Citation
Gonadal steroid modulation of opioid effects on gonadotropin secretion in the rat

Material Information

Title:
Gonadal steroid modulation of opioid effects on gonadotropin secretion in the rat
Creator:
Gabriel, Steven M
Publication Date:
Language:
English
Physical Description:
xiv, 199 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Dosage ( jstor )
Hormones ( jstor )
Hypothalamus ( jstor )
Luteinization ( jstor )
Morphine ( jstor )
Neurons ( jstor )
Opiates ( jstor )
Rats ( jstor )
Secretion ( jstor )
Sex hormones ( jstor )
Dissertations, Academic -- Pharmacy -- UF ( mesh )
Endorphins -- physiology ( mesh )
Gonadotropins -- secretion ( mesh )
Gonads -- physiology ( mesh )
Pharmacy thesis Ph.D ( mesh )
Steroids -- physiology ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1984.
Bibliography:
Bibliography: leaves 168-198.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
Steven M. Gabriel.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
030351839 ( ALEPH )
11998752 ( OCLC )
ACL2162 ( NOTIS )
AA00004891_00001 ( sobekcm )

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Full Text











GONADAL STEROID NODOLATIOI CF OPICID EFFECTS ON
GONADCTROPIN SECREIION IN THE BAT






BY


STEVEN 8. GABRIEL


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF PLCEICA IN
PARTIAL fULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF ECCTCB OF PHILCSCPHY



UNIVERSITY OF FLORIDA


1984




GONADAL STEROID MODULATION CF OFICIE EFFECTS ON
GONADOTROPIN SECRETION IN THE RAT
BY
STEVEN fl. GAERIEI
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOl
OF THE UNIVERSITY CF F1CEIEA IN
PARTIAL FULFILLMENT CF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR CF PHILOSOPHY
UNIVERSITY OF FLORIDA
1984


This dissertation is dedicated to Dr, Theodore J, Cicero.
His hard work and dedication tc science enccugaged me tc
pursue graduate studies in neuroendocrinology.


ACKNOWLEDGMENTS
It is difficult in this small space to acknowledge all of
the individuals who have assisted me in my graduate studies
during the past 4 years at the University of Florida. This
work would not have been possible without the advice,
support and encouragement of ay mentor. Dr, James W.
Simpkins. Jim's approach to graduate education allowed me to
act more as a collaborator in the design and execution of
experiments, rather than simply as his student. I wish to
thank Dr. Satya Kalra who also provided invaluable support
and advice in the development of my research project. The
other faculty members who have served on my dissertation
committee, Drs. Steven Childers, Michel Katcvich, Daniel
Sharp and William Thatcher, have imparted much needed advice
and knowledge in the laboratory and classrcctt. Many ether
faculty members have assisted me in my research, including
Drs. Pushpa Kalra, Kerry Estes, Hartmut Derendorf, Ken
Slcan, George Torosian and M.S.A, Kumar.
There is a great feeling cf ccmradery in cur department
and I would like to acknowledge some of the staff and
graduate students who have been so encouraging ever the
years. June O'Meara and Fran Eanniello have been helpful
often in the past and have always been tolerant cf ity many
in


errors. The assistance of Gayle Geegan, Becky Thro, Sharon
Layfield, and Becky Hamilton was greatly appreciated. I
would like to thank Dr. Ed Scltis, who was cheerful under
any circumstance, and Lee Ann Berglund and Cathy Smith who
will soon be taking over the reins in the lab.
Many of my friends outside of school have made life more
bearable. I would like to thank Robert Logan, who always
managed to cheer me up when life got me down. I would also
like to thank my rocmates, Scott Tokum and John Heintz, who
have been loyal friends over the years.
This dissertation was written using the computing
facilities of the Northeast Regional Data Center, and
incorporated the programs contained within the Statistical
Analysis System.
IV


TABLE CF CC NTESTS
PAGE
ACKNOHLEDGNENT S .................... iii
LIST OF TABLES ix
LIST OF FIGURES ..................... x
ABSTRACT xii
CHAPTER
I. INTRODUCTION 1
II. REVIES OF THE LITERATURE .............. 5
Historical .................. 6
Early Observations ............. 6
The Development of Neuroendocrinology ... 8
Neuroanatcmical Relationships .... 13
Anatomy of the Pituitary Gland ....... 13
General Hypothalamic Anatomy ........14
Anatomy of the luteinizing Hormone-Releasing
Hormone Neuronal Systems ..19
Anatomy of the Eonoaminergic Neuronal Systems 21
Noradrenergic pathways ...........21
Dopaminergic pathways ...........22
Epinephrine-containing pathways ...... 23
Serotonergic Pathways ...........23
Anatomy of Endogenous Cpicid Peptide Containing
Neuronal Systems ............24
Beta-endorphin-ccntaining neurons 24
Enkephalin-containing neurons ....... 25
Dynorphin-ccntaining neurons ........26
Steroid Concentrating Neurons in the Brain . 27
Neuroanatcmical Interactions .. 28
Patterns of Gonadotropin Secretion ...... 29
Hales 29
Females ..................30
Monoaminergic Control of Gonadotropin Secretion 32
Norepinephrine ...............34
Epinephrine ........ 37
Dopamine ..................38
v


Serotonin ................. 38
Endogenous Opicid Peptides and the Control of
Gonadotropin Secretion ......... 39
Reproductive Pharmacology of Opicids .... 40
Physiological Inhibition cf Gonadotropin
Secretion by Opicids .........42
Multiple Opioid Receptors ......... 43
Opioid-Mcnoaminergic Interactions 44
Opioid-Gonadal Steroid Interactions .... 45
Rationale ...................48
III. GENERAL MATERIALS AND METHODS 49
Animals ...............49
Dissection of Brain Tissue .. ...51
Gonadal Steroid Treatment ...........51
Treatment with Morphine or Naloxone ...... 52
Measurement of Catecholamines Indolaaines and
Metabolites ...............53
Hormone Radioimmunoassays ...........55
Luteinizing Hormone and Follicle Stimulating
Hormone ...............55
Luteinizing Hormone Releasing Hcrmone ... 56
Testosterone ................57
Statistical Analysis .... ..57
IV. THE EFFECTS OF CHBCNIC MOEPHINE TREATMENT ON
TESTOSTERONE NEGATIVE FEEDBACK IN CASTRATED
MALE RATS ................. 58
Introduction ............53
Experimental ................. 59
Experiment 1................59
Experiment 2................60
Experiment 3................60
Results ....................61
Effects of Time After Castration on the
Serum IH and Hypothalamic LHfiH
Responses to T and morphine ..... 61
The Effects of Chronic Morphine Treatment on
the LH Secretory and MBH IHRH
Responses to Graded Coses of T. ... 64
Effects of T and Morphine Treatment on the
Eituitary Responsiveness to LH8H ... 70
Discussion ............ 71
V.THE INFLUENCE OF CHBCNIC MCBEHINE TREATMENT ON THE
NEGATIVE FEEDBACK BEGULATICN OF GONADCTECEIK
SECRETION BV GO NACA L STEROIDS ....... 76
Introduction .................76
Experimental .................77
vi


Chronic Morphine and Gonadal Steroid
Treatments ..77
Evaluation of Pituitary Responsiveness to
LHfiH 78
Results . ... ...... ....78
Discussion ..................88
VI. THE EFFECTS OF CHRONIC MCBPHINE AND TESTOSTERONE
TREATMENT ON CATECHCIAMINE AND INDCLAKINE
METABOLISM AND GCNAECTBOPIN SECBETION IN
MALE EATS ...94
Introduction .................94
Experimental .................95
Results .................... 96
Serum LH and FSH 96
NE and NME ........ 100
DA and DOPAC ............... 100
5HT 5HIAA and HVA 101
Discussion ................. 105
VII. MODULATION OF ENDOGENOUS OPIOID INFLUENCE ON
LUTEINIZING HORMONE SECRETION EY ESTBOGEN
AND PROGESTERONE 110
Introduction ................ 110
Experimental 111
Experiment 1 .............. 112
Experiment 2........ .. 113
Experiment 3............... 113
Results ............ 114
Effects of Naloxone Administration During
the Estrous Cycle cn LH Release . 114
Effects of Naloxone on LH Release in Steroid
- Pretreated 0variectomized Rats . 116
The Effects of P on Naloxone Induced LH
Release on Prcestrus ........ 117
Discussion ................. 120
VIII. A DECLINE IN ENDOGENOUS OPIOID INFLUENCE DURING THE
STEROID INDUCED EYEERSECRETION OF
LUTEINIZING HORMONE IN THE RAT ...... 125
Introduction ................ 125
Experimental ......... 126
Results ................... 127
Discussion ... .............. 131
IX. THE EFFECTS OF CHRONIC MORPHINE TREATMENT ON THE
FEEDBACK ACTIONS OF ESTROGEN ON GONADOTROPIN
SECRETION IN OVAHIECTCMIZED BATS ..... 135
Introduction ................ 135
Experimental ................ 137
vii


Experiment 1. ....... ....... 137
Experiment 2 138
Experiment 3............... 138
Experiment 4............... 139
Experiment 5............... 139
Results ................... 140
Discussion ................. 149
X. GENERAL DISCUSSION 154
BIBLIOGRAPHY ......... 168
BICGEAEHICAL SKETCH 199
vm


LIST CF TABLES
TABLE PAGE
1. Effects of la Vivo Morphine and T Pretreatment on In
Vitro LH Belease from Hemisectioned Pituitarios
(LH release rate: ng/mg pituitary tissue/ hour) 71
2. The Effects of IHBH (100 ng/100 g E,W.# s.c.) on
serum LH Levels in Castrated Bats Treated
Chronically kith Morphine and/or Gonadal Steroids. 87
3. The Effects of Gonadal Steroids and Morphine
Treatment on 5HT 5HIAA and HVA Concentrations in
the MBH and POA 104
4. Effects of LHBH (75 ng/100 gm B.i., s.c.) on LH
secretion in ovariectomized steroid treated rats. 130
5. lhe Effects of IHBH on LH Hypersecretion in
E2-treated Ovariectomized rats. ........ 148
IX


LIST OF FIGUBES
FIGURE IAGE
1. Serum LH concentrations in rats treated with
morphine or T at the time of castration or two
weeks after castration. .............62
2. MBH LHBH concentrations in rats treated with
morphine or T at the time of castration or two
weeks after castration .............63
3. The effects of graded doses of T produced by various
sizes of T implants on serum T and IH in
morphine-treated and placebo-treated male rats
castrated for two weeks .............67
4.Relationship between LH and T levels in morphine-
treated and placebo-treated male rats castrated
for two weeks ..................68
5. The effects of various sized T implants on MBH IHBH
concentrations in morphine-treated and placebo-
treated male rats castrated for two weeks. ... 69
6. The effects of simultaneous morphine and 5 mm T
implants on LH secretion in rats which had beeD
orchidectomized two weeks previously ...... 80
7. The effects of simultaneous treatment with morphine
and various doses of E2 on LH secretion in rats
which had teen orchidectomized two weeks
previously. .... ...............81
8. The effects of simultaneous treatment with morphine
and various doses of E2 on FSE secretion in rats
which had been orchidectcmized two weeks
previously ...................82
9. The effects of simultaneous treatment with morphine
and various doses of DHT on IH secretion in rats
which had teen orchidectcmized two weeks
previously ...................84
x


10. The effects of simultaneous treatment with morphine
and various doses of CHT cn FSH secretion in rats
which had been castrated two weeks previously. 85
11. Interaction between morphine and T on IB secretion
in rats which were orchidectomized twc weeks
previously. ..................97
12. Interaction between morphine and 1 cn FSH secretion
in rats which had been castrated two weeks
previously. ...................99
13. Concentrations of NE and NHE in the MBH and EOA-AE
of intact rats and orchidectomized rats given
combinations of morphine and T, ....... 102
14. Concentrations of DA and DOPAC in the MBH and PCA-AH
of intact rats and orchidectomized rats given
combinations of morphine and I ........ 103
15. The effects of naloxone (2 mg/kg) on serum LB levels
at various times during the estrcus cycle. . 115
16. The effects of naloxone (2 mg/kg) on serum LE in
ovariectomized rats treated with FB (lower panel)
or E3 plus E (upper panel). .......... 118
17. Effects of P (5 mg Frog) on naloxone (2 mg/kg) -
induced LH secretion cn proestrus. ...... 119
18. The effects of naloxone injection on LB secretion in
ovariectomized rats receiving EB cr EBP
treatment. 129
19. The effects of continuous morphine exposure and
various doses of E2 on serum IH and FSH levels in
ovariectoaized rats. ...... 141
20. The effects of continuous morphine exposure on
midafternoon LH and FSH hypersecreticn induced by
E2 implantation in ovariectomized rats. .... 144
21. The effects of continuous morphine exposure cn IH
and FSH secretion in E2 implanted ovariectomized
rats. ..................... 145
22. The effects of continuous morphine exposure on LE
and FSH secretion in cvariectcmized rats. ... 146
xi


Abstract of Dissertation Presented tc the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Ehilosophy
GONADAL STEROID MODULATION Cl CEICID EFFECTS ON
GONADOTROPIN SECRETICN IN THE RAT
By
Steven M. Gabriel
December 1984
Chairman: Dr. James W. Simpkins
Major Department: Pharmacy
The interactions between opiates and gonadal steroids in
the control of the gonadotropins, luteinizing hormone (1H),
and follicle stimulating hormone (FSH), were investigated in
the rat. Male rats were chronically treated with morphine as
a means of providing continuous opiate receptor stimulation,
fihen initiated at the time of castration, both morphine
treatment and testosterone (T) replacement prevented post
castration LH hypersecretion and hypothalamic LH-releasing
hormone (LHRH) depletion. However, only T reversed these
changes when treatments were initiated two weeks after
orchidectomy. Further, in rats which had been castrated for
Xll


two weeks, morphine enhanced the ability cf T to inhibit
gcnadotrcpin secretion and blocked the T-induced
accumulation of LH EH in the hypothalamus. At physiological
doses, it was found that 17-beta-estradicl (E2) but net
5-alpha-dihydrotestcstercne (DH1), similarly interacted with
morphine to suppress gcnadotrcpin release. Further, the
ability cf morphine to interact with 1 in the suppression of
LH release could not be explained by changes in hypothalamic
norepinephrine, dopamine cr serotonin metabolism.
Female rats were injected with the opiate antagonist,
naloxone. Naloxone stimulated LH release at all times
tested during the estrus cycle and following estradiol
benzoate (EB) treatment to ovariectcmized rats, indicating
that endogenous opioid peptides (EOE) inhibit LH secretion
throughout the estrus cycle, and during the preestreus and
EB-induced LH surges. During LH hypersecretion, induced by
progesterone {P) treatment to proestrous or EE-treated
ovariectcmized rats, nalexone was unable tc stimulate IH
release, indicating that EOF may contribute to the advanced
onset and increased magnitude cf the LH surge seen following
P treatment.
In a final study, E2-treated ovariectomized rats were
given morphine treatment. morphine enhanced both the
negative and positive feedback effects cf E2 cn gcnadotrcpin
release in these animals. In summary, the work presented in
this dissertation indicates that EOF Flay an important rcle
xm


in the regulation c£ gonadal steroid feedback in male
female rats by modifying the sensitivity of the brain
circulating gonadal steroids.
and
tc
xiv


CHAPTER I
INTRODUCTION
Opiates have been used by cld world cultures for many
centuries. Opium is the dried latex from the unripe seed
pods of the plant Papa ver somnif erum. It was probably first
discovered by the ancient Mesopotamians (Beynolds and
Randal, 1957). The word 'opium* is derived from the Greek
word for juice, and was mentioned by Theophrastus in the
third century B.C. The Romans, Scribcnius largus and Galen
both described the medicinal uses of opium. In another
ancient reference tc opium, the Kama Sutra states that
Indian maidens and wives are forbidden to use opium in any
form until after menopause, as the use of the drug prevents
pregnancy (Kruger et al., 1941). Throughout the middle ages,
Arabs used opium for medicinal purposes. They introduced
the drug to China, where it was used tc treat dysentery and
other ailments. Its use in Europe increased during the
sixteenth century, where opium was used for the treatment of
diarrhea and pain, and as an adjunct tc surgery.
Raw opium contains over 20 different alkaloids. Surturner
isolated and described morphine in 18C6 (Leake, 1975).
Morphine comprises ever 10% of the dry weight of opium. Its
structure was proposed in 1925 by Gulland and Rcbinscn, and
1


2
finally synthesized in 1952 by Gates and Ischudi (see Leake,
1975), Other less abundant alkaloids such as narceine,
codeine, and thebaine were isolated in the early nineteenth
century. Improved organic synthesis techniques in the
twentieth century resulted in a flurry cf semi-synthetic
opiate and synthetic opiate-like compounds being produced.
In an effort to design a less addictive opiate, the partial
agonist, nalorphine, was introduced. Although analgesic and
less addictive than morphine, its dysphoric properties
precluded its widespread application. Ihis search for a less
addictive opiate also led to the synthesis cf naloxone, an
opiate antagonist relatively devoid cf analgesic and ether
opiate agonist actions,
The modern use of opiates also led to its misuse. Great
Britain secured a market for its Indian opium by addicting a
large population in China (Kane, 1881). The disputes that
developed between Great Britain and China over this trade
were referred to as the Great Cpium Wars of the eighteenth
and nineteenth centuries. Chinese immigrants introduced
opium socking into the United States where it spread to the
general population. Opiate abuse continued to increase in
this country after the development of the hypodermic needle
and the introduction of injectable morphine to relieve the
pain resulting from battle injuries received during the
Civil War ODonnell and Ball, 1966).


3
Many authors commented on opiate abuse at this time. In
1881, Kane described the physiological ccnseguences of
opiate abuse. Included in this discussion were the effects
of opiate abuse on the sexual organs. Opium smokers
exhibited "disgraceful conduct and initially considerable
sexual stimulation, although the completion of the sex act
was delayed". Several months cf abuse impaired both desire
and power. To further illustrate the decline in the sexual
ability of opiate addicts, population statistics were cited.
During the height cf the Great Cpium liars, China's annual
population increase was found to fall frcm 6% to under 1S.
It was even suggested that the cpium trade could be used as
a means of controlling China's overpopulation!
The deleterious effects of opiates and ether central
nervous system depressants on reproductive function
continued to be reported in the medical literature during
the twentieth century. While narcotic addicts appeared to
commit fewer violent crimes such as rape (Finestone, 1957),
men were found to be impotent and women showed inhibited sex
drives and amenorrhea. The first controlled clinical studies
by Azizi et al. (1973) and Gaulden et al. (1964) reported
depressed testosterone (T) levels in male herein acd
methadone users and diminished sexual function in women
narcotic addicts, respectively.
The numerous physical effects of opiates were postulated
to be the result of an interaction with specific central


4
nervous system receptors. In 1973, several laboratories
independently reported the existence of opiate receptors in
the nervous systems of mammals (Fert and Snyder, 1973;
Terenius, 1973). Soon to follow these discoveries has the
isolation of endogenous opioid peptides (EOF), Hughes et al,
(1975) described the two pentapeptides, leucine-enkephalin
and methionine-enkephalin, while Li and Chung (1976)
reported the isolation of beta-endorphin. Following these
discoveries, researchers began to identify the mechanisms
through which opiates exerted their antigonadal effects. It
soon became evident that opiates interfered with a normally
operating EOP influence on reproductive hormone secretion.
The effects of opiates on the regulation of reproductive
hormones, luteinizing hormone (IH) and follicle stimulating
hormone (FSIi) is the subject of this dissertation.


CHAPTER II
REVIEW OF THE LITERATURE
This chapter will survey the major work with respect to
the neuroendocrine control of gcnadotrcpin secretion. This
will include the tasic anatomy and physiology of the
hypothalamo-hypophyseal unit and neurcpharmacclogical
studies which contributed to the concept that an opioid
inhibitory component controlling gonadotropin secretion.
Emphasis will be placed on LH secretion, although important
differences between LH and FSH secretion will be discussed.
The field contains a wealth of literature and several
reviews were employed as starting points and to highlight
major trends and areas of agreement. This includes
historical reviews by Garrisson (1929), Leake (1975) and
Hedvei (1982). The descriptions of general hypcthalamo-
hypophyseal anatomy were derived from Adams et al. (1965),
Daniel (1966), Halasz (1969), Jenkins (1978), Ezrin ( 1979)
and Palkovitz and Zaborsky (1979). Detailed reviews of the
various neuronal systems can be found in Dahlstrom and Fuxe
(1964), Fuxe and Understedt (1966), Cooper et al. (1978),
Hoore and Bloom (1978 and 1979), Sternberger and Hoffman
(1978), iatson et al. (1980) and Palkovitz (1981).
Information regarding luteinizing hormone releasing hormone


6
(LHSH) biochemistry, steroid concentrating neurons, and
opioid peptides and their receptors may be found in reviews
by Naftolin at al. (1975), Sar and Stumpf (1975), Bloom et
al. (1978), McEwen et al. ( 1979), Childers (198C) and Kartin
(1581), Jutisz et al, (1983). Hany excellent reviews
concerning the role of monoaminergic neurons in regulating
gonadotropin secretion have appeared over the years. Among
the most complete are Coppola (1971), Heiner and Ganong
(1578), Barraclough and Wise (1982), S. Kalra and Kalra
(1983) and Simpkins et al., (1984). Finally, the
pharmacological and physiological effects of opiate
alkaloids and opioid peptides on gonadotropin secretion have
been presented by Meites et al. (1979), Cicero ( 1980a and
1980b) and S.,Kalra et al. (1980). fchile the literature
discussed will focus primarily on studies in the rat, where
appropriate, other animals will be discussed. The relation
of these studies and the present work to the regulation of
gonadotropin secretion in humans will be addressed in
Chapter X.
Historical
Early Observations
Although modern endocrinology has yet to complete its
first century, observations on endocrine function have been
made throughout history. Relief carvings, figurines, and
drawings from prehistoric, Egyptian, Babylonian,
and later


7
Greek, Roman and Eenissance artisans appear to depict
medical conditions (Garrisson, 1929). Sene of these
artifacts have been interpreted as illustrating such
illnesses as female endocrine system obesity, geiter and
gigantisism. Goiter was endemic among ancient cultures and
the Chinese prescribed an appropriate treatment cf iedine-
rich seaweed. While the Chinese, Hindus, and Egyptians all
described diabetes mielitis, the Greek Arctaec cf Kappadckia
coined the term 'diabetes' in the second century A.D.
Reproductive function was a primary ccncern of the
ancients. The effects of castration on animals and humans
have been known since prehistory, while the first
ovariectomies in humans and hysterectomies on farm animals
were performed by the Egypticns and Hebrews, respectively.
Huch later in the eighteenth century, the results cf
experimental removal of the gonads wculd be pioneered by
John and William Hunter of England (Medvei, 1982).
The discovery of gynecological instruments among Roman
ruins indicated some degree of medical sophistication during
this period. Theories of reproduction were somewhat less
advanced. The Hippocratic view that sperm arose from all
tissues of the body to be stored in the testes was
challenged by Aristotle who held that the right testes
produced sperm destined to become males while the left
testes produced sperm destined tc become females.
Additionally, Aristotle felt that the male provided all the


8
determining characteristics while the female provided merely
a fertile enviroment for the development of the fetus,
A more modern view of the factors involved in
reproduction awaited the development of the microscope in
the seventeenth century. The initial works of Fallopic,
deGraff, Leydig and many other scientists are reflected in
our anatomical vocatulary. Leeuwenhoek first described the
presence of "little animals of the semen" in 1677 and 200
years later Hertwig demonstrated the unicn cf the sperm and
the evurn.
Recognition of the importance of the ovary in maintaining
reproduction was the result of the work of many scientists.
At the turn of this century, Walter Heap described the
reproductive cycle in females and related the reproductive
changes of the estrous cycle tc these cccuring in the
menstrual cycle. The changes in vaginal cytology
characteristic of the estrous cycle were first described by
Long and Evans of America in the early twentieth century,
while at the same time Hitschman and Adler cf Vienna
described the cyclical changes in the uterine endometrium
which occur during the menstrual cycle.
The Development of Neurcendocrinology
The communication between various tissues of the body via
substances released into the circulation is one of the
central concepts of endocrinology. In the seventeenth and


9
eighteenth centuries Albrect von Haler and Frederick Buych
recognized that the body contains ductless glands which
release their contents into the blood. Claud Bernard
refered to this process as internal secretion in 1855, In
1902, after his discovery of secretin, Ernest Starling
coined the term 'hormone* to describe the active contents of
internal secretions. Many hormones were first recognized by
the ability of organ extracts to exert physiological effects
on animals in vivo or effects on isolated tissues in vitro.
Among the first hormones isolated were the gcnadal steroids,
testosterone (T), 17-beta-estradiol (E) and progesterone
(P), which were found to be responsible for maintaining
reproductive function in males and females.
One ductless organ which was found to be of major
importance in maintaining normal homeostasis was the
pituitary glaud. Its function was long debated and, until
1838 when Bathke demonstrated the non-neural origin of the
anterior portion, it was considered by many to he a
vestigial portion of the train. The pituitary gland was
found to secrete substances necessary for normal tody
growtn, the maintenance of reproduction, the initiation of
lactation, and the restoration of atropied thyroid and
adrenal tissue. In the 194C's C.H. Li and collegues
isolated and synthesized the two gonadotropic hormones,
luteinizing hormone and follicle stimulating hormone (IH and
FSH, respectively; li et al. ,
1940 and 1949)


10
A functional relationship between the brain and the
pituitary gland was evident to many scientists. Galen's view
that the pituitary drained phlegm from the brain tc the
nascpharnyx was refuted by the anatomist Schreiber in 1660.
Schreiber's contemporary. Bichard Lcwer proposed that
substances from the ventricles perfused from the brain to
the pituitary where they "percolated into the circulation.
This view is quite similar to cur current views on
neuroendocrine function.
In this century it was observed that many hypothalamic
lesions and tumors disrupted endocrine function. Experiments
showed that severing the pituitary connection with the brain
atrophied both the thyroid and the adrenal glands. The
transplantation of the pituitary gland to the renal capsule
or the anterior chamber of the eye produced similar
degeneration of the thyroid, adrenals and testes, tut the
ovarian corpera ltea was maintained in rats. It was
apparent that the brain exerted both stimulatory and
inhibitory influences on the pituitary. Additionally, this
interaction between the brain and the pituitary seemed to be
dependent on the preservation of the connection between the
pituitary and the hypothalamus,
Joseph Lietaud first described the pituitary stalk and
its relationship to the brain in 1742. It was not until 1930
that Pepa and Fielding clearly described the vascular link
between the two organs as a portal system, however. It is


11
unfortunate that the two scientist incorrectly proposed that
the direction of blood flow in the portal system was from
the pituitary to the hypothalamus Housay el al. (1935) and
Wislocki and King (1936) quickly rectified this error.
Several observations led to the development of cur
current understanding of neuroendocrine relationships. Among
the foremost was the discovery of neurosecretion fcy
magnocellular neurons in the paraventricular and supraoptic
nuclei by Scharrer and Scharrer (1940). These neurons
released oxytocin and vasopressin from nerve terminals in
the neural lobe of the pituitary into the general
circulation. Harris and associates performed numerous
studies which demonstrated the importance of the
hypothalamus in regulating anterior pituitary function.
These included endocrine changes following the electical
stimulation of the hypothalamus. In 1948, Harris proposed
the "cheaoreceptor hypothesis to explain the control of
anterior pituitary function by hypothalamic hormones
released into the portal circulation. This concept remains
one of the cornerstones of neuroendocrine thought.
Much of the work of neuroendocrinologists over the past
30 years has concerned the demonstration, isolation and
characterization of these hypothalamic hormones. Ealasz et
al., (1962) used the term hypcphysiotropic area to describe
the regions of the hypothalamus that would support pituitary
grafts. Using in vitro assays, releasing activity vas


12
demonstrated in hypothalamic extracts for thyroid
stimulating hormone (Shitusava et al., 1956), LH (McCann et
al., 1960), prolactin (Eeites et al., 1960), FSB (Igarshi
and McCann, 1964; Mittler and Beites, 1964), and growth
hormone (Deuben and Meites, 1964). Hypothalamic release
inhibiting* activity vas shown for prolactin (Talwalker et
al., 1961; Pasteis, 1961) and growth hormone (Krulich et
al, I 960).
The isolation of these releasing factors proved to be
more difficult. Because these factors were present in very
small amounts, sensitive biochemical and bicassay techniques
as well as large amounts of tissue were necessary. Many
incorrect claims have keen put forth over the years. The
first releasing factor successfully isolated was the
tripeptide, thyrotropin releasing hormone. It was found to
stimulate both prolactin and thyroid stimulating hormone
release and its structure was simultaneously reported in the
laboratories of Andrew Schally and Boger Guillemin (Scahlly
et al. 1969; Burgus et al., 1969). Schally's group also
described the next releasing factor. It was a decapeptide
which stimulates the release of both IH and FSH, tut because
other factors appear to also regulate FSH release, it has
been termed luteinzing hormone releasing hormone (LEBE,
Martsuc et al., 1971). The competition to isolate
hypothalamic releasing factors continued as Guillemin*s
laboratory reported the seguence of a growth hormone


13
re lease-inhib tiny factor, now called somatostatin (Erazeau
et al. 1973). The rivalry between these two laboratories did
not gc unrecognized in the scientific community. In 1977,
together with Rosalyn Yallow, Roger Guillemin and Andrei
Schally received the Nobel Prize in Physiology. The drive
to isolate releasing factors continues today. Corticotropin
releasing factor was isolated by Wylie Vale, Guillemin's
collegue at the Saulk Institute (Vale et al., 1981). In the
following year Vale and Guilleain simultaneously reported
the sequence for a growth hcrione releasing factor
(Guilleain et al., 1982, J. Bivier et al., 1982)
Neuroanatoaical Relationships
Anatomy of the Pituitary Gland
The pituitary gland lies within a portion of the spencid
bone called the sella tursica. It is positioned telow the
mid-ventral portion of the train and is encased in an
extension of the cerebral meninges known as the sellar
diaphragm. The two major anatomical divisions of the
pituitary gland, the neurohypophysis or posterior pituitary
and the adenohypophysis cr anterior pituitary, have distinct
embryological origins. The neurohypophysis is derived frcm
neural ectoderm and contains the nerve terminals cf
mag noce 11ular neurcus of the hypothalamus. It is involved
in the neurosecretion of vasopressin and cxytocin. The
adenohypophysis is derived from an envagination of the


14
stromal ectoderm called hathkes pouch and has do direct
neural connection with the brain. It constitutes 80X of the
weight of the pituitary gland. Another less prominent
division of the pituitary gland is the intermediate lote.
While derived from Hathke's poach, it does receive
innervation from the hypothalamus and is often included in
dissections with the neurchypophysis. Together these two
lobes are termed the neurointezmediary lobes (NIL). While
distinct in the rat, the intermediate lobe is only well
developed in humans during pregnancy.
The major anatomical division of the adenohypophysis is
the pars distalis. Approximately 5S of the cells of the
pars distalis are gonadotropin producing cells. It is still
uncertain whether a separate type of gcndadctroph exists of
LH and FSH. There is evidence that some endocrine
manipulations, such as ovariectomy, produce two distinct
populations of gonadotropes.
General Hy othalaaic Anatomy
The hypothalamus constitutes the ventral portion of the
diencephalon. Its many neural connections with other
portions of the diencephalon, limbic system and brainstem
highlight its important role in endocrine and autonomic
homeostasis. The boundaries of the hypothalamus are defined
as the ventral surface of the train extending from the
rostral border of the optic chiasm to the mammillary todies


15
caudally, and laterally to the hippocampal sulcus and optic
tracts, Ihe dorsal order of the hypothalamus is recognized
as the anterior commissure and lamina terminalis rcstrally
to the hypothalamic sulcus and cerbral agueduct caudally. In
rats the hypothalamus constitutes 1% c£ the weight of the
brain.
An important landmark evident frcm the midventral surface
of the brain is a prominence containing the infundibulum and
tuber cinerium which together constitute the median
eminence. This is the sole anatomical connection between the
hypothalamus and the pituitary gland. A unique vascular
system supplies this hypothalamo-hypophyseal unit.
Hypophyseal arteries branch off the Circle cf Willis tc
perfuse the various hypothalamic regions. The most important
aspect of the hypothalamic circulation is the vascular
supply to the median eminence and arcuate nucleus. Arteries
in this area form a capillary network containing multiple
anastomoses which is refered to as the primary plexus cr
palisadic zone, A portion of this configuration contains
numerous nerve endings bordering these vascular spaces and
is called the external layer of the median eminence,
Because these capillaries do not contain fenestrations, the
median eminence is not included in the blood brain barrier
and is considered cne of the brain*s circumventricular
organs


16
The veins of the median eminence collect tc for a portal
system which supplies blood to a secondary capillary plexus
in the pituitary. Venous flow from the pituitary is achieved
through sinuses adjacent to the adenohypophysis. This
hypothalamo-hypophyseal portal system is the primary tlocd
supply tc the pituitary gland. Its existence provides a
means for neurosecretory products of the hypothalamus to
reach the adenohypophysis in high concentrations undiluted
by the general circulation. While the direction of bleed
flow is from the hypothalamus to the pituitary, this system
may be more complex than previously thought. Bergland and
Page (1979) have proposed several other pathways through
which elements in this portal system may interact.
Cell bodies of the hypothalamus are distributed in three
major gray regions, the anterior, intermediate, and
posterior areas. The anterior and intermediate regions are
the most important in the regulation cf anterior pituitary
hormone secretion. Hypothalamic nuclei are generally located
bilaterally on either side of the third ventricle. The
exceptions to this rule are the arcuate nucleus and its
closely associated median eminence which are located at the
ventral border of the third ventricle surrounding the
infundibular recess. The anterior hypothalamic area includes
the preoptic area located rostral to the optic chiasm.
Consequently this area is often collectively refered tc as
the preoptic area-anterior hypothalamus (EGA-AH). Important


17
nuclei in this region include the suprachiasmatic nucleus
which is immediately dorsal to the optic chiasm, the
anterior hypothalamic nucleus located dcrsalateral tc the
suprachiasmatic nucleus, the paraventricular nucleus which
constitutes the rostral preoptic area tc the dcrsalateral
wall of the third ventricle, the supraoptic nucleus, and the
organum vasculosum of the laaina terminalis which is a
circunventricular organ located at the anterior ventral
third ventricle. The preoptic and and supraoptic nuclei
appear to be particularly important in the cyclic release of
gonadotropins in the rat, but not necessarily in the human
(Hillarp, 1949, Halasz, 1969, and Krey et al., 1975).
The intermediate and posterior areas are often grouped
into a tissue section refered to as the medial basal
hypothalamus (JiBH). Nuclei of the intermediate
hypothalalamus include the lateral hypothalamic nucleus,
ventralateral nucleus, dcrsalmedial nucleus and the arcuate
and median eminence nuclei. Lesions of the median eminence
or arcuate nucleus disrupt gonadotropin secretion and result
in a less of reproductive cycles (Goodman and Knotil, 198 1).
The nuclei of the hypothalamus have numerous afferent and
efferent connections. Seven afferent tracts impinge upon
the hypothalamus. The medial forebrain bundle contains
tracts originating in both the olfactory cortex and the
brainstem while the stria terminalis originates in the
amygdala. Both tracts terminate in the hypothalamus,


18
midbrain, anterior cosiissure and preoptic area.
Corticomedullary fibers include the fornix and
corticchypothalamic tracts which originate in the
hippocampus and frontal cortex, respectively. Afferents from
the medial thalamic nuclei and subthalamus reach the
hypothalamus via the periventricular regions. The mammillary
bodies receive inputs from the ascending spinal tracts, the
brainstem and the anterior thalamic nucleus. Finally, photic
input to the suprachiasmatic nucleus is received via a
direct retino-hypothalamic tract.
Several efferent pathways have been described as
originating in the hypothalamus. Two tracts, the
supraoptico-hypophyseal and the tubero-hypophyseal,
terminate in the NIL. The fasciculus mammillary princeps
terminate in the anterior thalamus and midtrain. A
periventricular fiber system returns inputs to the midbrain
thalamic nuclei from the posterior, tufceral and supraoptic
hypothalamic areas. While the primary direction cf all
these pathways is either afferent or efferent, it is net
absolute. In addition, many smaller pathways cannot be
identified without imsunocytochemical technigues. The
relation of these tracts as well as intrahypothalamic
pathways to the neurotransaitter systems will be discussed
in the following sections.


19
Ana tom y of the 1 uteinizi ng Horaone-E eleas ing Hormone
Neuronal Systems
Many problems associated with iirmunccy tcchemical
techniques have precluded a complete description cf the
distribution of LHEH neurons. Among these problems is the
lack of an amino acid sequence for a IHEH precursor cr its
mBNA sequence which could be used tc visualize IHBH-
containing perikaryia. Many antibodies to LHBE are
conformationally restricted or require cne cr both terminal
ends of the LHEH molecule for recognition. This can prevent
the visualization cf peptide-ccntaining cell bodies. Tc
demonstrate LHEH in neuronal perikaryia many studies have
employed cholchicine or barbiturate pretreatment, cr
deafferentation (Sternberger and Hoffman, 1978).
Several generalizations may be made regarding the
distribution of LHEH neurons in the rodent brain. The
neurons are bipolar or fusiform and have distributions net
limited to the traditional nuclear boundaries of the
hypothalamus. Nerve terminals are heavily concentrated in
the external layer of the median eminence, which fits the
role of LHEH as a hypothalamic releasing hormone. At least
two distinct neuronal pathways appear to exist:
1. a tuberoinfundibular pathway with cell bodies in the
arcuate nucleus projecting to the median eminence.
2. a preoptico-tuberal pathway with cell bodies in the
medial preoptic zone that project to the median
eminence


20
Although LHBH perikaryia have been visualized in the
arcuate nucleus of several species, many groups have had
difficulty demonstrating their presence in the rat
(Sternterger and Hoffman, 1978). However, deafferentaticn
spares 2C% to 3QX of LHBH concentrations in the MEH and does
not disrupt basal LH secretion (Blake and Sawyer, 1974; S,
Kalra, 1976; Brownstein et al., 1977; Sopor and Weik, 1980).
Since this does not seem to be due to incomplete lesions,
there appears to be some LHBH neurons inside the area of the
cut.
A recent study ty Kelly et al. (1982) reports the
presence of LHBH perikaryia in the MEH, especially the
lateral arcuate nucleus and median eminence. This study
employed a specific LHBH antisera which was net
ccnformationally restricted, and used rigorous fixation
procedures on saggital train sections to facilitate the
visualization of the cell bodies. These neurons and others
located in the retrochiasmatic nucleus, precptic area, and
organum vasculosum of the lamina terminalis, terminated in
the extern*1 layer of the median eminence.
Other recent studies have expanded the potential
distribution of LHBH cell bodies in the brain. These include
septal projections and preoptic projections to the organum
vasculosum of the lamina terminalis, the olfactory bulb and
midbrain central gray (Dluzen and Bamerez, 1981; Witkin et
al., 1982; Shivers et al,, 1983b).


