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

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Gonadal steroid modulation of opioid effects on gonadotropin secretion in the rat
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xiv, 199 leaves : ill. ; 29 cm.
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Gabriel, Steven M
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Gonadotropins -- secretion   ( mesh )
Steroids -- physiology   ( mesh )
Endorphins -- physiology   ( mesh )
Gonads -- physiology   ( mesh )
Pharmacy thesis Ph.D   ( mesh )
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Thesis:
Thesis (Ph. D.)--University of Florida, 1984.
Bibliography:
Bibliography: leaves 168-198.
Statement of Responsibility:
Steven M. Gabriel.
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Typescript.
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Vita.

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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































This dissertation is dedicated to Dr. Theodore J. Cicero.

His hard vork and dedication to 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 my 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, Micheal Katcvich, Daniel

Sharp and William Thatcher, have imparted much needed advice

and knowledge in the laboratory and classrccm. Many cther

faculty members have assisted me in my research, including

Drs. Pushpa Kalra, Kerry Estes, Hartaut Derendorf, Ken

Slcan, George Torosian and MS..A. Kumar.

There is a great feeling cf comradery in cur department

and I would like to acknowledge some of the staff and

graduate students who have been so encouraging over the

years. June O'Meara and Fran Panniello have been helpful

often in the past and have always been tolerant of my many


iii








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 Saith who

will soon be taking over the reins in the lat.

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 roomates, Scott lokus 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.















TABLE OF CONTENTS


FAGE

ACKNOWLEDGMENTS . iii

LIST OF TABLES . .. ix

LIST OF FIGURES . . x

ABSTRACT . .. xii


CHAPTER

I. INTBOCOCTION . . .. 1

II. REVIEW OF THE LITERATURE a a a a a 5

Historical . 6
Early Observations . 6
The Development of eeuroendocrinology 8
Neuroanatomical RelaticnshiFs 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 a a 22
Epinephrine-containing pathways 23
Serotcnergic Pathways a a a a a a a a 23
Anatomy of Endogenous Opicid Peptide Containing
Neuronal Systems ,a .. .a 24
Beta-endorphin-ccntaining neurcns 24
Enkephalin-containing neurons .. 25
Dynorphin-ccntaining neurcns a 26
Steroid Concentrating neurons in the Brain 27
Neuroanatcaical Interactions . 28
Patterns of Gonadotropin Secretion 29
Males aaaa aa a.. 29
Fesales . . 30
Females 30
Monoaminergic Ccntrcl cf Gcnadotropin Secretion 32
Norepinephrine a . a 34
Epinephrine 37
Dopamine a . 38








Serotonin . 38
Endogenous Opicid Peptides and the Control of
Gonadotropin Secretion 39
Reproductive Pharmacclogy of Opicids 40
Physiological Inhibition of Gonadotropin
Secretion by Opicids . 42
Multiple Opioid Beceptors 43
Opioid-Mcnoaminergic Interactions 44
Opioid-Gonadal Steroid Interactions 45
Rationale .* . 48

III. GENERAL MATERIALS ABD METHCDS . 49

Animals .. 49
Dissection of Brain Tissue a 51
Gonadal Steroid Treatment 51
Treatment with Morphine or Naloxone 52
Measurement of Catecholamines Indolamines and
Metabolites . 53
Hormone Radioiamunoassays . 55
Luteinizing Hormone and Follicle Stimulating
Hormone a 55
Luteinizing Hormone Releasing Hormone 56
Testosterone a 57
Statistical Analysis . 57

IV. THE EFFECTS OF CHRONIC MORPHINE TBIATMENT ON
TESTOSTERONE NEGATIVE FEEDBACK IN CASIRATED
MALE RATS . 58

Introduction a.. 58
Experimental a a a a a a 59
Experiment 1 . 59
Experiment 2 a & a .a 60
Experiment 3 .. a ... 60
Results .a a a a a a 61
Effects of Time After Castration on the
Serum IH and Hypothalamic LHRB
Responses to I and morphine 61
The Effects of Chronic Morphine Treatment on
the LH Secretcry and MBH LHRH
Responses to Graded Doses of T. 64
Effects of T and Morphine Treatment cn the
Pituitary Responsiveness to LBBB 70
Discussion .. .. 71

V. THE INFLUENCE OF CHRONIC MORPHINE TREATMENT ON THE
NEGATIVE FEEDBACK REGULAIICN OF GONADCTBCPEI
SECRETION BY GONABEA STEECIDS a 76

Introduction a a a a a a a a . 76
Experimental a a a a a a a a. a a .a 77








Chronic Iorphine and Gonadal Steroid
Treatments . 77
Evaluation of Pituitary Responsiveness to
LHBH . ..... 78
Results .. a . 78
Discussion . . 88

