hypothesis that TIDA neurons are subject to inhibitory
modulation via D1 receptors. If this hypothesis is true then SKF38393 should
lower DOPAC concentrations in the median eminence and increase plasma
prolactin whereas, pre-treatment with SCH39166 should block or attenuate these

SKF38393-induced changes.

153

 

Methods
Brain tissue for neurochemical analysis and blood for radioimmuno assay

of prolactin were handled as described in the General Methods section. The
lower limit of prolactin detection in these assays was 150 pg/ 100 pl of plasma

and the interassay coefficient of variation was 10%.

Drugs
SCH39166 (Dr. A. Barnett Scherng-Plough, Bloomfield, NJ) was dissolved in

50% ethanol (v/v), while SKF 38393 (Research Biochemicals International,
Natick, MA) was dissolved in 0.1% ascorbic acid. Both drugs were administered
as indicated in the appropriate figure legends, and dosages were calculated based

on their respective salts.

Resufls

The effect of incremental doses of the D1 antagonist SCH39166 on DOPAC
concentration in the median eminence is shown in Figure 35. SCH39166
induced a dose-related increase in the concentration of DOPAC in the median
eminence that was significant at 1 and 3 mg/ kg. Administration of 3 mg
SCH39166/ kg induced a time-related increase in median eminence DOPAC
concentration that was significant by 1 h and returned to control levels by 16 h
(Figure 36). In contrast, the D1 agonist SKF38393 significantly reduced the

concentration of DOPAC in the median eminence at all doses tested ( Figure

154

 

 

37A), and significantly increased plasma prolactin levels at these doses ( Figure
37B). As shown in Figure 38 administration of 20 mg SKF38393/ kg reduced
the concentration of DOPAC in the median eminence and increased plasma
prolactin. Pre-treatment with 3 mg SCH39166/ kg increased the concentration

of DOPAC in the median eminence per re and attenuated both the SKF38393

 

induced decrease in this metabolite and the increase in plasma prolactin.

 

 

155

36-

301

24-

18 - ._T_

12‘

DOPAC (ng/mg protein)

 

 

 

 

 

VEH 0.3 1.0
SCH39166 (mg/kg)

 

Figure 35 Dose-response effects of SCH39166 on the concentrations of DOPAC in the
median eminence. Rats were injected with either 0.3, 1 or 3 mg SCH39166/ kg; s.c. or its
50% ethanol vehicle (1 ml/ kg, s.c.) 120 min prior to decapitation. Columns represent means
and vertical lines 1 SEM of DOPAC concentrations in the median eminence from 7-9 rats.
*Values from SCH39166-treated rats (solid columns) that are significantly different (p <
0.05) from vehicle-treated rats(open column).

156

 

18-

 

 

 

3.?
93
O
512-
U)
E
\
C)
5
2 6-
D.
O
D
0 III I I I
012 4 8 16

Hours after SCH39166

Figure 36 Time-course of effects of SCH39166 on the concentrations of DOPAC in the
median eminence. Rats were injected with SCH39166 (3 mg/ kg, s.c.) at 1, 2, 4, 8 or 16 h or
its 50% ethanol vehicle (1 ml/ kg; s.c.) 1 h prior to decapitation (time 0). Circles represent
means and vertical lines 1 SEM of DOPAC concentrations in the median eminence from 7-9

male rats. The filled circle represents a value that is significantly different (p < 0.05) from
time 0.

157

 

 

 

DOPAC (ng/mg protein)

 

Prolactin (ng/ml)

 

 

VEH 5.0 10.0 20.0
SKF38393 (mg/kg)

Figure 37. Effects of SKF38393 on the concentrations of DOPAC in the median eminence
and prolactin in plasma. Rats were injected with 5,10 or 20 mg SKF38393/kg; i.p. or its
0.1% ascorbic acid vehicle (1 ml/ kg; i.p.) 60 min prior to decapitation. Panel A: Columns
represent means and vertical lines 1 SEM of DOPAC concentrations in the median
eminence from 7-8 male rats. *Values from SKF38393-treated rats (solid columns) that are
significantly different (p <0.05) from vehicle-treated controls (open column). Panel B:
columns represent means and vertical lines 1 SEM of plasma prolactin concentrations in 7-8
male rats. *Values from SKF38393-treated rats (solid columns) that are significantly
different (p < 0.05) from vehicle-treated controls (open column).

158

A 25 . E Saline

 

 

 

 

 

 

 

A - SKF38393
.E
2 20 -
9 ._'L. ..
a _'r_
g 15 . *
B:
5 1o ..
O
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o 5 ‘
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Vehicle SCH39166
B
12 -

 

Prolactin (ng/ml plasma)
0)

 

   

 

 

Vehicle SCH39166

 

Figure 38 SCH391663 blocks the SKF38393-induced decrease in the median eminence
DOPAC concentrations. Rats were injected with SCH 39166 (3 mg/ kg; s.c.) or its 50 %
ethanol vehicle (1 ml/ kg, s.c.) 120 min, and with either SKF38393 (20 mg/ kg) or its 0.1%
ascorbic acid vehicle (1 ml/ kg, i.p.) 60 min prior to decapitation. Panel A: columns represent
means and vertical lines 1 SEM of DOPAC concentrations in the median eminence from 7-
8 rats. Panel B: columns represent means and vertical lines 1 SEM of plasma prolactin
concentrations from 7-8 rats.* Values from SKF38393-treated rats (solid columns) that are
significantly different (p < 0.05) from vehicle-treated controls (open columns). ** Values
from SCH39166+SKF38393-treated rats (solid columns) that are significantly different from
SKF38393+vehicle-treated controls (solid columns).