21
Anatomy of the Monoaainergic Neuronal Systems
The gradual improvement of analytical procedures has
resulted in a fairly accurate description of the
distribution of monoaminergic neurcns and their terminal
beds in the central nervous system. Concentrations of the
monoamines can also be determined in small nuclear regions
using the "punch technique" of Palkovitz (1981), In
addition, with the use of antitedies generated against
catecholamine synthetic enzymes such as phenylethanolamine-
n-methyltransferase, which converts norepinephrine {NE) to
epinephrine (EPI), these two neuronal systems can te more
easily differentiated.
Noradrenergic pathways
Vcogt (1954) first demonstrated the presence of NE in the
brain. It now appears that the hypothalamus receives a rich
afferent innervation from NE-ccntaining neurcns. Evidence
favoring an external source for NE in the hypothalamus
includes the depletion of 70% to 90% of hypothalamic NE
after complete deafferentation (Weiner et al., 1972), The
residual NE seen after deafferentation or brainstem lesions
appears to be due to glial cells concentrating NE and to
collateral reinnervation, since no NE-containing perikaryia
have been visualized in the hypothalamus (Palkovitz, 1981),
Noradrenergic neurons are clustered in 5 major cell
groups in the brainstem the lateral reticular nucleus, the


22
solitary tract nucleus, the ventral pcntine nucleus, the
locus ceruleus, and the mesencephalic reticular formation.
These cell groups innervate the hypothalamus through three
major tracts the ventral noradrenergic bundle, and the
ventral and dorsal periventricular noradrenergic bundles.
The ventral noradrenergic bundle joins the medial fcrebrain
bundle prior to innervating the hypothalamus and appears to
be the most important of the three tracts in regulating
anterior pituitary hormone secretion. Although cell todies
that contribute to the ventral noradrenergic bundle are
primarily located in the lateral reticular nucleus, the
interconnections between all the various cell groups and
their ability to form collateralizations must be stressed.
Dopaminergic pathways
Several distinct dopaminergic pathways are found in the
brain. The largest consists cf dopamine (DA)-containing
neurons in the zona compacta of the substantia nigra and
ventral tegmentum. From these neurons arise the
nigralstriatal and mesocortical-aesoliabic CA systems.
However, neither of these systems appear to innervate the
hypothalamus significantly (Weiner et al., 1972).
The DA innervation of the hypothalamus arises primarily
from intrahypotha la sic DA systems. The tuberoinfunditular EA
system consists of cell todies in the arcuate nucleus which
project to the median eminence. Axon collaterals terminate


23
in the arcuate nucleus, ventralateral nucleus and
premamaillary nuclei. Included in this system is the
tuberohypophyseal DA tract which contains cell todies in the
arcuate and periventricular nuclei that project to the
neurohypophysis and intermediate lobes of the pituitary
(Moore and Bloom, 1978; Palkovitz, 1981). lastly, a poorly
defined collection of DA-containing neurons in the zona
incerta, dorsalmedial subthalamus and posterior hypothalamus
form the incerto-hypothalanic DA system. These cells project
to the dorsal anterior hypothalamus, and the paraventricular
and lateral septal nuclei (Bjcrklund et al., 1975).
Epinephrine-containing pathways
Of the three major catecholamines, EPI has the narrowest
distribution. Two EPI-containing cell groups are
intermingled with NE-containing cells in the lateral
tegmentum and dorsal medulla. In addition to other areas,
these cells project to the hypothalamus via the medial
fcrabrair bundle (Moore and Bloom, 1979; Palkovitz, 1981).
Serotonergic Pathways
There appears to be a widespread distribution of
serotonin (5BT) terminals throughout the brain. Clusters of
5HT-containing perikaryia are restricted to the midline
brainstem raphe nuclei. Ascending fibers course through the
medial fcrebrain bundle and innervate the hypothalamus,


24
particularly the suprachiasmatic nucleus (Kuhar et al.,
1972). In addition, there is evidence for SET perikaryia
within the hypothalamus (Fuxe and Onderstedt, 1968),
An atomy of Endogenous_Gpioid Peptide CcntainingNeurcnal
Systems
At present three distinct families cf endogenous cpicid
peptides (EOP) have been characterized. Each appears to have
its can unique precursor molecule, anatomical distribution
and receptor subtypes. Because of the difficulties in
producing specific antibodies which distinguish between
these three groups of molecules and in visualizing peptide-
containing cell bodies, the description of ECP neuronal
systems is far from complete.
Beta-endorphin-containing neurcns
Beta-endorphin is derived from proopiomelanocortin (PCMC)
and has teen colocalized in cells with ether PCKC-derived
molecules like adrenocorticotropin, melanocyte stimulating
hormone, and beta-lipotrepin (Mains et al., 1977). It is not
clear whether all ECMC-containing neurons release the same
degradation products, or whether as Batson et al. (1980)
suggest, several distinct PCEC-neuronal populations exist,
each releasing a characteristic set cf molecules. One
clearly defined beta-endorphin pathway is agreed upon (Bloom
et al,, 1978; Finley et ai, 1981a). Fusiform neurons are
contained in the tuberal hypothalamus extending from the


25
lateral arcuate nucleus to the lateral hypothalamic border.
Fibers project to the anterior hypothalamus and septum here
the pathway reverses direction and follows the stria
terminalis to terminate in the dcrsal raphe, locus ceruleus
and central gray. Arborizations are found throughout the
POA-AH and MBH including the median eminence and arcuate
nucleus, Electronmicroscopic studies have revealed local
interactions between POMC-ccntaining neurcns in the
hypothalamus (Kiss and Williams, 1983).
Enkeph alin-con tai ni c g neurons
Enkephalins are derived from a precursor molecule which
contains methionine-enkephalin, cartcxy-terminal extended
methionine-enkephalin and leucine-enkephalin. While teta-
endorphin-containing neurcns have a fairly distinct pathway,
enkephalin-containing neurons are widely distributed
throughout the brain. The highest concentrations cf
methionine-enkephalin are found in the striatum, followed by
the hypothalamus. Dense methionine-enkephalin innervation
has been found in the external layer of the median eminence
(Watson et al., 1980). Enkephalin-ccntaining cells are
generally interneurcns although several short pathways have
been described.
1. A dense collection of methionine-enkephalin-
containing cell todies in the central amygdala
project to the bed nucleus of the stria terminalis
(Cuello and Paximus, 1978).


26
2. Magnocellular neurons of the paraventricular nuclei
contain methionine-enkephalin acd project to the
neurohypophysis (Bossier et al., 1979).
3. A preopticotuberal pathway with cell todies in the
POA-AH projects to the median eminence (Finley et
al., 1981b).
4. Hethionine-enkephalin-ccntaining perikeryia in the
anterior hypothalamus terminate in the septal area
(Sakanaka et al., 1982).
Dynorphin-containinq neurons
Dynorphin is derived from the prcdynorphin-alpha-
necendcrphin precursor along with leucine-enkephalin and
carboxy-terminal extended leucine-enkephalin molecules
(Goldstein et al., 1979; Kakadani et al., 1982).
Consequently, there is some difficulty in distinguishing
between leucine-enkephalin contained within prodynorphin-
and proenkephalin-ccntaining neurons. Of the three families
of EOP, the distribution of dynorpnin-containing neurons is
the most restricted. A supracptico-nenrchypcphyseal pathway
appears to be distinct from a similar methionine-enkephalin
containing pathway. Some of these supraoptic neurons also
contain vasopressin and corticotropin releasing factor and
project to the median eminence in addition to the
neurchypophysis (Watson et al., 1982a,b; Both et al. 1983).


27
In addition to the brain, EOP are fcund in the pituitary
gland. Beta-endorphin coexists with other ECKC molecules in
adenohypophyseal corticotropes and is released inte the
blood by stimuli which also stimulate adrenocorticctropin
secretion (C. Bivier et al., 1962). Ihe anterior pituitary
and intermediate lobes contains some of the highest
concentrations of methionine-enkephalin in the body (Elccm
et al., 1977; Kumar et al., 1979). Because of the presence
of EOP and their receptors in the pituitary, their potential
influence on anterior pituitary hormone release cannot he
discounted (Simantcv and Snyder, 1978) .
Steroid Concentrating Neurons in the Brain
While it is recognized that neurons may serve a target
sites for gonadal steroids, the identities of these steroid
concentrating neurons are not known. Specific cytoplasmic
and nuclear receptors for androgens, estrogens, and
progestins as well as the steroid metabolic enzymes 5-alpha-
reductase and aroaatase have been characterized in the train
and hypothalamus (Massa et al., 1972; Naftolin et al., 1975;
Sar and Stumpf, 1975; McEwen et al., 1979). The electrical
activity and morphological characteristics cf neurons in the
hypothalamus can be altered ty estrogens 1983; loran-Allerand et al., 1983).


28
Neuroanatomical Interactions
Although the distributions of LHBH, monoamine, and ECE-
containing neurons show considerable overlap, evidence for a
direct anatomical interaction between these systems in the
hypothalamus is lacking. The anatomical relationships
between peptide-containing and noradrenergic neurons have
been most intensely studied. Methionine-enkephalin
containing nerve terminals do appear to synapse on
catecholamine perikaryia and axons (legar et al, 1983;
Schwartz, 1979). While reports of such an interaction in th
hypothalamus are lacking, Hoffman et al. (1982) presented
evidence for noradrenergic terminals synapsing with LHEH
cell todies en passant. Considering the clcse proximity of
many nerve terminals in the median eminence, a diffuse
nonsynaptic interaction between any of these systems is
possible. This is particularly reasonable considering the
extended half-life of beta-endorphin (Eloom et al., 1978),
which would allow its diffusion to adjacent nerve terminals
even in the absence of classical synaptic contacts.
Improved anatomical methods may further resolve the
neurcanatomica1 relationships in the hypothalamus. Eonkleiv
et al. (1981) compared beta-endorphin and LHEH
immunoreactivity in adjacent brain slices and found beta-
endorphin containing neurons and terminals to be more widely
distributed than these containing LHBti in the MBH. Double
immunostaining procedures have not been employed. Ey


29
comparing autoradiography and immunccytcchemistry Shivers et
al. (1983a) reported that LHEH neurons do not appear to
concentrate estradici in their nuclear regions. This
contrasts with the report that some neurcns in the
hypothalamus that are electrically sensitive to estrogen do
stain for LHRH (Kelly and Bonkleiv, 1S82).
Patterns of Gonadotropin Secretion
Gonadotropin secretion is inherently pulsatile. Whether
absolute levels of LH and FSH are determined at a single
time point across several animals or within a single animal
over time, these levels represent the summation of pulsatile
discharges of hormone which vary in freguency and amplitude.
Because of the longer half-life of FSE (Coble et al., 1969)
pulsatile LH secretio is most frequently studied.
Males
In the male, LH secretion is characterized by hourly low
amplitude pulses which are temporally related to periodic T
episodes (Ellis and Desjardins, 1982). Although LH levels
remain fairly constant throughout the day, T levels are
highest in the midafterncon and lowest at about midnight.
Peak levels of P and FSH coincide with midnight, while IHRH
concentrations in tne MBH fluctuate shewing lowest levels
between 1100 h and 1600 h and peak concentrations at 1900 h
through 0800 h (P. Kalra and Kalra, 1977b).


30
The renoval of gcnadal steroid feedback by gonadectomy is
followed by marked changes in the hypcthalasic-pituitary-LH
axis. Within hours of gonadectomy in the male LH secretion
is increased, and by two weeks after castration displays
characteristic high amplitude pulses with a frequency of 20
to 30 minutes (Badger et al., 1978; Galle, 1980a),
Concentrations of LHBH decline in the MBH while the
pituitary responsiveness to 1BE and receptors for the
decapeptide increase (P. Kalra and Kalra, 1977; Nansel et
al., 1979; Conne et al,, 1982), All of these effects are
reversed by T,
While FSH secretion shows a similar response tc
castration, T replacement is much less effective in
returning FSH to gcnadal-intact levels (Mahesh et al.,
1975). This is in agreement with reports that other gonadal
and hypothalamic factors also regulate FSH secretion (McCann
et al. 1983) .
Females
In the female rat low levels of gonadotropin secretion
are interrupted by a preovulatory discharge cf LH and FSH
every 4 to 5 days. Gonadotropin levels during the preestrous
surge may be 5 to 20 times greater than basal levels.
Whether basal or surging, LH and FSH levels in the female
rat are the result of pulsatile discharges of hormone
(Gallo, 1981a,b). Prior to the preestrous gonadotropin


31
surge is a period o follicular development characterized by
increasing estrogen titers (P. Kalra and Kalra, 1977a).
These sustained estrogen levels permit the expression of a
daily signal for LH release to be tiled to the midafterncon
(Legan et al., 1975). Superimposed upon this cyclic pattern
of LH and estrogen secretion is a circadian variation in P
secretion by the adrenal and ovary. Peak P levels occur in
the evening prior to midnight (S. Kalra and Kalra, 1974a).
The largest of these P rhythms is on proestrus, when it may
contribute to the preovulatory release of gonadotropins.
This proestrous LH surge is preceded by a period of IHEH
accumulation in the MBH and accompanied by a decline in
levels of the decapeptide (S. Kalra and Kalra, 1981). The
decline in MBH LHRH levels appears to be an indication of
enhanced LHRH release (Sarkar et al., 1 976). Alsc
accompanying this preovulatory interval is a marked increase
in the LH secretory response to LHRH and an increase in
pituitary LHRH binding sites (Cooper et al., 1973; Aiyer et
al., 1974; Savoy-Moore et al., 1980).
When the feedback effects of gonadal steroids are removed
as a result of ovariectomy, the response of the pituitary to
LHRH immediately increases (C coper et al., 1 975).
Luteinizing hormone levels gradually increase over a three
week period, the result of increased LH pulse amplitude and
a small increase in LH pulse frequency (Ueick et al., 1981).


32
The reinitiation of negative and positive feedback in
ovariectomized rats by gonadal steroid treatment can serve
as a controlled method for studying gonadotropin secretion.
Immediately after the injection of estrogen or the
implantation of estrogen containing capsules to
ovariectomized rats, IH levels decline and the pituitary
becomes refractory to LHBH (Vilchez-Martinez et al., 1974).
Two days of continuous estrogen exposure enhances the
response of the pituitary to LHBH atd induces a diffuse
midafternoon LH surge. This afternoon IH surge repeats for
several days if estrogen titers remain elevated (legan et
al., 1975)
The injection of P several hours before the onset of the
LH surge further increases the pituitary response tc LHBH,
enhances the magnitude cf the resultant LH surge, and
advances its onset (Aiyer et al., 1976; Kalra et al,, 1981).
Concentrations of LHBH in the MEH increase prior to and
decline during the period of IH hypersecretion in a fashion
similar to that seen on proestrus (S. Kalra and Kalra,
1979).
Monoaminergic Control of Gonadotropin Secretion
Of the major neurotransmitters, the influence cf the
monoamines in controlling gonadotropin secretion has been
most extensively studied. These neurotransmitters are also
important in EOP regulation cf hormone output. The following


33
section will briefly review our current understanding of
monoaminergic regulation cf LH and FSH secretion. While
many trends are apparent in this area, it must be cautioned
that few monoaminergic drugs are specific fcr cne
neurotransmitter or receptor. flather, it is the integration
of many studies and confirmations of those works that
provide an accurate description of the role each
neurotransaitter plays in the control cf gonadotropin
release.
Many studies have evaluated the activity cf monoaminergic
neurons in various reproductive states, especially the
catecholamine neurons (Weiner, 1974; Cooper et al., 1978).
Methods cf estimating catecholamine neuronal activity which
do not disturb the steady-state include the rates cf
synthesis of NE or CA from trace amounts of their
radiolabeled precursor, tyrosine, or the disappearance cf
trace amounts of added radiolabeled catecholamine. This
technique can also be employed fcr the evaluation cf 5HT
neuronal activity using radiolabeled tryptophan or 5H1.
Problems associated with this technique include the
inability to evaluate small tissue sections, unegual
distribution of the radiolabeled amino acid, and nonspecific
uptake of the label by nontargeted cells.
A primary non-steady-state method of evaluating
catecholamine activity includes the measurement cf NE or DA
depletion following the blockade cf the rate limiting envyroe


34
in catecholamine synthesis, tyrosine hydroxylase. With this
method greater neuronal activity is indicated by increased
rates of catecholamine depletion following the inhibition cf
synthesis. Using this method catecholamines can be evaluated
in small nuclear regions using sensitive analytical
techniques. With the high dosages required to inhibit
catecholamine synthesis, some nonspecific effects may occur.
Additionally, it cannot be assumed that catecholamine neuron
will behave similarly under ncn-steady-state conditicns.
Fortunately, studies using different methods often agree.
A more recent method of evaluating mcncaminergic neurcnal
activity includes the measurement of amine and metabolite
using amperometric methods. Ihese techniques have net been
used extensively in neuroendocrine studies, however.
Norepinephi ine
A large body cf evidence suggests that central
noradrenergic neurons control LH and FSH secretion. Sawyer
and collegues originally showed that a variety of centrally
acting adrenergic agents influence the ovulatory release of
gonadotropins (Everett et al., 1949; Sawyer, 1952;
Barraclough and Sawyer, 1957). Further, these effects were
not elicited at the level of the pituitary gland (Weiner and
Ganong, 1978). Most investigators today agree that central
noradrenergic neurons display both stimulatory and
inhibitory influences on the release of gonadotropins.


35
The activity of noradrenergic neurons appears to change
in concert with several IH secretory states. Following
ovariectomy or castration the concentration of NE in the
hypothalamus increases (Donoso et al. 1967). This suggests
an increase in NE ¡netatclisai and hence activity using
several techniques (Anton-Tay and Burtman, 1968; Anton-Tay
et al., 1970; Coppola, 1971; Kizer et al.,1974; Simpkins et
al,, 1980), Increased NE neuronal activity has also been
noted in the hypothalamus prior to LH hypersecretion on
proestrus or following gonadal stercid treatment to
ovariectcmized rats (Zschaeck and Burtaan, 1973; Loftstrcm,
1977; Munaro, 1977; Simpkins et al., 1979), These studies
argue for a stimulatory role for noradrenergic systems
controlling LH hypersecretion.
Many pharmacological studies support a role for NE in
regulating gonadotropin release. Adrenergic agents applied
systemically or intraventricularly are presumed to interact
with catecholamine receptors or alter monoaminergic neuronal
activity. The blockade of NE synthesis with DA-beta-
hydrcxylase (DBH) inhibitors suppresses pulsatile LH release
and LH hypersecretion induced by endogenous steroids prior
to ovulation, electrical stimulation of the hypothalamus and
gonadal steroid administration (P. Kalra et al, 1972; S,
Kalra and McCann, 1973; Drouva and Gallo, 1 976a; Gncdde and
Schuiling, 1976), This blockade of NE synthesis is overcome
by the pretreatment with dihydrcxyphenylserine which dees


36
not require DBH for its metabolism to NE, but not fcy 1-DCFA,
a precursor in catecholamine synthesis. Additionally, alpha-
adrenergic antagonists inhibit LH release in castrated and
gonadal steroid treated rats, while neurotoxic agents such
as 6-hydroxy-DA can prevent prcestrcus LH release (P. Kalra
et al., 1972; Gnodde and Schuiling, 1976; Kartinovic and
McCann, 1977). Together these data argue for a stimulatory
role for ME on gonadotropin release.
The administration of NE has varying effects on LE
release depending cn the mode of administration and
experimental paradigm employed. While in vitro evidence
suggests that NE can stimulate LHfiH secretion from
hypothalamic fragments via an alpha-adrenergic mechamisit
(Ojeda et al., 1982; Miyake, 1983) evidence from in vivo
studies indicate that LH secretion may represent a balance
of both stimulatory and inhibitory inputs. Intraventricular
administration of NE inhibits IH release in ovariectomized
rats (Gallo and Drouva, 1979). It is not certain whether
this effect is mediated by one particular adrenergic
receptor, if it is localized at a site within or cutside the
hypothalamus, or if the inhibition occurs through LEBE
neurons (Caceres and Taleisnik, 1S80, 1982; Leung et al.,
1981, 1982).
In ovariectomized rats pretreated with gcnadal steroids
the intraventricular administration cf NE stimulates IH
release (Krieg and Sawyer, 1976; Vijayan and McCann, 1978;


37
Gallo and Drouva, 1979), It would appear that in an
ovariectcmized animal, the inhibitory effects of NE on IH
release predominate, while in the presence of gonadal
steroids, stimulatory modes cf NE en LH release are
primarily operative.
Despite many studies, the nature cf the coupling of the
noradrenergic neuron to the IHEH-containing neuron is a
subject of debate. LH secretion is most vigorous when NE is
infused in a pulsatile fashion and desensitization ensues
with a continuous NE infusion (Gallo, 1S82). Yet, a recent
study by Estes et al. (1982) suggest that a single dose of
the alpha-adrenergic agonist, clonidine, stimulates
pulsatile LH release for several hours. This would suggest
that noradrenergic neurons have a permissive effect on IHBH
pulsation, rather than being the driving force behind each
individual pulse. This diffuse functional relationship fits
the general lack of a direct anatomical connection between
the two systems.
Epinephrin
The recent development of inhibitors of EFI synthesis has
allowed the differentiation of effects which could be
attributed to either NE or or EPI-ccntaining neurons. Cn a
molar basis EPI is more potent than either NE or DA in
eliciting H release when injected into the ventricles cf
gonadal steroid treated ovariectomized rats (Vijayan and


38
McCann, 1978)- Epinephrine might be important in mediating
the positive feedback effects of gonadal steroids in female
rats (Adler et al., 1928; S. Kalra 1983), It is not clear
however what role EPI may play in regulating LH release in
ovariectomized rats or in male rats (Regro-Villar et al.,
1979; Crowley et al, 1982; Crowley and Terry, 1981),
Dopamine
A great deal of conflicting evidence exists concerning
the role of DA in regulating IH and FSH release in male and
female rats. In vitro studies suggest that DA stimulates
LHfiH release from hypothalamic tissue fragments (Schneider
and McCann 1969; Rotsztejn et al., 1977). The activity of DA
neurons appears to be enhanced after castration in the ECA-
AH and prior to gonadal steroid-induced LH hypersecretion in
the MBH of female rats (Simpkins et al., 1979, 1980). While
this might argue for a stimulatory role for DA neurons on LH
release, DA itself does not consistently induce LH secretion
when injected into the ventricles (Drcuva and Gallo, 1976a;
Krieg and Sawyer, 1976; Vijayan and McCann, 1978; Gallo and
Drouva, 1979).
Serotonin
The importance of serotonergic neurons in the overall
regulation of gonadotropin release is unclear.
Intraventricularly administered 5HT stimulates or inhibits


39
LH release depending on the dose employed (Kamberi et al.,
1970; Cramer and Porter, 1973), Studies with the neurotoxin,
5,7-dihydroxytryptamine, suggest that serotonergic neurons
stimulate LH release (Wuttke et al, 1978, Van der Kar et
al., 1980). While it is uncertain whether 5HT-ccntaining
neurons control LH release in ovariectomized rats (Gallo,
1980b), several authors suggest that the stimulating effects
of estrogen and P on LH release may be influenced by
serotonergic neurons (Iyengar and Habii, 1983; Walker and
Wilson, 1983; Chen et al., 1S84) Since many serotonergic
drugs also act upon catecholamine neurons or their
receptors, more information must accumulate on this subject
before a more definitive assessment can be made.
Endogenous Opioid Peptides and the Control of Gonadotropin
Secretion
The first experimental evidence that opioids are
inhibitory to reproductive function was Barraclough and
Sawyer*s (1954) observation that morphine blocked ovulation
in the rat. It was later verified that this blockade was due
to an inhibition of the proestrous gcnadctrcin surge (Pang
et al., 1977). At this time Cicero et al. (1975a, b)
demonstrated that chronic treatment with morphine induced
changes in the reproductive system of the male rat similar
to the effects of narcotic abuse in men, i.e. depressed
serum 1 and diminished secondary sex organ function. These
studies indicated that the effects of opiates on


40
reproductive function could be assessed in a laboratory
setting.
Reproductive Pharaacolcqy of Opioids
The antigonadotropic effects of opiate administration are
ultimately exerted at the level of the hypothalamus through
the inhibition of LHBH release. Many careful studies have
eliminated other possible sites of action (Cicero 1980a).
Opiate administration has no effect on the metabolism of T,
its clearance from the blood, or its fate at target organs.
Further the effects of morphine are not exerted at the level
of the testes either by effecting 3 synthesis or the
response of the leydig cell to gonadotropins. At the
pituitary gland, morphine dees not alter the release or
synthesis of LH or the response of the gonadotrope to LHBH.
In one study however, leucine-enkephalin acutely inhibited
the LH secretory response to IHEH (May et al., 1979).
Several lines of evidence suggest that cpiates exert
their antigonadotropic actions by inhibiting LHBH release.
The increased LHBH concentrations in hypophyseal portal
plasma which accompanies the proestrous gonadotropin surge
are prevented by morphine treatment (Ching, 1983). Also,
opiate agonists and antagonists can modulate the release of
LHBH from in vitro hypothalamic incubations (Botsztejn et
al., 1978; Drouva et al. 1981; Wilkes and Yen, 1981). In
one study the LH stimulatory actions of an opiate antagonist


41
was prevented by treatment with an LHRH antagonist analogue
(Blank and Roberts, 1982).
It is likely that the central site for the opiate-LHBH
interaction is within the hypothalamus (S. Kalra, 1981).
However, some investigators have found IH-inhibitory effects
of opioids administered in the amygdala and brainstem
(Parvizi and Ellendcrf, 1980; lakoski and Gebhart, 1981 and
1982) .
Most studies of opioid effects on reproductive function
measure serum LH levels as an index of LHBH output. The
acute effects of opioids on IH secretion satisfy the
criteria for mediation by an opiate receptor (Cicero,
1980a)
1.
In general, all opioids depress serum LH levels. This
has been found to be true for both opiate alkaloids
and opioid peptides administered systemically as well
as opioid peptides administered intraventriculariy
(Bruni et al., 1977; Cicero, 1980b; Johnson and
Rosencrans, 1981; Kinoshita et al,, 1981; Kato et
al., 1982; Ehanot and Wilkinson, 1983; Leadem and
Kalra, 1983; Markc and Rcmer, 1983;
2. The relative potency of opiates in suppressing IH
release parallels their pharmacological efficacy in
other preparations such as the displacement of
tritiated opiates, the ability to inhibit the
contraction of guinea pig illeum, and analgesia
(Cicero e t al, ,
1976; Cicero, 1980a)


42
3. The effects cf opiates on LH secretion are reversed
by opiate antagonists like naloxone or naltrexone
(Pang et al., 1974; Cicerc et al. 1976, Bruni et
al. 1977., Huraki et al., 1980).
4. Levarotatory isomers of opiate alkaloids are far more
potent in inhibiting LH secretion than dextrorotatory
isomers (Cicero et al. 1976).
Physiological Inhibition of Gonadotropin Secretion by
Opioids
Although the existence of opiate receptors in the brain
and hypothalamus and the presence of a pharmacologic
response to stimulation of these receptors suggest opioid
pathway which effects IH secretion, this does not, per se,
verify that EOP normally act to inhibit LH release. If
physiologically released EOP do act to inhibit LH secretion,
then blockade of opiate receptors with a narcotic antagonist
should reverse this inhibition. The ability of naloxone, on
its own, to stimulate LH and FSH secretion is the most
persuasive and most freguently verified evidence favoring
an EOP inhibition cf LH secretion (Meites et al., 1979;
Cicero, 1980b; S. Kalra et al., 1980; Ferin et al., 1984).
If blockade of EOP activity with an opiate receptor
antagonist elicits LH secretion, then sequestering ECP with
an appropriate antibody might produce the same effect.
Antibodies to both beta-endorphin and dynorphin have been
found to stimulate IH secretion (Schulz et al., 1981; Forman
et al., 1983)


43
The mechanisms Mediating opiate antagonist induced IE
secretion are not known. Although naloxcne occupies cpicid
receptors and prevents the ongoing actions of EOP, naloxone
only transiently stimulates LH secretion, and like opiate
agonists, tolerance develops to its effects on LH secretion
(Owens and Cicero, 1981). Another recent study suggests that
the stimulation of LH secretion following naloxone injection
may reflect prior opiate agonist activity, rather than
simple displacement of an opiate agonist frcm its receptor
(Cicero et al,, 1983b). In this study a single injection of
morphine enhanced the ability of naloxone to elicit LH
release for several hours after morphine had been cleared
from the brain.
Multiple Opioid Receptors
Several classes of opioid receptors appear to exist.
While different classifications are used, there appear to be
at least three distinct opioid receptors, temed mu, delta,
and kappa. These receptors do not have widely divergent
binding affinities, thus distinguishing between the three
classes with specific agonists and antagonists has proven
difficult Martin, 1981). These three classes may share a
common high affinity binding component (Hahn and Pasternak,
1982). It is this high affinity component that appears to
mediate the analgesic actions of morphine. In relation to
the various opioid alkaloids and peptides, the mu-opioid


44
receptor appears to mediate the effects cf zcrphine and its
cogeners. The delta-opicid receptor appears to be mere
specific for the enkephalins, while the kappa-opioid
receptor appears tc be more specific for dynorphin
(Childers, 1980; Chavkin et al., 1982). Another receptor,
called epsilon, has been proposed based on receptor binding
studies with beta-endorphin (Law et al., 1979).
Several studies have utilized opiate alkaloids and opioid
peptides to discern which receptor sottype may mediate the
effects of opioids en 1H secretion. Opioid inhibition of LH
secretion appears to involve both a bu and kappa-cpicid
receptor component (Cicero et al., 1983c; Gabriel and
Simpkins, 1983; Leadem and Kalra, 1983; Pfieffer et al,,
1983). This agrees with studies which show that antibodies
to beta-endorphin and dynorphin, but not methionine-
enkephalin, stimulate LH secretion (Schulz et al., 1981;
Forman et al., 1983),
Opioid-Monoaminergic Interactions
The interaction between opioid and monoanine-ccntaining
neurons has been investigated in several systems (Schwartz,
1979; Kuchinsky, 1977). Several recent studies have
characterized a possible adrenergic interaction with cpicids
in the control of gonadotropin secretion. The stimulatory
effects of naloxone on LH secretion are prevented by
adrenergic antagonists, DBH inhibitors and EPI synthesis


45
inhibitors (S. Kalra, 1981; S. Kalra and Simpkins, 1981; Van
Vugt et al. 1981; S. Kalra and Crowley, 1982; Schulz et
al., 1982; Koh et al, 1983; Adler and Crowley, 1984).
Additionally, the acute administration of opiate antagonists
appears to modulate the activity of catecholamine neurons in
the hypothalamus (Adler and Crowley, 1984). It appears that
adrenergic neurons may influence 1H secretion without an
intermediary opioid interaction since the inhibition of IH
secretion seen following morphine is reversed by subsequent
treatment with clonidine or the intraventricular EFI
injection (S. Kalra and Simpkins, 1981; S. Kalra and Gallo,
1983) .
In addition to both NE and EPI-containing neurons, ECE
have teen shown to interact with dopaminergic and
serotonergic neurons (Van Loon and De Scuza, 1978; Gudelsky
and Porter, 1979). There have been reports that the effects
of opiates and EOP cn LH secretion are influenced by each of
these Bcnoaminergic neurcnal systems (Eotsztejn et al.,
1978; Ieiri et al., 1980b).
Opioid-Gonadal Steroid Interactions
While it is apparent that ECP-containing neurons act to
suppress LH release, the function of this inhibitory input
is not well understood. Some researchers have presented
evidence that EOP may relay the feedback signals of gonacal
steroids in the brain. Morphine, like 1,
can prevent the


46
post-castration rise in serum LH, while naloxone and 1 are
mutually antagonistic on LH secretion in cichidectcmized
rats. Similarly estrogens can inhibit naloxone's stimulation
of LH secretion in cvariectomized rats (Blank et al. 1979;
and 1980; Cicero et al., 1980; Sylvester et al. 1982; Van
Vugt et al., 1982)
If opioid neurons do relay the feedback signals of the
gonadal steroids on LH secretion, then it can be expected
that the pharmacological efficacy of opicid agonists and
antagonists will vary under differing reproductive states.
In prepubertal female rats, when LH levels are markedly
suppressed! naloxone is highly effective in stimulating LH
release (Blank et al., 1979). Ihe efficacy of naloxoce in
stimulating LH secretion appears to vary diurnally and
diminish after castration (Blank and Mann, 1981; Cicerc et
al., 1983). Opioid agonists are more effective in
prepubertal rats compared to pubertal rats, and in rats
castrated acutely versus rats castrated several weeks
(Bhanot and Wilkinson, 1983; Cicero et al., 1982a; Wilkinson
and Bhanot, 1983).
An alternate means of evaluating the potential
involvement of EOP in mediating gonadal steroid feedback
would be to assess changes in ICE concentrations or their
release under varying steroid milieus Peptide release wculd
be the most preferable estimate of neuronal activity. To
date, only beta-endorphin has teen evaluated in the


47
hypojhyseal portal plasma of non-human primates. Cf
interest, beta-endorphin levels decline precipitously
following ovariectomy and daring menstruation (Ferin et al.,
1984), This would imply that the activity of hypothalamic
beta-endorphin-containing neurons depends cn cvarian
factors, particularly F.
Tissue levels of EOP show changes which may be relevant
to gonadotropin secretion. Both beta-endorphin in the septum
and medial preoptic area and methicnine-enkephalin in the
flBH and POA-AH display circadian variations in tissue
concentrations that parallel changes in LHBH levels in the
hypothalamus of the male rat (Kumar et al., 1982; S. Kalra
et al., 19 1b; Kerdelhue et al., 1973). Orchidectcmy dees
not appear to alter beta-endorphin levels in the
hypothalamus but decreased levels of both beta-endorphin and
methionine-enkephalin are found in the Nil and anterior
pituitary following castration (lee et al., 1980; Fong et
al., 1982; Petraglia et al., 1982; Yoshikawa et al.,
1983a,b). Beta-endorphin levels in the hypothalamus appear
to increase as male rats approach puberty (lee et al.,
1980).


48
Bationale
From the literature presented it appears that EOP may
function as one of several neurotransuitte rs that regulate
the release of the gonadotropins, LH and FSH. Based on the
pharmacological effects of opioid agonists and antagonists
during various reproductive states, and the ability of
gonadal steroid alterations tc modify EOP levels in the
brain and pituitary it appears that ECP-containing neurons
respond to changes in the steroid milieu. These changes may
reflect alterations in EOP neuronal activity which mediate
the feedback effects of gonadal stercids cn gonadotropin
secretion.
This thesis will present a series of pharmacological
investigations of opioid neurons in male and female rats. In
the male, EOP appear to suppress LH secretion, but the
nature of this inhibition is not well understood. These
studies will evaluate the feedback effects of the gonadal
steroids on LH and FSH secretion in the presence of
continuous opiate receptor stimulation with morphine. In the
female rat, the extent of EOP inhibition of IE secretion has
not been fully assessed. Naloxone was used tc evaluate the
potential EOP inhibition of IH secretion present during the
estrous cycle and following gonadal steroid treatment tc
ovariectcmized rats. Finally, the feedback effects of
gonadal steroid treatment on gonadotropin secretion in
females was evaluated in the presence of continuous opiate
receptor stimulation with morphine.


CHAPTER III
GENERAL MATERIALS AMD METHODS
Anisis
The laboratory rat was chosen as the experimental animal
in these studies. Adult male and female S-D rats were
obtained from Charles Rivers Breeding Laboratories in
Wilmington, Massachusetts. Animals weighed 180 to 220 grams
upon arrival and were allowed several days to adjust to the
animal quarters before initiating an experiment. The rat
colony was maintained in a light (lights on 0500 h through
1900 h) and temperature (26 1 C.) controlled room with
food and water provided ad litituw.
Reproductive status of female rats was verified by
microscopic examination of vaginal lavages (Ingram, 1956).
Rats which displayed two consecutive 4-day estrcus cycles
were chosen for studies employing gonadal intact female
rats. The normal sequence of cell morphology in the vaginal
smear consists of lavages containing cornified epithelial
cells (estrus), followed by two days of predominately
leukocytic smears (diestrus I and diestrus II), which is
then followed by a day in which the lavages contain
nucleated epithelial cells (prcestrus). The cornified
epithelium and leukocytic smears are characteristic of a
49


50
gonadal steroid milieu
dominated
by
estrogens and
progestins,
respectively.
Surgical
procedures
consisted
of
subcutaneous
implantation of drugs or steroids and bilateral gonadectcmy
performed under light ether anesthesia. Hale rats were
orchidectomized by exteriorizing the testicles through a
midline ventral incision. Female rats were ovariectomized by
a bilateral dorsal approach. Animals were monitored for
post-surgical wound healing.
Two methods were employed for collecting blood. In most
experiments, trunk blood was collected by decapitation.
Decapitations were completed within 30 seconds of removal of
each rat from its home cage. In studies employing LHBH
injection, blood samples were collected by cardiac puncture
under light ether anesthesia. All blood samples were
collected in a room separate from the animal guarters. Sera
was separated from trunk blood by centrifugation (1000 X g)
for 15 minutes while jugular and cardiac blood samples were
centrifuged in a microcentrifuge for two minutes. All sera
were stored at -20 C. for later hormone analysis by
radioimmunoassay (BIA).