VI. THE EFFECTS OF CHRONIC MOBPHINE AND TESTOSTERONE
TREATMENT ON CATECBOLAMINE AND INDCLABIiE
METABOLISM AND GONAECTROPIN SECRETION IN
MALE RATS . . 94

Introduction . 94
Experimental . 95
Results .* a 96
Serum LH and FSH . 96
NE and NME . .. 100
DA and DOPAC . 100
5HT 5HIAA and HVA . 101
Discussion a a a a . 105

VII. MCDULATION OF ENDCGENOUS OPICID INFLUENCE ON
LUTEINIZING HORMONE SECRETION EY ESTROGEN
AND PRCGESTEBONE. .... 110

Introduction a a 110
Experimental . 111
Experiment 1 . 112
Experiment 2 113
Experiment 3 .. .. 113
Results . 114
Effects of Naloxone Adainistration During
the Estrous Cycle cn LH Release 114
Effects of Naloxone on LB Release in Steroid
Pretreated Ovariectomized Bats 116
The Effects of P on Baloxone Induced LB
Release on Prcestrus 117
Discussion a o a a 120

VIII. A DECLINE IN ENDOGENOUS OPIOID INFLUENCE DURING THE
STEROID INDUCED BYEERSECRETICN OF
LUTEINIZING HORMONE IN THE RAT 125

Introduction a a 125
Experimental .a .a a 126
Results a. . .a 127
Discussion a. .a a a a a a a 131

IX. THE EFFECTS OF CHRONIC MORPHINE TREATMENT ON TEE
FEEDBACK ACTIONS OF ESTROGEN ON GONAECTBCEII
SECRETION IN OVARIECTCHIZED RATS 135

Introduction . 135
Experimental A . a a 137


vii









Experiment 1 a. & 137
Experiment 2 . 138
Experiment 3 a 138
Experiment 4 . 139
Experiment 5. v .& a 139
Results . 140
Discussion . a 149

1. GENERAL DISCUSSICO . 154

BIBLIOGRAPHY a a a 168

BICGBAPHICAL SKETCH . 199











































viii














LIST OF TABLES


TABLE PAGE

1. Effects of In Vivo Morphine and T Pretreatment on In
Vitro LH Release from Bemisectioned Pituitaries
(LH release rate: ng/mg pituitary tissue/ hcur) 71

2. The Effects of IHBH (100 ng/100 g EV.., s.c.) on
serum LH Levels in Castrated Bats Treated
Chronically with Morphine and/or Gonadal Steroids. 87

3. The Effects of Gonadal Steroids and Mcrphine
Treatment on 5HT 5HIAA and HVA Concentrations in
the MBH and POA. . .. 104

4. Effects of LHRH (75 ng/100 gm B.N., s.c.) on LB
secretion in ovariectomized steroid treated rats. 130

5. The Effects of IHBH on LH Hypersecretion in
E2-treated Ovariectomized rats. . 148














LIST OF FIGURES


FIGURE 1AGE

1. Serum LH concentrations in rats treated with
morphine or T at the time of castration or two
weeks after castration. .. 62

2. MBH LHRH 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 I 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 sale rats castrated
for two weeks . . 68

5. The effects of various sized T implants on MBBH IHB
concentrations in morphine-treated and placeto-
treated male rats castrated fcr two weeks. 69

6. The effects of simultaneous morphine and 5 am T
implants on LH secretion in rats which had been
orchidectomized two weeks previously 80

7. The effects of simultaneous treatment with morphine
and various doses of E2 on LB secretion in rats
which had been orchidectomized two weeks
previously. . . 81

8. The effects of simultaneous treatment with morphine
and various doses of E2 on FSB 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 IB secretion in rats
which had been orchidectcaized two weeks
previously . . 84








10. The effects of simultaneous treatment with morphine
and various doses of DHT cn FSB 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 two weeks
previously. . ,* 97

12. Interaction between morphine and I on FSH secretion
in rats which had been castrated two weeks
previously. . 99

13. Concentrations of BE and NBB in the HBH and POA-AE
of intact rats and orchidectomized rats given
combinations of morphine and T. 102

14. Concentrations of DA and DOPAC in the BBH 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 estrous cycle. 115

16. The effects of naloxone (2 mg/kg) on serum LE in
ovariectomized rats treated with EB (lower panel)
or EB plus E (upper panel). .w. 118

17. Effects of P (5 ag Prog) on naloxone (2 mg/kg) -
induced LB secretion on jroestrus. 119

18. The effects of naloxone injection on LB secretion in
ovariectomized rats receiving EB cr EBP
treatment. a. a a a a 129

19. The effects of continuous morphine exposure and
various doses of E2 on serum IH and FSH levels in
ovariectomized rats. a. 141

20. The effects of continuous morphine exposure on
midafternoon LH and FSB hypersecretion induced by
E2 implantation in ovariectoaized 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 cvariectcaized rats. .. .. 146














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy



GONADAL STEBOID MODULATION CF OPICIC EFFECTS ON
GONADOTROPIN SECRETICN IN IHE RAI


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, luteinizimg hormone (IH),

and follicle stimulating hormone (FSH), were investigated in

the rat. Male rats were chronically treated with scrphine as

a means of providing continuous opiate receptor stimulation.