159

 

 

Discussion

The D1 antagonist SCH39166 induces a stimulatory effect on TIDA
neurons as demonstrated by the dose- and time-related increase in the median
eminence DOPAC concentration. By definition an antagonist binds to a receptor
with high affinity but lacks intrinsic activity or efficacy. This is the case for
SCH39166 (Chipkin 1988; Wamsley et al, 1991; Tice at al, 1994). The
conclusion drawn when administration of an antagonist induces an increase in
activity as shown for SCH39166 is that the drug is interfering with tonic
inhibitory effects mediated by the D1 receptor. This finding supports the
hypothesis that the D1 receptor plays an inhibitory role in the regulation of
TIDA neuronal activity and suggests that this inhibition is tonically active.

These inhibitory effects are rather unexpected given the findings that
direct effects of D1 receptor activation are usually stimulatory i.e., increases in
CAMP and intracellular Ca";+ concentrations (jakson and Westlind-Danielsson
1994). Thus, the direct effect of D1 receptor activation might be expected to be
stimulatory. While it is possible that the D1 receptor mediating this effect is
coupled via an inhibitory second messenger system, it is more likely the result of
an indirect action. Thus, D1 receptors could act indirectly by tonically activating
a neuronal system that inhibits TIDA neurons. In this way, the D1 receptor-

mediated inhibition would be analogous to the D2 receptor-mediated activation

160

 

...'- ,d-c-j

 

 

of TIDA which also occurs indirectly via inhibition of dynorphinergic neurons
(Chapter 6).

Incremental doses of the D1 receptor agonist SKF38393 significantly
reduced the basal activity of TIDA neurons as demonstrated by the reduction in
the concentration of DOPAC in the median eminence. Although D1 receptor-
mediated inhibition of TIDA neurons is tonic, the further reduction in activity
observed after SKF 38393 suggests that this inhibition is not maximal. Although,
Berry and Gudelsky (1990) reported that there was only a trend towards
reduction in activity, SKF 38393 at these doses did not significantly reduce
tyrosine hydroxylase activity in TIDA neurons except in one experiment where
the 20 mg/ kg dose reduced dopamine synthesis in the median eminence.

These results not withstanding, the biological importance of the
statistically significant decrease in the metabolic activity of TIDA neurons after
administration of the D1 agonist can be appreciated by looking at the effects on
plasma prolactin. At all dosages tested SKF38393 significantly increased the
concentration of prolactin in plasma. This finding is in agreement with others
who have reported similar elevations in prolactin after administration of D1
agonists (Saller and Salama 1989). Since there is no evidence to support a direct
effect of SKF38393 on lactotrophs in the anterior pituitary (due to absence of
D1 receptors; Cocchi et al, 1987), the elevation in prolactin is taken as functional

evidence for inhibition of TIDA neurons which regulate prolactin release.

161

 

 

 

Pro-treatment with the D1 antagonist blocked the SKF38393-induced
decrease in both metabolic and functional TIDA neuronal activity. This is
demonstrated by the blockade of SKF38393-induced changes in the
concentration of DOPAC in the median eminence and prolactin in plasma.
These agonist/ antagonist studies suggest that the effects of SCH39166 and
SKF38393 are both mediated by D1 receptors. However, these compounds
induce opposite effects on the activity of TIDA neurons and this leaves open to
question whether or not these effects are mediated at the same D1 receptor site.
However, in vitro binding studies as well as in viva data suggest that at these doses
these drugs are specific for the D1 receptor family (Sibley et al, 1982; Chipkin et
a], 1988). Although, there is a possibility that these drugs could be working on
D1 receptors in different sites or possibly D1 versus D5 receptors within the D1
receptor family, but the pharmacological tools to distinguish between D1 and DS
receptors are not yet available.

In conclusion, the results of studies presented in this chapter reveal that
TIDA neurons are inhibited following activation of D1 receptors, and that under

basal conditions these neurons are tonically regulated by inhibitory D1 receptors.

162

 

 

lg.»

 

Chapter Nine
Opposing Roles for 01 and 02 Dopaminergic Receptors in the
Regulation of Tuberoinfundibular Dopaminergic Neuronal
Activity.

The major ascending nigrostriatal and mesolimbic DA neuronal systems
in the brain are regulated by DA receptor-mediated mechanisms; i.e. DA
agonists reduce, whereas DA antagonists increase impulse flow and the
neurochemical activities of these neurons (Bunney et al, 1987). These effects are
chiefly mediated by inhibitory D2 and / or D3 DA autoreceptors located on axon
terminals, dendrites and neuronal cell bodies which function to maintain a
homeostatic balance between impulse flow-induced neurotransmitter release and
its replenishment by de nova synthesis (Elsworth and Roth, 1997). In contrast,
TIDA neurons lack DA autoreceptors, and are unresponsive to acute
administration of non-selective DA agonists (e.g. apomorphine and
bromocriptine) and antagonists (e.g. haloperidol; Moore 1987).