51
Dissection of Brain tissue
Brains were rapidly removed and placed with their dorsal
surface on ice. Tissue sections containing the MEH and POA-
AH were removed using fine iris scisscrs. Cuts for the KBH
fragment were made at the posterior torder of the optic
chiasm, then caudally at the level of the mammillary tedies,
2 mm laterally at the hippocampal sulcus, and 2 mm telow the
ventral surface of the hypothalamus. The bcundries of the
POA-AH tissue slice were the caudal borders of the olfactory
tubercles to the posterior border of the cptic chiasm.
Additional cuts were placed 2 mm lateral to the midline and
approximately 2 mm from the dorsal surface cf the PCA-AH at
the level of the anterior commissure.
Gonada1 Steroid Treatment
Gonadal steroids were obtained frea Steraloids Inc.,
Milton, N.J. and administered as subcutaneous implants or as
injections. Implants consisted of Silastic tubing (1.57 mm
i.d., 3.17 mm o.d.) of lengths ranging frem 2.5 mm tc 30 mm.
Capsules were filled with either crystalline forms of I,
5-alpha-dihydrotestcstercne (DHT) or 17-beta-estradicl (E2)
or E2 dissolved in sesame seed oil. The implants were sealed
at both ends with Silastic adhesive and allowed to dry at
room temperature for 24 to 48 hours. Before use, these
implants were soaked in phosphate buffered saline for 48
hours. These implants provide sustained bleed levels cf


52
gonadal steroids fox several weeks. In the male rat these
implants reversed post-castraticn LH hypersecretion at
physiologically relevant dosages (P. Kalra and Kalra, 1980).
The implantation of crystalline E2 in ovariectomized rats
provided sustained E2 levels which immediately reduced IH
secretion and stimulates a daily signal for the midafternoon
release of LH (Legan et al., 1975).
Two ether methods for stimulating midafternoon IH
hypersecretion in ovariectomized rats employed the injection
of estradiol benzoate (EB) or the sequential administration
of EB plus P. Rats which were ovariectomized two weeks
previously were injected with 7.5 ug of EB dissolved in 100
micrcliters of oil at 1000 h. This treatment produced a fall
in LH secretion followed by a midafternoon LH surge two days
later. If 5 mg of P dissolved in 100 micrcliters of oil were
injected into these rats 48 hours after EB treatment, a more
pronounced LH surge with an earlier onset results. ether
endocrine changes accompanying this treatment are discussed
in Chapter II.
Treatment with Morphine or Naloxone
Morphine dependency was produced by subcutaneous
implantation of one pellet containing 75 mg morphine (free
base, Berk, St. Louis, MO), 37.5 mg micrccrystalline
cellulose (Avisil, EMC Corporation, Philadelphia, PA,), 0,56
mg Cab-o-sil (Cabot Corporation, Boston,
MA) and 1, 13 mg


53
magnesium sterate (Fisher Chemical Co, Fair Lawn, N.J.). Two
days later, two additional morphine pellets were implanted.
The pellets were compounded in this laboratory. This
treatment regimen produced morphine dependency as measured
by several tests of analgesia and withdrawal (Gibscn and
Tingstad, 1970; Simpkins et al., 1983b). Control animals
received placebo pellets which were formulated with an
additional 75 mg, Avisil rather than morphine free base.
Naloxone HCl (Dupont Pharmaceuticals, Garden City, N.J.)
was dissolved in normal saline and administered
subcutan eously.
fleasureneit of Catecholamines Indolamines and Metabolites
Concentrations of NE, DA, SHT, the NE metabolite
normetanephrine (NNE) the EA metabolites dihydroxyphenyl-
acetic acid (DOPAC) and homovanillic acid (HVA) and the 5HT
metabolite 5-hydroxy-indolacetic acid (5HIAA) were measured
by ampercmetric methods following their separation by high-
pressure liguid chromatography using a modifacticn of the
procedure described by Kichaud et al. (1981). The
separation was accomplished using reverse phase
chromatography across an IBE 1C-18 (15 cm X 4.6 mm, 5
micrometer particle size) with a mobile phase composed of 8?
methanol, 0,2 mM octyl sodium sulfate, 0.1 M. NaH2 EC4 and
0.1 mH EETA at pH 2.9. The flew rate was varied from 0.5
ml/min for the first 7.5 minutes of separation, 0.7 ml/min


54
from 7.5 through 23 minutes, 2.C ml/rnin from 23 to 39
minutes and 3,0 ml/min thereafter, through 57 minutes. This
procedure allowed the elution of catecholamines, indclamines
and their metabolites in standards and samples within a one
hoar period. The times for eluticn {in crder) for HE, NliE,
DA, DOPAC, 5HIAA and HVA were 5.5, 13, 17.5, 21, 28.5, 33.5
and 36 minutes respectively. The detection of amines,
indolamines and their metabolites was accomplished with an
electrochemical detector (IBH,model IC 9533) set with the
potential difference between the working electrode and a
reference AgAgC12 electrode of 0.9 volts and a current
generated at 20 nA/mV.
Tissue sections containing the BEH and POA-AE were
dissected from brain tissue as described above. The
fragments were homogenized with a tissue sonicator in 0.4 N.
perchloric acid containing 1 mgS EDTA at a weight:volume
ratio of 1 ag/10 microliters. Average weights of these
tissues were 19.0 0.5 mg for BEH and 20.2 0.6 mg for
POA-AH.
To each 20 microliter of sample of MBH and POA-AE tissue
homogenate was added 2 ng dihydrcxybenzo-acetic acid (DHEA)
as an internal standard. The concentration of each amine,
indolamine, and metabolite was determined by the peak height
ratio of the compound to DHEA in relation to a standard
curve of peak-height ratios for that particular
catecholamine, indolamine, or metabolite. The sensitivity of


55
this assay was less than 100 pg for HE, DA, 5HT, and their
metabolites.
Heraone Radiolaaunoassays
Luteinizing Hormone and Follicle Stimulating Eorncne
Serua and medium saaples were assayed for LH and FSH
using the kits provided by the National Institutes of
Arthritis, Diabetes and Digestive and Kidney Diseases
(NIADDK). The rabbit-derived antisera used were NIADDK-anti-
rLH-S-7 for the LH assay and NIAEDK-anti-rFSH-S-11 for the
FSH assay, fiadioiodinatiens were perferaed in our laboratory
using standard procedures for a chloraaine-T iodination with
gel filtration to separate free iodine fres hcrmone-bcund
iodine.
Because these studies were performed ever a considerable
period of time, LH values were determined in relation to two
LH reference standards provided by the NIADDK. The later LH
reference preparation, IH-RE-2, was 61 times more potent
than the original LH-RP-1. To aid in the comparison of IH
values across experiments, all IH values were expressed
relative to the original LH-RP-1 standard. The figure or
table legends will note when this conversion was made. FSH
values were expressed in relation to the reference standard,
FSH-RP-2. The intra-assay variation for the LH and FSH
assays, determined by the coefficient of variation for 10
replicates of pooled castrate serum which inhibited the


56
binding of the radiolabeled hormone 40* to 60% was 6.85? and
6.3%, respectively. The inter-assay variation, determined
from successive pooled serum in assays performed over a 6
month period was 11% and 8% for IH and FSH, respectively.
The sensitivity of these assays, defined as a the amount of
standard hormone reguired to inhibit the binding of the
radiolabeled hormone by 20% was C.90 eg (LH-BP-1) for IH and
0.40 ng for FSH,
Luteinizing Hormone Releasing Hormone
Tissue sections containing the MEH and POA-AH were
homogenized in 2 ml of 0.1 N. HCl and supernates were
analyzed for LHRH using FIA methods described previously (S.
Kalra, 1976). Acid supernates were neutralized with 2 K.
NaOH during the assay procedure. Synthetic IH8H obtained
from Beckman Co. (Palo Alto, CA.) was used as the reference
standard and for iodinaticn. Mcnoradioicdinated LHEH was
employed as described previously (Nett and Adams, 1977).
Rabbit antibodies against LHRH were purchased from Miles
Laboratories (Elkhart, IN). The minimum sensitivity for this
assay was 2 pg per tube and was estimated as the
concentration of LHRH which inhibited the total labeled
binding by 10%. Concentrations of LEFH were expressed in
terms of tissue sample (i.e. ng per MBH or PCA-AH).


57
Testcsterone
Serura T levels ere analyzed according to procedures
described previously (P. Kalra and Kalra, 1982).
Statistical Analysis
For most experiments, analysis of variance with Student
Neuman Keuls tests ere used tc evaluate the significant
differences between treatment groups. Where appropriate,
Student-t tests were also used. In studies which injected
LHRH, paired-t analyses were employed tc determine the
significant effects of LHRH injection. To further evaluate
data in Chapter 4, the regression analysis programs
contained in the Statistical Analysis System package offered
by the Northeast Regional Data Center were utilized. In all
studies a significance level of p < 0.05 was required.


CHAPTEB IV
THE EFFECTS OP CH8CNIC MCBPHINE TBEATKENT ON TESTCS1EBONE
NEGATIVE EEDBACK IN CASTBA1ED ALE BATS
Introduction
Tne feedback effects of gonadal steroids cn LH secretion
are believed to be mediated by the hypothalamus and
pituitary (Drouin and labrie, 1976; S.. Kalra and Kalra,
1983). Becent investigations from several laboratories show
that morphine or ECP can acutely suppress IH release in
intact and gonadectcmized rats (Meites et al. 1979; Cicero,
1980; Kinoshita et al. 1981; leadem and Kalra, 1983).
Interestingly, gonadal steroids have also been found to
modify EOP levels in various sites within the hypothalamus
and the secretion of beta-endorphin in the hypophyseal
portal system (Barden et al., 1961a; Sardlaw et al., 1982;
Wehrenberg et al., 1982). Further, acute blockade of central
opiate receptors with narcotic antagonists transients
reverses the inhibitory feedback effects of 1 cn LH release
(Cicero et al., 1980). Since gonadal steroid treatment and
opiate receptor stimulation suppress LH release and ECF-
producing neurons are found in the vicinity of IHBH neurons,
it is logical to suspect that ECE-containing neurons may
either mediate the feedback effects of gonadal steroids or
58


59
that they may act through similar hypothalamic mechanisms to
decrease LH release (P. Kalra and Kalra, 1980; Hatson et
al., 1980; Sar and Stumpf, 1975; Shivers et al., 1983b). The
following study compares continuous opiate receptor
stimulation with morphine to 1 replacement on LH secretion
in castrated rats# and evaluates the feedback sensitivity of
T on LH secretion in the presence of chronic morphine
treatment.
Experimental
In these studies castrated rats were treated chronically
with morphine pellets and tubing containing crystalline T.
Experiment 1
In the first study, rats received either chronic morphine
treatment, replacement T therapy (two 15 mm tubes) or
control treatment (placebo pellets or empty tubes) which
commenced either at the time cf or two weeks after
castration. Animals were killed by decapitation after 7 days
of treatment. Serum was analyzed for LH and T, while the
brains were rapidly removed and dissected for analysis of
MBH and AH-POA LHBH concentrations.


60
Experiment 2
In the second study, rats were castrated and twc weeks
later received either control pellets or implants of
morphine, T, or morphine plus T. Testcsteicne-containing
tubes were either 2.5, 5.0, or 10.0 mm in length. Animals
were killed by decapitation 4 days later. Serum was analyzed
for LH and T, while brains were rapidly removed and tissues
dissected for analysis of LHRH concentrations.
Experiment 3
Bats which had been castrated two weeks previously
received either control, 5 mm T, or morphine plus 5 mm T
implants. After 4 days, rats were killed by decapitation,
anterior pituitaries were removed, hemisectioned, and
preincubated in control mediux (minimal essential medium
25 mM Hepes, pH 7.2, Gibco Inc,, Grand Island, NY) for one
hour at 37 C. Fresh control medium or medium containing 1
X 10~7 M. LHBH was then added to the hemipituitary
incubation medium and the incubation continued for an
additional hour. Medium was stored at -20 C for later
analysis of LH levels


61
Results
Effects of Time After_Castrati cn cn the Serui LH and
Hypothalamic LHBH Bespcnses tc 1 and morphine
Figure 1 illustrates serum IH concentrations in rats
treated with either T or morphine immediately after or tiro
weeks after castration. Serum T levels attained fcy the two
15 mm implants were 2161 12C pg/ml in rats treated
immediately after castration and 2439 129 pg/ml in rats
receiving T two weeks after castration. These levels are in
the range observed normally in intact male rats (P. Kalra
and Kalra, 1980). As evident from Figure 1, when treatment
was started at the time of castration {short-term castrate)
both morphine and T prevented the pcst-castration
hypersecretion of IH (p < 0.05). However, when the
initiation of treatment was delayed for two weeks after
castration (long-term castrate), unlike T, morphine was no
longer effective in suppressing IH secretion.
Levels of LHBH in the MBH of rats treated with T or
morphine either at the time of castration or two weeks after
castration are shown in Figure 2. Beth I and morphine
prevented the post-castration decline in MBH LEBE
concentrations if the treatments commenced at the time cf
castration (p < 0.05). Two weeks after castration, however,
only T was effective in stimulating LHBH accumulation in the
MBH.
Levels of LHBH in the AH-POA cf castrated rats treated
with 1 or morphine were unaffected by any experimental
treatment (data not shown).


62
^Control
HTestosterone
Short Term Long Term
Castrate Castrate
Figure 1: Serum LH concentrations in rats treated with
morphine or T at the time of castration or two
weeks after castration.
Short-term castrate = treatment started
immediately after orchidectcmy. long-term
castrate = treatment started two weeks post-
castraticn. p denotes < C.05 vs. control. LH
concentrations among the control groups were not
significantly different and were therefore
pooled.


63
Control
Testosterone
Castration Castration
Figure 2: MBH LHRH concertraticns in rats treated with
morphine or T at the time of castration or two
weeks after castration
Short-term castration = treatment started
immediately after orchidectcny. long-term
castration = treatment started two weeks post-
castration. p denotes < 0.05 vs. control. WEH
LHBH concentrations among the control groups were
not significantly different and were therefore
pooled.


64
The Effects of Chronic Morphine Treatment on the LB
Secretory and MBH LBSH Responses to Graded Ceses of 1.
Figure 3 illustrates serum T levels attained from various
sized implants in rats castrated fer two weeks treated
additionally with either morphine or placebo pellets. Lew
serum levels of T (<100 pg/al) were detected in morphine or
placebo-treated rats receiving only sham implants. There was
a progressive increase in serum T levels as the size of the
implant increased in both morphine and placebo-treated
groups (p < 0.05). Chronic morphine treatment did not affect
serum T levels attained by any of these implants when
compared to placebo-treated groups,
Serum Lfi concentrations in rats treated with morphine or
placebo pellets in combination with graded doses of T are
also shown in Figure 3. In placebo-treated rats, lew
circulating levels of T (355 20 pg/ml, 2.5 mm implant)
caused a slight but nonsignificant increase in serum LH.
Further increases in serum T levels produced a decrease in
serum LH secretion, with significant suppression of LH
levels seen at 1.18 0.07 ng/ml of T (10 mm implant). As
observed in Experiment 1, morphine treatment alone did net
significantly change serum LH levels in castrated male rats.
However, it influenced the LH response to T treatment. In
rats treated with beth morphine and T, the LH response curve
to T shifted to the left such that a 50S suppression of


65
serum LH was observed with the 2.5 mm T implant aud nearly
complete suppression of serum LH was seen with 5 mm T
implants.
To further evaluate the interaction between morphine and
T, the data from this experiment were grouped according to
different levels (Figure 4):
1. less than 199 pg/ml, representing ineffective
implants, or T levels found in sham-implanted
(castrated) animals;
2. between 200 and 499 pg/al, representing T levels
which have no effect on 1H or IHHH concentrations;
3. between 500 and 999 pg/al, representing T levels
which stimulate LHBH accumulation in the MEH tut have
little effect on LH secretion; and
4. greater than 1000 pg/al, representing T levels which
consistently suppress LH secretion (E. Kalra and
Kalra, 1982).
As evident in both placebo and morphine-treated rats, IH
levels declined progressively as a function of serum 1
levels. However the T-induced reduction of LH concentrations
was greatly enhanced in morphine-treated rats (p < 0.05).
The LH response curve to graded doses of T was shifted to
the left in morphine-treated rats with maximal inhibiticn
occuring at 622 44 pg/ml. In some of these morphine-
treated rats, near baseline LH levels were fcund with T
concentrations as low as 420 pg/ml serum. In contrast. tut


66
in agreement with previous studies (P. Kalra and Kalra,
1982), significant depression of serum IH concentrations was
only apparent with T concentrations cf greater than 600
pg/ml. Serum T concentrations necessary tc elicit a 50$
reduction in LH secretion were 300 pg/ml in morphine-treated
rats and 960 pg/ml in placebc-treated rats.
The data from Experiment 2 were further subjected to
regression analysis using the logarithm cf serum IH
concentration as a dependent variable, placebo or morphine
treatment as an independent variable and the logarithm cf
serum T concentration as a covariant. The resultant
regression model was found to be highly significant (p <
0.C01) as were the drug treatment (placebo or morphine ), T
treatment, and the interaction of drug with T (p < 0.01, for
each).
The effects of simultaneous T plus chrcnic morphine
treatment on LHRH concentrations in the MBH are shewn in
Figure 5. In placebo-implanted rats, T exposure for 4 days
resulted in an accumulation cf LHBH in the MBH (p < 0.05).
As has been noted previously, this accumulation of LBBH in
the MBH occured at T levels lower than that reguired tc
inhibit LH secretion (5 mm T implants, Figure 5 vs. 10 mm
implants. Figure 4; P. Kalra and Kalra, 1982). It can also
been seen from Figure 5 that in the presence cf morphine, T
was unable to cause any significant increases in LHB E stores
in the MEH


Serum LH (ng/ml)
67
5 mm T
10 mm T
implants
implants
Castrate
2.5 mm T
levels
implants
T 1 1 1 1 1
o 200 400 600 800 1000
Serum Testosterone (pg/ml)
Figure 3: The effects of graded doses of T produced ty
various sizes of T implants on serum T and IH in
morphine-treated and placetc-treated male rats
castrated for two weeks
The vertical and horizontal tars represent mean
standard error for serum LH and T concentrations,
respectively. Numbers in parentheses represent
the number of rats in each treatment group. *
denotes p < 0.01 vs. sham-implanted control
group; the dagger symbol denotes p < 0.05 vs.
placebo-implanted group at the same dose cf T.


68
Serum Testosterone (pg/ml)
Figure 4: Relationship between LH and T levels in morphine-
treated and placebo-treated male rats castrated
for two weeks
In the interest of clarity in presenting the
relationship between the LH response and 1
levels, the rats were blocked into 4 groups
according to serum T levels; < 199 pg/ml, between
200 and 499 pg/ml, 500 and 999 pg/ml, or > 1000
pg/ml. Number in parentheses represent the number
of rats in each T level. The vertical and
horizontal error tars represent mean standard
error for the IH and T concentrations,
respectively. denotes p < 0.01 vs. T < 199
pg/ml group; the dagger symbol denotes p < 0.05
vs. placebo-implanted group at the same T level.


LHRH
(% of Placebo Sham)
69
150-
100-
50-
*
Implant Size
0.0 mm (Sham)
02.5 mm
5.0 mm
10.0 mm
Placebo Morphine
Figure 5: The effects of various sized T implants on HBE
LHBH concentrations in morphine-treated and
placebo-treated male rats castrated for two
weeks.
LHRH concentrations were determined per HBH
tissue section and expressed relative to sham-
implanted HBH LHRH (control) concentrations.
denotes p < 0.C5 vs. sham-implanted grcup.


70
Effects of T aad Morphine Treatment cn the Pituitary
Respcnsivecess to LHRH
Testosterone levels achieved ty the 5 01m implants were
similar in placebo and morphine-treated rats. Once again
this T treatment failed to reduce serum LH ccncentraticns in
placebo treated rats but together with morphine pellets
reduced serum LH concentrations to baseline levels (Talle 1,
legend, p < 0,05)
The effects of the in vivo T or morphine plus T treatment
on the in vitro release of LH fret pituitary incubations are
also shown in Table 1. Incubation of pituitarios for cne
hour with 1 X 10~7 M. LHRH significantly increased LH
concentrations to levels above that seen in control medium
(p < 0.05). However, neither T cr morphine plus T treatment
in vivo significantly altered the baseline or the LEBE
stimulated levels of LH release,


71
TABLE 1
Effects of In Vivo Morphine and T Pretreatuent on In Vitro
LB Release iron Hemisectioned Fituitaries (IE release rate:
ng/ag pituitary tissue/ hour)
In vitro
treatment
In vivo treataent
Control T Morphine + T
Control 1,583 313 957 51 1,393 i 166
1 X 10-7 a. Lhbh 3, 930 555 3,682 27 5 4,588 285
delta-LH 2,346 1 666 2,612 362 3,194 425
Serum I levels achieved by the 5 mm T implants were 683 89
ng/ml for morphine-iaplanted and 636 73 pg/ml for
placebo-treated rats. Serum IH concentrations were
225 33 ng/ml in castrated (placebo plus sham treated) rats,
227 61 ng/ml in 5 uimT implanted rats, and 16 1 ng/ml in
5 mm I plus morphine treated rats.
Discussion
These studies reveal a putative underlying interaction
between opiates and T on LH release. As in the case cf T
implants, placement of morphine pellets immediately after
castration prevented the post-castration rise in serum IH
and the decline in IBGH concentrations. This extends the
observations of previous studies (Cicero et al., 1980; Van
Vuyt et al., 1982). However, in contrast to the expected
suppression of LH release and stimulation cf LEBE
concentrations in the MBH after T implantation, LH and its


72
releasing factor were unaffected by morphine treatment
initiated two weeks after castration. Apparantly, cpiate
receptor stimulation does not mimic the actions of 1 on the
hypothalamic-pituitary-LH axis under all circumstances as
has been suggested (Cicero et al., 1980; Van Vugt et al.,
1982).
The inability of morphine pellets to suppress IH release
in rats which had been castrated for two weeks is surprising
in view of the observation that administration of morphine
or opioids systemically or ECf intraventricularly promptly
suppressed LH release in gonadectcaized rats (Cicero et al.,
1980; Kinoshita et al., 1981; leadem and Kalra; 1S83). It
is quite possible that a similar decrease in LH release may
occur soon after morphine pellets are placed in two-week
castrated rats. Accordingly then, this IH suppression must
be transient because with sustained supply of morphine these
rats appear to overcome the inhibition and LH secretion
seemingly occured unaiated 4 to 7 days later. Rhile this may
be a plausible explanation for the absence of LH suppression
in long-term castrated rats, it should be noted that acutely
orchidectomized rats were unable to override the effects of
sustained morphine supply. This and previous reports of a
differential LH response to opioid administration which is
dependent upon the post-castration interval is intriguing
(Cicero et al. 1 982a; Bhanot and Wilkinson, 1983). The
ability of testosterone to inhibit LH release diminishes


73
with tine after castration (Cicero et al., 1S82a). It is
possible that similar mechanises may underlie the loss in
effectiveness of both androgens and cpicids.
In addition to the findings that in long-term castrated
rats morphine is either ineffective cr its suppressive
effects dissipate rapidly, the action of morphine appears to
manifest itself in a different form. This is shewn by the
observation that low concentrations of T, while failing to
exert any impact on LH release cn their cwn, were highly
effective in suppressing LH release in morphine-treated
rats. The LH response curve to graded dcses of T was shifted
to the left (Figures 3 and 4) in morphine-treated rats with
maximal inhibition cccuring at T concentrations of 622 i 44
pg/ml serum. In some morphine-treated rats, near baseline LH
levels were seen with T concentrations as low as 42C pg/ml.
Furthermore, it appears that serum I levels needed to
achieve a 50X reduction in serum LH levels were three times
lower in morphine-treated rats than it control rats.
Evidently morphine treatment concurrently with T rendered
rats mere responsive towards T feedback action. T has been
shown to decrease pituitary responsiveness to LHEH (Drouin
and Labrie, 1976; P. Kalra and Kalra, 1980). It is pcssitle
that morphine may interact synergistically with T at the
level of gonadotropes to suppress pituitary responsiveness
to endogenous LHRH stimulation and thereby produce a marked
decrease in serum LH levels. However, as shown by the dcse


74
employed in the Experiment 3 and previous studies (Cicero et
al., 1977; Wiesner et al., 1984), there was no evidence of
modification by morphine of LHBH action at the pituitary
level. Thus, one can assume that morphine acts at higher
centers, possibly at the preoptico-tuberal pathway where the
distribution of androgen concentrating, EOF, and LH£H-
producing neurons and opiate receptors overlap (Sarr and
Stumpf, 1975; Watson et al., 198C; S. Kalra, 1981; Shivers
et al., 1983b).
Precisely how T and chronic morphine interact to inhibit
Lfl secretion is not known. The apparent inability of
morphine to reduce IH secretion after two weeks of
castration would suggest a T requirement for this effect of
morphine. It is possible that after continuous morphine
exposure, neuronal systems regulating LHRH release may be
more responsive to T, so that extremely low serum T titers
can suppress LH release. Considering the ability cf chrcnic
morphine to block the accumulation of LHBE in the HBH
following exposure, it is possible that chronic morphine
suppressed the activity of the IHRH neuron at several steps
in the secretory process.
While the LHRH neuron may be a likely site for the
interaction between T and morphine, ether explanations are
possible. Earlier work has suggested that gonadal steroids
may modify brain opiate receptors but this possibility has
been disputed (Hahn and Fishman,
1979; Cicero et al.,


75
1983a). The possibility remains, however, that the
intracellular processing of the opioid signal reguires the
presence of androgens. Also other neurotransmitters, such
as NE, have been shown to interact with the opiates in
effecting LH secretion (S. Kalra and Simpkins, 1981) The
potential involvement of monoamines in mediating the
interaction between morphine and T will be explored in the
following chapter.


CHAPTER V
THE INFLUENCE OF CHRONIC MOREHINE TREATMENT CN THE NEGATIVE
FEEDEACK REGULATION CF GONADCIRCPIN SECRETION BY GONADAL
STEROIDS
Introduction
The negative feedback effects of testicular hormones on
gonadotropin secretion appear to be exerted at the level of
the hypothalamus and the pituitary (Drouin and Lafcrie, 1576;
Franchimont et al. 1979; S, Kalra and Kalra, 1983). In the
male rat, three major gonadal steroids, T, DHT and E2 have
been implicated in the feedback regulation of LH secretion,
and to a lesser extent, FSH secretion (P. Kalra and Kalra,
1980; D'Agata et al., 1981; Sherins et al., 1982; McCann et
al., 1983; Nishihara and Takahashi, 1983). ¡¡hile E2 and CUT
can be formed intracellularly from T in many neurcendccrine
tissues, all three of these gonadal steroids are present in
the circulation in sufficient concentrations to influence IH
secretion (Massa et al., 1972; Naftolin et al., 1975; P.
Kalra and alra, 1977, 1980, 1981, 1982).
While the neuroendocrine substrates which mediate the
feedback effects of steroids on gonadotropin are not known,
it is interesting that a close anatomical relationship
exists between steroid concentrating, LHBE and EOP-
containing neurons (McEwen et al., 1979; Watson et al.,
7


77
1980; Shivers et al., 1983b), ECP neuronal systems have teen
implicated in the central regulation of LH secretion, and a
considerable amount of pharmacologic evidence suggests that
EOP play a role in modulating the negative feedback effects
of gonadal steroids on LH release in the male (Cicero et
al., 1980; Van Vugt et al, 1982). As was seen in Chapter
IV, chronic opiate receptor stimulation with morphine, while
ineffective in inhibiting LH release cn its cwn, enhanced by
3-fold the negative feedback effects cf 1. The present study
extends these observations by comparing the effects on LH
and FSH secretion cf T, DHT and E2 in male rats treated
chronically with morphine.
Experimental
Chronic Morphine and Gonadal Steroid Treatments
Groups of placebo or mcrphine-treated rats were
simultaneously exposed to either sham implants or one cf the
three gonadal steroids at various dosages. All treatments
lasted for 4 days, after which animals were sacrificed by
decapitation between 1100 h aud 1300 h. Serum from trunk
blood was stored at -20 C. for subseguent analysis cf IH
and FSH by BIA. The stercid treatments were;
1. 5 mm tubes packed with crystalline T;
. 7.5 mm tubes packed with crystalline EHT or CHT which
had been diluted with cholesterol on a weight;weight
ratio of 1:1 or 1:3; and
2


78
3. E2 dissolved into sesame seed cil at a concentration
of 300 micrograms/ml and filled into 7.5 am tubes or
£2 diluted in oil to concentrations of 150 or 75
micrograms/ml and then placed into tubes of 5.0 m in
len th.
Evaluation of Pituitary Responsiveness to LHEB
Based on the results of the first series of experiments,
groups of placebo or chrcnic mcrphine treated rats were
simultaneously exposed to either sham, 5 mm T, 7.5 mm CET or
7.5 ma E2 (300 micrcgrams/al) implants. After 4 days cf the
above treatments, rats then received a single injection cf
LHHH (100 ng/100 g B.W., s.c.). Blood samples were obtained
by cardiac puncture under light ether anesthesia, prior to,
and 30 minutes after LHRH injection. This dose of LHEH was
based on earlier work (Lu et al., 1980). Serum was
separated by centrifugation and stored at -20 C. for
analysis of LH by HIA,
Results
The effects of simultaneous morphine plus T
administration are shown in Figure 6. While 5 mm T implants
alone reduced serum LH concentrations by greater than 40X,
this effect was not significant. Chronic morphine treataent
alone did not affect serum LH levels in castrated rats.
However, as was noted in Chapter IV, the combination of 5 mm


79
T plus morphine treatment reduced serum LH ccncentraticns by
90% to levels seen in intact male rats (p < 0.05; P, Kalra
and Kalra, 1977fc).
The consequences of chronic morphine exposure on serum IH
and FSH levels in castrated rats concurrently exposed tc
various dosages of E2 are shewn in Figures 7 and 8,
respectively. Chronic morphine did not alter serum LE
concentrations in animals receiving sham implants.
Similarly, E2 treatment alone failed tc significantly reduce
LH levels. In contrast, the ccmbinaticn of morphine
treatment plus 5 mm E2 (150 micrograas/ml) or 7.5 mm E2 (300
micrograms/ml oil) reduced serum LH ccncentraticns (p <
0.05). The highest E2 dosage (7.5 aa at 30C micrcgrams/m1)
alone produced a non-significant 25% reduction in LH
concentrations, while the combination of morphine plus the
same E2 dosage caused a greater than 75% reduction in serum
LH levels p < 0.05).
A significant effect of chronic morphine on the response
of FSH to E2 was observed. Although E2 alone did not reduce
serum FSH concentrations at any of the doses evaluated, the
combination of 7.5 mm E2 (300 micrograms/ml) plus merphine
significantly reduced FSH levels by 30% relative to sham-
implanted rats and 18% relative to placebo-treated rats at
the same E2 dosage (p < 0.05) .
The effects of chronic morphine administration on the
response of LH and FSH to various dosages of DHT in


80
Figure 6: The effects of simultaneous morphine and 5 mm T
implants on LH secretion in rats which had teen
orchidectomized two weeks previously
* denotes p <0.05 compared tc shan-implanted
rats; the dagger symbol denotes p < 0.05 when
compared to placebo-implanted rats.


81
600-
E
\
o>
c 400
& 200
I
Bm
x
~
't&;
WB
WM
>
Sil %
WM.
fj|
IM
mm.
SHAM
IMPLANT
I
£;p
5mmE2 5mm E2
(75jug/ml) (I50jug/ml)
Placebo
Morphine
7.5mm E2
(300jug/ml)
Figure 7: The effects of simultaneous treatment with
morphine and various doses of E2 on LH secretion
in rats which had been orchidectomized two weeks
previously.
E2 was dissolved in oil at the concentrations in
parenthesis and filled into tubes of the lengths
noted in the figure. IH was determined using the
LH-BP-2 reference standard and expressed relative
to LH-BP-1 (LH-BP-1 = 61 X LH-BP-2). denotes p
< 0.05 vs. sham-implanted rats; the dagger symbol
denotes p < 0.05 vs. placebo-implanted rats at
the same E2 dose.


82
Figure 8: The effects of simultaneous treatment with
morphine and various deses cf E2 cn FSH secretion
in rats which had been orchidectomized twc weeks
previously
E2 was dissolved into oil and filled into tutes
of lengths as noted in the figure. denotes p <
0.05 vs. sham-implanted rats; the dagger symbol
denotes p < 0.05 vs. placeto-implanted rats at
the same E2 dose.


83
castrated rats are shown in Figures 9 and 10, respectively.
As previously shown in Figures 6 and 7, chronic morphine
treatment was without effect on serum LH levels in sham-
implanted rats. The implantation of DHT alcne (7.5 mm DHT
crystals) significantly reduced serum 1H concentrations ty
63X, while the combination of chronic morphine plus EHT (at
1:1) or 7.5 mm DHT reduced IH levels 39* and 83*. The
combination of morphine plus DHT was not significantly mere
effective in inhibiting LH levels than DHT alone at any DHT
dosage, however. Chronic morphine and DHT had variable
effects on FSH levels. Morphine treatment caused a slight
elevation (16*) in serum FSH in sham-implanted rats, in
contrast to similarly exposed animals shewn in Figure 8.
Treatment with DHT alone was ineffective in inhibiting FSH
at any dose used; however, 7.5 mm DHT (at 1:1) caused a
slight increase in serum FSH levels (9*, p < 0.05). Mcrphine
treatment with 7,5 mm DHT reduced serum FSH levels 43*. This
reduction in serum FSH concentrations was significant
relative to sham-implanted rats and to placebo-impanted rats
at the same DHT dosage.
The effects of combinations of chronic morphine plus
steroid treatments cn the in yivc LH secretory response to
LHEH injection are shown in Table 2. Prior to LHEH
administration, the various mcrphine plus steroid treatments
produced similar effects cn LH levels as was seen in Figures
6, 7 and 9. Chronic morphine treatment was without effect


84
Figure 9: The effects of simultaneous treatment with
morphine and various doses cf DHT cn LB secretion
in rats which had been orchidectcmized twc weeks
pre viously
Crystalline DHT or DHT which had been diluted cn
a weight:weight basis with cholesterol was packed
into tubes 7.5 mm in lengths as ncted in the
figure. Serum 1H was determined using the LE-KP-2
reference standard and expressed relative tc IH-
RP-1. (LH-RP-1 = 61 X IH-8E-2). denotes p <
0.05 vs. sham-implanted rats; the dagger symbol
denotes p < 0.05 vs. placebo-implanted rats at
the same DHT dcse.


85
Figure 10; The effects of simultaneous treatment with
morphine and various doses cf DHT on FSH
secretion in rats which had been castrated two
weeks previously.
Crystalline DHT or DHT which had been diluted on
a weight:weight ratic with chclestercl was
packed into tubes 7.5 mm in length as noted in
the figure. denotes p < C.05 vs. sham-
implanted rats; the dagger symbol denotes p <
0.05 vs. placebo-implanted rats at the same EHT
dose.


Full Text
GONADAL STEROID MODULATION CF OFICIE EFFECTS ON
GONADOTROPIN SECRETION IN THE RAT
BY
STEVEN H. GAERIEI
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY CF FLCEIEA IN
PARTIAL FULFILLMENT CF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR CF PHILOSOPHY
UNIVERSITY OF FLORIDA
1984

This dissertation is dedicated to Dr, Theodore J, Cicero.
His hard work and dedication tc science enccugaged me tc
pursue graduate studies in neuroendocrinology.

ACKNOWLEDGMENTS
It is difficult in this small space to acknowledge all of
the individuals who have assisted me in ay graduate studies
during the past 4 years at the University of florida. This
work would not have been possible without the advice,
support and encouragement of ay mentor. Dr, James W.
Simpkins. Jim's approach to graduate education allowed me to
act more as a collaborator in the design and execution of
experiments, rather than simply as his student. I wish to
thank Dr. Satya Kalra who also provided invaluable support
and advice in the development of my research project. The
other faculty members who have served on my dissertation
committee, Drs. Steven Childers, Micheál Katcvich, Daniel
Sharp and William Thatcher, have imparted much needed advice
and knowledge in the laboratory and classrcctt. Many ether
faculty members have assisted me in my research, including
Drs. Pushpa Kalra, Kerry Estes, Hartmut Derendorf, Ken
Slcan, George Torosian and M.S.A, Kumar.
There is a great feeling cf ccmradery in cur department
and I would like to acknowledge some of the staff and
graduate students who have been so encouraging ever the
years. June O'Meara and fran Eanniello have been helpful
often in the past and have always been tolerant cf ity many
in

errors. The assistance of Gayle Geegan, Becky Thro, Sharcn
Layfield, and Becky Hamilton was greatly appreciated. I
would like to thank Dr. Ed Scltis, wbc was cheerful under
any circumstance, and Lee Ann Berglund and Cathy Smith who
will soon be taking over the reins in the lab.
Many of my friends outside of school have made life more
bearable. I would like to thank Robert Logan, who always
managed to cheer me up when life got me down. I would also
like to thank my rocmates, Scott Tokum and John Heintz, who
have been loyal friends over the years.
This dissertation was written using the computing
facilities of the Northeast Regional Data Center, and
incorporated the programs contained within the Statistical
Analysis System.
IV

TABLE CF CC NTESTS
PAGE
ACKNOHLEDGNENT S iii
LIST OF TABLES ix
LIST OF FIGÃœHES ..................... x
ABSTRACT xii
CHAPTER
I. INTRODUCTION 1
II. REVIES OF THE LITERATURE .............. 5
Historical .................. 6
Early Observations ............. 6
The Development of Neuroendocrinology ... 8
Neuroanatcmical Relationships .... 13
Anatomy of the Eituitary Gland ....... 13
General Hypothalamic Anatomy ........14
Anatomy of the luteinizing Hormone-Releasing
Hormone Neuronal Systems ..19
Anatomy of the Eonoaminergic Neuronal Systems . 21
Noradrenergic pathways ...........21
Dopaminergic pathways ...........22
Epinephrine-containing pathways ...... 23
Serotonergic Pathways ...........23
Anatomy of Endogenous Cpicid Peptide Containing
Neuronal Systems ............24
Beta-endorphin-ccntaining neurons 24
Enkephalin-containing neurons ....... 25
Dynorphin-ccntaining neurons ........26
Steroid Concentrating Neurons in the Brain , . 27
Neuroanatcisical Interactions 28
Patterns of Gonadotropin Secretion ...... 29
Hales ..29
Females ..................30
Monoaminergic Control of Gonadotropin Secretion 32
Norepinephrine ...............34
Epinephrine ........ 37
Dopamine ..................38
v

Serotonin ................. 38
Endogenous Opicid Peptides and the Control of
Gonadotropin Secretion ......... 39
Reproductive Pharmacology of Opicids .... 40
Physiological Inhibition cf Gonadotropin
Secretion by Opicids .........42
Multiple Opioid Receptors ......... 43
Opioid-Mcnoaminergic Interactions 44
Opioid-Gonadal Steroid Interactions .... 45
Rationale ...................48
III. GENERAL MATERIALS AND METHODS . 49
Animals ...............49
Dissection of Brain Tissue .. ...51
Gonadal Steroid Treatment ...........51
Treatment with Morphine or Naloxone ...... 52
Measurement of Catecholamines Indolamines and
Metabolites ...............53
Hormone Radioimmunoassays ........... 55
Luteinizing Hormone and Follicle Stimulating
Hormone ...............55
Luteinizing Hormone Releasing Hcrmone ... 56
Testosterone ................57
Statistical Analysis .... ..57
IV. THE EFFECTS OF CHBCNIC MOEPHINE TREATMENT ON
TESTOSTERONE NEGATIVE FEEDBACK IN CASTRATED
MALE RATS ................. 58
Introduction ............53
Experimental ................. 59
Experiment 1................59
Experiment 2................60
Experiment 3................60
Results ....................61
Effects of Time After Castration on the
Serum IH and Hypothalamic LHfiH
Responses to T and morphine ..... 61
The Effects of Chronic Morphine Treatment on
the LH Secretory a£d MBH IHRH
Responses to Graded Coses of T. ... 64
Effects of T and Morphine Treatment on the
Eituitary Responsiveness to LH8H ... 70
Discussion ............ 71
V.THE INFLUENCE OF CHBCNIC MCBEHINE TREATMENT ON THE
NEGATIVE FEEDBACK REGULATION OF GONADCTECEIN
SECRETION BV GO NACA L STEECIDS ....... 76
Introduction .................76
Experimental .................77
vi

Chronic Morphine and Gonadal Steroid
Treatments ..77
Evaluation of Pituitary Responsiveness to
LHfiH 78
Results . . . , . ....y....... ....78
Discussion ..................88
VI. THE EFFECTS OF CHRONIC MCBPHINE AND TESTOSTERONE
TREATMENT ON CATECHOLAMINE AND INDCLAMINE
METABOLISM AND GCNAECTFOPIN SECRETION IN
MALE EATS ...94
Introduction .....94
Experimental .................95
Results .................... 96
Serum LH and FSH 96
NE and SHE ........ 100
DA and DOPAC ............... 100
5HT 5HIAA and HVA 101
Discussion ................. 105
VII. MODULATION OF ENDOGENOUS OPIOID INFLUENCE ON
LUTEINIZING HORMONE SECRETION EY ESTBOGEN
AND PROGESTERONE 110
Introduction ................ 110
Experimental 111
Experiment 1 .............. . 112
Experiment 2............... 113
Experiment 3............... 113
Results . ............. 114
Effects of Naloxone Administration During
the Estrous Cycle cl LH Release . . 114
Effects of Naloxone on LH Release in Steroid
- Pretreated 0variectomized Rats . . 116
The Effects of P on Naloxone - Induced LH
Release on Prcestrus ........ 117
Discussion ................. 120
VIII. A DECLINE IN ENDOGENOUS OPIOID INFLUENCE DUBING THE
STEROID - INDUCED EYEERSECBETICN OF
LUTEINIZING HORMONE IN THE RAT ...... 125
Introduction ................ 125
Experimental ......... 126
Results ................... 127
Discussion ... .............. 131
IX. THE EFFECTS OF CHRONIC MORPHINE TREATMENT ON TEE
FEEDBACK ACTIONS OF ESTROGEN ON GONADOTROPIN
SECRETION IN OVAHIECTCHIZED BATS ..... 135
Introduction ................ 135
Experimental ................ 137
vii