When 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. Bowever, only T reversed these

changes when treatments were initiated two weeks after

orchidectomy. Further, in rats which had been castrated for


xii








two weeks, morphine enhanced the ability of T to inhitit

gonadotrcpin secretion and blocked the T-induced

accumulation of LHBH in the hypothalamus. At physiological

doses, it was found that 17-beta-estradicl (E2), but not

5-alpha-dihydrotestcstercne (DHT), similarly interacted with

morphine to suppress gonadotrcpin release. further, the

ability cf morphine to interact with I 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 ovariectomized rats, indicating

that endogenous opioid peptides (EOP) inhibit LH secretion

throughout the estrus cycle, and during the Ircestrcus and

EB-induced LH surges. During LH hypersecretion, induced by

progesterone (P) treatment to proestrous or EB-treated

ovariectomized rats, naloxone was unable tc stimulate LH

release, indicating that EOF may contribute to the advanced

onset and increased magnitude of the LB 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 of E2 cn gonadotrcpin

release in these animals. In summary, the work presented in

this dissertation indicates that EOP play an important role


xiii








in the regulation cf gonadal steroid feedback in sale and

female rats by modifying the sensitivity of the brain tc

circulating gonadal steroids.


xiv














CHAPTER I
INTRODUCTION


Opiates have been used by old world cultures for many

centuries. Opium is the dried latex from the unripe seed

pods of the plant Pagaver somniferum. It was probably first

discovered by the ancient Eesopotaxians (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 to 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 over 10% of the dry weight of opium. Its

structure was proposed in 1925 by Gulland and Rcbinscn, and






2
finally synthesized in 1952 by Gates and Ischudi (see Leake,

1975). Other less abundant alkalcids 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. Ihis search for a less

addictive opiate also led to the synthesis of naloxone, an

opiate antagonist relatively devoid cf analgesic and other

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 acrphine to relieve the

pain resulting from battle injuries received during the

Civil War O'Donnell and Ball, 1966).








Many authors coesented on opiate abuse at this time. In

1881, Kane described the physiological consequences 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 of 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 of the Great Cpium Bars, China's annual

population increase was found to fall frc 6% to under 1%.

It was even suggested that the opium trade could be used as

a means of controlling China's overpopulation!

The deleterious effects of opiates and cther 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 sale heroin and

methadone users and diminished sexual function in wcoen

narcotic addicts, respectively.

The numerous physical effects of cpiates were postulated

to be the result of an interaction with specific central








nervous system receptors. In 1973, several laboratories

independently reported the existence of opiate receptors in

the nervous systems of mammals (Pert and Snyder, 1973;

Terenius, 1973). Soon to follow these discoveries was the

isolation of endogenous opioid peptides (EOP). 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 cn reproductive hormone secretion.

The effects of opiates on the regulation of reproductive

hormones, luteinizing hormone (IB) and follicle stimulating

hormone (FSH), is the subject cf this dissertation.














CHAPTER II
REVIEW OF THE LITERATURE


This chapter will survey the major work with respect to

the neuroendocrine control of gonadotropin 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 LB secretion, although important

differences between LH and PSH secreticn 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 hypothalamo-

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 (1968), Ccoper et al. (1978),

Boore and Bloom (1978 and 1979), Sternberger and Boffaan

(1978), matson et al. (1980) and Palkovitz (1981).

Information regarding luteinizing hormcne releasing hcracne








(LHRH) biochemistry, steroid concentrating neurons, and

opioid peptides and their receptors may be found in reviews

by Naftolin et al. (1975), Sar and Stumpf (1975), Eloom et

al. (1978), McEwen et al. (1979), Childers (1980) and Martin

(1981), Jutisz et al. (1983). Many excellent reviews

concerning the role of monoaminergic neurons in regulating

gonadotropin secretion have appeared over the years. Among

the most complete are Coppola (1971), Weiner and Ganong

(1978), 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 Heites et al. (1979), Cicero (1980a and

1980b) and S. Kalra et al. (1980). While 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 1.