Utilization of newer more selective agonists which differentiate between
the D1 and D2 receptor subfamilies (Schwartz et al, 1992;]ackson and Westlind-
Danielsson, 1994) has revealed that TIDA neurons are subject to regulation by
DA receptors. Indeed, acute administration of quinelorane with preferential

affinity for the D2 receptor family increases the activity of TIDA neurons (Eaton
163

 

5' ‘3;

 

 

et al, 1993). Since apomorphine also interacts with D2 receptors it is curious that
this compound has no effect on the activity of TIDA neurons (Demarest and
Moore 1979). One possible explanation for this observation is that
apomorphine also interacts with both known receptors in the D1 family (D, and
D5). Currently available agonists and antagonists do not distinguish between
these two receptors. In addition, the D5 receptor is poorly expressed in rat brain
when compared with the D, receptor (Meador-Woodruff at al, 1992; Tiberi at al,
1991). For these reasons the inibitory effect on TIDA neurons as documented
by Berry and Gudelsky (1991) and confirmed in the previous chapter of this
dissertation are likely mediated by the D, receptor in rats. Thus, opposing effects
mediated by D, and D2 receptors on TIDA neuronal activity may lead to no net
measurable change when both receptors are activated.

The purpose of experiments described in this chapter was to test the
hypothesis that the lack of response of TIDA neurons to non-selective DA
agonists and antagonists is due to the simultaneous activation of inhibitory D,
and stimulatory D2 receptors. To test this hypothesis, the effects of quinelorane,
in the presence of either the selective D, receptor agonist SKF 38393 or the non-
selective DA agonist apomorphine were examined on the concentration of
DOPAC in the median eminence. If the lack of effect of non-selective DA

drugs on TIDA neurons is due to opposite effects from simultaneous activation

164

 

1F. ..

of D, )5 and D2 receptors then SKF 38393 should reverse quinelorane-induced
increases in the activity of TIDA neurons. Likewise, apomorphine should block
the stimulatory effect of quinelorane on these neurons.

Changes in the expression of immediate early gene products (i.e., c-Fos
and F os-related antigens (FRA’s)) represent an additional index of change in the
activity of specific neuronal populations. The appearance of these transcription
factors has been used as markers to identify neurons in which activity has
changed in response to drug treatments or alterations in physiological state
(Hoffman et al, 1993). Thus, immunocytochemical localization of Fos/FRA can
be used to visualize individual neurons that are active. One disadvantage is the
lack of specificity of this index since changes in Fos expression have been
demonstrated in response to a wide array of stimuli in a variety of cell types
(Hughes and Dragunow 1995). Thus, in a mixed neuronal population it is
important to specifically identify the neuronal cell type in which changes in
Fos/FRA expression occur.

Two populations of tyrosine hydroxylase—immunoreactive (TH-IR)
neurons have been described in the arcuate nucleus (ARC). These have been
identified based upon their neurochemical phenotypes, and the size and location
of their cell bodies in the dorsomedial (DM) and ventrolateral (VL) ARC (Everitt

at al, 1986). The expression of Fos/FRA in TH-IR neurons in these sub-

165

Tl

 

'r

divisions of the ARC is subject to differential regulation by gonadal steroids. In
females, estrogen stimulates Fos/FRA in the DM-ARC whereas in males
testosterone inhibits the expression of Fos/FRA in both the DM- and VL-ARC
(Cheung at al, 1997b).

The purpose of studies in this chapter is to determine if pharmacological
manipulations that induced neurochemical changes in TIDA neurons also cause
similar changes in immediate early gene expression in these neurons. To this
end, the effects of quinelorane alone and in combination with SKF 38393 were
determined on the co-localization of TH- and Fos/FRA-IR in the dorsomedial
and ventrolateral subdivisions of the arcuate nucleus. If the pharmacological
effects of these DA agonists alter the activity of TIDA neurons at the level of
the genome then quinelorane should activate and SKF 38393 reduce the number

of neurons expressing F 05 / FRA-IR.

Methods

The activity of TIDA neurons was determined by measuring dopamine
metabolism (concentration of DOPAC) in the terminals DA neurons in the
median eminence. In addition, dual irnmunohistochemistry for TH and
Fos/FRA was used to quantify the frequency with which TH-IR and Fos/Fra—
IR were co-localized in neurons of the arcuate nucleus. Details of these

procedures can be found in the General Methods.
166

 

[hugs
The D2 selective agonist quinelorane (Dr. M.M. Foreman, The Eli Lilly

Co., Indianapolis, IN) dissolved in 0.9% saline, D, agonist SKF38393 (Research
Biochemicals International, Natic, MA) was dissolved in 0.1% ascorbic acid, and
the non-selective agonist apomorphine (Sigma Chemical Co. St Louis, MO) was
dissolved in distilled water. All drugs were administered as indicated in the
appropriate figure legends. Dosages for all drugs were calculated based on their

respective salts.