Experiment 1 .............. . 137
Experiment 2 138
Experiment 3............... 138
Experiment 4............... 139
Experiment 5.. .............. 139
Results ........... 140
Discussion ................. 149
X. GENERAL DISCUSSION . 154
BIBLIOGRAPHY . ......... 168
BICGEAEHICAL SKETCH 199
viii

LIST CF TABLES
TABLE PAGE
1. Effects of la Vivo Morphine and T Pretreatment on In
Vitro LH Belease from Hemisectioned Pituitarios
(LH release rate: rg/mg pituitary tissue/ hour) . 71
2. The Effects of IHBH (100 ng/100 g E,W.# s.c.) on
serum LH Levels in Castrated Bats Treated
Chronically hith Morphine and/or Gonadal Steroids. 87
3. The Effects of Gonadal Steroids and Morphine
Treatment on 5HT 5HIAA and HVA Concentrations in
the MBH and POA 104
4. Effects of LHBH (75 ng/100 gm B.S., s.c.) on LH
secretion in ovariectomized steroid treated rats. 130
5. lhe Effects of IHBH on LH Hypersecretion in
E2-treated Ovariectomized rats. ........ 148
IX

LIST OF FIGUBES
FIGURE IAGE
1. Serum LH concentrations in rats treated with
morphine or T at the time of castration or two
weeks after castration. .............62
2. MBH LUfifl concentrations in rats treated with
morphine or T at the time of castration or two
weeks after castration .............63
3. The effects of graded doses of T produced by various
sizes of T implants on serum T and LH in
morphine-treated and placebo-treated male rats
castrated for two weeks .............67
4.Relationship between LH and T levels in morphine-
treated and placebo-treated male rats castrated
for two weeks ..................68
5. The effects of various sized T implants on MBH IHBH
concentrations in morphine-treated and placebo-
treated male rats castrated for two weeks. ... 69
6. The effects of simultaneous morphine and 5 mm T
implants on LH secretion in rats which had beeD
orchidectomized two weeks previously ...... 80
7. The effects of simultaneous treatment with morphine
and various doses of E2 on LH secretion in rats
which had teen orchidectomized two weeks
previously. .... ...............81
8. The effects of simultaneous treatment with morphine
and various doses of E2 on FSE secretion in rats
which had been orchidectcmized two weeks
previously ...................82
9. The effects of simultaneous treatment with morphine
and various doses of DHT on IH secretion in rats
which had teen orchidectomized two weeks
previously ...................84
x

10. The effects of simultaneous treatment with morphine
and various doses of CHT cn FSH secretion in rats
which had been castrated two weeks previously. . 85
11. Interaction between morphine and T on IB secretion
in rats which were orchidectomized twc weeks
previously. . ..................97
12. Interaction between morphine and 1 cn FSH secretion
in rats which had been castrated two weeks
previously. ...................99
13. Concentrations of NE and NHE in the MBH and EOA-AE
of intact rats and orchidectomized rats given
combinations of morphine and T, , ....... 102
14. Concentrations of DA and DOPAC in the MBH and PCA-AH
of intact rats and orchidectomized rats given
combinations of morphine and I ........ 103
15. The effects of naloxone (2 mg/kg) on serum LB levels
at various times during the estrcus cycle. . . 115
16. The effects of naloxone (2 mg/kg) on serum LE in
ovariectomized rats treated with FB (lower panel)
or E3 plus E (upper panel). .......... 118
17. Effects of P (5 mg Prog) on naloxone (2 mg/kg) -
induced LH secretion cn proestrus. ...... 119
18. The effects of naloxone injection on LB secretion in
ovariectomized rats receiving EB cr EBP
treatment. 129
19. The effects of continuous morphine exposure and
various doses of E2 on serum IH and FSH levels in
ovariectoaized rats. ...... 141
20. The effects of continuous morphine exposure on
midafternoon LH and FSH hypersecreticn induced by
E2 implantation in ovariectomized rats. .... 144
21. The effects of continuous morphine exposure cn IH
and FSH secretion in E2 implanted ovariectomized
rats. ..................... 145
22. The effects of continuous morphine exposure on LE
and FSH secretion in cvariectcmized rats. ... 146
xi

Abstract of Dissertation Presented tc the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Ehilosophy
GONADAL STEROID MODULATION Cl CEICIE EFFECTS ON
GONADOTROPIN SECRETICN IN THE RAT
By
Steven M. Gabriel
December 1984
Chairman: Dr. James W. Simpkins
Major Department: Pharmacy
The interactions between opiates and gonadal steroids in
the control of the gonadotropins, luteinizing hormone (1H),
and follicle stimulating hormone (FSH), were investigated in
the rat. Male rats were chronically treated with morphine as
a means of providing continuous opiate receptor stimulation,
fihen initiated at the time of castration, both morphine
treatment and testosterone (T) replacement prevented post¬
castration LH hypersecretion and hypothalamic LH-releasing
hormone (LHRH) depletion. However, only T reversed these
changes when treatments were initiated two weeks after
orchidectomy. Further, in rats which had been castrated for
Xil

two weeks, morphine enhanced the ability cf T to inhibit
gcnadotrcpin secretion and blocked the T-induced
accumulation of LH EH in the hypothalamus. At physiological
doses, it was found that 17-beta-estradicl (E2) , but net
5-alpha-dihydrotestcstercne (DH1), similarly interacted with
morphine to suppress gcnadotrcpin release. Further, the
ability cf morphine to interact with T in the suppression of
LH release could not be explained by changes in hypothalamic
norepinephrine, dopamine or serotonin metabolism.
Female rats were injected with the opiate antagonist,
naloxone. Naloxone stimulated LH release at all times
tested during the estrus cycle and following estradiol
benzoate (EB) treatment to ovariectcmized rats, indicating
that endogenous opioid peptides (EOE) inhibit LH secretion
throughout the estrus cycle, and during the preestreus and
EB-induced LH surges. During LH hypersecretion, induced by
progesterone {P) treatment to proestrous or EE-treated
ovariectcmized rats, nalexone was unable tc stimulate IH
release, indicating that EOF may contribute to the advanced
onset and increased magnitude cf the LH surge seen following
P treatment.
In a final study, E2-treated ovariectomized rats were
given morphine treatment. morphine enhanced both the
negative and positive feedback effects cf E2 cn gcnadotrcpin
release in these animals. In summary, the work presented in
this dissertation indicates that EOF Flay an important rcle
xm

in the regulation c£ gonadal steroid feedback in male
female rats by modifying the sensitivity of the train
circulating gonadal steroids.
and
tc
xiv

CHAPTER I
INTRODUCTION
Opiates have been used by cld world cultures for many
centuries. Opium is the dried latex from the unripe seed
pods of the plant Papa ver somnif erum. It was probably first
discovered by the ancient Mesopotamians (Beynolds and
Sandal, 1957). The word 'opium* is derived from the Greek
word for juice, and was mentioned by Theophrastus in the
third century B.C. The Romans, Scribcnius largus and Galen
both described the medicinal uses of opium. In another
ancient reference tc opium, the Kama Sutra states that
Indian maidens and wives are forbidden to use opium in any
form until after menopause, as the use of the drug prevents
pregnancy {Kruger et al., 1941). Throughout the middle ages,
Arabs used opium for medicinal purposes. They introduced
the drug to China, where it was used tc treat dysentery and
other ailments. Its use in Europe increased during the
sixteenth century, where opium was used for the treatment of
diarrhea and pain, and as an adjunct tc surgery.
Raw opium contains over 20 different alkaloids. Surturner
isolated and described morphine in 18C6 (Leake, 1975).
Morphine comprises ever 10% of the dry weight of opium. Its
structure was proposed in 1925 by Gulland and Rcbinscn, and
1

2
finally synthesized in 1952 by Gates and Ischudi (see Leake,
1975), Other less abundant alkaloids such as narceine,
codeine, and thebaine were isolated in the early nineteenth
century. Improved organic synthesis techniques in the
twentieth century resulted in a flurry of semi-synthetic
opiate and synthetic opiate-like compounds being produced,
in an effort to design a less addictive opiate, the partial
agonist, nalorphine, was introduced. Although analgesic and
less addictive than morphine, its dysphoric properties
precluded its widespread application. Ibis search for a less
addictive opiate also led to the synthesis of naloxone, an
opiate antagonist relatively devoid cf analgesic and ether
opiate agonist actions,
The modern use of opiates also led to its misuse. Great
Britain secured a market for its Indian opium by addicting a
large population in China (Kane, 1881). The disputes that
developed between Great Britain and China over this trade
were referred to as the Great Cpium Wars of the eighteenth
and nineteenth centuries. Chinese immigrants introduced
opium smoking into the United States where it spread to the
general population. Opiate abuse continued to increase in
this country after the development of the hypodermic needle
and the introduction of injectable morphine to relieve the
pain resulting from battle injuries received during the
Civil War 0*Donnell and Ball, 1966).

3
Many authors commented on opiate abuse at this time. In
1881, Kane described the physiological ccnseguences of
opiate abuse. Included in this discussion were the effects
of opiate abuse on the sexual organs. Opium smokers
exhibited "disgraceful conduct and initially considerable
sexual stimulation, although the completion of the sex act
was delayed". Several months cf abuse impaired both desire
and power. To further illustrate the decline in the sexual
ability of opiate addicts, population statistics were cited.
During the height cf the Great Cpium liars, China's annual
population increase was found to fall frcm 6$ to under 1K.
It was even suggested that the cpium trade could be used as
a means of controlling China's overpopulation I
The deleterious effects of opiates and ether central
nervous system depressants cn reproductive function
continued to be reported in the medical literature during
the twentieth century. While narcotic addicts appeared to
commit fewer violent crimes such as rape (Finestone, 1957),
men were found to be impotent and women showed inhibited sex
drives and amenorrhea. The first controlled clinical studies
by Azizi et al. (1973) and Gaulden et al. (1964) reported
depressed testosterene (T) levels in male herein aEd
methadone users and diminished sexual function in wemen
narcotic addicts, respectively.
The numerous physical effects of cpiates were postulated
to be the result of an interaction with specific central

4
nervous system receptors. In 1973, several laboratories
independently reported the existence of opiate receptors in
the nervous systems of mammals (Fert and Snyder, 1973;
Terenius, 1973). Soon to follow these discoveries has the
isolation of endogenous opioid peptides (EOF), Hughes et al,
(1975) described the two pentapeptides, leucine-enkephalin
and methionine-enkephalin, while Li and Chung (1976)
reported the isolation of beta-endorphin. Following these
discoveries, researchers began to identify the mechanisms
through which opiates exerted their antigonadal effects. It
soon became evident that opiates interfered with a normally
operating EOP influence on reproductive hormone secretion.
The effects of opiates on the regulation of reproductive
hormones, luteinizing hormone (IH) and follicle stimulating
hormone (FSH), is the subject of this dissertation.

CHAPTER II
REVIEW OF THE LITERATURE
This chapter will survey the major work with respect to
the neuroendocrine control of gcnadotrcpin secretion. This
will include the basic anatomy and physiology of the
hypothalamo-hypophyseal unit and neurcpharmacclogical
studies which contributed to the concept that an opioid
inhibitory component controlling gonadotropin secretion.
Emphasis will be placed on LH secretion, although important
differences between LH and FSH secretion will be discussed.
The field contains a wealth of literature and several
reviews were employed as starting points and to highlight
major trends and areas of agreement. This includes
historical reviews by Garrisson (1929), Leake (1975) and
Hedvei (1982). The descriptions of general hypcthalamo-
hypophyseal anatomy were derived from Adams et al. (1965),
Daniel (1966), Halasz (1969), Jenkins (1978), Ezrin ( 1979)
and Palkovitz and Zaborsky (1979). Detailed reviews of the
various neuronal systems can be found in Dahlstrom and Fuxe
(1964), Fuxe and Understedt (1966), Cooper et al. (1978),
Hoore and Bloom (1978 and 1979), Sternberger and Hoffman
(1978), iatson et al. (1980) and Palkovitz (1981).
Information regarding luteinizing hormone releasing hormone

6
(LHSH) biochemistry, steroid concentrating neurons, and
opioid peptides and their receptors may be found in reviews
by Naftolin at al. (1975), Sar and Stumpf (1975), Bloom et
al. (1978), McEwen et al. ( 1979), Childers (198C) and Kartin
(1581), Jntisz et al, (1983). Hany excellent reviews
concerning the role of monoaminergic neurons in regulating
gonadotropin secretion have appeared over the years, Among
the most complete are Coppola (1971), Heiner and Ganong
(1578), Barraclough and Wise (1982), S. Kalra and Kalra
(1983) and Simpkins et al., (1984). Finally, the
pharmacological and physiological effects of opiate
alkaloids and opioid peptides on gonadotropin secretion have
been presented by Meites et al. (1979), Cicero ( 1980a and
1980b) and S.,Kalra et al. (1980). fchile the literature
discussed will focus primarily on studies in the rat, where
appropriate, other animals will be discussed. The relation
of these studies and the present work to the regulation of
gonadotropin secretion in humans will be addressed in
Chapter X.
Historical
Early Observations
Although modern endocrinology has yet to complete its
first century, observations on endocrine function have been
made throughout history. Relief carvings, figurines, and
drawings from prehistoric, Egyptian, Babylonian,
and later

7
Greek, Roman and Benissance artisans appear to depict
medical conditions {Garrisson, 1929). Seme of these
artifacts have been interpreted as illustrating such
illnesses as female endocrine system obesity, geiter and
gigantisism. Goiter was endemic among ancient cultures and
the Chinese prescribed an appropriate treatment cf iedine-
rich seaweed. While the Chinese, Hindus, and Egyptians all
described diabetes mielitis, the Greek Arctaec cf Kappadckia
coined the term 'diabetes' in the second century A.D.
Reproductive function was a primary concern of the
ancients. The effects of castration on animals and humans
have been known since prehistory, while the first
ovariectomies in humans and hysterectomies on farm animals
were performed by the Egypticns and Hebrews, respectively.
Huch later in the eighteenth century, the results cf
experimental removal of the gonads wculd be pioneered by
John and William Hunter of England (Medvei, 1982).
The discovery of gynecological instruments among Roman
ruins indicated some degree of medical sophisticatioE during
this period. Theories of reproduction were somewhat less
advanced. The Hippocratic view that sperm arose from all
tissues of the body to be stored in the testes was
challenged by Aristotle who held that the right testes
produced sperm destined to become males while the left
testes produced sperm destined tc become females.
Additionally, Aristotle felt that the male provided all the

8
determining characteristics while the female provided merely
a fertile enviroment for the development of the fetus,
A more modern view of the factors involved in
reproduction awaited the development of the microscope in
the seventeenth century. The initial works of Fallopic,
deGraff, Leydig and many other scientists are reflected in
our anatomical vocatulary, Leeuwenhoek first described the
presence of "little animals of the semen" in 1677 and 200
years later Hertwig demonstrated the unicn cf the sperm and
the cvus.
Recognition of the importance of the ovary in maintaining
reproduction was the result of the work of many scientists.
At the turn of this century, Walter Heap described the
reproductive cycle in females and related the reproductive
changes of the estrous cycle tc these cccuring in the
menstrual cycle. The changes in vaginal cytology
characteristic of the estrous cycle were first described by
Long and Evans of America in the early twentieth century,
while at the same time Hitschman and Adler cf Vienna
described the cyclical changes in the uterine endometrium
which occur during the menstrual cycle.
The Development of Neurcendocrinology
The communication between various tissues of the body via
substances released into the circulation is one of the
central concepts of endocrinology. In the seventeenth and

9
eighteenth centuries Albrect von Haler and Frederick Buych
recognized that the body contains ductless glands which
release their contents into the blood. Claud Bernard
refered to this process as internal secretion in 1855, In
1902, after his discovery of secretin, Ernest Starling
coined the term 'hormone* to describe the active contents of
internal secretions. Many hormones were first recognized by
the ability of organ extracts to exert physiological effects
on animals in vivo or effects on isolated tissues in vitro.
Among the first hormones isolated were the gcnadal steroids,
testosterone (T), 17-beta-estradiol (E) and progesterone
(P), which were found to be responsible for maintaining
reproductive function in males and females.
One ductless organ which was found to be of major
importance in maintaining normal homeostasis was the
pituitary glaud. Its function was long debated and, until
1838 when Bathke demonstrated the non-neural origin of the
anterior portion, it was considered by many to he a
vestigial portion of the train. The pituitary gland was
found to secrete substances necessary for normal tody
growtn, the maintenance of reproduction, the initiation of
lactation, and the restoration of atropied thyroid and
adrenal tissue. In the 194C's C.H. Li and collegues
isolated and synthesized the two gonadotropic hormones,
luteinizing hormone and follicle stimulating hormone (IH and
FSH, respectively; li et al. ,
1940 and 1949)

10
A functional relationship between the brain and the
pituitary gland was evident to many scientists. Galen's view
that the pituitary drained phlegm from the brain tc the
nascpharnyx was refuted by the anatomist Schreiber in 1660.
Schreiber's contemporary. Bichard Lcwer proposed that
substances from the ventricles perfused from the brain to
the pituitary where they "percolated” into the circulation.
This view is quite similar to cur current views on
neuroendocrine function.
In this century it was observed that many hypothalamic
lesions and tumors disrupted endocrine function. Experiments
showed that severing the pituitary connection with the brain
atrophied both the thyroid and the adrenal glands. The
transplantation of the pituitary gland to the renal capsule
or the anterior chamber of the eye produced similar
degeneration of the thyroid, adrenals and testes, tut the
ovarian corpera lútea was maintained in rats. It was
apparent that the brain exerted both stimulatory and
inhibitory influences on the pituitary. Additionally, this
interaction between the brain and the pituitary seemed to be
dependent on the preservation of the connection between the
pituitary and the hypothalamus,
Joseph Lietaud first described the pituitary stalk and
its relationship to the brain in 1742. It was not until 1930
that Pepa and Fielding clearly described the vascular link
between the two organs as a portal system, however. It is

11
unfortunate that the two scientist incorrectly proposed that
the direction of blood flow in the portal system was from
the pituitary to the hypothalamus Housay el al. (1935) and
Wislocki and King (1936) quickly rectified this error.
Several observations led to the development of cur
current understanding of neuroendocrine relationships. Among
the foremost was the discovery of neurosecretion fcy
magnocellular neurons in the paraventricular and supraoptic
nuclei by Scharrer and Scharrer (1940). These neurons
released oxytocin and vasopressin from nerve terminals in
the neural lobe of the pituitary into the general
circulation. Harris and associates performed numerous
studies which demonstrated the importance of the
hypothalamus in regulating anterior pituitary function.
These included endocrine changes following the electical
stimulation of the hypothalamus. In 1948, Harris proposed
the "cheaoreceptor hypothesis” to explain the control of
anterior pituitary function by hypothalamic hormones
released into the portal circulation. This concept remains
one of the cornerstones of neuroendocrine thought.
Much of the work of neuroendocrinologists over the past
30 years has concerned the demonstration, isolation and
characterization of these hypothalamic hormones. Ealasz et
al., (1962) used the term hypcphysiotropic area to describe
the regions of the hypothalamus that would support pituitary
grafts. Using in vitro assays, releasing activity vas

12
demonstrated in hypothalamic extracts for thyroid
stimulating hormone (Shitusawa et al., 1956), LH (McCann et
al., 1960), prolactin (Eeites et al., 1960), FSB (Igarshi
and McCann, 1964; Mittler and Beites, 1964), and growth
hormone (Deuben and Meites, 1964). Hypothalamic release
inhibiting* activity was shown for prolactin (Talwalker et
al., 1961; Pasteéis, 1961) and growth hormone (Krulich et
al, I 960).
The isolation of these releasing factors proved to be
more difficult. Because these factors were present in very
small amounts, sensitive biochemical and bicassay techniques
as well as large amounts of tissue were necessary. Many
incorrect claims have teen put fcrth over the years. The
first releasing factor successfully isolated was the
tripeptide, thyrotropin releasing hormone. It was found to
stimulate both prolactin and thyroid stimulating hormone
release and its structure was simultaneously reported in the
laboratories of Andrew Schally and Boger Guillemin (Scafally
et al. , 1969; Burgus et al., 1969). Schally's group also
described the next releasing factor. It was a decapeptide
which stimulates the release of both IH and FSH, tut because
other factors appear to also regulate FSH release, it has
been termed luteinzing hormone releasing hormone (LEBE,
Martsuc et al., 1971). The competition to isolate
hypothalamic releasing factors continued as Guillemin*s
laboratory reported the seguence of a growth hormone

13
re lease-inhibí tiny factor, now called somatostatin (Erazeau
et al. 1973). The rivalry between these two laboratories did
not gc unrecognized in the scientific community. In 1977,
together with Rosalyn Yallow, Roger Guillemin and Andrew
Schally received the Nobel Prize in Physiology. The drive
to isolate releasing factors continues today. Corticotropin
releasing factor was isolated by Wylie Vale, Guillemin's
collegue at the Saulk Institute (Vale et al., 1981). In the
following year Vale and Guilleain simultaneously reported
the sequence for a growth hcrione releasing factor
(Guilleain et al., 1982, J. Bivier et al., 1982)
Neuroanatoaical Relationships
Anatomy of the Pituitary Gland
The pituitary gland lies within a portion of the spencid
bone called the sella tursica. It is positioned telow the
mid-ventral portion of the train and is encased in an
extension of the cerebral meninges known as the sellar
diaphragm. The two major anatomical divisions of the
pituitary gland, the neurohypophysis or posterior pituitary
and the adenohypophysis cr anterior pituitary, have distinct
embryological origins. The neurohypophysis is derived from
neural ectoderm and contains the nerve terminals cf
mag noce 11ular neurcus of the hypothalamus. It is involved
in the neurosecretion of vasopressin and cxytocin. The
adenohypophysis is derived from an envagination of the

14
stromal ectoderm called hathke’s pouch and has nc direct
neural connection with the brain. It constitutes 80X of the
weight of the pituitary gland. Another less prominent
division of the pituitary gland is the intermediate lote.
While derived from Hathke's peach, it does receive
innervation from the hypothalamus and is often included in
dissections with the neurchypophysis. Together these two
lobes are termed the neurointermediary lobes (NIL). While
distinct in the rat, the intermediate lobe is only well
developed in humans during pregnancy.
The major anatomical division of the adenohypophysis is
the pars distalis. Approximately 5S of the cells of the
pars distalis are gonadotropin producing cells. It is still
uncertain whether a separate type of gendadetroph exists of
LH and FSH. There is evidence that some endocrine
manipulations, such as ovariectomy, produce two distinct
populations of gonadotropes.
General Hy othalaaic Anatomy
The hypothalamus constitutes the ventral portion of the
diencephalon. Its many neural connections with other
portions of the diencephalon, limbic system and brainstem
highlight its important role in endocrine and autonomic
homeostasis. The boundaries of the hypothalamus are defined
as the ventral surface of the train extending from the
rostral border of the optic chiasm to the mammillary todies

15
caudally, and laterally to the hippocampal sulcus and optic
tracts. The dorsal ¿order of the hypothalamus is recognized
as the anterior commissure and lamina terminalis rcstrally
to the hypothalamic sulcus and cerbral agueduct caudally, In
rats the hypothalamus constitutes 1X c£ the weight of the
brain.
An important landmark evident frcm the midventral surface
of the brain is a prominence containing the infundibulum and
tuber cinerium which together constitute the median
eminence. This is the sole anatomical connection between the
hypothalamus and the pituitary gland. A unique vascular
system supplies this hypothalamo-hypophyseal unit.
Hypophyseal arteries branch off the Circle cf Willis tc
perfuse the various hypothalamic regions. The most important
aspect of the hypothalamic circulation is the vascular
supply to the median eminence and arcuate nucleus. Arteries
in this area form a capillary network containing multiple
anastomoses which is refered to as the primary plexus cr
palisadic zone, A portion of this configuration contains
numerous nerve endings bordering these vascular spaces and
is called the external layer of the median eminence,
Because these capillaries do not contain fenestrations, the
median eminence is not included in the blood brain barrier
and is considered cne of the brain*s circumventricular
organs

16
The veins of the median eminence collect tc fori a pcrtal
system which supplies blood to a secondary capillary plexus
in the pituitary. Venous flow from the pituitary is achieved
through sinuses adjacent to the adenohypophysis. This
hypothalamo-hypophyseal portal system is the primary tlocd
supply tc the pituitary gland. Its existence provides a
means for neurosecretory products of the hypothalamus to
reach the adenohypophysis in high concentrations undiluted
by the general circulation. While the direction of bleed
flow is from the hypothalamus to the pituitary, this system
may be more complex than previously thought. Bergland and
Page (1979) have proposed several other pathways through
which elements in this portal system may interact.
Cell bodies of the hypothalamus are distributed in three
major gray regions, the anterior, intermediate, and
posterior areas. The anterior and intermediate regions are
the most important in the regulation cf anterior pituitary
hormone secretion. Hypothalamic nuclei are generally located
bilaterally on either side of the third ventricle. The
exceptions to this rule are the arcuate nucleus and its
closely associated median eminence which are located at the
ventral border of the third ventricle surrounding the
infundibular recess. The anterior hypothalamic area includes
the preoptic area located rostral to the optic chiasm.
Consequently this area is often collectively refered tc as
the preoptic area-anterior hypothalamus (EGA-AH). Important

17
nuclei in this region include the suprachiasmatic nucleus
which is immediately dorsal to the optic chiasm, the
anterior hypothalamic nucleus located dcrsalateral tc the
suprachiasmatic nucleus, the paraventricular nucleus which
constitutes the rostral preoptic area tc the dcrsalateral
wall of the third ventricle, the supraoptic nucleus, and the
organum vasculosum of the laaina terminalis which is a
circunventricular organ located at the anterior ventral
third ventricle. The preoptic and and supraoptic nuclei
appear to be particularly important in the cyclic release of
gonadotropins in the rat, but not necessarily in the human
(Hillarp, 1949, Halasz, 1969, and Krey et al., 1975).
The intermediate and posterior areas are often grouped
into a tissue section refered to as the medial basal
hypothalamus (JiBH). Nuclei of the intermediate
hypothalalamus include the lateral hypothalamic nucleus,
ventralateral nucleus, dcrsalmedial nucleus and the arcuate
and median eminence nuclei. Lesions of the median eminence
or arcuate nucleus disrupt gonadotropin secretion and result
in a less of reproductive cycles (Goodman and Knotil, 198 1).
The nuclei of the hypothalamus have numerous afferent and
efferent connections. Seven afferent tracts impinge upon
the hypothalamus. The medial forebrain bundle contains
tracts originating in both the olfactory cortex and the
brainstem while the stria terminalis originates in the
amygdala. Both tracts terminate in the hypothalamus,

18
midbrain, anterior commissure and preoptic area.
Corticomedullary fibers include the fornix and
corticchypothalamic tracts which originate in the
hippocampus and frontal ccrtex, respectively. Afferents from
the medial thalamic nuclei and subthalamus reach the
hypothalamus via the periventricular regions. The mammillary
bodies receive inputs from the ascending spinal tracts, the
brainstem and the anterior thalamic nucleus. Finally, photic
input to the suprachiasmatic nucleus is received via a
direct retino-hypothalamic tract.
Several efferent pathways have been described as
originating in the hypothalamus. Two tracts, the
supraoptico-hypophyseal and the tubero-hypophyseal,
terminate in the NIL. The fasciculus mammillary princeps
terminate in the anterior thalamus and midtrain. A
periventricular fiber system returns inputs to the midbrain
thalamic nuclei from the posterior, tufceral and supraoptic
hypothalamic areas. While the primary direction cf all
these pathways is either afferent or efferent, it is net
absolute. In addition, many smaller pathways cannot be
identified without imsunocytochemical technigues. The
relation of these tracts as well as intrahypothalamic
pathways to the neurotransaitter systems will be discussed
in the following sections.

19
Ana tom y of the 1 uteinizi ng Horaone-E eleas ing Hormone
Neuronal Systems
Many problems associated with iirmunccy tcchemical
techniques have precluded a complete description cf the
distribution of LHEH neurons. Among these problems is the
lack of an amino acid sequence for a IHEH precursor cr its
mBNA sequence which could be used tc visualize IHBH-
containing perikaryia. Many antibodies to LHBE are
conformationally restricted or require cne cr both terminal
ends of the LHEH molecule for recognition. This can prevent
the visualization cf peptide-ccntaining cell bodies. Tc
demonstrate LHEH in neuronal perikaryia many studies have
employed cholchicine or barbiturate pretreatment, cr
deafferentation (Sternberger and Hoffman, 1978).
Several generalizations may be made regarding the
distribution of LHEH neurons in the rodent brain. The
neurons are bipolar or fusiform and have distributions net
limited to the traditional nuclear boundaries of the
hypothalamus. Nerve terminals are heavily concentrated in
the external layer of the median eminence, which fits the
role of LHEH as a hypothalamic releasing hormone. At least
two distinct neuronal pathways appear to exist:
1. a tuberoinfundibular pathway with cell bodies in the
arcuate nucleus projecting to the median eminence.
2. a preoptico-tuberal pathway with cell bodies in the
medial preoptic zone that project to the median
eminence

20
Although LHBH perikaryia have been visualized in the
arcuate nucleus of several species, many groups have had
difficulty demonstrating their presence in the rat
(Sternfcerger and Hoffman, 1978). However, deafferentaticn
spares 2C% to 3054 of LHBH concentrations in the WEH and does
not disrupt basal LH secretion (Elake and Sawyer, 1974; S,
Kalra, 1976; Brownstein et al., 1977; Sopor and Weik, 1980).
Since this does not seem to be due to incomplete lesions,
there appears to be some LHBH neurons inside the area of the
cut.
A recent study ty Kelly et al. (1982) reports the
presence of LHBH perikaryia in the HEH, especially the
lateral arcuate nucleus and median eminence. This study
employed a specific LHBH antisera which was net
ccnformationally restricted, and used rigorous fixation
procedures on saggital train sections to facilitate the
visualization of the cell bodies. These neurons and others
located in the retrochiasmatic nucleus, precptic area, and
organum vasculosum of the lamina terminalis, terminated in
the extern«1 layer of the median eminence.
Other recent studies have expanded the potential
distribution of LHBH cell bodies in the brain. These include
septal projections and preoptic projections to the organum
vasculosum of the lamina terminalis, the olfactory bulb and
midbrain central gray (Dluzen and Bamerez, 1981; Witkin et
al., 1982; Shivers et al,, 1983b).

21
Anatomy of the Monoaminergic Neuronal Systems
The gradual improvement of analytical procedures has
resulted in a fairly accurate description of the
distribution of monoaminergic neurcns and their terminal
beds in the central nervous system. Concentrations of the
monoamines can also be determined in small nuclear regions
using the "punch technique" of Palkovitz (1981). In
addition, with the use of antitedies generated against
catecholamine synthetic enzymes such as phenylethanolamine-
n-methyltransferase, which converts norepinephrine {NE) to
epinephrine (EPI), these two neuronal systems can te more
easily differentiated.
Noradrenergic pathways
Vcogt (1954) first demonstrated the presence of NE in the
brain. It now appears that the hypothalamus receives a rich
afferent innervation from NE-ccntaining neurcns. Evidence
favoring an external source for NE in the hypothalamus
includes the depletion of 70% to 90% of hypothalamic NE
after complete deafferentation (Weiner et al., 1972). The
residual NE seen after deafferentation or brainstem lesions
appears to be due to glial cells concentrating NE and to
collateral reinnervation, since no NE-containing perikaryia
have been visualized in the hypothalamus (Palkovitz, 1981).
Noradrenergic neurons are clustered in 5 major cell
groups in the brainstem - the lateral reticular nucleus, the

22
solitary tract nucleus, the ventral pcntine nucleus, the
locus ceruleus, and the mesencephalic reticular formation.
These cell groups innervate the hypothalamus through three
major tracts - the ventral noradrenergic bundle, and the
ventral and dorsal periventricular noradrenergic bundles.
The ventral noradrenergic bundle joins the medial fcrebrain
bundle prior to innervating the hypothalamus and appears to
be the most important of tte three tracts in regulating
anterior pituitary hormone secretion. Although cell todies
that contribute to the ventral noradrenergic bundle are
primarily located in the lateral reticular nucleus, the
interconnections between all the various cell groups and
their ability to form collateralizations must be stressed.
Dopaminergic pathways
Several distinct dopaminergic pathways are found in the
brain. The largest consists cf dopamine (DA)-containing
neurons in the zona compacta of the substantia nigra and
ventral tegmentum. From these neurons arise the
nigralstriatal and mesocortical-aesoliabic CA systems.
However, neither of these systems appear to innervate the
hypothalamus significantly (Weiner et al., 1972).
The DA innervation of the hypothalamus arises primarily
from intrahypotha la sic DA systems. The tuberoinfunditular EA
system consists of cell todies in the arcuate nucleus which
project to the median eminence. Aron collaterals terminate

23
in the arcuate nucleus, ventralateral nucleus and
premaaBiliary nuclei. Included in this system is the
tuberohypophyseal DA tract which contains cell todies in the
arcuate and periventricular nuclei that project to the
neurohypophysis and intermediate lobes of the pituitary
(Moore and Bloom, 1978; Palkovitz, 1981). lastly, a poorly
defined collection of DA-containing neurons in the zona
incerta, dorsalmedial subthalamus and posterior hypothalamus
form the incerto-hypothalanic DA system. These cells project
to the dorsal anterior hypothalamus, and the paraventricular
and lateral septal nuclei (Bjcrklund et al., 1975).
Epinephrine-containing pathways
Of the three major catecholamines, EPI has the narrowest
distribution. Two EPI-containing cell groups are
intermingled with NE-containing cells in the lateral
tegmentum and dorsal medulla. In addition to other areas,
these cells project to the hypothalamus via the medial
fcrabrair bundle (Moore and Bloom, 1979; Palkovitz, 1981).
Serotonergic Pathways
There appears to be a widespread distribution of
serotonin (5BT) terminals throughout the brain. Clusters of
5HT-containing perikaryia are restricted to the midline
brainstem raphe nuclei. Ascending fibers course through the
medial fcrebrain bundle and innervate the hypothalamus,

24
particularly the suprachiasmatic nucleus (Kuhar et al.,
1972). In addition, there is evidence for SET perikaryia
within the hypothalamus (Fuxe and Onderstedt, 1968),
An atomy of Endogenous_ Gpi oid Peptide CcntainingNeurcnal
Systems
At present three distinct families cf endogenous cpicid
peptides (EOP) have been characterized. Each appears to have
its can unique precursor molecule, anatomical distribution
and receptor subtypes. Because of the difficulties in
producing specific antibodies which distinguish between
these three groups of molecules and in visualizing peptide-
containing cell bodies, the description of ECP neuronal
systems is far from complete.
Beta-endorphin-containing neurons
Beta-endorphin is derived from proopiomelanocortin (PCMC)
and has teen colocalized in cells with ether PCKC-derived
molecules like adrenocorticotropin, melanocyte stimulating
hormone, and beta-lipotrepin (Mains et al., 1977). It is not
clear whether all FCMC-containing neurons release the same
degradation products, or whether as Batson et al. (1980)
suggest, several distinct PCKC-neuronal populations exist,
each releasing a characteristic set cf molecules. One
clearly defined beta-endorphin pathway is agreed upon (Bloom
et al,, 1978; Finley et al., 1981a). Fusiform neurons are
contained in the tuberal hypothalamus extending from the

25
lateral arcuate nucleus to the lateral hypothalamic border.
Fibers project to the anterior hypothalamus and septum «here
the pathway reverses direction and follows the stria
terminalis to terminate in the dcrsal raphe, locus ceruleus
and central gray. Arborizations are found throughout the
POA-AH and SBH including the median eminence and arcuate
nucleus, Electronmicroscopic studies have revealed local
interactions between POMC-ccntaining neurcns in the
hypothalamus (Kiss and Williams, 1983).
Enkeph alin-con tai ni n g neurons
Enkephalins are derived from a precursor molecule which
contains methionine-enkephalin, cartcxy-terminal extended
methionine-enkephalin and leucine-enkephalin. While teta-
endorphin-containing neurcns have a fairly distinct pathway,
enkephalin-containing neurons are widely distributed
throughout the brain. The highest concentrations of
methionine-enkephalin are found in the striatum, followed by
the hypothalamus. Dense methionine-enkephalin innervation
has been found in the external layer of the median eminence
(Watson et al., 1S60). Enkephalin-ccntaining cells are
generally interneurcns although several short pathways have
been described.
1. A dense collection of methionine-enkephalin-
containing cell todies in the central amygdala
project to the bed nucleus of the stria terminalis
(Cuello and Paximus, 1978).

26
2. Magnocellular neurons of the paraventricular nuclei
contain methionine-enkephalin and project to the
neurohypophysis (Bossier et al., 1979).
3. A preopticotuberal pathway with cell todies in the
POA-AH projects to the median eminence (Finley et
al., 1981b).
4. Methionine-enkephalin-ccntaining perikeryia in the
anterior hypothalamus terminate in the septal area
(Sakanaka et al., 1982).
Dynorphin-containinq neurons
Dynorphin is derived from the prcdynorphin-alpha-
necendcrphin precursor along with leucine-enkephalin and
carboxy-terminal extended leucine-enkephalin molecules
(Goldstein et al., 1979; Kakadani et al., 1982).
Consequently, there is some difficulty in distinguishing
between leucine-enkephalin contained within prodynorphin-
and proenkephalin-ccntaining neurons. Of the three families
of EOP, the distribution of dynorpnin-containing neurons is
the most restricted. A supracptico-neurchypcphyseal pathway
appears to be distinct from a similar methionine-enkephalin
containing pathway. Some of these supraoptic neurons also
contain vasopressin and corticotropin releasing factor and
project to the median eminence in addition to the
neurchypophysis (Watson et al., 1982a,b; Roth et al. , 1983).

27
In addition to the brain, EOP are fcund in the pituitary
gland. Beta-endorphin coexists with other ECKC molecules in
adenohypophyseal corticotropes and is released inte the
blood by stimuli which also stimulate adrenocorticctropin
secretion (C. Bivier et al., 1962). Ihe anterior pituitary
and intermediate lobes contains some of the highest
concentrations of methionine-enkephalin in the body (Elccm
et al., 1977; Kumar et al., 1979). Because of the presence
of EOP and their receptors in the pituitary, their potential
influence on anterior pituitary hormone release cannot he
discounted (Simantcv and Snyder, 1978) .
Steroid Concentrating Neurons in the Brain
While it is recognized that neurons may serve a target
sites for gonadal steroids, the identities of these steroid
concentrating neurons are not known. Specific cytoplasmic
and nuclear receptors for androgens, estrogens, and
progestins as well as the steroid metabolic enzymes 5-alpha-
reductase and aroaatase have been characterized in the train
and hypothalamus (Massa et al., 1972; Naftolin et al., 1975;
Sar and Stumpf, 1975; McEwen et al., 1979). The electrical
activity and morphological characteristics cf neurons in the
hypothalamus can be altered ty estrogens 1983; loran-Allerand et al., 1983).