Historical

Earlj Observations

Although modern endocrinology has yet to complete its

first century, observations cn endocrine function have been

made throughout history. Relief carvings, figurines, and

drawings from prehistoric, Egyptian, Babylonian, and later








Greek, Roman and Eenissance artisans appear to depict

medical conditions (Garrisson, 1929). Some of these

artifacts have been interpreted as illustrating such

illnesses as female endocrine system obesity, gciter and

gigantisism. Goiter was endemic among ancient cultures and

the Chinese prescribed an appropriate treatment cf iodine-

rich seaweed. While the Chinese, Hindus, and Egyptians all

described diabetes melitis, the Greek Arctaec of 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 Egyptions and Hebreis, respectively.

Much later in the eighteenth century, the results of

experimental removal of the gonads would be pioneered ty

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 sales while the left

testes produced sperm destined to become females.

Additionally, Aristotle felt that the male provided all the






8

determining characteristics while the female provided merely

a fertile environment 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 vocabulary. Leeuwenhock first described the

presence of "little animals of the semen" in 1677 and 200

years later Hertwig demonstrated the union of the sperm and

the ovum.

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 to those ccuring 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 of Vienna

described the cyclical changes in the uterine endometrium

which occur during the menstrual cycle.



The Development of Neuroendocrinology

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








eighteenth centuries Albrect von Baler and Frederick Buych

recognized that the body contains ductless glands which

release their contents into the blood. Claud Bernard

referred 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 sales and females.

One ductless organ which was found to be of major

importance in maintaining normal hcaeostasis was the

pituitary gland. 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 be a

vestigial portion of the train. The pituitary gland was

found to secrete substances necessary for normal body

growth, the maintenance of reproduction, the initiation of

lactation, and the restoration of atropied thyroid and

adrenal tissue. In the 1940's C.H. Li and colleagues

isolated and synthesized the two gonadotropic hormones,

luteinizing hormone and follicle stimulating hormone (LH and

FSH, respectively; li et al., 1940 and 1949).








A functional relationship between the brain and the

pituitary gland was evident to many scientists. Galen's view

that the pituitary drained phlegm frc the brain to the

nasopharnyx was refuted by the anatomist Schreiber in 1660.

Schreiber's contemporary, Richard Lcwer proposed that

substances from the ventricles perfused from the brain to

the pituitary where they percolatedd" into the circulation.

This view is quite similar to our 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 chapter of the eye produced similar

degeneration of the thyroid, adrenals and testes, but the

ovarian corpera lutea 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 Popa 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 hypcthalaaus Housay el al. (1935) and

Wislocki and King (1936) quickly rectified this error.

Several observations led to the development of cur

current understanding of neurcendccrine relationships. Among

the foremost was the discovery of neurosecretion ty

magnocellular neurons in the paraventricular and supraoptic

nuclei by Scharrer and Scharrer (1940). These neurons

released oxytocin and vasopressin frcm 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 electrical

stimulation of the hypothalamus. In 1948, Harris proposed

the chemoreceptorr hypothesis" to explain the control of

anterior pituitary function by hypothalamic horcmnes

released into the portal circulation. This concept remains

one of the cornerstones of neurcendocrine thought.

Much of the work of neuroendocrinologists over the past

30 years has concerned the deacnstraticn, isolation and

characterization of these hypothalamic hormones. Balasz et

al., (1962) used the term hypophysiotropic area to describe

the regions of the hypothalamus that would support pituitary

grafts. Using in vitro assays, releasing activity was








demonstrated in hypothalamic extracts for thyroid

stimulating hormone (Shibusawa et al., 1956), LH (BcCann et

al., 1960), prolactin (Heites et al., 1960), FSB (Igarshi

and McCann, 1964; Mittler and Meites, 1964), and growth

hormone (Deuben and Meites, 1964). Bypothalamic release

inhibiting-'activity was shown for prolactin (Talwalker et

al., 1961; Pasteels, 1961) and growth hcracne (Krulich et

al, 1968).

The isolation of these releasing factors proved tc 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 been 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 cf tcth IH and FSH, but because

other factors appear to also regulate FSH release, it has

been termed luteinzing hormone releasing hormone (LERE,

Martsuo et al., 1971). The competition to isolate

hypothalamic releasing factors continued as Guillemin's

laboratory reported the sequence of a growth hormone






13
release-inhibiting factor, now called somatostatin (Brazeau

et al.1973). The rivalry between these two laboratories did

not cg unrecognized in the scientific community. In 1977,

together with Rosalyn Yallow, Boger Guillesin and Andreb

Schally received the Nobel Prize in Physiology. The drive

to isolate releasing factors continues today. CorticotrcFin

releasing factor was isolated by Wylie Vale, Guillemin's

college at the Saulk Institute (Vale et al., 1981). In the

following year Vale and Guillemin simultaneously reported

the sequence for a growth hocrone releasing factor

(Guillemin et al., 1982, J. Bivier et al., 1982)