Resufls

Given the inhibitory effects of SKF 38393 on DOPAC concentration in
the median eminence this drug was tested for its ability to block increases in this
metabolite following administration of the D2 receptor agonist quinelorane. As
shown in Figure 39, administration of 2 mg SKF38393/ kg completely reversed
the quinelorane-induced increase in median eminence DOPAC, and 20 mg
SKF 38393 / kg significantly reduced DOPAC concentration compared to vehicle
controls. To determine if simultaneous activation of D1 and D2 receptors could
explain the lack of effect on the concentration of DOPAC in the median

eminence following administration of apomorphine, rats were treated with
quinelorane 100 lig/ kg and apomorphine 2 mg/ kg. As shown in figure Figure

40, apomorphine had no effect per re, but blocked the ability of quinelorane to

167

 

Ti

 

 

increase DOPAC concentration in the median eminence. There was a
significant interaction between quinelorane and apomorphine on median
eminence DOPAC concentrations in this study (p < 0.05).

The number of cells containing both TH-IR and Fos/FRA-IR was
quantified and the results are presented in Figure 41. SKF 38393 (20 mg/ kg, i.p.)
had no effect per re but was able to block the quinelorane—induced increase in the
number of TH-IR cells expressing Fos/FRA in the DM-ARC whereas, none of

these drug treatments had any effect in the VL-ARC.

168

 

 

 

 

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!: Saline
E 12 - - Qurnelorane
0)
§
3 9 - ——T—
E
B)
5 .
O 6
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n.
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Vehicle 02 2 20
SKF38393 (mg/kg)

Figure 39 Dose-response effect of SKF38393 on DOPAC concentrations in the median
eminence quinelorane-treated rats. Rats were injected with quinelorane (100 ilg/ kg; i.p.) or
its 0.9% saline vehicle (1 ml/ kg; i.p.) at 60 min, and with 0.2, 2 or 20 mg SKF38393/ kg, i.p.,
or its 0.1% ascorbic acid vehicle (1 ml/ kg, i.p.) 60 min prior to decapitation. Columns
represent means and vertical lines 1 SEM of DOPAC concentrations in the median
eminence sampled from 6—8 rats. *Values from quinelorane-treated rats (solid columns) that
are significantly different (p < 0.05) from vehicle-treated controls (open column). “Values
from SKF38393-treated rats that are significantly different from vehicle-treated controls.

169

 

 

 

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A 1: Vehicle
.E _ Apomorphine t
s 9.
9 T
a **
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B: 6‘ T
5
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3‘. 3.
O
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0

Saline Quinelorane

Figure 40 Effects of apomorphine on DOPAC concentrations in the median eminence of
vehicle- and quinelorane-treated rats. Rats were injected with quinelorane (100 ug/ kg; i.p.)
or its 0.9% saline vehicle (1 mg/ kg; i.p.) at 60 min, and with apomorphine (2 mg/ kg; s.c.) or
0.9% saline 30 min prior to decapitation. Columns represent means and vertical lines 1 SEM
of DOPAC concentrations in the median eminence sampled from 6-8 rats. *Values from
quinelorane-treated rats that are significantly different (p < 0.05) from saline-treated
controls. ** Values for apomorphine-treated rats (solid columns) that are significantly
different (p < 0.05) mm quinelorane-treated rats (open columns).

170

 

DM-ARC

 

 

 

 

 

 

 

 

 

 

 

 

 

30- *
a) =Vehicle
SE 24., _Ouinelorane
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59$ 12'
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Vehicle SKF38393

Figure 41 Effect of SKF38393 on quinelorane-induced changes in the number of perikarya
in the ARC co-expressing TH-IR and Fos/FRA-IR. Rats were injected with quinelorane
(100 ilg/ kg; i.p.) or its 0.9% saline vehicle (1 ml/ kg i.p.) at 90 min, and with SKF38393 (20
mg/ kg; i.p.) or its 0.1 °/o ascorbic acid vehicle (1 ml/ kg; i.p.) 90 min prior to perfusion.
Columns represent means and vertical lines 1 SEM of the number of TH-IR neurons co-
expressing Fos/FRA-IR in the DM- or VL-ARC of 5 male rats. *Values from quinelorane-
treated rats (solid columns) that are significantly different (p < 0.05) from vehicle-treated
controls (open columns).

171

 

Discussion

Administration of the D, agonist blocked quinelorane-induced increases in
metabolic activity of TIDA neurons. These findings are in agreement with those
of Berry and Gudelsky (1991) who demonstrated that D, receptors function to
inhibit TIDA neurons activated by a variety of stimuli. Indeed, activation of D,
receptors reduced both the basal activity of these neurons as well as quinelorane-
induced increases. Thus, it appears that the opposite effects of D1 and D2
agonists on TIDA neurons cancel one another when activated simultaneously.
These data lend support to the basic thesis of this chapter i.e., that non-selective
agonists such as apomorphine have no effect on the activity of TIDA neurons
because of concurrent activation of both D1 and D2 receptors.

One possible way to test this hypothesis would be to examine the effects
of apomorphine in combination with a D, antagonist. Following blockade of D1
receptors, apomorphine should behave as a D2 receptor selective agonist similar
to quinelorane. The problem with this approach is that D, antagonists elevate
TIDA neuronal activity due to the tonic D, receptor-mediated inhibitory
influences described in the previous chapter. An alternative approach to address
this question would be to activate the D2 receptors by pretreatment with
quinelorane and then test the ability of apomorphine to alter the activity of

TIDA neurons. In this scenario apomorphine should behave as a D, agonist

172

 

"i

 

 

since quinelorane will already be interacting with D2 receptors. This was the
case, and under these conditions apomorphine had no effect per re, but reversed
quinelorane-induced increases in the activity of TIDA neurons. Taken together,
these results suggest that the lack of effect of non-selective agonists on TIDA
neurons is due to the opposing roles for D, and D2 receptors in the regulation of
the activity of their activity.