28
Neuroanatomical Interactions
Although the distributions of LHBH, monoamine, and ECF-
containing neurons show considerable overlap, evidence for a
direct anatomical interaction between these systems in the
hypothalamus is lacking. The anatomical relationships
between peptide-containing and noradrenergic neurons have
been most intensely studied. Methionine-enkephalin
containing nerve terminals do appear to synapse on
catecholamine perikaryia and axons (legar et al», 1983;
Schwartz, 1979). While reports of such an interaction in th
hypothalamus are lacking, Hoffman et al. (1982) presented
evidence for noradrenergic terminals synapsing with LHEH
cell todies en passant. Considering the clcse proximity of
many nerve terminals in the median eminence, a diffuse
nonsynaptic interaction between any of these systems is
possible. This is particularly reasonable considering the
extended half-life of beta-endorphin (Eloom et al., 1978),
which would allow its diffusion to adjacent nerve terminals
even in the absence of classical synaptic contacts.
Improved anatomical methods may further resolve the
neurcanatomica1 relationships in the hypothalamus. Eonkleiv
et al. (1981) compared beta-endorphin and LHEH
immunoreactivity in adjacent brain slices and found beta-
endorphin containing neurons and terminals to be more widely
distributed than these containing LHBH in the HBH. Double
immunostaining procedures have not been employed. Ey

29
comparing autoradiography and immunccytcchemistry Shivers et
al. (1983a) reported that LHEH neurons do not appear to
concentrate estradici in their nuclear regions. This
contrasts with the report that some neurcns in the
hypothalamus that are electrically sensitive to estrogen do
stain for LHRH (Kelly and Bonkleiv, 1S82).
Patterns of Gonadotropin Secretion
Gonadotropin secretion is inherently pulsatile. Whether
absolute levels of LH and FSH are determined at a single
time point across several animals or within a single animal
over time, these levels represent the summation of pulsatile
discharges of hormone which vary in freguency and amplitude.
Because of the longer half-life of FSE (Coble et al., 1969)
pulsatile LH secretio is most frequently studied.
Hales
In the male, LH secretion is characterized by hourly low
amplitude pulses which are temporally related to periodic T
episodes (Ellis and Desjardins, 1982). Although LH levels
remain fairly constant throughout the day, T levels are
highest in the midafterncon and lowest at about midnight.
Peak levels of P and FSH coincide with midnight, while IBRH
concentrations in tne MBH fluctuate shewing lowest levels
between 1100 h and 1600 h and peak concentrations at 1900 h
through 0800 h (P. Kalra and Kalra, 1977b).

30
The removal of gcnadal steroid feedback by gonadectomy is
followed by marked changes in the hypcthalasic-pituitary-LH
axis. Within hours of gonadectomy in the male LH secretion
is increased, and by two weeks after castration displays
characteristic high amplitude pulses with a frequency of 20
to 30 minutes (Badger et al., 1978; Galle, 1980a),
Concentrations of LHBH decline in the MBH while the
pituitary responsiveness to 1BEÜ and receptors for the
decapeptide increase (P. Kalra and Kalra, 1977; Nansel et
al., 1979; Conne et al,, 1982), All of these effects are
reversed by T,
While FSH secretion shows a similar response tc
castration, T replacement is much less effective in
returning FSH to gcnadal-intact levels (Mahesh et al.,
1975). This is in agreement with reports that other gonadal
and hypothalamic factors also regulate FSH secretion (McCann
et al. , 1983) .
Females
In the female rat low levels of genadotrepin secretion
are interrupted by a preovulatory discharge cf LH and FSH
every 4 to 5 days. Gonadotropin levels during the preestreus
surge may be 5 to 20 times greater than basal levels.
Whether basal or surging, LH and FSH levels in the female
rat are the result of pulsatile discharges of hcrmcne
(Gallo, 1981a,b). Prior to the preestrous gonadotropin

31
surge is a period oí follicular development characterized by
increasing estrogen titers (P. Kalra and Kalra, 1977a).
These sustained estrogen levels permit the expression of a
daily signal for LH release to be tiled to the midafterncon
(Legan et al., 1975). Superimposed upon this cyclic pattern
of LH and estrogen secretion is a circadian variation in P
secretion by the adrenal and ovary. Peak P levels occur in
the evening prior to midnight (S. Kalra and Kalra, 1974a).
The largest of these P rhythms is on proestrus, when it may
contribute to the preovulatory release of gonadotropins.
This proestrous LH surge is preceded by a period of IHBH
accumulation in the MBH and accompanied by a decline in
levels of the decapeptide (S. Kalra and Kalra, 1981). The
decline in MBH LHRH levels appears to be an indication of
enhanced LHRH release (Sarkar et al., 1 976). Alsc
accompanying this preovulatory interval is a marked increase
in the LH secretory response to LHRH and an increase in
pituitary LHRH binding sites (Cooper et al., 1973; Aiyer et
al., 1974; Savoy-Moore et al., 1980).
When the feedback effects of gonadal steroids are removed
as a result of ovariectomy, the response of the pituitary to
LHRH immediately increases (C coper et al., 1 975).
Luteinizing hormone levels gradually increase over a three
week period, the result of increased LH pulse amplitude and
a small increase in LH pulse freguency (tfeick et al., 1981).

32
The reinitiation of negative and positive feedback in
ovariectomized rats by gonadal steroid treatment can serve
as a controlled method for studying gonadotropin secretion.
Immediately after the injection of estrogen or the
implantation of estrogen containing capsules to
ovariectomized rats, IH levels decline and the pituitary
becomes refractory to LHBH (Vilchez-Martinez et al., 1974).
Two days of continuous estrogen exposure enhances the
response of the pituitary to LHBH atd induces a diffuse
midafternoon LH surge. This afternoon IH surge repeats for
several days if estrogen titers remain elevated (legan et
al., 1975)
The injection of P several hours before the onset of the
LH surge further increases the pituitary response tc LHBH,
enhances the magnitude cf the resultant LH surge, and
advances its onset (Aiyer et al., 1976; Kalra et al,, 1981).
Concentrations of LHBH in the MEH increase prior to and
decline during the period of IH hypersecretion in a fashion
similar to that seen on proestrus (S. Kalra and Kalra,
1979).
Monoaminergic Control of Gonadotropin Secretion
Of the major neurotransmitters, the influence cf the
monoamines in controlling gonadotropin secretion has been
most extensively studied. These neurotransmitters are also
important in EOP regulation cf hormone output. The following

33
section «ill briefly review our current understanding of
monoaminergic regulation cf LH and FSH secretion. While
many trends are apparent in this area, it must be cautioned
that few monoaminergic drugs are specific fcr one
neurotransmitter or receptor. flather, it is the integration
of many studies and confirmations of those works that
provide an accurate description of the role each
neurotransmitter plays in the control cf gonadotropin
release.
Many studies have evaluated the activity cf monoaminergic
neurons in various reproductive states, especially the
catecholamine neurons (Weiner, 1974; Cooper et al., 1978).
Methods cf estimating catecholamine neuronal activity which
do not disturb the steady-state include the rates cf
synthesis of NE or CA from trace amounts of their
radiolabeled precursor, tyrosine, or the disappearance cf
trace amounts of added radiolabeled catecholamine. This
technique can also be employed fcr the evaluation cf 5HT
neuronal activity using radiolabeled tryptophan or 5H1.
Problems associated with this technique include the
inability to evaluate small tissue sections, unegual
distribution of the radiolabeled amino acid, and nonspecific
uptake of the label by nontargeted cells.
A primary non-steady-state method of evaluating
catecholamine activity includes the measurement cf NE or DA
depletion following the blockade cf the rate limiting enzyme

34
in catecholamine synthesis, tyrosine hydroxylase. With this
method greater neuronal activity is indicated by increased
rates of catecholamine depletion following the inhibition cf
synthesis. Using this method catecholamines can be evaluated
in small nuclear regions using sensitive analytical
techniques. With the high dosages required to inhibit
catecholamine synthesis, some nonspecific effects may occur.
Additionally, it cannot be assumed that catecholamine neuron
will behave similarly under ncn-steady-state conditicns.
Fortunately, studies using different methods often agree.
A more recent method of evaluating mcncaminergic neurcnal
activity includes the measurement of amine and metabolite
using amperometric methods. Ihese techniques have net been
used extensively in neuroendocrine studies, however,
Norepinephi ine
A large body cf evidence suggests that central
noradrenergic neurons control LH and FSH secretion. Sawyer
and collegues originally showed that a variety of centrally
acting adrenergic agents influence the ovulatory release of
gonadotropins (Everett et al., 1949; Sawyer, 1952;
Barraclough and Sawyer, 1957). Further, these effects were
not elicited at the level of the pituitary gland (Seiner and
Ganong, 1978). Most investigators today agree that central
noradrenergic neurons display both stimulatory and
inhibitory influences on the release of gonadotropins.

35
The activity of noradrenergic neurcns appears to change
in concert with several IH secretory states. Following
ovariectomy or castration the concentration of NE in the
hypothalamus increases (Donoso et al. , 1967). This suggests
an increase in NE metabolism and hence activity using
several techniques (Anton-Tay and Burtman, 1968; Anton-lay
et al., 1970; Coppola, 1971; Kizer et al.,1974; Simpkins et
al,, 1980). Increased NE neuronal activity has also been
noted in the hypothalamus prior to LH hypersecretion on
proestrus or following gonadal steroid treatment to
ovariectcmized rats (Zschaeck and Burtman, 1973; Loftstrcm,
1977; Munaro, 1977; Simpkins et al., 1979). These studies
argue for a stimulatory role for noradrenergic systems
controlling LH hypersecretion.
Many pharmacological studies support a role for NE in
regulating gonadotropin release. Adrenergic agents applied
systemically or intraventricularly are presumed to interact
with catecholamine receptors or alter monoaminergic neuronal
activity. The blockade of NE synthesis with DA-beta-
hydrcxylase (DBH) inhibitors suppresses pulsatile LH release
and LH hypersecretion induced by endogenous steroids prior
to ovulation, electrical stimulation of the hypothalamus and
gonadal steroid administration (P. Kalra et al, 1972; S,
Kalra and McCann, 1973; Drouva and Gallo, 1 976a; Gncdde and
Schuiling, 1976), This blockade of NE synthesis is overcome
by the pretreatment with dihydrcxyphenylserine which dees

36
not require DBH for its metabolism to NE, but not fcy 1-DCFA,
a precursor in catecholamine synthesis. Additionally, alpha-
adrenergic antagonists inhibit LH release in castrated and
gonadal steroid treated rats, while neurotoxic agents such
as 6-hydroxy-DA can prevent prcestrcus LH release (P. Kalra
et al., 1972; Gnodde and Schuiling, 1976; Martinovic and
McCann, 1977). Together these data argue for a stimulatory
role for ME on gonadotropin release.
The administration of NE has varying effects on LE
release depending cn the mode of administration and
experimental paradigm employed. Hhile in vitro evidence
suggests that NE can stimulate LHfiH secretion from
hypothalamic fragments via an alpha-adrenergic mechamisit
(Ojeda et al., 1982; Miyake, 1983) , evidence from in vivo
studies indicate that LH secretion may represent a balance
of both stimulatory and inhibitory inputs. Intraventricular
administration of NE inhibits IH release in ovariectomized
rats (Gallo and Drouva, 1979). It is not certain whether
this effect is mediated by one particular adrenergic
receptor, if it is localized at a site within or cutside the
hypothalamus, or if the inhibition occurs through LEBE
neurons (Caceres and Taleisnik, 1S80, 1982; Leung et al.,
1981, 1982).
In ovariectomized rats pretreated with gcnadal steroids
the intraventricular administration cf NE stimulates IH
release (Krieg and Sawyer, 1976; Vijayan and McCann, 1978;

37
Gallo and Drouva, 1979), It would appear that in an
ovariectcmized animal, the inhibitory effects of NE on IH
release predominate, while in the presence of gonadal
steroids, stimulatory modes cf NE ce LH release are
primarily operative.
Despite many studies, the nature cf the coupling of the
noradrenergic neuron to the IHEH-containing neuron is a
subject of debate. LH secretion is most vigorous when NE is
infused in a pulsatile fashion and desensitization ensues
with a continuous NE infusion (Gallo, 1S82). Yet, a recent
study by Estes et al. (1982) suggest that a single dose of
the alpha-adrenergic agonist, clonidine, stimulates
pulsatile LH release for several hours. This would suggest
that noradrenergic neurons have a permissive effect on IHBH
pulsation, rather than being the driving force behind each
individual pulse. This diffuse functional relationship fits
the general lack of a direct anatomical connection between
the two systems.
Epinephrin
The recent development of inhibitors of EPI synthesis has
allowed the differentiation of effects which could be
attributed to either NE or or EPI-ccntaining neurons. Cn a
molar basis EPI is more potent than either NE or DA in
eliciting H release when injected inte the ventricles cf
gonadal steroid treated ovariectomized rats (Vijayan and

38
McCann, 1978)- Epinephrine might be important in mediating
the positive feedback effects of gonadal steroids in female
rats (Adler et al., 1928; S. Kalra 1983), It is not clear
however what role EPI may play in regulating LH release in
ovariectomized rats or in male rats (Regro-Villar et al.,
1979; Crowley et al., 1982; Crowley and Terry, 1981),
Dopamine
A great deal of conflicting evidence exists concerning
the role of DA in regulating IH and FSH release in male and
female rats. In vitro studies suggest that DA stimulates
LHfiH release from hypothalamic tissue fragments (Schneider
and McCann 1969; Rotsztejn et al., 1977). The activity of DA
neurons appears to be enhanced after castration in the ECA-
AH and prior to gonadal steroid-induced LH hypersecretion in
the MBH of female rats (Simpkins et al., 1979, 1980). While
this might argue for a stimulatory role for CA neurons on LH
release, DA itself does not consistently induce LH secretion
when injected into the ventricles (Drcuva and Gallo, 1976a;
Krieg and Sawyer, 1976; Vijayan and McCann, 1978; Gallo and
Drouva, 1979).
Serotonin
The importance of serotonergic neurons in the overall
regulation of gonadotropin release is unclear.
Intraventricularly administered 5HT stimulates or inhibits

39
LH release depending on the dose employed (Kamberi et al.,
1970; Cramer and Porter, 1973), Studies with the neurctoxin,
5,7-dihydroxytryptamine, suggest that serotonergic neurons
stimulate LH release (Wuttke et al», 1978, Van der Kar et
al., 1980). While it is uncertain whether 5HT-ccntaining
neurons control LH release in ovariectomized rats (Gallo,
1980b), several authors suggest that the stimulating effects
of estrogen and P on LH release may be influenced by
serotonergic neurons (Iyengar and Habii, 1983; Walker and
Wilson, 1983; Chen et al., 1S84). Since many serotonergic
drugs also act upon catecholamine neurons or their
receptors, more information must accumulate on this subject
before a more definitive assessment can be made.
Endogenous Opioid Peptides and the Control of Gonadotropin
Secretion
The first experimental evidence that opioids are
inhibitory to reproductive function was Barraclough and
Sawyer*s (1954) observation that morphine blocked ovulation
in the rat. It was later verified that this blockade was due
to an inhibition of the proestrous gcnadctrcin surge (Pang
et al., 1977). At this time Cicero et al. (1975a, b)
demonstrated that chronic treatment with morphine induced
changes in the reproductive system of the male rat similar
to the effects of narcotic abuse in men, i.e. depressed
serum 1 and diminished secondary sex organ function. These
studies indicated that the effects of opiates on

40
reproductive function could be assessed in a laboratory
setting.
Reproductive Pharaacolcqy of Opioids
Tiie antigonadotrcpic effects of opiate administration are
ultimately exerted at the level of the hypothalamus through
the inhibition of LHBH release. Many careful studies have
eliminated other possible sites of action (Cicero 1980a).
Opiate administration has no effect on the metabolism of T,
its clearance from the blood, or its fate at target organs.
Further the effects of morphine are not exerted at the level
of the testes either by effecting 3 synthesis or the
response of the leydig cell to gonadotropins. At the
pituitary gland, morphine dees not alter the release or
synthesis of LH or the response of the gonadotrope to LHBH.
In one study however, leucine-enkephalin acutely inhibited
the LH secretory response to IHEH (May et al., 1979).
Several lines of evidence suggest that cpiates exert
their antigonadotropic actions by inhibiting LHBH release.
The increased LHBH concentrations in hypophyseal portal
plasma which accompanies the proestrous gonadotropin surge
are prevented by morphine treatment (Ching, 1983). Also,
opiate agonists and antagonists can modulate the release of
LHBH from in vitro hypothalamic incubations (Botsztejn et
al., 1978; Drouva et al. , 1981; Wilkes and Yen, 1981). In
one study the LH stimulatory actions of an opiate antagonist

41
was prevented by treatment with an LHRH antagonist analogue
(Blank and Roberts, 1982).
It is likely that the central site for the opiate-LHBH
interaction is within the hypothalamus (S. Kalra, 1981).
However, some investigators have found IH-inhibitory effects
of opioids administered in the amygdala and brainstem
(Parvizi and Ellendcrf, 1980; lakoski and Gebhart, 1981 and
1982) .
Most studies of opioid effects on reproductive function
measure serum LH levels as an index of LHBH output. The
acute effects of opioids on IH secretion satisfy the
criteria for mediation by an opiate receptor (Cicero,
1980a).
1. In general, all opioids depress serum LH levels. This
has been found to be true for both opiate alkaloids
and opioid peptides administered systemically as well
as opioid peptides administered intraventriculariy
(Bruni et al., 1977; Cicero, 1980b; Johnson and
Rosencrans, 1981; Kinoshita et al,, 1981; Kato et
al., 1982; Ehanot and Wilkinson, 1983; Leadem and
Kalra, 1983; Harkc and Rcmer, 1983;
2. The relative potency of cpiates in suppressing IH
release parallels their pharmacological efficacy in
other preparations such as the displacement of
tritiated opiates, the ability to inhibit the
contraction of guinea pig illeum, and analgesia
(Cicero e t al, ,
1976; Cicero, 1980a)

42
3. The effects cf opiates on LH secretion are reversed
by opiate antagonists like naloxone or naltrexone
(Pang et al., 1974; Cicerc et al., 1976, Bruni et
al. , 1977., Huraki et al., 1980).
4. Levarotatory isomers of opiate alkaloids are far more
potent in inhibiting LH secretion than dextrorotatory
isomers (Cicero et al. , 1976).
Physiological Inhibition of Gonadotropin Secretion by
Opioids
Although the existence of opiate receptors in the brain
and hypothalamus and the presence of a pharmacologic
response to stimulation of these receptors suggest opioid
pathway which effects IH secretion, this does not, per se,
verify that EOP normally act to inhibit LH release. If
physiologically released EOP do act to inhibit LH secretion,
then blockade of opiate receptors with a narcotic antagonist
should reverse this inhibition. The ability of naloxone, on
its own, to stimulate LH and FSH secretion is the most
persuasive and most freguently verified evidence favoring
an EOP inhibition cf LH secretion (Meites et al., 1979;
Cicero, 1980b; S. Kalra et al., 1980; Ferin et al., 1984).
If blockade of EOP activity with an opiate receptor
antagonist elicits LH secretion, then sequestering ECE with
an appropriate antibody might produce the same effect.
Antibodies to both beta-endorphin and dynorphin have been
found to stimulate IH secretion (Schulz et al., 1981; Forman
et al., 1983)

43
The mechanisms Mediating opiate antagonist induced IE
secretion are not known. Although naloxcne occupies cpicid
receptors and prevents the ongoing actions of EOP, naloxone
only transiently stimulates LH secretion, and like opiate
agonists, tolerance develops to its effects on LH secretion
{Owens and Cicero, 1981). Another recent study suggests that
the stimulation of LH secretion following naloxone injection
may reflect prior opiate agonist activity, rather than
simple displacement of an opiate agonist frcm its receptor
(Cicero et al,, 1983b). In this study a single injection of
morphine enhanced the ability of naloxone to elicit LH
release for several hours after morphine had been cleared
from the brain.
Multiple Opioid Receptors
Several classes of opioid receptors appear to exist.
While different classifications are used, there appear to be
at least three distinct opioid receptors, temed mu, delta,
and kappa. These receptors do not have widely divergent
binding affinities, thus distinguishing between the three
classes with specific agonists and antagonists has proven
difficult Martin, 1981). These three classes may share a
common high affinity binding component (Hahn and Pasternak,
1982). It is this high affinity component that appears to
mediate the analgesic actions of morphine. In relaticn to
the various opioid alkaloids and peptides, the mu-opioid

44
receptor appears to mediate the effects cf zcrphine and its
cogeners. The delta-opicid receptor appears to be mere
specific for the enkephalins, while the kappa-opioid
receptor appears tc be more specific for dynorphin
(Childers, 1980; Chavkin et al., 1982). Another receptor,
called epsilon, has been proposed based on receptor binding
studies with beta-endorphin (Law et al., 1979).
Several studies have utilized opiate alkaloids and opioid
peptides to discern which receptor sottype may mediate the
effects of opioids en 1H secretion. Opioid inhibition of LH
secretion appears to involve both a nu and kappa-cpicid
receptor component (Cicero et al., 1983c; Gabriel and
Simpkins, 1983; Leadea and Kalra, 1983; Pfieffer et al,,
1983). This agrees with studies which show that antibodies
to beta-endorphin and dynorphin, but not methionine-
enkephalin, stimulate LH secretion (Schulz et al., 1981;
Forman et al., 1983),
Opioid-Monoaainergic Interactions
The interaction between opioid and monoanine-ccntaining
neurons has been investigated in several systems (Schwartz,
1979; Kuchinsky, 1977). Several recent studies have
characterized a possible adrenergic interaction with opioids
in the control of gonadotropin secretion. The stimulatory
effects of naloxone on LH secretion are prevented by
adrenergic antagonists, DBH inhibitors and EPI synthesis

45
inhibitors (S. Kalra, 1981; S. Kalra and Simpkins, 1981; Van
Vugt et al. , 1981; S. Kalra and Crowley, 1982; Schulz et
al., 1982; Koh et al. , 1983; Adler and Crowley, 1984).
Additionally, the acute administration of opiate antagonists
appears to modulate the activity of catecholamine neurons in
the hypothalamus (Adler and Crowley, 1984). It appears that
adrenergic neurons may influence 1H secretion without an
intermediary opioid interaction since the inhibition cf IH
secretion seen following morphine is reversed by subsequent
treatment with clonidine or the intraventricular EFI
injection (S. Kalra and Simpkins, 1981; S. Kalra and Gallo,
1983) .
In addition to both NE and EPI-containing neurons, ECF
have teen shown to interact with dopaminergic and
serotonergic neurons (Van Loon and De Souza, 1978; Gudelsky
and Porter, 1979). There have been reports that the effects
of opiates and EOP on LH secretion are influenced by each cf
these Bcnoaminergic neuronal systems (Eotsztejn et al.,
1978; Ieiri et al., 1980b).
Opioid-Gonadal Steroid Interactions
While it is apparent that ECP-containing neurons act to
suppress LH release, the function of this inhibitory input
is not well understood. Some researchers have presented
evidence that EOP may relay the feedback signals of gonacal
steroids in the brain. Morphine, like 1,
can prevent the

46
post-castration rise in serum LH, while naloxone and 1 are
mutually antagonistic on LH secretion in cichidectcmized
rats. Similarly estrogens can inhibit naloxone's stimulation
of LH secretion in cvariectomized rats (Blank et al. , 1979;
and 1980; Cicero et al., 1980; Sylvester et al. , 1982; Van
Vugt et al., 1982)
If opioid neurons do relay the feedback signals of the
gonadal steroids on LH secretion, then it can be expected
that the pharmacological efficacy of opicid agonists and
antagonists will vary under differing reproductive states.
In prepubertal female rats, when LH levels are markedly
suppressed! naloxone is highly effective in stimulating LH
release (Blank et al., 1979). Ihe efficacy of naloxore in
stimulating LH secretion appears to vary diurnally and
diminish after castration (Blank and Mann, 1981; Cicerc et
al., 1983). Opioid agonists are more effective in
prepubertal rats compared to pubertal rats, and in rats
castrated acutely versus rats castrated several weeks
(Bhanot and Wilkinson, 1983; Cicero et al., 1982a; Wilkinson
and Bhanot, 1983).
An alternate means of evaluating the potential
involvement of EOP in mediating gonadal steroid feedback
would be to assess changes in ICE concentrations or their
release under varying steroid milieus Peptide release wculd
be the most preferable estimate of neuronal activity. To
date, only beta-endorphin has teen evaluated in the

47
hypophyseal portal plasma of non-human primates. Cf
interest, beta-endorphin levels decline precipitously
following ovariectomy and during menstruation (Ferin et al.,
1984), This would imply that the activity of hypothalamic
beta-endorphin-containing neurons depends cn cvarian
factors, particularly F.
Tissue levels of EOP show changes which may be relevant
to gonadotropin secretion. Both beta-endorphin in the septum
and medial preoptic area and methicnice-enkephalin in the
flBH and POA-AH display circadian variations in tissue
concentrations that parallel changes in LHBH levels in the
hypothalamus of the male rat (Kumar et al., 1982; S. Kalra
et al., 19 1b; Kerdelhue et al., 1973). Orchidectcmy dees
not appear to alter beta-endorphin levels in the
hypothalamus but decreased levels of both beta-endorphin and
methionine-enkephalin are found in the Nil and anterior
pituitary following castration (lee et al., 1980; Fong et
al., 1982; Petraglia et al., 1982; Yoshikawa et al.,
1983a,b). Beta-endorphin levels in the hypothalamus appear
to increase as male rats approach puberty (lee et al.,
1980).

48
Bationale
From the literature presented it appears that EOP may
function as one of several neurotransuitte rs that regulate
the release of the gonadotropins, LH and FSH. Based on the
pharmacological effects of opioid agonists and antagonists
during various reproductive states, and the ability of
gonadal steroid alterations tc modify EOP levels in the
brain and pituitary it appears that ECP-containing neurons
respond to changes in the steroid milieu. These changes may
reflect alterations in EOP neuronal activity which mediate
the feedback effects of gonadal stercids cn gonadotropin
secretion.
This thesis will present a series of pharmacological
investigations of opioid neurons in male and female rats. In
the male, EOP appear to suppress LH secretion, but the
nature of this inhibition is not well understood. These
studies will evaluate the feedback effects of the gonadal
steroids on LH and FSH secretion in the presence of
continuous opiate receptor stimulation with morphine. In the
female rat, the extent of EOP inhibition of IE secretion has
not been fully assessed. Naloxone was used tc evaluate the
potential EOP inhibition of IH secretion present during the
estrous cycle and following gonadal steroid treatment tc
ovariectcmized rats. Finally, the feedback effects of
gonadal steroid treatment on gonadotropin secretion in
females was evaluated in the presence of continuous opiate
receptor stimulation with morphine.

CHAPTER III
GENERAL MATERIALS AMD METHODS
Anisáis
The laboratory rat was chosen as the experimental animal
in these studies. Adult male and female S-D rats were
obtained from Charles Rivers Breeding Laboratories in
Wilmington, Massachusetts. Animals weighed 180 to 220 grams
upon arrival and were allowed several days to adjust to the
animal quarters before initiating an experiment. The rat
colony was maintained in a light (lights on 0500 h through
1900 h) and temperature (26 ± 1° C.) controlled room with
food and water provided ad lititum.
Reproductive status of female rats was verified by
microscopic examination of vaginal lavages (Ingram, 1956).
Rats which displayed two consecutive 4-day estrcus cycles
were chosen for studies employing gonadal intact female
rats. The normal sequence of cell morphology in the vaginal
smear consists of lavages containing cornified epithelial
cells (estrus), followed by two days of predominately
leukocytic smears (diestrus I and diestrus II), which is
then followed by a day in which the lavages contain
nucleated epithelial cells (prcestrus). The cornified
epithelium and leukocytic smears are characteristic of a
49

50
gonadal steroid milieu
dominated
by
estrogens and
progestins.
respectively.
Surgical
procedures
consisted
of
subcutaneous
implantation of drugs or steroids and bilateral gonadectcmy
performed under light ether anesthesia. Hale rats were
orchidectomized by exteriorizing the testicles through a
midline ventral incision. Female rats were ovariectomized by
a bilateral dorsal approach. Animals were monitored for
post-surgical wound healing.
Two methods were employed for collecting blood. In most
experiments, trunk blood was collected by decapitation.
Decapitations were completed within 30 seconds of removal of
each rat from its home cage. In studies employing LHEH
injection, blood samples were collected by cardiac puncture
under light ether anesthesia. All blood samples were
collected in a room separate from the animal guarters. Sera
was separated from trunk blood by centrifugation (1000 X g)
for 15 minutes while jugular and cardiac blood samples were
centrifuged in a microcentrifuge for two minutes. All sera
were stored at -20° C. for later hormone analysis by
radioimmunoassay (BIA).

51
Dissection of Brain tissue
Brains were rapidly removed and placed with their dorsal
surface on ice. Tissue sections containing the MEH and POA-
AH were removed using fine iris scisscrs. Cuts for the KBH
fragment were made at the posterior torder of the optic
chiasm, then caudally at the level of the mammillary tedies,
2 mm laterally at the hippocampal sulcus, and 2 mm telow the
ventral surface of the hypothalamus. The bcundries of the
POA-AH tissue slice were the caudal borders of the olfactory
tubercles to the posterior border of the cptic chiasm.
Additional cuts were placed 2 mm lateral to the midline and
approximately 2 mm from the dorsal surface cf the PCA-AH at
the level of the anterior commissure.
Gonada1 Steroid Treatment
Gonadal steroids were obtained fren Steraloids Inc.,
Milton, N.J. and administered as subcutaneous implants or as
injections. Implants consisted of Silastic tubing (1.57 mm
i.d., 3.17 mm o.d.) of lengths ranging frem 2.5 mm tc 30 mm.
Capsules were filled with either crystalline forms of I,
5-alpha-dihydrotestcstercne (DHT) or 17-beta-estradicl (E2)
or E2 dissolved in sesame seed oil. The implants were sealed
at both ends with Silastic adhesive and allowed to dry at
room temperature for 24 to 48 hours. Before use, these
implants were soaked in phosphate buffered saline for 48
hours. These implants provide sustained bleed levels cf

52
gonadal steroids fox several weeks. In the male rat these
implants reversed post-castraticn LH hypersecretion at
physiologically relevant dosages (P. Kalra and Kalra, 1980).
The implantation of crystalline E2 in ovariectomized rats
provided sustained E2 levels which immediately reduced IH
secretion and stimulates a daily signal for the midafternoon
release of LH (Legan et al., 1975).
Two ether methods for stimulating midafternoon IH
hypersecretion in ovariectomized rats employed the injection
of estradiol benzoate (EB) or the sequential administration
of EB plus P. Rats which were ovariectomized two weeks
previously were injected with 7.5 ug of EB dissolved in 100
micrcliters of oil at 1000 h. This treatment produced a fall
in LH secretion followed by a midafternoon LH surge two days
later. If 5 mg of P dissolved in 100 micrcliters of oil were
injected into these rats 48 hours alter EB treatment, a more
pronounced LH surge with an earlier onset results. ether
endocrine changes accompanying this treatment are discussed
in Chapter II.
Treatment with Morphine or Naloxone
Morphine dependency was produced by subcutaneous
implantation of one pellet containing 75 mg morphine (free
base, Berk, St. Louis, MO), 37.5 mg micrccrystalline
cellulose (Avisil, EMC Corporation, Philadelphia, PA,), 0,56
mg Cab-o-sil (Cabot Corporation, Boston,
MA») and 1, 13 mg

53
magnesium sterate (Fisher Chemical Co. Fair Lawn, N.J.). Two
days later, two additional morphine pellets were implanted.
The pellets were compounded in this laboratory. This
treatment regimen produced morphine dependency as measured
by several tests of analgesia and withdrawal (Gibscn and
Tingstad, 1970; Simpkins et al., 1983b). Control animals
received placebo pellets which were formulated with an
additional 75 mg. Avisil rather than morphine free base.
Naloxone HCl (Dupont Pharmaceuticals, Garden City, N.J.)
was dissolved in normal saline and administered
subcutan eously.
fleasureneit of Catecholamines Indolamines and Metabolites
Concentrations of NE, DA, SHT, the NE metabolite
normetanephrine (NNE) , the EA metabolites dihydroxyphenyl-
acetic acid (DOPAC) and homovanillic acid (HVA) and the 5HT
metabolite 5-hydroxy-indolacetic acid (5HIAA) were measured
by ampercmetric methods following their separation by high-
pressure liguid chromatography using a modifacticn of the
procedure described by Kichaud et al. (1981). The
separation was accomplished using reverse phase
chromatography across an IBE 1C-18 (15 cm X 4.6 mm, 5
micrometer particle size) with a mobile phase composed of 8?
methanol, 0,2 mM octyl sodium sulfate, 0.1 N. NaH2 EC4 and
0.1 mM ECTA at pH 2.9. The flew rate was varied from 0.5
ml/min for the first 7.5 minutes of separation, 0.7 ml/min

54
from 7.5 through 23 minutes, 2.C ml/win from 23 to 39
minutes and 3,0 ml/min thereafter, through 57 minutes. This
procedure allowed the elution of catecholamines, indclamines
and their metabolites in standards and samples within a one
hour period. The times for elution {in order) for NE, NflE,
DA, DOPAC, 5HIAA and HVA were 5.5, 13, 17.5, 21, 28.5, 33.5
and 36 minutes respectively. The detection of amines,
indolamines and their metabolites was accomplished with an
electrochemical detector (IBH,model IC 9533) set with the
potential difference between the working electrode and a
reference AgAgC12 electrode of 0.9 volts and a current
generated at 20 nA/mV.
Tissue sections containing the HEH and POA-AE were
dissected from brain tissue as described above. The
fragments were homogenized with a tissue sonicator in 0.4 N.
perchloric acid containing 1 mgS EDTA at a weight:volume
ratio of 1 ag/10 microliters. Average weights of these
tissues were 19.0 ± 0.5 mg for MEH and 20.2 ± 0.6 mg for
POA-AH.
To each 20 microliter of sample of MBH and POA-Afl tissue
homogenate was added 2 ng dihydrcxybenzo-acetic acid (CHEA)
as an internal standard. The concentration of each amine,
indolamine, and metabolite was determined by the peak height
ratio of the compound to DHEA in relation to a standard
curve of peak-height ratios for that particular
catecholamine, indolamine, or metabolite. The sensitivity of

55
this assay was less than 100 pg for HE, DA, 5HT, and their
metabolites.
Hcr mone Radiolanunoassays
Luteinizing Hormone and Follicle Stimulating Hormone
Serua and medium saaples were assayed for LH and FSH
using the kits provided by the National Institutes of
Arthritis, Diabetes and Digestive and Kidney Diseases
(NIADDK). The rabbit-derived antisera used were NIADDK-anti-
rLH-S-7 for the LH assay and NIAEDK-anti-rFSH-S-11 for the
FSH assay, Radioiodinaticns were performed in our laboratory
using standard procedures for a chloraaine-T iodination with
gel filtration to separate free iodine fres hormone-bound
iodine.
Because these studies were performed ever a considerable
period of time, LH values were determined in relation to two
LH reference standards provided by the NIADDK. The later LH
reference preparation, IH-RE-2, was 61 times more potent
than the original LH-RP-1. To aid in the comparison of IH
values across experiments, all IH values were expressed
relative to the original LH-RP-1 standard. The figure or
table legends will note when this conversion was made. FSH
values were expressed in relation to the reference standard,
FSH-RP-2. The intra-assay variation for the LH and FSH
assays, determined by the coefficient of variation for 10
replicates of pooled castrate serum which inhibited the

56
binding of the radiolabeled hormone 40* to 60% was 6.85? and
6.3%, respectively. The inter-assay variation, determined
from successive pooled serum in assays performed over a 6
month period was 11% and 8% for IH and FSH, respectively.
The sensitivity of these assays, defined as a the amount of
standard hormone reguired to inhibit the binding of the
radiolabeled hormone by 20% was C.90 eg (LH-BP-1) for IH and
0.40 ng for FSH,
Luteinizing Hormone Releasing Hormone
Tissue sections containing the MEH and POA-AH were
homogenized in 2 ml of 0.1 N. HCl and supernates were
analyzed for LHRH using RIA methods described previously (S.
Kalra, 1976). Acid supernates were neutralized with 2 K.
NaOH during the assay procedure. Synthetic LHHH obtained
from Eeckman Co. (Palo Alto, CA.) was used as the reference
standard and for iodinaticn. Mcnoradioicdinated LHEH was
employed as described previously (Nett and Adams, 1977).
Rabbit antibodies against LHRH were purchased from Miles
Laboratories (Elkhart, IN). The minimum sensitivity for this
assay was 2 pg per tube and was estimated as the
concentration of LHRH which inhibited the total labeled
binding by 10%. Concentrations of LEEH were expressed in
terms of tissue sample (i.e. ng per MBH or PCA-AH).

57
Testcsterone
Serum T levels «ere analyzed according to procedures
described previously (P. Kalra and Kalra, 1982).
Statistical Analysis
For most experiments, analysis of variance with Student
Neuman Keuls tests «ere used tc evaluate the significant
differences between treatment groups. Where appropriate,
Student-t tests were also used. In studies which injected
LHRH, paired-t analyses were employed tc determine the
significant effects of LHRH injection. To further evaluate
data in Chapter 4, the regression analysis programs
contained in the Statistical Analysis System package offered
by the Northeast Regional Data Center were utilized. In all
studies a significance level of p < 0.05 was required.

CHAPTEB IV
THE EFFECTS OP CH8CNIC MCBPHINE TBEATKENT ON TESTCS1EBONE
NEGATIVE ÍEEDBACK IN CASTRA1ED «ALE RATS
Introduction
Tne feedback effects of gonadal steroids cn LH secretion
are believed to be mediated by the hypothalamus and
pituitary (Drouin and labrie, 1976; S.. Kalra and Kalra,
1983). Recent investigations from several laboratories show
that morphine or ECP can acutely suppress IH release in
intact and gonadectcmized rats (Meites et al. , 1979; Cicero,
1980; Kinoshita et al. , 1981; leadem and Kalra, 1983).
Interestingly, gonadal steroids have also been found to
modify EOP levels in various sites within the hypothalamus
and the secretion of beta-endorphin in the hypophyseal
portal system (Barden et al., 1961a; Sardlaw et al., 1982;
Rehrenberg et al., 1982). Further, acute blockade of central
opiate receptors with narcotic antagonists transients
reverses the inhibitory feedback effects of 1 cn LH release
(Cicero et al., 1980). Since gonadal steroid treatment and
opiate receptor stimulation suppress LH release and ECF-
producing neurons are found in the vicinity of IHBH neurons,
it is logical to suspect that ECE-containing neurons may
either mediate the feedback effects of gonadal steroids or
58

59
that they may act through similar hypothalamic mechanisms to
decrease LH release (P. Kalra and Kalra, 1980; Hatson et
al., 1980; Sar and Stumpf, 1975; Shivers et al., 1983b). The
following study compares continuous opiate receptor
stimulation with morphine to 1 replacement on LH secretion
in castrated rats, and evaluates the feedback sensitivity of
T on LH secretion in the presence of chronic morphine
treatment.
Experimental
In these studies castrated rats were treated chronically
with morphine pellets and tubing containing crystalline T.
Experiment 1
In the first study, rats received either chronic morphine
treatment, replacement T therapy (two 15 mcc tubes) or
control treatment (placebo pellets or empty tubes) which
commenced either at the time cf cr twc weeks after
castration. Animals were killed by decapitation after 7 days
of treatment. Serum was analyzed for LH and 1, while the
brains were rapidly removed and dissected for analysis of
MBH and AH-POA LHBH concentrations.