Neuroanatomical Selationships

Anatoay of the Pituitary Gland

The pituitary gland lies within a portion of the syencid

bone called the sella tursica. It is positioned below the

aid-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 or anterior pituitary, have distinct

embryological origins. The neurohypophysis is derived from

neural ectoders and contains the nerve terminals cf

magnocellular neurons of the hypothalamus. It is involved

in the neurosecretion of vasopressin and caytocin. The

adenohypophysis is derived from an envagination of the








stromal ectoderm called Rathke's pouch and has nc direct

neural connection with the brain. It constitutes 80% of the

weight of the pituitary gland. Another less prominent

division of the pituitary gland is the intermediate Icte.

While derived from Rathke's pouch, it does receive

innervaticn from the hypothalamus and is often included in

dissections with the neurohypophysis. Together these two

lobes are termed the neurointeraediary lobes (NIL). lhile

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 5% of the cells of the

pars distalis are gcnadotropin 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 _H othalamic 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 autcncaic

hoaeostasis. 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 border of the hypothalamus is recognized

as the anterior commissure and lamina terminalis rostrally

to the hypothalamic sulcus and cerbral aqueduct caudally. In

rats the hypothalamus constitutes 1% of the weight of the

brain.

An important landmark evident frca the aidventral 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 of Uillis 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 referred to as the primary pleius 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 form 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 blood

supply to 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 blccd

flow is from the hylothalaaus 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 of 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 hypcthalaaic area includes

the preoptic area located rostral to the optic chiasm.

Consequently this area is often collectively referred to as

the preoptic area-anterior hypothalamus (POA-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 to the

suprachiasmatic nucleus, the paraventricular nucleus which

constitutes the rostral preoptic area to the dorsalateral

wall of the third ventricle, the supraoptic nucleus, and the

organum vasculosum of the lamina terminalis which is a

circuaventricular 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 postericr areas are often grcuped

into a tissue section referred to as the medial basal

hypothalamus (MBH). 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 loss of reproductive cycles (Goodman and Knobil, 1981).

The nuclei of the hypothalamus have numerous afferent and

efferent connections. Seven afferent tracts impinge upcn

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,








midbrain, anterior comaissure and preoptic area.

Corticomedullary fibers include the fcrnix 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 masmillary

bodies receive inputs from the ascending spinal tracts, the

brainstem and the anterior thalamic nucleus. Finally, photic

input to the suprachiassatic 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 princess

terminate in the anterior thalamus and aidbrain. A

periventricular fiber system returns inputs to the midbrain

thalamic nuclei from the posterior, tuberal and supraoptic

hypothalamic areas. While the primary direction cf all

these pathways is either afferent or efferent, it is not

absolute. In addition, many smaller pathways cannot be

identified without immunocytochemical techniques. The

relation of these tracts as well as intrahypothalamic

pathways to the neurotransmitter systems will be discussed

in the following sections.








Anatomy of the luteinizing Hormone-Beleasing bcrmone
Neuronal Systems

Many problems associated with inmunccytochemical

techniques have precluded a complete description of the

distribution of LHBH neurons. Among these problems is the

lack of an amino acid sequence for a IHBH precursor or its

mRNA sequence which could be used to visualize LHBH-

containing perikaryia. Many antibodies to LHRB are

conformationally restricted or require one cr both terminal

ends of the LHRH molecule for recognition. This can prevent

the visualization of peptide-ccntaining cell bodies. Tc

demonstrate LHRH 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 LHRH neurons in the rodent brain. The

neurons are bipolar or fusifcrm and have distributions nct

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 LHRH as a hypothalamic releasing hormone. At least

two distinct neuronal pathways appear to exist:

1. a tuberoinfunditular 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.








Although LHRH perikaryia have been visualized in the

arcuate nucleus of several species, zany groups have had

difficulty demonstrating their presence in the rat

(Sternberger and Hoffaan, 1978). However, deafferentaticn

spares 20% to 30% of LHBH concentrations in the MEB and does

not disrupt basal LB secretion (Blake and Sawyer, 1974; S.

Kalra, 1976; Brownstein et al., 1977; Sopor and Weik, 1980).

Since this does not sees to be due to incomplete lesions,

there appears to be some LHRH neurons inside the area of the

cut.

A recent study by 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 LHEH antisera which was not

conformationally 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 retrochiassatic nucleus, precptic area, and

organus vasculosum of the lamina terminalis, terminated in

the external layer of the median eminence.