The neurochemical information suggests that two converging pathways
modulate the activity of TIDA neurons. 1) The stimulatory path mediated by the
action of dopamine at D2 receptors is dependent upon afferent neuronal input to
the mediobasal hypothalamus and this effect is mediated, in part, by reducing
tonic inhibition of TIDA neurons via the action of dynorphin at kappa/OP2
receptors. 2) The inhibitory pathway is mediated by an action of dopamine at D1
receptors, perhaps by an indirect mechanism that is dependent upon the
augmentation of an inhibitory system. Based on this information it is tempting
to speculate that inhibitory dynorphinergic neurons may serve as the target that
mediates these inhibitory D, )5 receptor effects. Thus, both the stimulatory and
inhibitory actions of dopamine on TIDA activity could be mediated indirectly via
D2 receptor inhibition and D, )5 stimulation of dynorphinergic neurons.

Neurochemical changes observed in the terminals of TIDA neurons were
reflected in changes in Fos/FRA expression in TH-IR neurons of the DM-ARC

with the caveat that only increases and not decreases in TIDA activity were

173

 

Ti

 

 

observed with this technique. While quinelorane increases the expression of
these immediate early gene products in TH-IR cells, SKF 38393 was without
effect per re, yet it completely blocked the quinelorane-induced increase in
Fos/FRA expression in these neurons. There are several related proteins that
the fos antiserum recognizes (Hoffman et al, 1994) and these authors have
demonstrated that in addition to the rapid inducible form of F as there are related
forms of this immediate early gene (FRA’s) that are constitutive. Thus, with
selection of the appropriate F 05 antiserum (Cambridge Research Biochemicals as
used here) for identification of F RA irnmunocytochemical localization of these
proteins permits not only assessment of stimulated activity, but also suppressed
function in these neurons. However the low basal activity of TIDA neurons in
males may account for our inability to measure decreases in F 05 / FRA expression
after administration of the D, agonist.

Although these studies failed to demonstrate the inhibitory effect of D, )5
agonists immediate early gene expression was useful for demonstrating the
stimulatory effect induced by D2 receptor activation. These results suggest, that
the opposing roles of D, and D2 receptors in the regulation of TIDA neurons
extend to alterations in gene expression and (perhaps more interestingly) that
only the TIDA neurons originating in the dorsomedial portion of the arcuate
nucleus are responsive to these treatments. These data suggest that in addition

to the neurochemical changes observed in the median eminence the activation of

174

 

is

DA receptors alters c-fos gene transcription, and synthesis of c-fos mRNA and
FRA proteins. These changes may in turn be related to regulation of tyrosine
hydroxylase in these neurons via the AP1 promoter. These results also suggest
that the activation of TIDA neurons occurs at perikarya or dendrites rather than
axon terminals.

In conclusion, data in this chapter support the hypothesis that the activity
of TIDA neurons can be modulated by the action of DA agonists at inhibitory
D, receptors and stimulatory D2 receptors. These opposing effects provide an
explanation for the lack of effect non-selective DA agonists on TIDA neuronal

activity.

175

 

kn.

 

 

Chapter Ten

General Summary and Discussion

This body of work has aimed to characterize DA receptor-mediated
regulation of TIDA neurons. Although these neurons are not regulated by pre-
or post-synaptic DA receptor mediated negative feedback in the way that other
DA neurons are, the development of drugs that distinguish between the D1 and
D2 receptor families has uncovered a heretofore unrecognized mode of
regulation of these neurons. These studies have served to demonstrate that D2
receptor activation stimulates, whereas D, receptor activation inhibits TIDA

neurons.

D2 Receptor-mediated activation of TIDA neurons

The schematic in Figure 42 depicts the hypothesized relationship between
D2 receptors and the intervening steps that lead to activation of TIDA neurons.
It was interesting to discover that the mechanism involves disinhibition of TIDA
neurons mediated by inhibition of tonically active dynorphinergic influences.
These inhibitory neurons are likely spared after mediobasal hypothalamic
deafferentation since the basal activity of TIDA neurons was not altered by this
procedure. In addition, in ritu hybridization experiments reveal that quinelorane
decreases prodynorphin mRNA in the arcuate nucleus while having no effect on

dynorphin message in the nearby ventromedial hypothalamic nucleus. Taken

176

 

 

together, these data suggest that the location of the dynorphinergic neurons

mediating this response is within the region circumscribed by the mediobasal

 

hypothalamic deafferentation.

 

177

   

 

D, Inhibition D2 Activation

I».m.~.~.\_.

 

 

TIDA
+
Prolactin
/\
DA

Figure 42 A schematic representation of the hypothetical relationship between inhibitory D1
and stimulatory D2 receptors on the regulation of TIDA neuronal activity. Abbreviations;
(DA) dopamine, (DYN) dynorphin, (DMN) dorsomedial hypothalamic nucleus, (VMN)
ventromedial hypothalamic nucleus.