60
Experiment 2
In the second study, rats were castrated and twc weeks
later received either control pellets or implants of
morphine, T, or morphine plus T. Testosterone-containing
tubes were either 2.5, 5.0, or 10.0 mm in length. Animals
were killed by decapitation 4 days later. Serum was analyzed
for LH and T, while brains were rapidly removed and tissues
dissected for analysis of LHRH concentrations.
Experiment 3
Bats which had been castrated two weeks previously
received either control, 5 mm T, or morphine plus 5 mm T
implants. After 4 days, rats were killed by decapitation,
anterior pituitaries were removed, hemisectioned, and
preincubated in control mediux (minimal essential medium ♦
25 inM Hepes, pH 7.2, Gibco Inc,, Grand Island, NY) for one
hour at 37° C. Fresh control medium or medium containing 1
X 10~7 M. LHBH was then added to the hemipituitary
incubation medium and the incubation continued for an
additional hour. Medium was stored at -20° C for later
analysis of LH levels

61
Results
Effects of Time After_Castrati cn cn the Serui LH and
Hypothalamic LHEH Responses te I and morphine
Figure 1 illustrates serum IH concentrations in rats
treated with either T or morphine immediately after or tiro
weeks after castration. Serum T levels attained fcy the two
15 mm implants were 2161 ± 12C pg/ml in rats treated
immediately after castration and 2439 ± 129 pg/ml in rats
receiving T two weeks after castration. These levels are in
the range observed normally in intact male rats (P. Kalra
and Kalra, 1980). As evident from Figure 1, when treatment
was started at the time of castration {short-term castrate)
both morphine and T prevented the pcst-castration
hypersecretion of IH (p < 0.05). However, when the
initiation of treatment was delayed for two weeks after
castration (long-term castrate), unlike T, morphine was no
longer effective in suppressing IH secretion.
Levels of LHRH in the MBH of rats treated with T or
morphine either at the time of castration or two weeks after
castration are shown in Figure 2. Beth I and morphine
prevented the post-castration decline in MBH LE8E
concentrations if the treatments commenced at the time cf
castration (p < 0.05). Two weeks after castration, however,
only T was effective in stimulating LHEH accumulation in the
MBH.
Levels of LHEH in the AH-POA cf castrated rats treated
with 1 or morphine were unaffected by any experimental
treatment (data not shown).

62
^Control
HTestosterone
Short Term Long Term
Castrate Castrate
Figure 1: Serum LH concentrations in rats treated with
morphine or T at the time of castration or two
weeks after castration.
Short-term castrate = treatment started
immediately after orchidectcmy. long-term
castrate = treatment started two weeks post-
castraticn. * p denotes < C.05 vs. control. LH
concentrations among the control groups were not
significantly different and were therefore
pooled.

63
â–¡ Control
N Testosterone
Castration Castration
Figure 2: MBH LHRH concertraticns in rats treated with
morphine or T at the time of castration or two
weeks after castration
Short-term castration = treatment started
immediately after orchidectcny. long-term
castration = treatment started two weeks post-
castraticn. * p denotes < 0.05 vs. control. HEH
LHBH concentrations among the control groups were
not significantly different and were therefore
pooled.

64
The Effects of Chronic Morphine Treatment on the LB
Secretory and MBH LBSH Responses to Graded Ceses of 1.
Figure 3 illustrates serum T levels attained from various
sized implants in rats castrated fer two weeks treated
additionally with either morphine or placebo pellets. Lew
serum levels of T (<100 pg/al) were detected in morphine or
placebo-treated rats receiving only sham implants. There was
a progressive increase in serum T levels as the size cf the
implant increased in both morphine and placebo-treated
groups (p < 0.05). Chronic morphine treatment did not affect
serum T levels attained by any of these implants when
compared to placebo-treated groups,
Serum Lfi concentrations in rats treated with morphine or
placebo pellets in combination with graded doses of T are
also shown in Figure 3. In placebo-treated rats, lew
circulating levels cf T (355 ± 20 pg/ml, 2.5 mm implant)
caused a slight but nonsignificant increase in serum LH.
Further increases in serum T levels produced a decrease in
serum LH secretion, with significant suppression of LH
levels seen at 1.18 ± 0.07 ng/ml of T (10 mm implant). As
observed in Experiment 1, morphine treatment alone did net
significantly change serum LH levels in castrated male rats.
However, it influenced the LH response to T treatment. In
rats treated with beth morphine and T, the LH response curve
to T shifted to the left such that a 50S suppression cf

65
serum LH was observed with the 2.5 mm T implant aud nearly
complete suppression of serum LH was seen with 5 mm T
implants.
To further evaluate the interaction between morphine and
T, the data from this experiment were grouped according to
different levels (Figure 4):
1. less than 199 pg/ml, representing ineffective
implants, or T levels found in sham-implanted
(castrated) animals;
2. between 200 and 499 pg/al, representing T levels
which have no effect on 1H or IHBH concentrations;
3. between 500 and 999 pg/al, representing T levels
which stimulate LHBH accumulation in the MEH tut have
little effect on LH secretion; and
4. greater than 1000 pg/al, representing T levels which
consistently suppress LH secretion (E. Kalra and
Kalra, 1982).
As evident in both placebo and morphine-treated rats, IH
levels declined progressively as a function of serum I
levels. However the T-induced reduction of LH concentrations
was greatly enhanced in morphine-treated rats (p < 0.05).
The LH response curve to graded doses of T was shifted to
the left in morphine-treated rats with maximal inhibiticn
occuring at 622 ± 44 pg/ml. In some of these morphine-
treated rats, near baseline LH levels were fcund with T
concentrations as low as 420 pg/ml serum. In contrast. tut

66
in agreement with previous studies (P. Kalra and Kalra,
1982), significant depression of serum IH concentrations was
only apparent with T concentrations cf greater than 600
pg/ml. Serum T concentrations necessary tc elicit a 5 0??
reduction in LH secretion were 300 pg/ml in morphine-treated
rats and 960 pg/ml in placebc-treated rats.
The data from Experiment 2 were further subjected to
regression analysis using the logarithm cf serum IH
concentration as a dependent variable, placebo or mcrphine
treatment as an independent variable and the logarithm cf
serum T concentration as a covariant. The resultant
regression model was found to be highly significant (p <
0.C01) as were the drug treatment (placebo or morphine ), T
treatment, and the interaction of drug with T (p < 0,01, for
each).
The effects of simultaneous T plus chrcnic mcrphine
treatment on LHRH concentrations in the MBH are shewn in
Figure 5. In placebo-implanted rats, T exposure for 4 days
resulted in an accumulation cf LHBH in the MBH (p < 0.05).
As has been noted previously, this accumulation cf LBBH in
the MBH occured at T levels lower than that reguired tc
inhibit LH secretion (5 mm T implants, Figure 5 vs. 10 mm
implants. Figure 4; P. Kalra and Kalra, 1982), It can also
been seen from Figure 5 that in the presence cf morphine, T
was unable to cause any significant increases in LHBE stores
in the MEH

Serum LH (ng/ml)
67
5 mm T
10 mm T
implants
implants
Castrate
2.5 mm T
levels
implants
T 1 1 1 1 1—
o 200 400 600 800 1000
Serum Testosterone (pg/ml)
Figure 3: The effects of graded doses of T produced by
various sizes of T implants on serum T and IH in
morphine-treated and placebo-treated male rats
castrated for two weeks
The vertical and horizontal tars represent mean ±
standard error for serum LH and T concentrations,
respectively. Numbers in parentheses represent
the number of rats in each treatment group. *
denotes p < 0.01 vs. sham-implanted control
group; the dagger symbol denotes p < 0.05 vs.
placebo-implanted group at the same dose of T.

68
Serum Testosterone (pg/ml)
Figure 4: Relationship between LH and T levels in morphine-
treated and placebo-treated male rats castrated
for two weeks
In the interest of clarity in presenting the
relationship between the LH response and 1
levels, the rats were blocked into 4 groups
according to serum T levels; < 199 pg/ml, between
200 and 499 pg/ml, 500 and 999 pg/ml, or > 1000
pg/ml. Number in parentheses represent the number
of rats in each T level. The vertical and
horizontal error tars represent mean ± standard
error for the IH and T concentrations,
respectively. * denotes p < 0.01 vs. I < 199
pg/ml group; the dagger symbol denotes p < 0.05
vs. placebo-implanted group at the same T level.

LHRH
(% of Placebo - Sham)
69
150-
100-
50-
*
Implant Size
â–¡ 0.0 mm (Sham)
02.5 mm
â–  5.0 mm
â–  10.0 mm
Placebo Morphine
Figure 5: The effects of various sized T implants on HBE
LHBH concentrations in morphine-treated and
placebo-treated male rats castrated for two
weeks.
LHRH concentrations were determined per HBH
tissue section and expressed relative to sham-
implanted HBH LHRH (control) concentrations. ♦
denotes p < 0.C5 vs. sham-implanted grcup.

70
Effects of T aad Morphine Treatment cn the Pituitary
Resrcnsivecess to LHRH
Testosterone levels achieved ty the 5 mm implants were
similar in placebo and morphine-treated rats. Once again
this T treatment failed to reduce serum LH ccncentraticns in
placebo treated rats but together with morphine pellets
reduced serum LH concentrations to baseline levels (Talle 1,
legend, p < 0,05)
The effects of the in vivo T or morphine plus I treatment
on the in vitro release of LH fret pituitary incubations are
also shown in Table 1. Incubation of pituitarios for cne
hour with 1 X 10~7 M. LHEH significantly increased LH
concentrations to levels above that seen in control medium
(p < 0.05). However, neither T cr morphine plus T treatment
in vivo significantly altered the baseline or the LEBE
stimulated levels of IH release,

71
TABLE 1
Effects of In Vivo Morphine and T Pretreatment on In Vitro
LH Release iron Hemisectioned Fituitaries (IE release rate:
ng/mg pituitary tissue/ hour)
In vitro
treatment
In vivo treataent
Control T Morphine + T
Control 1,583 ± 313 957 ± 51 1,393 i 166
1 X 10-7 a. LHRH 3, 930 ± 555 3,682 ± 27 5 4,588 ± 285
delta-LH 2,346 1 666 2,612 ± 362 3,194 ± 425
Serum I levels achieved by the 5 mm T implants were 683 i 89
ng/ml for morphine-implanted and 636 ± 73 pg/ml for
placebo-treated rats. Serum IH concentrations were
225 ± 33 ng/ml in castrated (placebo plus sham treated) rats,
227 ± 61 ng/ml in 5 uimT implanted rats, and 16 ± 1 ng/ml in
5 mm I plus morphine treated rats.
Discussion
These studies reveal a putative underlying interaction
between opiates and T on LH release. As in the case cf T
implants, placement of morphine pellets immediately after
castration prevented the post-castraticn rise in serum IH
and the decline in IHGH concentrations. This extends the
observations of previous studies (Cicero et al., 1980; Van
Vuyt et al., 1982). However, in contrast to the expected
suppression of LH release and stimulation cf LERE
concentrations in the MBH after T implantation, LH and its

72
releasing factor were unaffected by morphine treatment
initiated two weeks after castration. Apparantly, cpiate
receptor stimulation does not mimic the actions of 1 on the
hypothalamic-pituitary-LH axis under all circumstances as
has been suggested (Cicero et al., 1980; Van Vugt et al.,
1982).
The inability of morphine pellets to suppress IH release
in rats which had been castrated for two weeks is surprising
in view of the observation that administration of morphine
or opioids systemically or ECf intraventricularly promptly
suppressed LH release in gonadectcaized rats (Cicero et al.,
1980; Kinoshita et al., 1981; Leadem and Kalra; 1983). It
is quite possible that a similar decrease in LH release may
occur soon after morphine pellets are placed in two-week
castrated rats. Accordingly then, this IH suppression must
be transient because with sustained supply of morphine these
rats appear to overcome the inhibition and LH secretion
seemingly occured unaiated 4 to 7 days later. While this may
be a plausible explanation for the absence of LH suppression
in long-term castrated rats, it should be noted that acutely
orchidectomized rats were unable to override the effects of
sustained morphine supply. This and previous reports of a
differential LH response to opioid administration which is
dependent upon the post-castration interval is intriguing
(Cicero et al. , 1 982a; Bhanot and Wilkinson, 1983). The
ability of testosterone to inhibit LH release diminishes

73
with tine after castration (Cicero et al., 1S82a). It is
possible that similar mechanisms may underlie the loss in
effectiveness of both androgens and cpicids.
In addition to the findings that in long-term castrated
rats morphine is either ineffective cr its suppressive
effects dissipate rapidly, the action of morphine appears to
manifest itself in a different form. This is shewn by the
observation that low concentrations of T, while failing to
exert any impact on LH release cn their cwn, were highly
effective in suppressing LH release in morphine-treated
rats. The LH response curve to graded dcses of T was shifted
to the left (Figures 3 and 4) in morphine-treated rats with
maximal inhibition cccuring at T concentrations of 622 1 44
pg/ml serum. In some morphine-treated rats, near baseline LH
levels were seen with T concentrations as low as 42C pg/ml.
Furthermore, it appears that serum T levels needed to
achieve a 50X reduction in serum LH levels were three times
lower in morphine-treated rats than ir control rats.
Evidently morphine treatment concurrently with T rendered
rats mere responsive towards T feedback action. T has been
shown to decrease pituitary responsiveness to LHEH (Drouin
and Labrie, 1976; P, Kalra and Kalra, 1980). It is pcssitle
that morphine may interact synergistically with T at the
level of gonadotropes to suppress pituitary responsiveness
to endogenous LHRH stimulation and thereby produce a marked
decrease in serum LH levels. However, as shown by the dose

74
employed in the Experiment 3 and previous studies (Cicero et
al., 1977; Wiesner et al., 1984), there was no evidence of
modification by morphine of LHBH action at the pituitary
level. Thus, one can assume that morphine acts at higher
centers, possibly at the preoptico-tuberal pathway where the
distribution of androgen concentrating, EOF, and 1H£H-
producing neurons and opiate receptors overlap (Sarr and
Stumpf, 1975; Watson et al., 198C; S. Kalra, 1981; Shivers
et al., 1983b).
Precisely how T and chronic morphine interact to inhibit
Lfl secretion is not known. The apparent inability of
morphine to reduce IH secretion after two weeks of
castration would suggest a T requirement for this effect of
morphine. It is possible that after continuous morphine
exposure, neuronal systems regulating LHRH release may be
more responsive to 1, so that extremely low serum T titers
can suppress LH release. Considering the ability cf chrcnic
morphine to block the accumulation of LHBE in the HBH
following exposure, it is possible that chronic morphine
suppressed the activity of the IHRH neuron at several steps
in the secretory process.
While the LHRH neuron may be a likely site for the
interaction between T and morphine, ether explanations are
possible. Earlier work has suggested that gonadal steroids
may modify brain opiate receptors but this possibility has
been disputed (Hahn and Fishman,
1979; Cicero et al.,

75
1983a). The possibility remains, however, that the
intracellular processing of the opioid signal reguires the
presence of androgens. Also other neurotransmitters, such
as NE, have been shown to interact with the opiates in
effecting LH secretion (S. Kalra and Simpkins, 1981) . The
potential involvement of monoamines in mediating the
interaction between morphine and T will be explored in the
following chapter.

CHAPTER V
THE INFLUENCE OF CHRONIC KOREHINE TREATMENT CN THE NEGATIVE
FEEDEACK REGULATION CF GONADCIECPIN SECRETION BY GONADAL
STEROIDS
Introduction
The negative feedback effects of testicular hormones on
gonadotropin secretion appear to be exerted at the level of
the hypothalamus and the pituitary (Drouin and Lafcrie, 1576;
Franchimont et al., 1979; S, Kalra and Kalra, 1983). In the
male rat, three major gonadal steroids, T, DHT and E2 have
been implicated in the feedback regulation of LH secretion,
and to a lesser extent, FSH secretion (P. Kalra and Kalra,
1980; D'Agata et al., 1981; Sherins et al., 1982; McCann et
al., 1983; Nishihara and Takahashi, 1983). ¡¡hile E2 and CUT
can be formed intracellularly from T in many neuroendocrine
tissues, all three of these gonadal steroids are present in
the circulation in sufficient concentrations to influence IH
secretion (Massa et al., 1972; Kaftolin et al., 1975; P.
Kalra and alra, 1977, 1980, 1981, 1982).
While the neuroendocrine substrates which mediate the
feedback effects of steroids on gonadotropin are not known,
it is interesting that a close anatomical relationship
exists between steroid concentrating, LHBE and EOP-
containing neurons (McEwen et al., 1979; Watson et al.,
7€

77
1980; Shivers et al., 1983b), ECP neuronal systems have teen
implicated in the central regulation of LH secretion, and a
considerable amount of pharmacologic evidence suggests that
EOP play a role in modulating the negative feedback effects
of gonadal steroids on LH release in the male (Cicero et
al., 1980; Van Vugt et al„, 1982). As was seen in Chapter
IV, chronic opiate receptor stimulation with morphine, while
ineffective in inhibiting LH release cn its cwn, enhanced ty
3-fold the negative feedback effects cf 1. The present study
extends these observations by comparing the effects on LH
and FSH secretion cf T, DHT and E2 in male rats treated
chronically with morphine.
Experimental
Chronic Morphine and Gonadal Steroid Treatments
Groups of placebo or mcrphine-treated rats were
simultaneously exposed to either sham implants or one cf the
three gonadal steroids at various dosages. All treatments
lasted for 4 days, after which animals were sacrificed by
decapitation between 1100 h aud 1300 h. Serum from trunk
blood was stored at -20° C. for subseguent analysis cf IH
and FSH by BIA. The stercid treatments were;
1. 5 mm tubes packed with crystalline T;
. 7,5 mm tubes packed with crystalline EHT or CHT which
had been diluted with cholesterol on a weight;weight
ratio of 1:1 or 1:3; and
2

78
3. E2 dissolved into sesame seed cil at a concentration
of 300 micrograms/ml and filled into 7.5 am tubes or
£2 diluted in oil to concentrations of 150 or 75
micrograms/ml and then placed into tubes of 5.0 mu in
len th.
Evaluation of Pituitary Responsiveness to LHEB
Based on the results of the first series of experiments,
groups of placebo or chrcnic morphine treated rats were
simultaneously exposed to either sham, 5 mm T, 7.5 mm CET or
7.5 ma E2 (300 micrcgrams/al) implants. After 4 days cf the
above treatments, rats then received a single injection cf
LHHH (100 ng/100 g B.W., s.c.). Blood samples were obtained
by cardiac puncture under light ether anesthesia, prior to,
and 30 minutes after LHRH injection. This dose of LHEH was
based on earlier work (Lu et al., 1980). Serum was
separated by centrifugation and stored at -20° C. for
analysis of LH by RIA,
Results
The effects of simultaneous morphine plus T
administration are shown in Figure 6. While 5 mm T implants
alone reduced serum LH concentrations by greater than 40X,
this effect was not significant. Chronic morphine treataent
alone did not affect serum LH levels in castrated rats.
mm
However, as was noted in Chapter IV, the combination of 5

79
T plus morphine treatment reduced serum LH ccncentraticns by
90% to levels seen in intact male rats (p < 0.05; P, Kalra
and Kalra, 1977fc).
The consequences of chronic morphine exposure on serum IH
and FSH levels in castrated rats concurrently exposed tc
various dosages of E2 are shewn in Figures 7 and 8,
respectively. Chronic morphine did not alter serum LE
concentrations in animals receiving sham implants.
Similarly, E2 treatment alone failed tc significantly reduce
LH levels. In contrast, the ccabinaticn cf morphine
treatment plus 5 mm E2 (150 micrograas/ml) or 7.5 mm E2 (300
micrograms/ml oil) reduced serum LH ccncentraticns (p <
0.05). The highest E2 dosage (7.5 am at 30C micrcgrams/m1)
alone produced a non-significant 25% reduction in LH
concentrations, while the combination cf morphine plus the
same E2 dosage caused a greater than 75% reduction in serum
LH levels p < 0.05).
A significant effect of chronic morphine on the response
of FSH to E2 was observed. Although E2 alone did not reduce
serum FSH concentrations at any of the doses evaluated, the
combination of 7.5 mm E2 (300 micrograms/xl) plus merphine
significantly reduced FSH levels by 30% relative to sham-
implanted rats and 18% relative to placebo-treated rats at
the sane E2 dosage (p < 0.05) .
The effects of chronic morphine administration on the
response of LH and FSH to various dosages of DHT in

80
Figure 6: The effects of simultaneous morphine and 5 mm T
implants on LH secretion in rats which had teen
orchidectomized two weeks previously
* denotes p <0.05 compared tc shan-implanted
rats; the dagger symbol denotes p < 0.05 when
compared to placebo-implanted rats.

81
600-
E
O)
c 400-
§j 200-
T
X
HH
in
¡§i
fiSsssi
Wm
Mi
SiiSW;
í^'já^íVfi
mm
mam
EflSsM
H
ɧÉ
üi.
SHAM
IMPLANT
I
Sip
533S&4;
• s
^-4ii
?M$r
ws <*V-:
5mmE2 5mm E2
(75jug/ml) (I50>jg/ml)
Placebo
Morphine
7.5mm E2
(300jug/ml)
Figure 7: The effects of simultaneous treatment with
morphine and various doses of E2 on LH secretion
in rats which had been orchidectomized two weeks
previously.
E2 was dissolved in oil at the concentrations in
parenthesis and filled into tubes of the lengths
noted in the figure. IH was determined using the
LH-BP-2 reference standard and expressed relative
to LH-BP-1 (LH-BP-1 = 61 X Lfl-BP-2). * denotes p
< 0.05 vs. sham-implanted rats; the dagger symbol
denotes p < 0.05 vs. placebo-implanted rats at
the same E2 dose.

82
Figure 8: The effects of simultaneous treatment with
morphine and various deses cf E2 cn FSH secretion
in rats which had been orchidectomized twc weeks
previously
E2 was dissolved into oil and filled into tutes
of lengths as noted in the figure. * denotes p <
0.05 vs. sham-implanted rats; the dagger symbol
denotes p < 0.05 vs. placeto-implanted rats at
the same E2 dose.

83
castrated rats are shown in Figures 9 and 10, respectively.
As previously shown in Figures 6 and 7, chronic morphine
treatment was without effect on serum LH levels in sham-
implanted rats. The implantation of DHT alone (7.5 mm DHT
crystals) significantly reduced serum 1H concentrations ty
63*, while the combination of chronic morphine plus EHT (at
1:1) or 7.5 mm DHT reduced IH levels 39* and 83*. The
combination of morphine plus DHT was not significantly mere
effective in inhibiting LH levels than DHT alone at any DHT
dosage, however. Chronic morphine and DHT had variable
effects on FSH levels. Morphine treatment caused a slight
elevatior (16*) in serum FSH in sham-implanted rats, in
contrast to similarly exposed animals shewn in Figure 8.
Treatment with DHT alone was ineffective in inhibiting FSH
at any dose used; however, 7.5 mm DHT (at 1:1) caused a
slight increase in serum FSH levels (9*, p < 0.05). Morphine
treatment with 7,5 mm DHT reduced serum FSH levels 43*. This
reduction in serum FSH concentrations was significant
relative to sham-implanted rats and to placebo-impanted rats
at the same DHT dosage.
The effects of combinations of chronic morphine plus
steroid treatments cn the in vivo LH secretory response to
LHEH injection are shown in Table 2. Prior to LHEH
administration, the various morphine plus steroid treatments
produced similar effects cn LH levels as was seen in Figures
6, 7 and 9. Chronic morphine treatment was without effect

84
Figure 9: The effects of simultaneous treatment with
morphine and various deses cf DHT on LB secretion
in rats which had been orchidectcmized twc weeks
pre viously
Crystalline DHT or DHT which had been diluted cn
a weight:weight basis with cholesterol was packed
into tubes 7.5 mm in lengths as ncted in the
figure. Serum 1H was determined using the 1E-BP-2
reference standard and expressed relative tc IH-
RP-1. (LH-RP-1 = 61 X IH-8P-2). * denotes p <
0.05 vs. sham-implanted rats; the dagger symbol
denotes p < 0.05 vs. placebo-implanted rats at
the same DHT dcse.

85
Figure 10; The effects of simultaneous treatment with
morphine and various doses cf DHT on FSH
secretion in rats which had teen castrated two
weeks previously.
Crystalline DHT or DHT which had been diluted on
a weight:weight ratic with chclestercl was
packed into tubes 7.5 mm in length as noted in
the figure. * denotes p < C.05 vs. sham-
implanted rats; the dagger symbol denotes p <
0.05 vs. placebo-implanted rats at the sane EHT
dose.

86
on serum LH levels in sham-implanted rats prior tc LHEH
injection. When compared to shan-implanted rats, 5 mm 1 was
without effect, while 7.5 mm E2 (300 micrograms/ml oil) and
7.5 mm DHT reduced serum LH levels by 57* and 69*,
respectively (p < 0.05). As expected, in these stercid-
treated rats, opiate receptor stimulation with morphine
further reduced LH concentrations prior to LHRH injection.
However, in 7.5 bit DHT implanted rats this additional
reduction was not significant.
LHRH injection stimulated LH release in all 8 treatment
groups (p 0.01). When compared tc placebo plus sham
implanted (castrate) controls, T exposure did not alter the
pituitary response to the decapeptide. Additionally,
although the combination of morphine plus T reduced LH
concentrations before LHRH injection, this combination did
not alter the pituitary response to LHRH. In rats treated
with E2 alone, pituitary responsiveness to LHBE was
increased significantly. Despite a reduction in LH
concentrations prior to IHRH injection in morphine plus
E2-implanted rats, the LH secretory response tc LHRH was
further enhanced. Finally, DHT alone diminished the
responsiveness of the pituitary tc LHRH and this reduction
in sensitivity was not modified by morphine exposure.

87
TABLE 2
The Effects of LHRH (100 ng/1C0 g B.R., s.c.) on serum IH
Levels in Castrated Bats Treated Chronically with Morphine
and/or Gonadal Steroids.
Pre-LHRH Post-LHBH delta-LHBH
LH (ng/Bil serum)
Placebo - Implanted Group:
Sham Implant
832
±
10S
2C49
±
122
1213
±
67
5 mm 1
622
±
176
1653
i
298
1 140
±
323
7.5 mm E2
355
±
791
2110
±
256
1732
±
195»
7.5 am DHT
253
±
1 1 31
1091
i
1971
835
t
1461
Morphine - Implan
ted
Group:
Sham Implant
683
±
54
2135
±
176
1451
±
183
5 mm T
162
±
751 2
1421
±
134
1250
±
91
7,5 mm E2
122
±
181 2
3281
±
4331 2
3 147
±
439» 2
7.5 mm DHT
89
±
181
965
±
143»
854
±
146»
E2 was dissolved in oil at a concentration of 300 micrcgrams/ml
and filled into tubes 7,5 mm in length. LH was determined
using the LH-HP-2 reference standard and expressed relative
to LH-HP-1 (LH-RP-1 = 61 X LH-BP-2) . » denotes p < 0,05 vs,
sham-implanted rats. 2 denotes p < 0.05 vs. placebo-implanted
rats within the same steroid treatment group.

88
Discussion
The results of the present study confirm the findings
presented in Chapter IV that, while ineffective on its own,
chronic morphine treatment greatly enhances the feedback
inhibition by T of LH secretion in rats which had been
castrated two weeks previously. This observation is extended
by the finding that chronic morphine also enhances the
negative feedback effects of E2 and DHT on LH and FSH
secretion. Since stimulation of opiate receptors by ECF
should similarly affect the gcnadctrcpin secretory
mechanism, these data argue for a major rcle of ECE-
containing neurons in regulating the sensitivity of the
hypothalamus to circulating gonadal steroids.
When initiated at the time of castration, chronic
morphine can block the subsequent rise in serum IH
concentrations (Chapter IV; Cicero et al., 1980; Van Vugt et
al., 1982). However, chronic opiate receptor stimulation
with morphine is unable to suppress gonadotropin levels in
rats castrated two weeks previously (Figures 1, 3, 4, and 6
- 10). This castration-induced loss in the ability of
opioids to inhibit LH secretion cannot be satisfactorily
explained by alterations in hypothalasic EOF levels (lee et
al,, 1980) or the number of opioid binding sites in the
brain (Cicero et al,, 1983a), Bather, this influence of
castration on the response of LH to ECP or morphine would

89
appear to result from a synergistic action between gonadal
steroids and opioids {Chapters IV and V; Cicero et al.,
1982, Ehanot and Wilkinson, 1983). Since mcrphine enhances
the negative feedback sensitivity of gonadal steroids cn
gonadotropins, the effectiveness of morphine in suppressing
LH soon after castration likely results from the persistence
of gonadal steroids in the circulation, or the gradual loss
of some steroid-dependent process.
The effects of morphine on gonadotropin secretion appear
to be mediated centrally, since the present results (Tables
1 and 2) and in vivo and in vitro studies indicate that
morphine and EOP do net act at the pituitary to inhibit IH
secretion (Cicero, 1980b; Weiscer et al., 1984). In
contrast, it is well documented that gonadal steroids can
act locally at the gonadotropes to alter their sensitivity
to LHHH (Drouin and labrie, 1976), At the dosages
administered, variable effects cf T, DHI and E2 on the IH
secretory response to IHBH were noted. These dosages were
chosen because they had little effect cn LH secretion when
administered alone, but decreased LH release when
administered with morphine. The 5 mm T implant, which
provides low physiological circulating T levels cf arcund
70C pg/ml in castrated rats (Chapter IV; Eamassa et al.,
1976; P. Kalra et al., 1982), did not alter pituitary
responsiveness to LHHH. T has teen shown to decrease the
response of the pituitary to IHBH, but at much higher doses

90
than were employed in this experiment (Verjans et al., 1974;
P. Kalra and Kalra., 1980). Thus the interaction between
morphine and T in suppressing Ifi release is not due to their
individual or combined action cn the pituitary.
Many studies suggest that DHT is more potent on a molar
basis than T in reducing LH secretion (Verjans et al., 1974;
Martini et al., 1979) . This is in part due to a direct
action on gonadotropes (Verjans et al., 1974; Verjans and
Eik-Nes, 1976, 1977). The less dramatic interaction between
DHT and morphine in reducing IH secretion may be due to the
overriding effects of DHT cn LHEH sensitivity.
Nevertheless, the combined effects of DHT on pituitary
responsiveness to LHHH and its interaction with opicids in
suppressing LH release, likely contribute to the ability of
DHT to inhibit LH release at concentrations lower than the
physiological range ( < 140 pg/ml; Coyotupa et al., 1974;
Saksena et al., 1978; Saksena and Lau, 1979; P, Kalra and
Kalra, 1980).
E2 treatment increased the LH secretory response to LHKH,
as has been demonstrated previously (Verjans and Eik-Nes,
1976; P. Kalra and Kalra; 1980). Since at the dosages used,
E2 alone did not modify serum LH levels, the alterations in
pituitary responsiveness are likely associated with reduced
LHHH release from the hypothalamus. In combination with
morphine, E2 substantially reduced serum LH concentrations
despite a marked increase in the ability of the pituitary to

91
respond to LHUH. Thus, under simultaneous treatment with
this steroid and morphine, release cf LHfiH from the
hypothalamus must he decreased to an extent greater than
would fce anticipated on the basis cf serum LH values alone.
The doses of E2 administered have been shown to produce
serum E2 levels in the range cf 15 to 30 pg/ml. Since E2
levels in male rats have been variously reported to range
from 10 to 50 pg/ml (Ewing et al,, 1977; Saksena et al.,
1978; Saksena and Lau 1979; Keel and Abney, 1980; E. Kalra
and Kalra 1981; Nishihara and Takahashi, 1983), alterations
in EGP neuronal activity within the hypothalamus could
interact with circulating E2 to physiologically modify IHBH
release from the MBH.
The effects of LfiBfl, gonadal steroids and opicids on FSH
secretion are, in general, less pronounced than their
effects cn LH release (Mahesh et al., 1375; Sherins et al,,
1 982; McCann et al., 1983). Similarly, the present data
show that the gonadal steroid-cpioid interaction is less
effective in suppressing FSH relative to LH release. Factors
other than LHHH and gonadal steroids are operative in the
regulation of FSH secretion (Steinberger and Steinfcerger,
1976; McCann et al., 1983; Mizunuma et al., 1983, Lumpkin et
al., 1964). This may have contributed tc the present
failure to document a synergistic interaction between
gonadal steroids and morphine in suppressing FSH secretion.
This relative refractoriness cf the FSH secretory mechanism

92
te chronic morphine with or without concurrent gonadal
steroid treatment, is consistent with previous experiments
which have used acute exposure to opiates {Eruni et al.,
1977; Eelitalia et al., 1981; Grossman et al., 1981; Hemming
et al. , 1982) .) .
All thr e of the gonadal steroids evaluated could te
involved in the regulation of LH secretion (P. Kalra and
Kalra, 1980, 1981, 1982; Nishihara and Takahashi, 1983). E2
appeared to interact most potently with morphine to inhibit
LH release. In contrast, DHT did not show a substantial
interaction with morphine and appeared to exert its primary
effect directly on the pituitary gland. This existance of a
hypothalamic feedback modulator for E2, in preference to
DHT, argues in favor of the aromatization of T to E2 or a
direct effect of circulating E2 on the hypothalamus as the
major steroid feedback mechanism regulating LHRH release in
the male rat. Additionally, a direct pituitary effect of
DHT to suppress LHRH responsiveness could further diminish
LH secretion. Thus, T, E2, and DHT may act in concert at
separate sites to achieve basal serum LH levels seen in male
rats.
The major finding of the present chapter is that chronic
morphine treatment may simulate the effects of activation of
opioid receptors by EOP and hence indicate a mechanism by
which EOP can interact with gonadal steroids to regulate LH
release. The low, physiological levels achieved by these

93
implants (P, Kalra and Kalra, 1980, 1981, 1982), indicates
that such an interaction may be an important mechanism by
which the sensitivity of the hypothalamus to steroids is
regulated. It is of interest that, in the rat, steroid
hormones can alter beta-endorphin concentrations (Sardlaw et
al., 1982), and in the monkey, the release of ECP is clearly
steroid dependent (Ferin et al., 1984)• By modifying
activity in one or more EOP neuronal systems, steroid
hormones could alter the sensitivity of the hypothalamus to
their own negative feedback effects.

CHAPTER VI
THE EfFECTS OF CHHCNIC MORPHINE AND TESTOSTERONE TREATMENT
ON CATECHOLAMINE AN I INDOLAMINE METABOLISM AND GO NADCTECEIN
SECRETION IN MALE EATS
Introduction
The release of the gonadotropins, LH and FSH, appears to
be under the primary stimulatory influence cf LHRE and
possibly a distinct FSH releasing factor (S. Kalra and
Kalra, 1983; McCann et al., 1983). In the male, the
feedback actions of gonadal steroids icdulate the release of
LHRH from the hypothalamus, as well as the response of the
pituitary to LIIRH (Ercuin and Lalrie, 1976; S. Kalia and
Kalra, 1983). The elucidation of the neurochemical
substrates responsible fcr LHRH output frcm the hypcthalamus
and the negative feedback effects of gcnadal steroids has
been a subject of investigation for several decades, Two
neuronal systems which appear to be intimately involved in
the release of LHRH are the catecholamines (P. Kalra et al.,
1972) and the EOP-ccntaining neurcns (Meites et al., 1979;
Cicero, 1980b; S. Kalra et al., 198C). Eoth cf these
neuronal systems have been implicated in the regulation cf
gonadal steroid feedback in the male (Cicerc et al., 1980;
Van Vugt et al., 1982). As was seen in the previous two
chapters, chronic morphine treatment, while unable tc
94

95
inhibit LH secretion cn its own, enhances the ability of T
to inhibit LH release in rats castrated for two weeks.
Several studies indicate that the opiates interact with
catecholamines and indolamines to effect IH secretion
(Rotsztejn et al,, 1978; Ieiri et al. , 1980a; S. Kalra and
Simpkins, 1981). In this chapter the metabolism of CA, NE
and 5HT was evaluated in castrated male rats treated with
morphine and T.
Experimental
Male rats which had been castrated two weeks previously
were treated with chronic morphine and 1 as described in the
General Materials and Methods section of this dissertation.
The treatment groups were: sham plus placebo treatment
(castrated rats), 5 mm T, chronic M, or morphine plus 5 mm T
treatments. For comparison, an additional group cf gonadal
intact rats and castrated rats receiving replacement levels
of T (30 mm T implants, approximately 2 ng/ml, Chapter IV)
were given sham plus placebo treatments. All treatments
lasted for 4 days, after which bleed was collected by
decapitation for LH and FSH analysis by RIA and brain tissue
containing the MBH and POA-AH tissue fragments were analyzed
for catecholamines, indolamines and their metabolites as
described in General Materials and Methods.

96
Results
Serum_IH and FSH
The effects of control, 5 bib T, morphine cr 5 mu I plus
morphine treatment cn LH secretion are presented in Figure
11. Analysis of variance revealed a significant effect of
both morphine and 5 mm T treatment (p < 0.05) as well as a
highly significant effect of morphine plus 1 treatment (p
<0.01), Morphine treatment alone was without effect when
compared to castrated rats (sham plus placebo treatment),
while 5 mm T treatment resulted in a slight, nonsignificant
lowering of LH levels. The combination cf morphine plus 5 mm
T resulted in a 65X reduction in serum LH levels when
compared to castrated rats (p < 0.05). Although treatment of
castrated rats with the morphine and 5 am T implants did not
return LH levels to those found in gcnadal intact animals,
treatment with 30 mm T implants alcne resulted in LH levels
lower than those found in intact male rats (figure legend, p
<0.05) .
Serum from the same samples were assayed for FSH, the
results of which are depicted in Figure 12, Both mcrphine
and 5 mm T, when administered alone were ineffective in
altering serum FSH concentrations in comparison to castrated
male rats (sham plus placebo treatment). The combination cf
5 mm 1 with morphine reduced FSH levels 3951 compared tc
castrated rats (p < 0,05). While FSH levels in castrated
rats receiving 5 mm T plus mcrphine implants or 30 mm T

97
O- 600
C£
I
X
? 400
a 200
LÜ
(J)
T
Placebo
Morphine
*
i
SHAM IMPLANT
5mm T
Figure 11: Interaction between morphine and T on Lfl
secretion in rats which were orchidectomized two
weeks previously.
LH concentrations in castrated rats receiving 5
mm T plus morphine treatment were significantly
greater than levels seen in castrated rats
receiving 30 mm T implants (58 ± 2.6 ng/ml) cr
in gonadal intact rats (78 ±4.6 ng/ml). IH
values were determined using the LH-EE-2
reference preparation and were expressed
relative to the LH-BP-1 reference standard (IH-
HP-1 = 61 X IH-RE-2), * denotes p < 0.05 vs.
castrated rats receiving sham implants.