Other recent studies have expanded the potential

distribution of LHRB cell bodies in the brain. These include

septal projections and preoptic projections to the organus

vasculosum of the lamina terainalis, the olfactory tulb and

midbrain central gray (Dluzen and Bamerez, 1981; Witkin et

al., 1982; Shivers et al., 1983b).








Anatomy of the Monoaminerqic Neuronal Systems

The gradual improvement of analytical procedures has

resulted in a fairly accurate description of the

distribution of monoaminergic neurons 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 antibodies generated against

catecholamine synthetic enzymes such as phenylethanolamine-

n-sethyltransferase, which converts ncrefinephrine (BE) to

epinephrine (EPI), these two neuronal systems can te more

easily differentiated.



Noradrenergic pathways

Voogt (1954) first demonstrated the presence of NE in the

brain. It now appears that the hypothalamus receives a rich

afferent innervation from HE-containing neurons. Evidence

favoring an external source for NE in the hypothalamus

includes the depletion of 70% to 90% cf hyFothalamic BE

after complete deafferentation (Weiner et al., 1972). The

residual NE seen after deafferentaticn or brainstem lesicns

appears to be due to glial cells concentrating NE and to

collateral reinnervation, since no BE-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








solitary tract nucleus, the ventral fcntine nucleus, the

locus ceruleus, and the mesencephalic reticular formation.

These cell groups innervate the hypothalamus through three

major tracts the ventral ncradrenergic 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 bodies

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.



Dopa ninergi2c_gathyas

Several distinct dopaminergic pathways are found in the

brain. The largest consists of dopamine (DA)-containing

neurons in the zona compact of the substantial nigra and

ventral tegmentum. From these neurons arise the

nigralstriatal and mesocortical-mesolimbic DA 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 intrahypothalamic DA systems. The tuberoinfunditular CA

system consists of cell bodies in the arcuate nucleus which

project to the median eminence. Axon collaterals terminate








in the arcuate nucleus, ventralateral nucleus and

premammillary nuclei. Included in this system is the

tuberohypophyseal DA tract which contains cell bodies in the

arcuate and periventricular nuclei that project to the

neurohypophysis and intermediate lobes of the pituitary

(Hoore and Bloom, 1978; Palkovitz, 1981). lastly, a poorly

defined collection of DA-containing neurons in the zona

incerta, dorsalaedial subthalamus and posterior hypothalamus

form the incerto-hylothalamic DA system. These cells project

to the dorsal anterior hypothalamus, and the paraventricular

and lateral septal nuclei (Bjorklund et al., 1975).



Epinephrine-containinq 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

forebrair bundle (Hoore and Bloom, 1979; Palkovitz, 1981).



Serotonergic Pathways

There appears to be a widespread distribution of

serotonin (5HT) terminals throughout the brain. Clusters of

5HI-containing perikaryia are restricted to the midline

brainstea raphe nuclei. Ascending fibers course through the

medial forebrain bundle and innervate the hypothalamus,








particularly the suprachiasmatic nucleus (Kuhar et al.,

1972). In addition, there is evidence for 5WT perikaryia

within the hypothalamus (Fuxe and Understedt, 1968).



Anatomy of Endogenous Opioid Pegtide Containing lneurcnal


At present three distinct families of endcgencus opicid

peptides (EOP) have been characterized. Each appears to have

its own 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 EOP neurcnal

systems is far from complete.



Beta-endorphin-containing neurons

Beta-endorphin is derived from prccpiomelanocortin (POMC)

and has been colocalized in cells with ether POCC-derived

molecules like adrenocorticotropin, melanocyte stimulating

hormone, and beta-lipotropin (Mains et al., 1977). It is not

clear whether all PCMC-containing neurons release the save

degradation products, or whether as Watson et al. (1980)

suggest, several distinct PCOC-neuronal populations exist,

each releasing a characteristic set cf aclecules. One

clearly defined beta-endorphin pathway is agreed upon (Bloom

et al., 1978; Finley et al., 1981a). Fusifora neurons are

contained in the tuberal hypcthalamus extending frcm the







25
lateral arcuate nucleus to the lateral hypothalamic border.

Fibers project to the anterior hyFothalamus and septum where

the pathway reverses direction and follows the stria

terminalis to terminate in the dcrsal raphe, locus ceraleus

and central gray. Arborizations are found throughout the

POA-AH and MBH including the median eminence and arcuate

nucleus. Electronaicroscopic studies have revealed local

interactions between PONC-containing neurcns in the

hypothalamus (Kiss and Williams, 1983).