178

Gonadal steroids are known to modulate the activity of TIDA neurons.
In male rats androgens tonically inhibit TIDA neurons (Gunnet et al, 1986;
Toney et al, 1991) and this effect is mediated, in part, by the actions of
dynorphin at kappa/OP2 receptors. Administration of either the kappa/OP2
receptor antagonist nor-BNI or anti-dynorphin antibodies induces an acute
increase in the activity of TIDA neurons in intact male but not female rats
(Manzanares et al., 1992B). This effect was completely reversed by the co-
administration of the kappa/OPZ-selective agonist U50,488 (Manzanares at al,
1992A).

The lack of kappa/OP2 mediated regulation of TIDA neurons in females
was initially of some concern, since our laboratory (Eaton at al, 1993) had
previously demonstrated that the quinelorane-induced activation of TIDA
occurs in both male and female rats. In females, the ability of quinelorane to
suppress circulating levels of prolactin (via a direct action at the anterior pituitary)
indirectly reduces the activity of TIDA neurons and thereby masks the
stimulatory action of this drug on these neurons. Thus, if the mechanism of
quinelorane—induced activation of TIDA neurons is mediated by inhibition of
dynorphinergic neurons, then a similar arrangement should exist in females.
Interestingly, the activity of TIDA neurons is reduced after ovariectomy and this
effect is mediated by the loss of estrogen-dependent positive feedback by

prolactin (T oney at al., 1992). In this situation the kappa/OP2 receptor

179

 

3T:
II}

antagonist nor-BNI is capable of activating these neurons (Manzanares at al,
1992B). Taken together, these data suggest that ovariectomy and the resulting
reduction in positive prolactin feedback unmasks a latent dynorphinergic
inhibitory pathway. This possibility is consistent with the observation that
treatment with prolactin antisera renders female rats sensitive to the stimulatory
effects of quinelorane (Eaton et al, 1993). The discovery that there is a gonadal
steroid insensitive population of dynorphinergic neurons that can account for
the disinhibition of TIDA neurons after activation of D2 receptors in male rats
suggests that this mechanism might also occur in female rats as well.

Future experiments could address this possibility by making use of
immunohistochemistry and / or in rim hybridization techniques that could allow
for the dynamic neuroanatomical mapping of dynorphinergic neurons in the
mediobasal hypothalamw. Thus, a hypothesis that quinelorane activates TIDA
neurons via a population of dynorphinergic neurons in both male and female rats
that is insensitive to the effects of gonadal steroids could be tested. If this
hypothesis is correct then quinelorane should reduce the activity of
dynorphinergic neurons. This effect ought to be evident in both male and

female castrated rats.

180

 

in
'l'

D1 receptor-mediated inhibition of TIDA neurons and prolactin secretion

The observation that the D, antagonist SCH39166 activates TIDA
neurons suggests that D, receptor-mediated inhibition is tonically active.
Considering the lack of effect of mediobasal hypothalamic deafferentation on
the basal activity of TIDA neurons in male rats (Barton et al, 1989, Chapter
Three) this finding suggests that the effect of this inhibitory circuit is located
with in the area circumscribed by the deafferentation. If D, receptor-mediated
inhibition of these neurons was dependent upon afferent input arising from
outside the mediobasal hypothalamus, then deafferentation should disrupt this
inhibitory tone and increase the basal activity of these neurons. Deafferentation
studies could be employed to explicitly test this hypothesis, as was done in the
D2 stimulatory pathway. If this hypothesis is correct then deafferentation should
have no effect on the ability of SCH39166 to increase the activity of TIDA
neurons.

In the studies describing D, inhibitory regulation of TIDA neurons,
decreases in the activity of these neurons coincided with elevated plasma
prolactin levels. This finding was taken as functional evidence that the reduced
activity of TIDA neurons was biologically significant and is consistent with
numerous studies that have demonstrated that endogenous dopamine released
from TIDA neurons is a prolactin inhibiting factor by an action at D2 receptors

on the anterior pituitary (Ben-Jonathan, 1985).

181

 

 

 

 

Enhanced prolactin secretion following activation of stimulatory D,
receptors on the anterior pituitary could be an alternate explanation for these
findings. Many studies have associated the action of D, receptor agonists with
elevated prolactin levels. A number of in vitra studies have suggested that
dopamine under appropriate conditions may stimulate prolactin secretion by an
action at stimulatory DA receptors located on the anterior pituitary. Indeed, this
effect has been demonstrated in primary cultures of anterior pituitaries from
both male and female rats (Shin, 1977; Denef at al, 1980; Kramer and Hopkins
1982; Burris at al, 1991; Hill at al, 1991). This was most elegantly demonstrated
in studies where application of the non-selective agonist apomorphine to
cultured pituitary cells inhibited prolactin secretion into the media. In contrast,
administration of the antagonist haloperidol had no effect unless combined with
apomorphine and this combination induced a 30% increase in prolactin release
(Denef at al, 1980). Thus, blocking the inhibitory D2 agonist actions of
apomorphine effectively converted it into a D, agonist.

These findings were taken as evidence of a second DA binding site that
was unique from that coupled to inhibition of prolactin secretion. Burris et al,
(1991) postulated that this receptor was related to the D2 receptor since the
highly selective D2 antagonist eticlopride (Seeman and Ulpian, 1988) was able to
block both the inhibitory and stimulatory actions of dopamine on prolactin

secretion in this system. However, the DA agonist bromocriptine alone or in the

182

 

 

 

presence of the D2 antagonist eticlopride failed to elicit an increase in the
stimulation of prolactin in these cells. Whereas in earlier studies bromocriptine
alone or in the presence of haloperidol increased the rate of prolactin secretion.