98
treatment were similar, both groups were signi
elevated when compared to gcnadal intact rats
legend) .
ficantly
(figure

99
X
fe
5
3
tr
LU
(f)
30 -
c\i
£L
o:
I
X
en
b> 20
c
10 -
JL
JL
i
j Placebo
n Morphine
*
i
SHAM IMPLANT
5mm T
Figure 12: Interaction between morphine and I cn FSH
secretion in rats which had been castrated two
weeks previously.
Serum FSH concentrations in rats receiving 5 mm
T plus morphine implants were similar to FSH
levels in 30 aim 1 implanted rats (15.7 ± C.7
ng/ml) , but both groups were significantly
greater than FSH levels in gonadal intact rats
(8.4 ± 0,4 ng/ntl). * denotes p < 0,05 vs.
castrated rats receiving sham implants.

100
K|_ana_NME
In the MBH of castrated rats, NE concentrations were
elevated when compared to gonadal intact male rats or
castrated rats receiving 30 mm 1 implants (Figure 13, p <
0.05), Treatment of castrated rats with 5 mm T or morphine
alone significantly reduced NE concentraticns. The
combination of morphine plus T, while reducing NE levels
compared to castrated rats, did not produce any additional
reduction in NE levels than T or morphine alcne. There were
no significant differences in NME levels in the MEH tetween
any of the treatment groups.
There were no differences in NE or NME concentrations in
the PC A-AH among any of the treatment groups.
DA and DOPAC
In the MBH of castrated rats, DA concentrations were
elevated in comparison to intact male rats or castrated rats
treated with 30 mm T implants (Figure 14, p < 0.C5).
Castrated male rats treated with 5 mm T alcne displayed
reduced DA concentrations in the MBH (p < 0.05). MEE DA
levels in rats receiving morphine alcne cr mcrphine with 5
mm T treatment were similar to DA concentrations in rats
given 5 mm T implants alcne. Mcrphine cr mcrphine plus T
treatment groups were not significantly lower than castrated
rats given control implants.

101
Levels of DOPAC in the MBH were also increased following
castration when compared to intact rats or castrated rats
given 30 mm T treatment (p < 0,05). khile both 5 mm 1 and
morphine alone reduced DOPAC levels relative to castrated
rats, this effect was only significant in rats given the
combination of morphine and 5 mm T.
Levels of DA and DCPAC were similarly affected in the
POA-AH. Two weeks after castration DA levels were increased
relative to both intact rats and castrated rats given 3C mm
T treatment, while DOPAC levels were increased when compared
to intact controls (p < 0.05). The treatment of castrated
rats with 5 mm T implants did not reduce DA or DOPAC levels
significantly, but morphine given alone or in combination
with 5 mm 1 did reduce DA levels in comparison to castrated
rats.
5HT 5HIAA and HVA
There were no significant effects of any treatment cn the
levels of 5HT, 5HIAA, or HVA in either the MBH or EOA-Afi
(Table 3).

102
Pigure 13: Concentrations of NE and NKE in the MEH and ECA-
AH of intact rats and orchidectcnized rats given
combinations of morphine and T.
The left-hand scale represents NE
concentrations, while the right-hand scale
represents NIUE concentrations, a - denotes p <
0,05 vs. intact male rats; fc - denotes p < 0.05
vs. castrated rats given 3C am T implants; c -
denotes p < 0,05 vs. castrated rats given
control treatments.
NME (ng/gm wet weight)

DA (ng/gm wet weight)
103
♦ M
Figure 14: Concentrations of EA and DCPAC in the MEH and
POA-AH cf intact rats and orchidectomized rats
given combinations of morphine and T
The left-hand scale represents EA
concentrations, while the right-hand scale,
which contains a fcreak, depicts DCPAC
concentrations, a - denotes p < 0.05 vs. gonadal
intact male rats; b - denotes p < 0.05 vs;
castrated rats given 30 mm 1 implants, c -
denotes p < 0.05 vs. castrated rats given
control implants.

104
TABLE 3
The Effects of Gonadal Steroids and Morphine Treatment cn
5HT 5HIAA and HVA Concentrations in the MEH and PCA,
MBH Region:
5HT 5HIAA HVA
(ng/gm wet tissue wt)
Intact
254
±
2
282
1
3
23
±
4
30 mm T
256
1
10
274
±
4
28
±
3
Castrate
252
±
6
275
±
9
26
±
3
5 mm T
255
±
4
289
i
2
31
±
3
M.
260
±
7
288
±
6
35
±
3
M. ♦ 5mm T
260
i
5
265
±
5
31
±
4
POA-AH Region:
Intact
252
±
5
309
±
9
44
±
5
30 mm T
265
±
5
309
±
9
43
±
3
Castrate
271
t
7
315
i
7
47
±
2
5 mm 1
267
±
5
314
±
7
43
±
3
M.
268
i
6
319
±
5
38
±
6
M» + 5 mm T
261
±
5
309
t
2
39
±
8
Castrated rats were given either 30 mm 1 implants, 5 mm T
implants, chronic morphine alone (M.) cr morphine plus 5 mm T
implants (i.e. M. e 5 mm T). Gonadal intact and castrated
rats were given sham plus placebo implants.

105
Discussion
The results of the present study demonstrate the
existence of a potent interaction between morphine and T in
the inhibitory feedtack regulaticn cf gcnadctropins. iihile
it appears that both morphine and T can alter catecholamine
metabolism, an interaction between the andrcgen and the
opiate cn catecholamine metabolism, similar to that
demonstrated for LH and FSH, was not found. Cn its own, the
5 mm T implant does not alter serum LH levels (Figures 1, 3,
4 and 6; P. Kalra and Kalra, 1982). These implants provide
circulating T levels of approximately 650 pg/ml and are
effective in restoring LHBH concentrations in the MBH and
median eminence to levels seen in intact male rats (Figures
3 and 5; P. Kalra and Kalra, 1982; F» Kalra et al., 1984).
Continuous morphine exposure dees net alter LH
concentrations in rats which were castrated two weeks
previously, in agreement with the castration-induced
attenuation in the inhibitory influence cf opiates on IH
secretion seen in the present work and in previous studies
(Figures 1 and 2; Cicero et al., 1982a; Ehanct and
Wilkinsor, 1983). As noted before, while each are
ineffective on their own, the combination of morphine plus 5
mm T potently inhibits LH release (Figures 3, 6 and 11).
This effect is apparently mediated centrally since the
effects cf morphine cannot be attributed to changes in the

106
response of the pituitary to LHEH or to morphine-induced
changes in T metabolism (Figure 3; Tables 1 and 2; Cicero,
1980b; Siesner et al. , 1984) .
The present study also demonstrates that the interaction
between morphine and T in inhibiting LH release is
generalized to include FSH release. This extends the
findings of the previous chapter that morphine in
combination with E2 or DHT can reduce FSH levels more
effectively than treatment with the steroid alone (Figures 8
and 10), The combination of 5 mm T and M, each ineffective
on their own, reduced FSH to levels seen in castrated rats
given 30 mm T implants. Morphine and EOP acutely inhibit LH
secretion (Meites et al,, 1979; Cicero, 1980b; Leaden and
Kalra, 1983), but are less effective in reducing FSH release
(Bruni et al. , 1977; Ieiri et al., 1980a; Lelitalia et al.,
1981; Grossman et al., 1981; Headings et al., 1982). Since
FSH has a longer half-life in plasma compared to LH (Cobel
et al., 1969), the present chronic morphine treatment
regimen may have contributed to the differences in this
study. The inability of 30 mm T implants to suppress FSH
levels in castrated rats to levels seen in gonadal intact
animals indicates that physiological replacement with T
(approximately 2 ng/ml serum) is not sufficient in returning
FSH to concentrations to basal levels, and supports the
existence of other gonadal factors which regulate FSE
release in addition to T (Steinberger and Steinberger, 1976;

107
Fraiichimont et al., 1979). It is not certain whether
treatment with morphine plus higher dcses of T than employed
here (eg. 15 mm or 10 mm T) would further reduce FSH
concentra tions.
Castration produced an increase in NE concentrations and
T replacement produced a dose-related decrease in NE levels.
Since a gonadal steroid reversible increase íe MBH NE levels
after gonadectomy has been noted in some (Eonoso et al. ,
1967) but not all studies (Simpkins et al., 1980), it is not
certain that this change in NE concentration reflects an
increase in NE turnover often noted in this region after
castration (Anton-lay and Siurtaan, 1968; Simpkins et al.,
1980). In the present study, NHE concentrations did net
change in concert with NE, suggesting that the observed
change in NE concentrations did net reflect NE release. In
this respect treatment with a 5 mm T implant or morphine
decreased MBH NE concentration without altering NME levels,
and the combination of 5 mm T with morphine did not further
changes NE or NME levels. Thus, it appears likely that the
interaction between gonadal steroids and morphine in
inhibiting LH release is not reflected by changes in NE
metabolism. This contrasts with experiments employing
noradrenergic acting agents which suggest that the effects
of opiates on LH release are mediated ty NE-containing
neurons (S. Kalra and Simpkins, 1981).

108
Interestingly, dopaminergic neurons in both the KBE and
POA-AH appeared to be stimulated by castration since levels
of both BA and DOPAC were increased two week after
castration. Further, T produced a dose-related decrease in
both BA and its major acid metabolite. The effects of T on
the incertohypothalamic BA system appears to be inhibitory
(Simpkins et al., 1980, 1983a). However the reported
effects of T replacement on KBH DA metabolism appears to
depend on the specific nuclear regicns sampled and the
methods used to estimate DA turnover (iuxe et al., 1978;
Simpkins et al., 1983a). Chronic morphine treatment reduced
activity in incertohypothalamic DA neurons but was
ineffective in altering DA metabolism in the MBH. Also, the
combination of morphine with T did net further reduce BA or
its metabolite more than the two administered alone. Thus,
the interaction between morphine and I in inhibiting LH and
FSH secretion is net clearly reflected by a change in BA
metabolism or activity. Using ether means of evaluating BA
neuronal systems it is still uncertain whether BA mediates
opioid effects on LH release (Bctsztejn et al., 1978;
Sirinathsinhji and Kartini, 1984).
Of the three neurotransmitters evaluated, 5BT was the
least responsive to any experimental treatment. Levels of
5HT and its acid metabolite, 5BIAA, were unaffected by
castration,? T treatment, or morphine alone and in
combination with 1. Some evidence exists for a role of

109
serotonergic neurons in the control c£ LH release and in
mediating opioid effects on 1H release (Ieiri et al.,
1980a). The present investigation argues against changes in
5HT metabolism as a stimulus for the interaction between
morphine and gonadal steroids in inhibiting gonadotropin
release. Similarly, the DA metabolite, HVA, was unaffected
by the experimental treatments in this study. In view cf the
marked changes in EA and DCEAC after several treatments
noted above, it would appear that HVA dees not serve as a
sensitive index of EA neuronal activity or metabolism.
Collectively these studies argue against a role for EA,
NE, or 5HT neuronal systems in mediating the potent
interaction between morphine and gonadal steroids in
inhibitng LH and FSH secretion. Presumably, then, this
steroid interaction is manifested at the level cf some ether
neuron, such as the LHSH neuron or the ECE containing
neuron.

CHAPTER VII
MODULATION OF ENDOGENOUS OPIOID INFLUENCE CN LUTEINIZING
HORMONE SECRETION BY ESTROGEN AND PROGESTERONE
Introduction
Considerable evidence indicates that ECP containing
neurons may exert an inhibitory influence on LHRH neurons in
the hypothalamus (Meites et al., 1979; Cicero, 1980b; S.
Kalra et al., 1980). Among the most persuasive evidence
suggesting a role for EOP in the inhibition cf LH secretion
is the observation that the blockade cf opiate receptors
with naloxone stimulates LH secretion. This is presumably
due to preventing an ongoing endogenous opioid inhibition of
LH secretion. Several studies suggest that the influence of
EOP on LH secretion may vary considerably during various
phases of reproduction and that the magnitude cf IH
stimulation after naloxone injection may accurately reflect
the existing suppressive influence of EOP. Nhile effective
in increasing serum LH levels in acyclic prepubertal
females, naloxone failed to stimulate LH release in adult
cycling rats (Blank et al., 1979). Such an observation
suggests that EOP are not operative after sexual maturation
in the female rat, in contrast to the adult male rat
In addition, estrogen treatment inhibited
(Cicero, 1980b)

111
stimulation of LH release by naloxone in immature female and
adult ovariectomized rats (Blank et al., 1979; Blank et al,,
1980). More recently, estrogen and F treatment have teen
shown to alter EOF levels in the hypothalamus and
hypophysial portal plasma, and there is evidence for
fluctuation during the estrous cycle in some opioid peptide
levels in the hypothalamus, pituitary and systemic
circulation (Dupont et al., 1980; Barden et al., 1981a;
Ishizuka et al., 1982; Knuth et al., 1984; Lim and Funder,
1984). These findings indicate that ovarian steroids may
modulate the influence of EOP on 1HHH neurons, and this may
constitute a mode of feedback action by gonadal steroids. In
the present study the potential ECF involvement in the
regulation of LH secretion during the rat estrous cycle and
in ovariectomized rats pretreated with gonadal steroids was
carefully evaluated. In these experiments the LH response
after naloxone treatment served as an indicator of potential
ECE influence on LH secretion.
Experimental
These studies employed normally cycling adult female rats
or rats which had been ovariectomized for two weeks before
being treated with EB or EB plus P.

112
Experiment 1
This experiment used cycling fesiale rats and compared the
LH response after naloxone treatment during the rising phase
of estrogen secretion (2000 h on diestrus II), at the tine
of maximal elevations in serum estrogen levels (0800 h and
1200 h on proestrus) , and before (14C0 h cn proestrus),
during (1600 h and 1800 h on proestrus), and after (0800 h
and 1400 h on estrus) the preovulatory IH surge.
Additionally, the IH response was evaluated during the
period of elevated P secretion cn diestrus I (0800 b; S.
Kalra and Kalra, 1974a). Rats were administered nalcxone (2
mg/kg in saline, s.c.) or saline (ccntrol) at these
designated times and killed by decapitation exactly 15 min
later. Trunk blood was collected, and serum was stored
frozen at -20° C, for subseguent determination of LH
concentrations by RIA. The dose of nalcxone employed in
this and subseguent experiments (2 mg/kg) is comparable to
that which has been shewn to elicit Dear maximal IH release
in male rats and has been used previously to evaluate
naloxone-induced LH responses in adult female rats (Eruni et
al., 1977; Blank et al», 1979; Blank et al.,
1980b; S. Kalra and Simpkins, 1981).
1980; Cicero,

113
Experiment 2
In this experiment, the influence of EB alone cr EE and P
on the naloxone-induced LH response was analyzed. Two weeks
after ovariectomy, rats received EB (7.5 micrcgrams, in oil,
s.c., at 1000 h). Two days later, they were divided into two
groups. One group received nc further steroid treatment (EB
group), while the second group received P (5 mg in cil/rat,
s.c.) at 1000 h (EBP group). The EE-treated rats were
further subdivided into five groups, which then received
either saline (control) or naloxone (2 mg/kg, s.c.) on day 2
at 1000, 1200, 1400, 1600, or 2000 h. Similarly, EEP-
treated rats were further subdivided into five groups, which
then received naloxone or saline on day 2 at 1200, 1400,
1600, 1800, or 2000 h. All animals were killed by
decapitation 15 min later, and serum obtained from trunk
blood was analyzed for LH levels ty RIA.
Experiment 3
In this study the influence of P administration on the
naloxone-induced LH response on proestrus was analyzed.
Proestrous rats received an injection cf P (5 mg/rat, s.c.)
at 0900 h. The naloxone-induced LH response in these rats
was examined at 1400 h and 160C h by administering naloxcne
(2 mg/kg, s.c.) or saline. A control group received oil
vehicle instead of P at 0900 h, followed ty naloxcne cr
saline at 1400 h. Eats were decapitated 15 min later, and

serum from trunk blood was stored frozen for later analysis
of LH by FIA,
Results
Effects of Naloxone Administration During the Estrcus Cycle
on L11 fielease
Naloxone administration elicited significant increases in
serum LH levels at every stage cf the estrous cycle studied
(Figure 15 . On diestrus II, LH levels increased by 211?
from control values after naloxone injection at 2000 k (p <
0.05). A similar magnitude cf response was evident the
following day at 0800 h and 12CC h and just before the
preovulatory LH release at 1400 h cn proestrus, In
addition, naloxone was effective in eliciting significant LH
release during the period of the preovulatory LB surge.
Serum LH levels in controls at 16C0 h and 1800 h were 1615 ±
386 and 1952 ± 381 ng/ml, respectively, whereas in naloxone-
treated rats, they were 4497 ± 767 and 3105 ± 460 ng/ml,
respectively, at those times (p < 0.05). After the IH surge
on proestrus, naloxone injection also elicited a 5-fold
increase in LH at 0800 h and a 10-fold increase at 1400 h on
estrus (p < 0.05). A group of rats (not shown in Figure 15)
was injected with naloxone or saline cn diestrus day 1 at
0800 h. Serum LH levels in saline-treated rats were 61.22 ±
10.87 ng/ml, whereas they were significantly elevated to
214.79 ± 27.68 ng/ml in naloxone-injected rats (p < 0.05).

115
PROESTRUS
Figure 15: The effects of nalcxcne (2 xg/kg) on serum LH
levels at various tines during the estrous
cycle.
The time of sacrifice, noted on the abscissa,
was 15 minutes after nalcxcne (NAL) or saline
(SAL) injection. Note the scale change fcr LH
concentrations in groups injected during the
preovulatory surge (1615 h and 1815 h on
proestrus). * denotes p < C.05 vs. saline
control at the same injection tine.
SERUM LH (ng/ml)

Effects cf Naloxone on LH Release in Steroid - Pretreated
Ovariectomized Eats
In agreement with previous reports, EB treatment elicited
a modest midafternocn IH surge, as indicated by elevated LH
levels at 1600 h in saline treated controls (Figure 16,
lower pannel; p < 0,05; P. Kalra et al., 1972; S. Kalra and
Kalra, 1979). In these rats, regardless cf the tine cf
administration, naloxone evoked a significant rise in serum
LH levels. Elevations in serum IH levels over the control
values varied between 48% and 73% before and after the
spontaneous LH surge. At 1600 h, during the EB-induced LH
surge, the naloxone-induced LH rise was not significantly
different from that at other times studied.
As expected, P treatment advanced and amplified the LH
surge in EB-primed rats (P. Kalra et al,, 1972), A rise in
serum LH levels was evident as early as 1400 h. Thereafter,
LH levels peaked rapidly at 1600 h and decreased
progressively at 1800 h and 2000 h. Also, P treatment
markedly altered the naloxone-induced LH response. iithin
two hours of P treatment, the naloxone-induced LH response
was significantly higher than that seen at this time in EB-
treated rats (Figure 16) or before P treatment at 1000 h (p
< 0.05). However, during the rising phase at 1400 h, the
peak phase at 1600 h, and the receding phase at 1800 h cf
the LH surge, naloxone failed to stimulate any further

117
increase in LH release. The ability cf naloxcne tc elicit LH
release was again evident at the end of the IH surge at 2000
h. The L3 response at this time was guite similar to that
seen before the onset cf the LH surge.
The Effects of P on Naloxcne - Induced LH Release on
Prcestrus
The injection of P on prcestrous ncrning advanced the
midafternoon LH surge (Figure 17). Serum LH concentrations
in P injected rats were significantly elevated by 1400 h,
while oil-injected rats showed no evidence cf an LH surge at
this time. Secretion of IH increased further at 1600 h in
these P-treated rats (p < 0.05). However, naloxcne-induced
LH responses observed in the oil-injected controls at 1400 h
(Figure 17) or 16C0 h (Figure 16) were abolished by P
pretreatment

SERUM LH ( ng/ml )
118
3455 3567
Figure 16: The effects cf naloxone (2 mg/kg) on seruio LE in
ovariectomized rats treated with EB (lower
panel) cr EB plus P (upper panel).
The time cf death on day 2 after EB treatment,
noted on the abscissa, was 15 minutes after
naloxone (NAL) or saline (SAL) injection. The EB
group treated with naloxone cr saline at 1000 h
is also presented as a control group in the EBP
panel, since it did net receive P on day 2 at
1000 h. * denotes p < 0.05 vs. saline control at
the same injection time.

SERUM LH (ng/ml)
119
Figure 17: Effects of P (5 mg Frog) on naloxone (2 mg/kg) -
induced LH secretion on prcestrus.
The time of death, noted on the abscissa, was 15
minutes after naloxone or saline injection.
Controls (inset) were treated with oil rather
than P at 09CC h and received saline or naloxone
at 1400 h. + denotes p < 0.05 vs. saline control
at the same injection time.

120
Discussion
These studies clearly demonstrate that naloxcne
administration can readily stimulate LH release at all
stages of the estrous cycle. Since the priaary action cf
naloxone is believed to involve the displacement of the EGP
from their receptors, it seems likely that this action led
to the stimulation of 1H release in this study (fleites et
al., 1979; Cicero, 1980b; S. Kalra et al., 1980). This
observation is in line with the view that during the esttous
cycle, EOP containing neurons, located within the
hypothalamus, exert a tonic inhibitory influence on LE
release (Rotsztejn et al., 1978, S. Kalra, 1981).
Blank et al. (1979) were able to elicit IB release with
naloxone (at 2.5 mg/kg) in prepubertal female rats. In adult
cycling rats, they failed to observe a stimulatory LH
response after a similar naloxone treatment. While the
reasons for the disparity between these data and the wcrk cf
Blank et al. (1979) are not apparent, it should be noted
that the basal serum LH levels in the previous work were two
to three times higher than the levels determined in this
experiment. Whether this high basal LH secretion rendered
the adult rats unresponsive to naloxone is uncertain.
Interestingly, Ieiri et al. (1980b) also found that a single
injection of naloxone on the afternoon of proestrus slightly
prolonged the duration of the LH surge.

121
Quite unexpectedly, the LH response after naloxone
treatment between diestros II and just before the
preovulatory LH release were similar. This finding is
interesting because it suggests that factors other than the
responsiveness of the pituitary to LHBH, which increases
progressively as a function of E2 secretion during this
period, may be operative after naloxone treatment (S. Kalra
and Kalra, 1974; Libertun et al., 1974; Martin et al., 1974;
Gabriel et al., 1S83). Evidence suggests that naloxone
treatment induces LHBH output from the hypothalamus (S.
Kalra, 1981; Wilkes and Yen, 1981; Blank and Boberts, 1982).
Therefore, it is likely that the uniform naloxone-induced LH
responses coincident with the progressive enhancement in
pituitary responsiveness to IHEH result from a corresponding
gradual decrease in naloxone-induced LHBH release.
Accordingly, it is predictable that late on diestrus II,
when the pituitary sensitivity to LHRH is lew, naloxone may
have elicited LHBH output at considerably higher levels than
later, at any time before the preovulatory LH release on
preestrus. This interesting possibility of a gradual
decrease in naloxone-induced LHBH release as estrogen
secretion increases between diestrus TI and proestrus would
require further documentation by direct measurement of 1HBH
levels in the hypophysial portal plasma. It could indicate
that over the 36 hour period approaching the preestrous IH
surge that there is a gradual decrease in the activity of
ECf-containing neurens controlling LH release.

122
In immature 22-day-old rats, EB treatment blocked the
stimulation of LH release by naloxone (Blank et al,, 1979).
Siailarly, Blank et al. (1580) reported that up to 4 hours
after an EB injection, naloxone was ineffective in
stimulating LH release in cvariectcmized adult rats.
However, as this study indicates, two days after EB
treatment, the ability of nalcxcne to stimulate LH secretion
returns. The injection of EB has been shown to exert
biphasic effects cn pituitary responsiveness to LHHH in
ovariectcmized rats (Libertun et al., 1974). After EE
treatment, there is initially a suppression for up to 6
hours, followed by an enhanced LH response to LEBE
injection. Thus the apparent contradictory observations cf
Blank et al. (1979 and 1980) and this study can be
reconciled on the basis of changing patterns of pituitary
responsiveness after EB injection. However this mcde cf
biphasic change in pituitary responsiveness does not explain
the uniform LH responses during the period between 2015 h on
diestrus II and 1415 h on proestrus in cycling rats. At
present it cannot be precluded that the rise in estrogen
secretion may attenuate naloxone-induced LHBH release ever
this interval.
kith respect to the effects of P, the data are consistent
with the observation that P advances and amplifies the LH
surge in EB-primed ovariectouized and proestrus rats (E.
Kalra et al., 1972; Sedmcnd, 1568). This study has further

123
found that two hours after P injection, the naloxone-induced
LH response was greatly enhanced in EE-primed rats. This may
be partly due to increased availability cf a peel cf
releasable LHEH and enhanced pituitary sensitivity to LBBB
at this time (Cooper et al., 1973; Aiyer et al., 1976;
Simpkins and Kalra, 1980). Interestingly, as the LE surge
began in these P-treated (EB-primed cr preestrus) rats,
naloxone was no longer able tc elicit LH release. This
refractory period began 9 hours after P injection and lasted
for almost 8 hours when LH was being continuously released
at high rates. The failure of naloxone to stimulate LE
release cannot be entirely due tc the possibility that the
hypothalamic-pituitary-LH axis is responding maximally
(Blake and Garner, 1980), because the refractory period was
seen even 4 hours after P injection, when LH secretion was
clearly not maximal. It should, however, be noted that the
endogenous P secretion that follows the LH rise on proestrus
failed to suppress the nalcxone-induced LH response (S.
Kalra and Kalra, 1974a). Under these conditions, either the
optimal time course of P action was net allowed before the
naloxone test or the endogenous E secretion failed to reach
the effective concentration attained after exogenous E
administra tion.
In the absence of P treatment, naloxone was highly
effective in eliciting LH release throughout the LE surge on
proestrus and in EB-treated rats. These observations are

124
interesting in two ways. First, they suggest that
considerable amounts of biologically active IHRH were net
being secreted during the Lfl surge on proestrus or in IE-
treated rats, and this was not the case after P treatment.
Second, the fact that naloxone can evoke further stimulation
of LH release indicates that a considerable degree of
inhibitory influence of ECP was present during the surge in
proestrus and EB-primed rats and cot in P-treated rats. It
appears, therefore, that P treatment may serve to curl the
inhibitory influence of EOP tc allow LHBH output tc cccur at
an optimal rate for amplificaticn of the IE surge. The
mechanism by which P exerts these effects is not known.
In summary, these studies show that naloxone can elicit
LH release in intact and estrogen-treated cvariectcmized
rats. Further, estregens may alter this Lfl response. In seme
circumstances, the alteration cf estrogen may reflect
pituitary sensitivity to LHBH at the time of naloxone
treatment, while in ethers it may reflect the degree cf
inhibitory influence of EOP on LHBE neurons in the
hypothalamus. While some degree of inhibitcry influence
continues to exist during the IH surge on proestrus and in
EB-treated rats, P treatment appears tc abolish this
influence in association with amplification cf the LE surge.
It is possible that the action cf excgencus P in advancing
and magnifying the IH surge may in part be due to
alterations in opicid activity before and during the
hypersecretion of IH,

CHAPTEB VIII
A DECLINE IN ENDOGENOUS OPIOID INFLUENCE DUBING THE STEBCID
- INDUCED HÍPEBSECBETION OF LUTEINIZING HOBKCNE IN THE BAT
Introduction
A variety of anatomical, physiological and
pharmacological evidence indicates a relé fcr EOP in the
regulation of LH secretion (Meites et al. , 1979; Cicerc,
1980b; S. Kalra et al. , 1980; Watson et al., 1980), While
morphine or EOP can suppress LH secreticn, the significance
of these pharmacological effects is uncertain (Cicerc et
al., 1976; Kinoshita et al., 1981; Leaden and Kalra, 1983).
However the capacity of the narcotic antagonist, naloxone,
when administered alone to elicit IH secreticn suggests a
tonic inhibitory role for EOP (Bruni et al., 1977; Cicerc et
al., 1981 . Further the magnitude of the LH secretory
response to naloxone may serve as an indication of the level
of ongoing opioid agonist activity exerted at the level of
the hypothalamic-pituitary-LH axis (Cicerc et al., 1983b).
As was suggested in the previous chapter, EOP-coctaining
neurons appear to tcnically inhibit IH secreticn throughout
the estrcus cycle of the rat, and ovarian steroids appear to
modulate this inhibitory activity. To further evaluate the
role of EOP in the phasic secretion cf LH, this chapter
125

126
examines the LH secretory response tc a wide range cf
naloxone dosages, prior to and during the period of LH
hypersecretion seen following the administration cf EE or EB
plus P to ova riectomized rats,
Exp> erimental
This study consists of two experiments, both cf which
employ models routinely used to simulate preovulatory IH
release in the female rat (as described in General Materials
and Methods). Two weeks after ovariectomy, rats receive 7.5
micrograas EB in oil (EB priming) at 1000 h. After two days
of EB treatment, a diffuse oidafternocn LH surge ensues with
peak LH levels achieved around 1600 h (Legan et al., 1975),
The adainistration of P (5 mg., s.c. in oil) 48 hours after
EB treatment (day 2, 1000 h, EBE priming) advances the onset
and increases the magnitude cf the resultant LH surge (P.
Kalra et al., 1972). The following three sampling times were
used in these EBP-treated rats:
1. during the basal LH secretion seen prior tc the onset
of the LH surge at 1200 h;
2. at the onset of the LH surge at 1400 h; and
3. during the period of maximal LH titers at 1530 h.
Additionally, for comparison, rats treated with EB alone
were sampled at 1600 h during the peak LH response to this
treatment regimen.

127
In the first experiment, groups of EB- and EEP-primed
rats were injected with saline vehicle cr naloxone HCl
dissolved in saline at dosages ranging from 0.1 to 15.0
mg/kg B.W. Blood was collected by decapitation at exactly 15
minutes after injection. The dose range of naloxone and the
sampling time were chosen on the basis of work dene in this
and other laboratories (Cicero et al. , 1981; S. Kalra and
Simpkins, 1981).
In the second experiment, the pituitary responsiveness to
LHHH injections (75 ng/100 gm B.H., s.c.) was evaluated in
rats pretreated with EB or EBP as described above. Bleed
samples (750 microliters) were obtained by cardiac puncture
prior to and 30 minutes after LHBH injection. Light ether
exposure was used as an anesthetic. The dosage of LHBH, as
well as the sampling interval, were based on previous work
done by in this and other laboratories (Chapter V; lu et
al., 1 980) .
Results
The effects of gonadal steroid treatment on serum IK
concentrations in ovariectomized rats are shewn in Figure
18. In EBP-treated rats treated with saline, IB
concentrations at all three time intervals were
significantly different from one another. Concentrations of
LH increased progressively between 1200 h and 1530 h
following P injection to EB-primed rats. Further, LE levels

128
at 1530 h in EBP-treated rats were acre than 4-times higher
than EB-treated rats at a similar time (1600 h; p < 0.05).
The effects of naloxone cn IH secretion during the
hormone surge induced fcy gonadal steroids in ovariectcmized
rats are also shown in Figure 18. All dosages of nalcxone
employed from 0.1 through 4.5 mg/Xg B.N. stimulated IH
release prior to the IH surge in EBP-treated rats at 1200 h
and during the LH surge in EB-primed rats at 1600 h. The
lowest effective dose (0.1 mg/Xg E.B.) stimulated IE
secretion 73% and 56% in EBP-treated rats at 1200 h and EB-
treated rats at 1600 h, respectively (p < 0.05). In
contrast, during the LH hypersecretion induced fcy EBP
priming to ovariectomized rats, higher dosages of naloxone
were required to stimulate LH levels further. In EBP-treated
rats at 1400, only the highest dose cf nalcxone (15 mg/Xg
B.B.) significantly increased IH levels while the 4.5 mg/Xg
B.W. dose of nalcxone was reguired tc significantly
stimulate LH levels at 1530 h.
The effects of LHHH administration (75 ng/100 g. B.S.,
s.c.) on LH secretion in gonadal steroid primed rats are
shown in Table 4. Prior to LHRH injection, LH
concentrations were elevated in EB-treated rats at 16CC h
and in EBP-treated rats at 1400 h and 1530 h relative to
EBP-treated rats at 1200 h (p < C.05). Paired t-analysis
revealed a significant stimulation of LH secretion after
LHBH injection in all 4 gonacal steroid treatment groups.

129
*
x
DOSE OF NALOXONE (mg/kg)
Figure 18: The effects cf nalcxcte injection on IH
secretion in ovariectomized rats receiving FE or
EBP treatment.
Data are expressed in terms of LH-RP-1 (LH-RP-1
= 61 X LH-BP-1) * denotes p < 0.05 vs saline.

130
The magnitude of the LH secretory response to LHRH (delta
LH) was similar in ESP-treated rats at 1200 h and 1400 h and
in EE-treated rats at 16C0 h. However, the LH response to
LHBH injection in rats treated with EE plus P at 1530 h was
greater than the response seen in all ether treatment groups
(p < 0.05) .
TABIE 4
Effects of LHRH (75 ng/100 gm B.i., s.c.) on LH secretion in
ovariectomized steroid treated rats.
Group Pre-LfiSH Post-LHSH delta-LEBE
LH (ng/ml serum)
EBP
1200
206
±
27
1372
i
190
1206
i
198
EBP
1400
358
±
281
1 162
±
184
803
i
184
EEP
1530
2006
i
406 1
5551
±
1329*
3538
i
1055»
EB
1600
375
±
25»
1311
t
153
934
±
153
Data are expressed in terms of LH-BP-1 (LH-BP-1 = 61 X IE-RP-2).
1 denotes p < 0.05 vs, EBP 1200 h.

131
Eiscussi.cn
The results of the present study provide clear evidence
that during the period cf LQ hypersecretion induced in
ovariectcmized rats by the seguential administration cf EE
plus P, a decline in the inhibitory influence of ECE en IH
secretion occurs. This is evidenced by the marked decline in
the ability of naloxone to elicit LH secreticn during this
steroid-induced LH surge. The present study further
indicates that the decline in the LH secretory response to
naloxone cannot be fully explained by changes in the
response of the pituitary to LHRH, cr to increased
variability in LH levels inherent to the onset of the LE
hypersecretory state.
As seen in Table 4, IHBH administration to EE-treated
rats (1600 h) caused a marked elevation in IH
concentrations, and P administration to these EE-priaed rats
(1530 h) further augmented the pituitary response tc IHFH,
as has been noted previously (Aiyer et al., 1976).
Additionally, during the progression cf the EBP-induced IH
surge, the response to LHEH was stable at 1200 h and 1400 h,
and increased at 1530 h, during the period cf the peak LH
response to gonadal steroid priming. These results suggest
that the heightened pituitary responsiveness to LERE at the
apex of the LH surge, as has teen noted by ethers, is a
consequence of LHRH self-priming (Elake and Garner, 1980)
and the direct action of gonadal steroids on the pituitary.

132
Regardless of the mechanism of this enhanced responsiveness,
it is clear that the diminished ability of nalcxoce tc
stimulate LH release during the EBE-induced LH surge is not
a consequence of a hypophyseal deficit.
In EB-treated rats at 1600 h and in EBP-treated rats at
1200 h, the LH secretory mechanism is extremely sensitive to
naloxone and comparable tc that previously reported in male
rats (Cicero et al., 1981). Nalcxcne appears to stimulate LH
release by blocking the ongoing actions of EOP (Eruni et
al., 1S77; Cicero et al,, 1981; S. Kalra and Simpkins,
1981). The extreme sensitivity of these animals tc naloxcne
would suggest that ongoing ECE neuronal activity plays an
inhibitory role in the period preceding the LH surge induced
by EBP treatment as well as in dampening the magnitude cf
the LH surge induced by EB priming alcne. These
observations as well as the findings of the previous chapter
that naloxone can stimulate LH secretion throughout the
estrcus cycle would support such an inhibitory role of ECP
on LH release in the female rat.
During the hypersecretion of LH induced by P
administration to EE-primed rats, the influence cf ECP on LH
secretion diminishes, as is evident by the relative
insensitivity of the LH secretory mechanism tc naloxone
injection. The high dose of naloxone reguired to stimulate
LH secretion in EBP-primed rats at 1400 h (15 mg/kg B.i.)
and at 1530 h (4.5 mg/kg B.fi.) suggests that the LH

133
secretory response observed in these animals may not be due
to the antagonism of opioid receptors. In the dose range of
5 through 20 mg/kg E.W., naloxone can have Dcn-cpicid and
opiate agonist activity (Sawynok et al., 1979), However, a
caution in interpreting the response tc naloxone duting IH
hypersecretion should be presented. Since the onset and
magnitude of steroid-induced IH surges vary among animals,
higher variability in LH levels would be expected during the
LH surge. Consequently, a proportionately larger response to
naloxone would be required to discern a significant increase
in serum levels of the hormone, However, this variability
alone cannot account for the lack cf response tc naloxone
during the EBP-induced LH surge. Only small chauges in the
mean LH response were observed during the IH surge at
naloxone dosages (0.1 and 0.5 mg/kg E.H.) which caused
nearly maximal LH secretory responses in EB-treated rats and
EBP-treated rats at 1200 h. Furthermore, other LH secretory
states which have an inherently high variation in mean LH
levels, such as the acutely orchidectomized male rat, are
highly sensitive to naloxone-induced LH secretion (Cicero et
al,, 15 81)
In EBP-treated rats, the resulting LH surge is earlier in
onset, higher in magnitude and shorter in duration than the
LH surge observed in EB-treated rats (E, Kalra et al», 1972;
Aiyer et al., 1976). The findings of this and the previous
chapter indicate that ECP may be involved in determining

134
these LH secretory responses tc gcnadal steroids. For a 4
hour period following E injection to EB-primed
ovariectomized rats, ECP inhibitory influence on LH
secretion persists and may be enhanced. During the ascending
and peak phase of EBP-induced IH hypersecretion, ECP
influence is lacking, suggesting that associated with this
LH surge is a decline in ECE neuronal activity or the
responsiveness of its post-synaptic effectors. Further, the
decline in the LH surge induced by EEF-treatment is
associated with a reinitiation of nalcxoEe-induced LH
secretion, suggesting that ECP may be involved also in the
termination of LH hypersecretion (Chapter VII). In contrast,
EOF neuronal systems appear to be active during the entire
extent of the EB-induced LH surge. It is reasonable tc
assume, therefore, that EOP may be involved in the late
onset, and low magnitude of EB-induced LH hypersecretion.
Methods cf measuring the release of various ECP prior to and
during steroid-induced IH hypersecretion are clearly needed
to test this hypothesis.