Enkephalin-containing neurons

Enkephalins are derived from a precursor molecule which

contains methionine-enkephalin, carboxy-terminal extended

methionine-enkephalin and leucine-enkephalin. While teta-

endorphin-containing neurons have a fairly distinct Fathway,

enkephalin-containing neurons are widely distributed

throughout the brain. The highest concentrations cf

methionine-enkephalin are found in the striatus, followed by

the hypothalamus. Dense methionine-enkephalin innervaticn

has been found in the external layer of the median eminence

(Watson et al., 1980). Enkephalin-ccntaining cells are

generally interneurons although several short pathways have

been described.

1. A dense collection of methionine-enkephalin-

containing cell bodies in the central amygdala

project to the bed nucleus of the stria terminalis

(Cuello and Paximus, 1S78).






26

2. Magnocellular neurons of the paraventricular nuclei

contain methionine-enkephalin and ricject to the

neurohypophysis (Bossier et al., 1979).

3. A preopticotuberal pathway with cell bodies in the

POA-AH projects to the median eminence (Finley et

al., 1981b).

4. Methionine-enkephalin-containing perikeryia in the

anterior hypothalamus terminate in the septal area

(Sakanaka et al., 1982).



Dynorphin-containing neurcns

Dynorphin is derived from the prodynorphin-alpha-

necendorphin precursor along with leucine-enkephalin and

carboxy-terminal extended leucine-enkephalin molecules

(Goldstein et al., 1979; sakadani et al., 1982).

Consequently, there is some difficulty in distinguishing

between leucine-enkephalin contained within prodynorphin-

and proenkephalin-containing neurons. Of the three families

of EOP, the distribution of dynorphin-containing neurons is

the most restricted. A supraoptico-neurchypclhyseal 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

neurohypophysis (Watson et al., 1982a,b; Roth et al., 1983).







27
In addition to the brain, EOP are found in the pituitary

gland. Beta-endorphin coexists with other 1CMC molecules in

adenohypophyseal corticotropes and is released into the

blood by stimuli which also stimulate adrenocorticotropin

secretion (C. Rivier et al., 1962). The 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 te

discounted (Simantov 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 aromatase have been characterized in the train

and hypothalamus (Massa et al., 1972; Naftolin et al., 1975;

Sar and Stumpf, 1975; McEven et al., 1979). The electrical

activity and morphological characteristics of neurons in the

hypothalamus can be altered ty estrogens (Pfaff and McEwen,

1983; loran-Allerand et al., 1983).









Neuroanatomical Interactions

Although the distributions of LHBB, 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 ncradrenergic 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 LBBH

cell bodies en passant. Considering the close 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

neuroanatomical relationships in the hypothalamus. Bonkleiv

et al. (1981) compared teta-endorjhin and LBRH

immunoreactivity in adjacent brain slices and found teta-

endorphin containing neurons and terminals tc be more widely

distributed than these containing LHRH in the MBH. Double

immunostaining procedures have not been employed. By







29
comparing autoradiography and immunccytochemistry Shivers et

al. (1983a) reported that LBEH neurons do not appear to

concentrate estradicl in their nuclear regions. This

contrasts with the report that some neurons in the

hypothalamus that are electrically sensitive to estrogen do

stain for LHBH (Kelly and Ronkleiv, 182).



Patterns of Gonadotrcpin Secretion

Gcnadotropin secretion is inherently pulsatile. Whether

absolute levels of LH and PSH 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 frequency and amplitude.

Because of the longer half-life of PSB (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 IHBH

concentrations in the MBB fluctuate showing 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 hyrcthalawic-pituitary-LH

axis. Within hours of gonadectomy in the male LB 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; Gallc, 1980a).

Concentrations of LHRH decline in the HBH while the

pituitary responsiveness to IBHI 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 such less effective in

returning FSH to gonadal-intact levels (Hahesh et al.,

1975). This is in agreement with reports that other gonadal

and hypothalamic factors also regulate FSH secretion (HcCann

et al., 1983).



Females

In the female rat low levels of gcnadotrcpin secretion

are interrupted by a preovalatory discharge of LH and FSH

every 4 to 5 days. Gonadotropin levels during the proestrous

surge may be 5 to 20 times greater than tasal levels.

whether basal or surging, LH and PSB levels in the female

rat are the result of pulsatile discharges of hcrucne

(Gallo, 1981a,b). Prior to the prcestrous gonadotrcpin







31
surge is a period of 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 timed to the aidafterncon

(Legan et al., 1975). Superimposed upon this cyclic pattern

of LB 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 IH surge is preceded by a period of H1BH

accumulation in the BBH and accompanied by a decline in

levels of the decapeptide (S. Kalra and Kalra, 1981). The

decline in RBH LHRB levels appears to be an indication of

enhanced LHRH release (Sarkar et al., 1976). Alsc

accompanying this preovulatory interval is a marked increase

in the LH secretary response to LHBH and an increase in

pituitary LHHH binding sites (Cooper et al., 1973; Aiyer et

al., 1974; Savoy-Hoore et al., 1980).