Much effort has also been directed at finding the “prolactin-stimulating”
DA receptor responsible for activation of prolactin secretion in vitra. In studies
utilizing prolactin secreting GH4C, cells expressing D2, D,’ or D5 receptors have
demonstrated that either D, or DS receptors will positively couple to prolactin
secretion, whereas the D2 receptor exhibits only an inhibitory effect on prolactin
secretion in these cells (Porter et al, 1994). While this study demonstrates that
D, receptors are competent to mediate this response in cell culture systems, the
assertion that these findings are relevant to the secretion of prolactin in viva is
weakened by the failure to unequivocally demonstrate D, receptors in the
anterior pituitary in rim. Thus, the prolactin stimulatory effect of dopamine in
these systems may be an artifact of removing pituitary cells from their normal in
rim environment possibly leading to the abnormal expression of a D, receptor
that responds to physiological levels of dopamine by increasing rather than
inhibiting prolactin secretion. If this is the case these culture studies may be
more relevant to the mechanisms underlying trans formation of normal anterior
pituitary lactotrophs to prolactin secreting tumor cells.

Messenger RNA for the D5 receptor has been measured in tissue from the

anterior pituitary (Porter at al, 1994) and this could be the receptor that is

183

 

 

 

expressed in these culture systems. However, the absence of functional D, or D5
receptor proteins in the rat anterior pituitary (Cocci at al, 1987; Albert at al,
1997) argues against involvement of these receptors in the in viva regulation of
prolactin secretion. Rather, the absence of compelling evidence for a stimulatory
DA receptor on the anterior pituitary suggests that the D, agonists and
antagonists are having their effect on prolactin secretion via a central action.

It is interesting to note that in sheep, central administration of SKF38393
increases prolactin secretion (Curlewis et al, 1994), possibly via an action in the
hypothalamic ventromedial nucleus (Curlewis et al, 1995a). It has been
postulated that D, receptors located in this region mediate photoperiod-induced
changes in prolactin secretion in this species (Curlewis at al, 1995b). In rats the
ventromedial nucleus contains a very low density of D1 receptors as identified by
specific irnmunocytochernical localization studies (Fremeau et al, 1991).
Furthermore, there is no evidence that DA neurons terminate in this region.
Thus, D1 receptors located here may be present but “non-functional” in rats
that, unlike sheep, do not respond to changes in photoperiod. Alternatively the
site of action of these D1 receptor agonists and antagonists could be determined
by comparing the effects of deafferentation with hypophysectomy. If the site of
action is within the mediobasal hypothalamm rather than on the pituitary itself,

then hypophysectomy alone should block both the stimulatory effects of

184

 

ii,

 

SCH39166 and inhibitory effects of SKF 38393. On the other hand, these effects

should not be disrupted by deafferentation of the mediobasal hypothalamus.

Interactions between D1 and D2 receptors in TIDA neuronal regulation
Researchers have long appreciated that there are important interactions
between the DA receptor subtypes found in the brain. Evidence for
synergistic/ cooperative interactions have been demonstrated in behavioral
studies where the full expression of stereotyped behaviors observed with non-
selective DA agonists can only be duplicated when D, and D2 receptor agonists
are combined (Walters et al, 1987). Parallel studies examining the effects of
these agents on neuronal firing rates in the striatum, and nucleus accumbens add
further support to a synergistic/ cooperative interaction between D, and D2
receptors (Walters et a1, 1987). Results obtained in studies descn'bed in this
dissertation lend evidence to there being an opposing interaction between D,
and D2 receptor families in the regulation of TIDA neuronal activity. The
discovery of drugs with greater selectivity for the various members of the DA
receptor family has uncovered a heretofore-unrecognized pattern of DA
regulation in these neurons. Furthermore, these results suggest that concurrent
activation of dopamine D, and dopamine D2 receptors nullify their respective
actions on TIDA neurons, which likely accounts for the inability of mixed

D, / D2 receptor agonists to have an acute effect on these hypothalamic

185

 

 

 

dopamine neurons. Thus, the activity of TIDA neurons is dependent upon the
relative balance of D, vs. D2 receptor influences.

This balance may be important in the regulation of prolactin in certain
physiological states. There is a circadian rhythmicity in the secretion of prolactin
that is associated with the rapid eye movement stage of sleep (Franz 1978). In
addtion, stressful situations are known to elevate prolactin (Gala 1990).
Although the physiological role for prolactin in these instances is poorly
understood it has been postulated that it may play a role in the maintenance of
normal immune system function (Gala 1990). In either of these examples
elevated prolactin may be the result of a shift in the balance of DA activity to
favor the D, inhibitory path thereby leading to elevated prolactin. If this
hypothesis is true then local application of D, agonists into the ventromedial
nucleus ought to inhibit TIDA neurons and increase prolactin secretion. In
addition, the activity of DA neurons in this nucleus ought to be elevated in
association with this sleep-related elevation in prolactin.