CHAPTEB IX
THE EFFECTS OF CHBCNIC MORPHINE IR EATMENT ON THE FEEDBACK
ACTIONS OF ESTROGEN ON GONADOTBCEIN SECRETION IN
OVARIECTOMIZED RATS
Introduction
The feedback effects cf the ovarian steroid, E2, on the
gonadotropins LH and FSH, are coordinated at the levels cf
the hypothala «us and pituitary (Fink, 1979; S. Kalra and
Kalra, 1983). E2 displays both inhibitory and stimulatory
effects cn gonadotropin secretion. Inhibitory, or negative
feedback effects, are seen during periods cf basal IH
secretion throughout the estrous cycle or immediately after
the injection of E2 to ovariectomized rats, while
stimulatory, or positive feedback effects, are witnessed
after periods of increased follicular estrogen secretifrcm
diestrus through on prcestrus or after more than 48 hours of
sustained E2 exposure to ovariectcmized rats (Vilchez-
Martinez et al., 1974; Legan et al,, 1975; Fink, 1979; £.
Kalra and Kalra, 1S83), In the ovariectomized rat, the
positive feedback effects of estrogen are expressed as a
daily signal for midafternoon IH hypersecretion which ensues
for several days if E2 titers are maintained at levels
similar to or greater than E2 levels seen cn proestrus
(Legan et al., 1975). The neuronal mechanises mediating this
LH hypersecretion are only partially understood.
135

136
The localization of EOP in brain regions closely
associated with steroid-concentrating neurons and LHBH-
containing neurons has sparked an investigation of their
role in the regulation of gonadotropin secretion (Meites et
al., 1S79; Cicero, 1980b; S. Kalra et al», 1980), It is not
certain whether tissue levels of EOP in the hypothalamus
change in response to alterations in the reproductive state
of the female rat (Kumar et al., 1980; Knuth et al., 1984).
A large body of pharmacologic evidence, however, indicates
that EOP act to inhibit IH secretion and possibly mediate
the feedback effects of E2 (S. Kalra, 1981; S. Kalra and
Simpkins, 1981, Sylvester et al., 1982).
As was shown in the two previous chapters, EOP appear to
inhibit LH secretion throughout the estrous cycle, and may
participate in the advanced onset and increased magnitude of
the LH surge seen after P treatment to proestrous and EE-
primed ovariectomized rats. Additionally, it was shewn that
chronic morphine treatment to castrated male rats enhanced
the negative feedback effects of T cn gonadotropin secretion
(Chapters IV through VI). This chapter concludes the
experimental work presented in this dissertation by
investigating the feedback effects of E2 on gonadotropin
release in ovariectcâ– ized rats treated with morphine.

137
Experimental
In these studies cvariectomized rats were treated with
morphine and E2 implants or appropriate control treatments
as described in the General Materials and Methods section of
this dissertation. All treatments were initiated two weeks
after ovariectomy.
Experiment 1
This study evaluated the inhibitory and stimulatory
effects of E2 on LH and FSH secretion in cvariectcmized rats
pretreated with morphine or placebo pellets. Ovariectomized
rats were given morphine or placebo pellets, followed two
days later at 1000 h by two additional implants. At the time
of the second morphine or placebo pellets, groups cf rats
received E implants at one of three dosages:
1. 5 mm long tubes packed with crystalline E2 diluted
with cholesterol on a (weight:weight) basis of 1:1;
hereafter referred to as 5 mm E2 (1:1);
2. 5 mm long tubes packed with crystalline E2; hereafter
referred to as 5 mm E2; and
3. 10 mm long tubes packed with crystalline E2;
hereafter referred to as 10 mm E2.
On the second day after E2 treatment, basal LH secretion
is seen at 1000 h while IH hypersecretion is observed at
1600 h (Legan et al., 1975). Groups cf E2-implanted rats
treated with morphine or placebo pellets were killed at

133
these two times. Trunk blood was collected for serum 1H and
FSH analysis by HIA.
Experiment 2
Based on the results oí Experiment 1, groups of
ovariectomized rats were given morphine or placebo pellets
plus one 5 mm E2 (1:1) implant as described in Experiment 1.
To obtain a more detailed time course of the E2-induced
gonadotropin surge, groups of animals were sacrificed at
1200 h, 1400 h, 1600 h, and 1800 h on the second day after
E2 implantation. Serum from trunk bleed was stored at -20°
C. for later analysis of 1H and FSH by EIA»
^Experiment 3
An LB surge can be initiated for several days after the
implantation of E2 capsules to ovariectomized rats if serum
E2 levels are maintained elevated (Legan et al., 1975). In
this experiment rats received morphine or placebo treatment
plus one 5 mm E2 (1:1) implant as described in Experiment 1.
Both morphine and E2 treatment were continued for an
additional two days, however. At this time (after 6 days of
morphine or placebo treatment and 4 days after receiving the
E2 implant) groups of rats were killed at 1C00 h and 1600 h.
Trunk blood was collected for later serum LH and FSB assays.

139
Iiperiment_4
To determine whether chronic morphine treatment alone
altered gonadotropin secretion in the absence cf E2
treatment, groups cf rats were treated with morphine or
placebo pellets plus sham capsules. After 4 days cf morphine
or placebo treatment, groups of rats were killed by
decapitation at 1000 h and 1600 h. Serum was stored at -20°
C, for later serum hormone analysis.
Experiment 5
To determine whether chronic morphine treatment
influenced the pituitary response to LHBH, groups cf rats
received morphine or placebo treatment plus 5 mm E2 (1:1)
capsules as described in Experiment 1. Cn the second day
after E2 treatment rats were given an injection of IHBH (75
ng/100 g. B.W., s.c.) at 1530 h. Blood samples (750
microliters) were obtained by cardiac puncture under light
ether anesthesia prior to, and 30 minutes after LHBE
injection. The dose of LHBH used and the sampling interval
were based on earlier experiments (Chapters V and VIII; lu
et al., 1980). Serum was analyzed for LH by BIA.

140
Results
The inhibitory and stimulatory effects of three different
E2 doses on LH and FSH release in placebo-implanted rats are
shown in Figure 19. At 1000 h, serum LH levels were belcw
225 ng/ml in these placebo-treated, E2-iaplanted rats. This
is comparable to LH levels shewn in earlier chapters
employing EB-treated ovariectomized rats (Figures 16 and
18). tiidaf ter noon elevations in serum LH were evident in all
three of these E2-implanted groups at 1600 h relative to
1000 h (p < 0.05). Of the three E2 dcses used, the 10 mm E2
implants caused the largest (6-fcld) increase in serum IH
above these observed at 1000 h. The FSH response to E2
implantation in placebo-treated rats was less pronounced
than that of LH (Figure 19, lower panel). Serum FSH levels
at 1000 h in placebo-treated rats were inhibited by the 5 mm
and 10 mm E2 implants compared to FSH levels seen following
5 mm E2 (1:1) implantation. In placebo-treated rats nene of
the E2 implants produced a significant elevation in
aidafternoon serum FSH concentrations.
The feedback effects of E2 cn serum LH and FSH
concentrations in the presence of chronic morphine treatment
are also shown in Figure 19. Treatment with morphine
further reduced LH levels at 1000 h by 655?, 58% and 50% in 5
mm E2 (1:1), 5 mm E2 and 10 mm E2-implanted rats,
respectively (p < 0.05). Surprizingly, concurrent treatment
with morphine plus E2 enhanced the midafternocn

SERUM SERUM
FSH (ng/ml) LH (ng/ml)
141
COLONY TIME (hrs)
Figure 19: The effects of continuous morphine exposure and
various doses of E2 cn serum LH and FSH levels
in ovariectomized rats.
Crystalline E2 or E2 diluted on a 1:1
(weight:weight) ratio with cholesterol was
packed into tubes as shewn in the figure. Eats
were sacrificed at the times shown 4 days after
morphine or placebo treatment and two days after
E2 implantation, LH levels were determined using
the LH-EP-2 reference standard and are expressed
relative to the LH-EP-1 standard (LH-BP-1 = 61 X
LH-RP-2). * denotes p < 0.C5 vs. placebo
treatment at the same time point; the dagger
symbol denotes p < 0.05 vs. 1000 h.

142
hypersecretion of If! seen at 1600 h. Exposure to morphine
enhanced LH levels 3-fold in 5 urn E2 (1:1)-treated rats and
2-fold in 10 mm E2-treated rats (p < C.05) when compared to
placebo-treated rats at this time. Although the 5 mm E2
implant caused a proportionately larger LH surge in morphine
than in placebo-treated rats (greater than 12-fold vs. mere
than 4-fold, respectively), peak LH levels at 1600 hours
were not significantly different from one another.
Chronic morphine treatment also altered the response of
FSH to E2 exposure (figure 19, lewer panel). Levels of FSH
were reduced relative to placebo-implanted controls at 1000
h in 5 mm E2 (1:1) and 10 mm E2-implanted rats, tut net in
the 5 mm E2-treated group (p < 0.05). Additionally,
midafternoon FSH levels in these morphine-treated rats
(1600) were significantly increased by the 5 sm E2 (1:1) and
10 mm E2 implants relative tc morning values (1000 h).
Figure 20 displays the time ccurse of the E2-induced LH
and FSH surges in placebo and morphine-treated rats. Serum
LH concentrations in placebo-implanted rats increased
greater than 4-fold over the period frem 1200 h tc 1800 h (p
< 0.05). Concentrations of FSH in the blood increased in a
similar, though more gradual fashion, ever this period (46%,
p < 0.05). Chronic morphine exposure to these 5 mm E2
(1:1)-implanted rats appeared tc advance the onset and
augment the magnitude of the midafterncon gonadetrepin
surges. Compared to 1200 h, serum LH concentrations in

143
morphine-treated animals were increased earlier (at 1600 h)
than similarly treated placeto-implanted rats. Serum IE
levels seen in morphine-treated rats at 1600 h and 1800 h
were twice placebo-implanted LH levels (p < C.05). Chronic
morphine treatment significantly reduced FSH levels prior to
(1200 h) and augmented FSH levels during (1800 h) the surge
induced by the 5 mm E2 (1:1) implant when compared to
ovariectomized placebo-implanted rats.
After 4 days of 5 mm E2 (1:1) exposure, midafternoon
(1600 h) hypersecretion of IH but not FSH was noted in
placebo-treated rats relative tc 1000 h (Figure 21, p <
0.05). fchile morphine treatment further reduced serum LE
levels at 1000 h (p < 0.05), treatment with the opiate did
not alter LH values at 1600 h. Further, chronic morphine did
not effect FSH levels at these two tixes.
Serum LH and FSH levels in ovariectomized rats receiving
4 days morphine treatment are shown in Figure 22. LH
concentrations in ovariectomized rats (placebo treatment) at
1000 h were 902 ± 33 ng/ml. At 1600 h these levels were
decreased by 30% (p < 0.05). Serum FSH concentrations in
ovariectcmized rats at 1000 h (32.4 ± 1,2) were similar to
these seen in the afternoon. After 4 days cf exposure tc
morphine, LH concentrations at 1000 h were similar to
placebo-implanted rats. Interestingly, morphine treatment
stimulated LH levels at 1600 h 39% compared to morphine-
treated rats at 1000 h, and 16% compared tc placebo-treated
rats at 16 10 h (p < 0.05).

144
Figure 20: The effects of continuous morphine exposure on
midafternoon LH and FSH hypersecretion induced
by E2 implantation in ovariectomized rats.
Crystalline E2 was diluted cn a 1:1
(weight:weight) ratio with cholesterol and
packed into tubes 5 bb in length. Animals vete
sacrificed at the times indicated after 4 days
of morphine or plácete treatment and 2 days
after 5 mm E2 (1:1) implantation. LH levels
were determined using the 1H-FP-2 reference
standard and are expressed relative to the LH-
RP-1 standard (LH-RP-1 = 61 X LH-BP-2) . *
denotes p < 0.05 vs. placebo treatment at the
same time point; the dagger symbol denotes p <
0.05 vs. 1200 h.

145
COLONY TIME (hrs)
Figure 21: The effects cf continuous morphine exposure cn
LH and FSH secretion in E2 implanted
ovariectomized rats.
Crystalline E2 was diluted on a 1:1
(weight:weight) ratio with cholesterol and
packed iuto tubes 5 mm in length. Animals were
sacrificed at the tives indicated after 6 days
of morphine or placebo-treatment and 4 days
after 5 aim E2 (1:1) implantation. LH levels were
determined using the LH-BE-2 reference standard
and are expressed relative to the LH-RP-1
standard (LH-BP-1 = 61 X LH-HP-2). * denotes p <
0.05 vs. placebo treatment at the same time
point; the dagger symbol denotes p < 0.05 vs.
1000 h.

146
Figure 22:
COLONY TIME (hrs)
The effects of continuous morphine exposure on
LH and FSH secretion in ovariectcmized rats.
Animals were killed at the times indicated after
4 days cf morphine cr placebo treatment. LH
levels were determined using the LH-RP-2
reference standard and are expressed relative tc
the LH-RP-1 standard (LH-EE-1 = 61 X LH-RP-2). *
denotes p < 0.05 vs. placebo treatment at the
same time point; the dagger symbol denotes p <
0.05 vs. 1000 h.

147
The effects of LHRH (75 ng/100 gin B,K,, £,c.) injecticn
on LH secretion in 5 nun E2 (1:1)-treated ovariectomized rats
given morphine or placebo implants are shown in Tatle 5.
Serum LH concentrations prior to LHRH injection in these 5
mm E2 (1:1)-treated rats were similar to LH levels in EE-
treated rats at 1600 h (Table 4), LHRH injection
significantly increased LH levels in both treatment groups
(p < 0.01)* In this study, morphine treatment did not
appear to influence LH levels before or after LHRH
injection.

148
TABLE 5
The Effects of LHHH on LH Hypersecretion in E2-treated
Ovariectomized rats.
Treatment: Pre-LHHH Post-LHRH delta-IHEH
LH (mg/ffil serum)
E2
E2 +
386 ± 30
383 ± 53
2196 ± 399 1488 ± 195
2574 ± 606 2191 ± 585
LH concentrations were determined using the IH-BP-2 reference
standard and are expressed relative tc the LH-RP-1 standard
(LH-RP-2 = 61 X LH-EP-2).

149
Discussion
The present study reveals an interaction between aorphine
and E2 on the feedback control of gonadotropin secretion in
the ovariectomized rat. In the presence cf chronic morphine
treatment both the inhibitory and stimulatory effects of E2
implantation on LH and FSH secretion are potentiated. Since
the effects of opiates on gonadotropin secretion are
mediated at a site within the central nervous system (Table
5; Cicerc, 1980b; S. Kalra, 1981; Wiesner et al., 1984) and
EÃœP likely exert effects similar to that cf morphine (leadem
and Kalra, 1983), the present study argues for an important
role of EOP-containing neurons in the regulation of the
cyclic release of LH and FSH.
The ability of E2 to inhibit IH release was enhanced by
concurrent treatment with morphine. Immediately after the
administration of estrogens to ovariectomized rats, a
decline in LHRH release combined with a decline in pituitary
responsiveness to the decapeptide causes a rapid decline in
LH levels (Vilchez-Martinez et al., 1974; Fink, 1979).
During basal LH release seen 48 hours after estrogen
treatment, pituitary responsiveness to LH EH is enhanced,
however (Vilchez-Martinez et al., 1974). The negative
feedback of E2 at this time is likely due to an inhibition
of LHBH release, and it would appear that morphine enhances
this effect. This interaction between morphine and F2 in

150
suppressing LH release is not merely additive, since after 4
days of treatment with the opiate there was no difference in
LH levels between morphine and placebo-treated, sham-
implanted (ovariectcmized) controls at 1C0C h (Figure 22).
These findings are similar to the observations of Chapters
IV through VI that while ineffective in inhibiting IH
release on its own, chronic morphine treatment enhances the
feedback sensitivity of T in male rats.
The lack of an effect of morphine in altering LB
secretion in gonadectomized male cr female rats is cf
interest since it indicates that the gonadal steroids are
required to maintain the responsiveness of the
neuroendocrine substrates mediating the LH response to
opiates. This castration-induced loss in the LH response to
opiates has been noted in Chapter 4 and by other
investigators, although the mechanism cf this response has
not been determined (Cicero et al., 1982a; Bhanot and
Wilkinson, 1983). Gonadal steroids dc net appear tc modify
opiate receptor numbers (Cicero et al., 1983a). Further,
cyclic fluctuations in gonadal steroids in intact female
rats, and physiological doses cf gonadal steroids in
ovariectcmized rats do not consistently modify EOF levels in
the hypothalamus (Kumar et al.* 1980; fcardlaw et al., 1980;
Knuth et al. , 1984). In monkeys however, beta-endorphin
release into hypophyseal portal blood is clearly steroid
dependent (Ferin et al., 1984). Finally, the interaction

151
between morphine and T in inhibiting LH release is not
reflected by changes in the metabolism cf EA, NE, or 5HT in
the MBH or POA-AH (Chapter IV).
The observation that in the presence of chronic morphine
treatment the stimulatory effects cf E2 cn IH release are
enhanced is most surprising, It is well known that when
given acutely, opiates block cvulaticn, and preovulatory and
gonadal steroid-induced IH hypersecretion (Earraclough and
Sawyer, 1955; Pang et al., 1977; S. Kalra and Simpkins,
1981). It is possible that chronic morphine treatment
reveals a stimulatory opiate component cn IH secretion in
these E2-treated rats. This stimulatory effect of morphine
on afternoon LH release was evident even in the absence of
E2 treatment to ovariectomized rats (Figure 21). ether
neurotransmitters, such as EA and NE have teen shewn to
display both stimulatory and inhibitory effects on IH
release (Drouva and Gallo, 1976a; Eotsztejn et al., 1977;
Gallo and Drouva, 1979). Additionally, several
investigators have observed stimulatory effects of opioids
on LH release, particularly at low dosages, but they have
not attempted to explain these effects (Pang et al., 1977;
Cicero et al., 1980; Hotta and Eartini, 1982; Sylvester et
al., 1982; Leadem and Kalra, 1983; Mittler et al., 1983;
Wilkinson and Bhanot, 1983). Piva et al. (1984) have
suggested that the stimulatory effects of intraventricularly
administered morphine may be due to an opiate receptor

152
different from the site which mediates the inhibitory
effects of morphine on LH release.
The LH surge in morphine dependent, E2-implanted rats was
enhanced in magnitude and advanced in onset (Figure 20). It
is well documented that F administration advances the time
of onset and increases the magnitude cf LH hypersecretion
seen cn proestrus or after E2 treatment to ovariectoaized
rats (Redmond, 1968; P. Kalra et al., 1972). Thus, chronic
morphine treatment may have resulted in the same neuronal
alteration as seen after P exposure. As was shown in Chapter
VIII, EOP inhibition of LH secretion, present two hours
after P injection to EE-treated rats, was diminished from 4
to 6 hours after P injection to these animals. In this
sense, the morphine treatment may have stimulated receptors
normally activated by EOP after F treatment. In morphine-
treated, E2-implanted rats however, opiate receptors were
being continuously stimulated during the interval that LH
hypersecretion was occuring. Whether morphine acted to
stimulate LH release at this time, or if its inhibitory
effects were transiently removed is net known.
The interaction between E2 and morphine in inhibiting LH
persisted for at least 4 days cf E2 exposure (Figure 22).
However, while the stimulatory effects cf E2 alone persisted
for 4 days, at this time morphine was no longer able to
enhance this midafternoon LH surge

153
While ovarian steroids clearly influence FSH release,
their effects on FSH are less pronounced than cn LH (Mahesh
et a 1. , 1975; Fink:, 1979). This supports the role of ether
ovarian factors regulating this gonadotropin (Lumpkin et
al., 1984). In this study E2 was also less effective in
modulating FSH levels. While chronic morphine treatment
produced smaller changes in FSH levels compared tc LH, its
influence was apparent. fiorphine stimulated both positive
and negative feedback responses at E2 doses which were
themselves unable tc alter FSH release. like LH, the time-
course and magnitude of the E2-induced FSH surge was beth
advanced and amplified, suggesting that the effects of
morphine and E2 on FSH release may be mediated by the same
neural mechanisms which regulate LH release. However,
neuronal factors other than LBRH may ultimately mediate this
effects of morphine and E2 on FSH release (McCann et al.,
1983) .
In conclusion, these studies indicate that chronic
morphine treatment enhances the negative and positive
feedback effects of £2 on gonadotropin release. This would
suggest an important role for ECE in modulating the feedback
actions of ovarian steroids. The existence of both
stimulatory and inhibitory influences of morphine on E2
feedback over such a brief time interval (6 heurs) indicates
that the modulation of gonadotropin secretion by ECP is more
complex than previously thought.

CHAPTER X
GENERAL DISCUSSION
The present work evaluated the interaction of opioids
with gonadal steroids in their regulation cf LH and FSH
secretion in the rat. The individual experiments were
organized into 6 studies - three in the Bale and three in
the female. The major findings drawn from these studies are
as follows.
1, Chronic morphine treatment, while unable to inhibit
gonadotropin release alone, enhances the negative
feedback sensitivity of T on gonadotropin release in
rats orchidectcmized two weeks previously.
This extends previous findings that castration results in
a loss of the inhibitory effects of cpicids on LH release,
and that ECP act to inhibit the release of LH and mediate
the feedback effects of gonadal steroids (Cicero et al. ,
1982a; Van Vugt et al,, 1982; Bhanot and Wilkinson, 1983).
The present studies suggest that in the presence cf opicid
receptor stimulation the central feedback inhibition of T on
LH secretion is enhanced. In this respect, ECP may serve as
the set point cr gcnadostat for T feedback.
It must be noted that chronic treatment with morphine
serves as a pharmacological prcbe to elucidate the normal
15 U

155
function of EOP-containing neurons. Opiate dependence
effects many neuronal systems in addition to the
neuroendocrine substrates determining LH release. It cannot
be assumed that the pattern of EOP release resembles that of
continuous opiate receptor stimulation with morphine. Yet,
this criticism can be argued for any pharmacological study
of EOP-containing neurons, including acute opioid
administration. Since gonadal steroids influence endocrine
events that are often expressed over a period of hours to
days, the subcutaneous morphine implants provide continuous
opiate receptor stimulation over a similar time frame.
2. At physiologically relevant doses, the ability of
morphine to enhance the LH inhibitory effects of T
and E2 are mere pronounced than those of CHT. low
circulating levels of DHL appear to reduce the IH
secretory response to IHEH in preference to centrally
inhibiting the release of LHBH.
It would appear that the feedback regulation of LE
release in the male is orchestrated by several gonadal
steroids acting at multiple loci within the hypothalamic-
pituitary-LH axis. This supports past studies which indicate
that £2 is important in the regulation of LH release in the
male (C'Agata et al., 1981; P. Kalra and Kalra, 1981;
Nishihara and lakahashi, 1983). The assumption that the 12
and DHI implants provided physiologically relevant doses is
based on work done by others (P. Kalra and Kalra, 1980,

156
1981). Since E2 and DHI in the hypothalamus and DHT in the
pituitary can be metabolized fro® T, it is not certain
whether the actions of E2 and DHT are normally mediated via
circulating levels of these steroids {Hassa et al, 1972;
Naftolin et al., 1975).
3. The interaction between chronic morphine and T in
inhibiting LH release in male rats is net clearly
reflected in changes in the metabolism of NE, Dfi, or
5HT in the MEH or EOA-AH.
Of the three neurotransmitters evaluated, DA metabolism
in the EOJ-AH was most responsive tc morphine and T
administration. An interaction between the two treatments
was not apparent, however. These studies support evidence
that 1 and the opiates are inhibitory to hypothalamic
dopaminergic activity (Gudelsky and Porter, 1979; Simpkins
et al., 1980), but do not support the notion that opiates
exert their effects cn the IHBH neuron by influencing Efl
activity (Rotsztejn et al., 1978).
While a large body of evidence suggests that NE
stimulates LHRH output from the hypothalamus, and mediates
opiate effects on IH release, the present data do not
support this contention (S. Kalra and Simpkins, 1981; Van
Vugt et al., 1981; Adler and Crowley, 1984). The
differences between this and previous studies may be related
to the chronic treatment paradigm employed in this werk.
While the measurement of NBE levels provides a noninvassive

157
measure of noradrenergic activity, being present in let*
concentrations in hypothalamic tissue, HUE may not be a
sensitive index of noradrenergic activity. While more
difficult to guantify, the NE metafcclite, 3-methcxy-
phenylethylene-glyccl, in its free and conjugated forms, may
be present in larger quantities and cculd serve as a better
metabolic indication of NE release in the rat (Elchisak and
Carlson, 1982).
4. The narcotic antagonist, naloxone, stimulates LH
release at all times during the rat estreus cycle,
including during the preestreus LH surge.
The mechanism through which naloxone stimulates LH
release is likely through antagonizing the ongoing actions
of EOP, since the application of appropriate ECP antibodies
also stimulates LH release (Schulz et al., 1981, Forman et
al., 1S83). These studies clarify earlier evidence, using
naloxone as an indication of opioid inhibition of LH
release, that EOP are important in the maturation cf the
central mechanisms controlling IB release, but are not
involved in regulating LH release in the adult female rat
(Blank et al., 1979). Bather, it appears that EOP are
important in maintaining basal LH release throughout the
estreus cycle and perhaps in limiting the magnitude of the
proestrous LH surge. A corollary to this finding is that
when appropriately administered, opioid agonists and
antagonists could modify the reproductive status cf the
animal (see below) .

158
5. The ability cf naloxone to stimulate IH release is
reduced during the hypersecretion of LH induced ty P
administration to prcestrcus cr EB-primed
ovariectoaized rats.
The ability of naloxone to stimulate IH release is very
sensitive prior to the EBP-induced LH surge and during the
EB-induced LH surge in cvariectomized rats. Minimal doses
reguired to elicit LH secretion are comparable to doses
needed to stimulate LH release in male rats (Cicero et al.,
1S81), It would appear that a considerable amount cf ECE
inhibition of LH release exists at these times in the female
rat and possibly during the period of basal LH release in
the estrous cycle.
In contrast, naloxone was relatively ineffective in
stimulating LH release during the period of hypersecretion
seen following the sequential administration cf EB plus £ to
ovariectoaized rats. Since doses of naloxone which elicited
LH release at this time were more than sufficient tc blcck
any ongoing EOP activity at their receptors, it is safe to
assume that opioid inhibition of LH release at this time was
diminished
The effects of naloxone on LH hypersecretion yields
information regarding the relation between gonadal steroid-
induced LH hypersecretion in ovariectcmized rats and the
proestrous LH surge. The sequential administration of EB
plus P to ovariectcmized rats is in several respects an

159
appropriate experimental model for the proestrous LB surge.
This includes the similarities in time course and magnitude
of the two LH surges, and the temporal changes in
hypothalamic tissue levels of LHBH and catecholamine
activity associated with this hormonal event (P. Kalra and
Kalra, 1971a; Lofstrom, 1977; Simpkins et al. , 1979; S.
Kalra and Kalra, 1979). However, LH hypersecretion can be
elicited by E2 and P administered to ovariectomized rats at
doses much lower than what are commonly used (McPherson and
Mahesh, 1979). Also, the steroid milieu during and after
the proestrous LH surge is not accurately reflected in this
model. Consequently, some investigators have proposed that
the LH surge induced by E2 or EB alone more precisely models
the neuroendocrine changes that occur on prcestxus
(Barraclcugh and Wise, 1982). This model ignores the
potential contribution of the increased P secretion that
accompanies the proestrous LH surge (S. Kalra and Kalra,
1974a). Ihile endogenous P levels cn proestrus dc not
compare to levels achieved by an exogenous P injection,
circulating steroid levels are net the only indication of
the effectiveness of a biclogical response. Since E2 induces
the synthesis of cytosolic E receptors (Rainbow et al.,
1982), rats in their late follicular phase might be highly
sensitive to changes in E titers.
In the present work, naloxone stimulated LB
hypersecretion during the proestrous and ovariectcmized EB-

160
induced IH surges, but was ineffective during Lfl
hypersecretion in ovariectomized EBP-treated rats. In this
respect, the LH surge in EB-treated rats mere accurately
reflects the inhibition of LH secretion by ECP seen during
the proestrus LH surge. A decline in EGP inhibition was
noted before the proestrous IH surge since as pituitary
responsiveness to LfiRH increases over the follicular phase,
naloxone-induced LH secretion remains constant (Cooper et
al., 1973; Aiyer et al., 1974 ; Gabriel et al., 1983; Chapter
VII). It is probable that a decline in EOP inhibition of LH
secretion may have occured during the proestrus LH surge
that was not indicated through the use of talcxcne as an
index of EOP neuronal activity.
6, Chronic morphine treatment enhances both the
inhibitory and stimulatory effects of E2 on
gonadotropin release in ovariectomized rats.
This extends the studies initiated in the male by shotting
that morphine acts to stimulate both the negative and
positive feedback actions of gonadal steroids in the female.
These findings suggest that EOP are important in modulating
the feedback actions of E2 in the female rat. It further
lends support to the notion that EOP are involved in the
advanced onset and increased magnitude of the LH surge seen
following P treatment to proestrous and EB-treated
ovariectomized rats, since morphine treatment acted in a
similar fashion in these E2-treated animals. Because

161
morphine and EOP appear to exert both inhibitory and
stimulatory effects on LH and FSH release, their role in
regulating gonadotropin secretion is more complex than
previously anticipated (Piva et al. , 1984).
The work presented in this dissertation supports the
contention that alterations in the activity cf ECP neurcns
modify the sensitivity of the hypothalamus to gonadal
steroids. This implicates ECP in the maintenance cf
reproductive endocrine homeostasis and in the regulation of
basal and cyclic gcnadotropin secretion. While these
studies employed the pharmacologic application of an opioid
agonist or antagonist to infer ECP neurcnal activity, the
measurement of EOP as an index of neuronal activity has not
been adequately studied in the rat. Quantifying ECP levels
in the hypophysial portal plasma or in a push-pull perfusate
would presumably serve as an index cf release. These
techniques though difficult to perform, have beeu reported
for other neuropeptides (Sarkar et al., 1976; Levine and
Ramirez, 1982; Kasting et al., 1S81) . A low post-surgical
success rate and an inevitable amount cf tissue damage must
be anticipated with these methcds.
It is difficult to infer neuronal activity based solely
on tissue levels of a neuropeptide. While ovariectomy dees
not appear to change beta-endorphin levels in the
hypothalamus, prolonged E2 treatment decreases levels cf
this peptide in the hypothalamus in a manner reversed by P

162
(Hardlaw et al., 1982). Although the doses of steroid
administered in this study were in the physiological range,
inadequate data on hormone levels, time of sacrifice, and
size of the tissue fragments makes this study difficult to
interpret. Changes in ECF levels in the hypothalamus during
the estrous cycle are also of interest. However, data cn
this topic are conflicting (Kumar et al., 1980; Wardlaw et
al., 1982; Barden et al. , 1981a; Knuth et al., 1984).
In contrast to the brain, EOP levels in the pituitary are
more clearly responsive to changes in the steroid milieu.
Beta-endorphin levels in the anterior pituitary decline
after ovariectomy, and increase in the plasma and NIL on the
afternoon of proestrus (Lee et al., 1980; Ishizuka et al.,
1982). While this agrees with the present pharmacologic
observations that EOP activity is altered after gcnadectcmy
and during the pericvulatory interval, opiates do not appear
to influence gonadotropin secretion at the level cf the
pituitary Chapters IV, V and IX; Cicero et al., 1980fc;
Kiesner et al., 1984). It is possible that these
hypophyseal EOP could influence gonadotropin secretion
centrally through an uucharacterized hypophyseal pathway
(Bergland and Page, 1979), or through an action of
circulating EOP in the plasma. It has been suggested that
EOP exert their actions by acting cn circumventricular
regions cf the brain, such as the median eminence. (Panerai
et al,, 1983). While methionine-enkephalin likely cannot act

163
through a plasma route, leta-endcrphin cculd (Bloom et al. ,
1978). Sather than being the cause of steroid-induced
changes in gonadotropin secretion, these alterations in
pituitary EOP levels likely reflect similar neural and
hormonal influences.
There are similarities between the mcde cf EOF influece
on gonadotropin secretion in humans and rodents. In adult
men, naloxone stimulates LH secretion, indicating that ECE
exert a tonic inhibition of IH secretion in males cf toth
species (Delitalia et al., 1983). Additionally, the ability
of opiates to influence LH secretion is diminished in
castrated men, and is restored after gonadal steroid
treatment (Foresta et al,, 1983a,b). The rat, therefore may
serve as an appropriate experimental model to study gcnadal
steroid interactions with opioids in the regulation of LH
secretion in males.
In humans and ncnhuman primates, cyclic changes in
gonadotropin secretion may be mere dependent on ovarian
rather than neural signals. This is evidenced by the
maintenance of menstrual cycles in roetkeys in the presence
of an LH8H stimulus which does not change in amplitude or
frequency (Knobil, 1980). More recent work in human and
nonhuman primates indicates that normal menstrual cyclicity
does depend on an LfiRH signal that varies in frequency and
amplitude, and that ECF are important in modifying this
signal (Ferin et al., 198h) In women, nalcxcne stimulates LH

164
secretion during the luteal and late follicular phases tut
not during the early follicular phase cf the menstrual cycle
(Quigley and Yen, 1980; Blankstein et al, , 1981; Snowden et
al., 1984). In primates, follicular phase IH secretion is
characterized by high frequency low amplitude LH pulses,
while luteal phase LH secretion is characterized ty lew
frequency high amplitude LH pulses (Van Vugt et al., 1984).
Although naloxone does not alter the pattern of LH secretion
during the early follicular phase, the infusion cf naloxone
during the luteal phase of the menstrual cycle causes an
increase in LH pulse frequency similar to that seen during
the follicular phase of the menstrual cycle. Evidently,
luteal P secretion modifies LH pulse amplitude by altering
EOP activity. Overriding this luteal phase pattern cf LH
secretion with exogenously administered LHBH disrupts the
menstrual cycle (Ferin et al., 1984), Finally, naloxone-
induced LH release returns in the late follicular phase cf
the menstrual cycle near the preovulatory LH surge (Ferin et
al., 1984). In primates as in rats, ECP neuronal activity
appears to be influenced by P and activated during the
petiovulatory period.
It appears that hypothalamic beta-endcrphin may be
involved in mediating these opioid-induced changes in LH
secretion during the menstrual cycle. Beth P and E2 appear
to influence beta-endorphin release into hypophyseal portal
vessels of monkeys (Ferin et al.,
1984) .
Following

165
menstruation or ovariectomy, bata-endorphin concentrations
in portal plasma are undetectable (tiehrenberg et al., 1982).
If beta-endorphin acts to inhibit LHBH output, then this
would explain the inability of naloxone to stimulate LH
release after luteclysis. Similarly, as £2 titers increase
during late follicular development the reinitiation of beta-
endorphin release would be evidenced by a resumption in
naloxone-induced LH release. At this time ECP inhibition of
LH release would aid in timing preovulatory LH release with
follicular maturation.
The present work helps to understand the deleterious
effects of opiate abuse on reproductive hormone secretion.
Azizi et al. (1973) were among the first to present clinical
evidence of depressed T levels in heroin addicts. 3n this
investigation, depressed T levels were seen in the presence
of normal LH secretion. Because opiates enhance the feedback
sensitivity of the hypothalamus to gonadal steroids, it is
reasonable to expect lower T levels to iraintain normal IH
output in these opiate dependent individuals. Similarly,
the present work clarifies reports in female narcotic
abusers (Gaulden et al,, 1964). Since opiate receptor
stimulation in females alters the magnitude and time course
of the LH surge, an appropriately timed heroin injection
could prevent preovulatory LH release. Prolonged opiate
abuse could cause inappropriate IH hypersecretion prior to
or after follicular maturation, leading to infertility and

166
improper luteal development. Additionally, it is of interest
that some of the antigonadotropic actions cf ether CNS
depressants may act through an cpioid mechanism (Cicerc et
al., 19826).
Both clinical and experimental werk suggest that ECE may
be important in seasonal, developmental, and pathological
changes in reproductive hcrmcne secretion. For example, in
hamsters, exposure to short photoperiods is characterized ty
testicular degeneration or anestrus due tc increased
sensitivity to gonadal steroid feedback (Sisk and Turek,
1983). The altered opioid recepten concentrations
associated with short days in hamsters might indicate opioid
involvement in this increased gcnadal stercid feedback
(Wilkinson et al. , 1983),
In a similar manner, increased sensitivity tc gcnadal
steroid feedback has been noted in prepubertal and aged rats
and after experimentally induced hyperprolactinemia (HcCann
et al., 1974; Gray et al., 1980; McNeilly et al., 1983). In
all cf these states, an alteration in ECP levels in the
hypothalamus has been noted (Steger et al., 1980; Barden et
al. , 1981b; Forman et al., 1981; Facchinetti et al., 1983;
Simpkins et al., 1984). It is of interest that since this
project was initiated several clinical and experimental
studies have suggested opioid involvement in the
reproductive dysfunction associated with these states
(Steger et al., 1980; Veldhuis et al., 1982; Beid et al.,
1983; Carter et al.,
1984; Simpkins et al., 1984)

167
If EOP participate in normal, developmental, and
pathological changes in in gcnadctropin secretion, then
appropriately administered opiates should modify
gonadotropin secretion in these states. Narcotic antagonists
should, therefore, be able to modify the effects of altered
EOP activity during reproductive senescence and
hyperprolactinemia (Eeid et al., 1983; Carter et al. , 198h).
Since, tolerance develops to the ability of naloxone to
stimilate LH secretion (Ovens and Cicero, 1981; Gabriel and
Simpkins, 1983), the pattern in vhich an opiate antagonist
is delivered is important. Similarly, an opiate agonist
could stimulate opiate receptors in a period of depressed
activity. In this case, the unvanted effects of the opiate
on other systems not involved in hormone secretion, such as
analgesia or respiration, must be considered.
In conclusion, these studies demonstrate that opiates
interact vith gonadal steroids in their regulation of
gonadotropin secretion, by increasing the sensitivity of the
hypothalamus to circulating gonadal steroids. This
indicates that EOP are important in the modulation of
gonadal steroid feedback in both males and females, and in
the regulation of basal and cyclic gonadotropin release.

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BIOGRAPHICAL SKETCH
It Has a cold and windy day in Anchorage when my parents
brought me back to the igloo. I knew that I had tc move
somewhere warmer. Host of my childhccd and adolescence was
spent in beautiful suburban Hazelwood, Hisscuri. A vacation
to Florida when I was 9 years old left a lasting impression.
Assisted by an academic scholarship and several liberal
Democratic education programs, I entered Washington
University in St. Louis in the fall cf 1975. My Artium
Baccarlarri in psychology was awarded in Kay, 1979. The next
year was spent buying a stereo and applying to graduate
school using the now famous Sun-Eelt Strategy. I moved to
Florida in August 1S80 tc meet my destiny.
199

I certify that I have read this study and that in my
opinion it conforms tc acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
mes H. Sinpkips, Chairman
ssociate Professor of Pharmacy
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
liche&J. J. Ka
Associate Pr
Pharmacy
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adeguate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
tuJluJ——
Satya P. Kalra
Professor of Obstetrics and
Gynecology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adeguate, in scope and guality, as
a dissertation for the degree of Doctor of Philosophy,
Steven £. Childers
Assistant Professor of Pharmacology
and Theraputics

I certify that I have read this study and that in ay
opinion it conforms tc acceptable standards of schclarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctcr cf Philosophy.
¿O.
c.
Daniel S. Sharp
Professor of Animal Science
This dissertation was submitted to the Graduate Faculty of
the College of Pharmacy and to the Graduate School, and
was accepted as partial fulfillment of the requirements for

UNIVERSITY OF FLORIDA
3 1262 08554 3774
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