The shift in balance toward D2 stimulation of TIDA activity is
independent of prolactin mediated feedback. The activation of these neurons by
prolactin results from a 12-hour delay that is dependent upon the activation of
neurotensin receptors (Hentschel et al, 1998). Quinelorane-induced activation of
the D2 stimulatory pathway is acute and not dependent on the action of

neurotensin. In fact, the stimulatory effects of prolactin and quinelroane on

186

 

 

 

TIDA neuronal activity are additive and this suggests an independent
pharmacological mechanism of action as shown in Figure 42. The balance of
DA activity may shift toward the D2 stimulatory pathway in situations following
suppression of these neurons during physiological states where prolactin has
been elevated i.e. stress, lactation, pre-ovulatory prolactin surge. The source of
dopamine in this situation may be the neurons in the dorsomedial hypothalamic
nucleus as electrical stimulation of this region has been shown to elevate TIDA
neuronal activity (Gunnett et al, 1988). Interestingly, this response to electrical
stimulation occurred even under conditions of hyperprolactinemia induced by
administration of haloperidol. These results suggest that as in the D2 stimulatory
pathway electrical stimulation of the dorsomedial nucleus is independent of
prolactin induced activation of TIDA neurons also.

Neurochemical experiments have not been useful for measuring changes
in DA synthesis and metabolism in the ventromedial and dorsomedial nuclei.
This problem may be the result of these systems being too small or the release of
dopamine too minute to be discernable by this method. In this situation,
dynamic neuroanatomical mapping by dual irnmunohistochemistry for FRA’s
and TH may be useful in determining the relevance of DA neurons in the
ventromedial or dorsomedial nuclei in the regulation of TIDA neurons.
Alternatively, this technique may suggest other brain regions that may be

important in the physiological regulation of these neurons.

187

 

T‘I'r'

 

‘t

The results of studies described in this dissertation suggest that as drugs
with greater selectivity for DA receptors become available it will be important to
determine their effect on TIDA neurons in order to anticipate any side-effects
due to alterations in prolactin secretion. Finally, this dopamine receptor
mediated regulation of TIDA neuronal activity may allow for an easy way to
determine the relative D1/D2 properties of new DA drugs as they are F‘

developed. ,

 

188

 

APPENDIX

189

 

 

Table 10 List of drugs used by genedc name chemical name (source) and vehicle solvent

 

 

 

 

 

 

 

 

 

 

 

 

 

 

used.
Generic Name Chemical name (Source) Solvent

Apomorphine 4H-Dibenzo[de,g]quinoline-10,1 1-diol,5,6,6a,7- 0.9% saline
tetrahydro-6-methyl- ,hydrochloride (Sigma Chemical
Co., St. Louis, MO)

DPDPE [D-Pen2, D-Pen5]enkephalin (Peninsula Laboratories, Distilled water
Belmont, CA.)

Naltrindole 17-cyclopropyl-methyl-6,7,2’,3’-indol morphinan (Dr. Distilled water
James H. Woods Department of pharmacology,
University of Michigan Medical school, Ann Arbor, MI)

Neurotensin PGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile- Distilled water
Lou-OH (Sigma chemical Co., St. Louis, MO)

Nor-BNI Nor-binaltorphimine hydrochloride hydrate (Research 0.03 % saline
Biochemical International, Natic, MA)

NSD 1015 3-Hydroxybenzylhydrazine dihydrochloride (Aldrich Distilled water
Chemical Co., Milwaukee, WI)

PD128907 [R-(+)-trans-3,4,4a,10b-tetrahydro-4—propyl— 2H,5H- Distilled water
[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol] (Dr. M. D.
Davis Parke-Davis Research, Ann Arbor MI)

PNU-95,666 5(R)-(methylamino)-2,4,5,6-tetrahydro~1H- 0.9% saline
imidazo[4,5,1-ij]-quinolin-2- onemaleate (Dr. P. Von
Voigtlander, The Upjohn Co., Kalamazoo, MI)

Quinelorane Trans-(-)-5,5a, 6,7,8,9,9a, 10-octahydro-6 0.9% saline
propylpyrirnido[4,5-nguinolin-2-amine dihydrochlorde
(Dr. M. M. Foreman, The Eli Lilly Co., Indianapolis,
IN)

SCH 39166 [(-)-trans-6,7,6a,a,9,13b-hexahydro-3-chloro-2-hydroxy- 50% ethyl
N-methyyl-SH benzo[d]naptho- {2,1-b}azepine (Dr. A. alcohol v/v
Barnett Schereng—Plouhg, Bloomfield, NJ)

SKF 38393 (i)-1-phenyl-2,4,5—tetrahydro-(1H)—3benzapine-7,8-diol 0.1% ascorbic
hydrochloride (Research Biochemicals International, acid
Natic, MA)

SR48692 Tricyclo[3.3.1.13,7]decane-2-carboxylic acid,2- [[[1-(7- 10% DMSO

 

chloro—4-quinolinyD-5- (2,6-dimethoxyphenyD-1H-
pyrazol-3-yl]carbonyl]amino] (Dr. Danielle Gully Sanofi
Recherche, Toulouse, France.)

 

 

 

190

 

 

 

Generic Name Chemical name (Source) Solvent

 

 

U-50,488 Trans-(i)—3,4-dichloro-N-Methyl-N—(2- [1- Distilled water
pyrrolindinyl] cyclohexyl) benzenacetamide (Dr. P. Von
Voigtlander, The Upjohn Co., Kalamazoo, MI)

 

 

 

191

 

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