When the feedback effects of gonadal steroids are remcved

as a result of ovariectomy, the response of the pituitary to

LHBH immediately increases (Ccoper et al., 1975).

Luteinizing hormone levels gradually increase over a three

week period, the result of increased LB pulse amplitude and

a small increase in LH pulse frequency (Weick et al., 1981).








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

ovariectcaized rats, LH levels decline and the pituitary

becomes refractory to LHBH (Vilchez-Hartinez et al., 1974).

Two days of continuous estrogen exposure enhances the

response of the pituitary to LBBH and induces a diffuse

midafternoon LH surge. This afternoon LH surge repeats for

several days if estrogen titers remain elevated (Legan et

al., 1975)

The injection of P several hours before the onset cf the

LH surge further increases the pituitary response to LHBH,

enhances the magnitude of the resultant LH surge, and

advances its onset (Aiyer et al., 1976; Kalra et al., 1981).

Concentrations of LHRH in the HBB increase prior to and

decline during the period of 1H hypersecretion in a fashion

similar to that seen on proestrus (S. Kalra and Kalra,

1979).



Honoaminerqic 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 of hormone output. The following








section will briefly review our current understanding of

monoaminergic regulation of LH and FSH secretion. hhile

many trends are apparent in this area, it must be cautioned

that few monoaminergic drugs are specific for cne

neurotransmitter or receptor. Bather, it is the integration

of many studies and confirmaticns of those works that

provide an accurate description of the role each

neurotransmitter plays in the control of gonadctropin

release.

Many studies have evaluated the activity cf moncaminergic

neurons in various reproductive states, especially the

catecholamine neurons (Weiner, 1S74; Cooper et al., 1978).

Methods of estimating catecholamine neuronal activity which

do not disturb the steady-state include the rates of

synthesis of NE or DA from trace amounts of their

radiolabeled precursor, tyrosine, or the disappearance of

trace amounts of added radiolabeled catecholamine. This

technique can also be employed for the evaluation cf 5HT

neuronal activity using radiolabeled tryptophan or 51.

Problems associated with this technique include the

inability to evaluate small tissue sections, unequal

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 of NE or DA

depletion following the blockade of 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 of

synthesis. Using this method catecholamines can be evaluated

in small nuclear regions using sensitive analytical

techniques. with the high dosages required to inhbitit

catecholamine synthesis, some nonspecific effects may cccur.

Additionally, it cannot be assumed that catecholanine neuron

will behave similarly under ncn-steady-state conditions.

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. These techniques have not been

used extensively in neuroendocrine studies, however.



NoreKpin9ePhine

A large body of evidence suggests that central

noradrenergic neurons control LH and PSB secretion. Sawyer

and colleagues originally showed that a variety of centrally

acting adrenergic agents influence the cvulatory release of

gonadotrcpins (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 stimulatcry and

inhibitory influences on the release of gonadotropins.







35
The activity of noradrenergic neurcns appears to change

in concert with several LH secretary 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 Vurtian, 1968; Anton-lay

et al., 1970; Coppola, 1971; Kizer et al.,1974; Siapkins 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

ovariectomized rats (Zschaeck and Wurtuan, 1973; Loftstrom,

1977; Nunaro, 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 neurcnal

activity. The blockade of NE synthesis with DA-beta-

hydroxylase (DBH) inhibitors suppresses pulsatile LB release

and LH hypersecreticn induced by endogenous steroids pricr

to ovulation, electrical stimulation of the hypothalamus and

gonadal steroid administration (P. Kalra et al, 1972; S.

Kalra and McCann, 1973; Drouva and Gallo, 1976a; Gncdde and

Schuiling, 1976), This blockade of IB synthesis is overcome

by the pretreatment with dihydrcxyphenylserine which does







36
not require DBH for its metabolism to NE, but not by 1-DCPA,

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 prcestrouE LH release (P. Kalra

et al., 1972; Gnodde and Schuiling, 1976; Hartinovic and

McCann, 1977). Together these data argue for a stimulatory

role for NE on gonadotropin release.

The administration of NE has varying effects on Lb

release depending on the mode of administration and

experimental paradigm employed. While in vitro evidence

suggests that NE can stimulate LHBB secretion from

hypothalamic fragments via an alpha-adrenergic mechamism

(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 IB 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 outside the

hypothalamus, or if the inhibition occurs through LBBE

neurons (Caceres and Taleisnik, 1980, 1982; Leung et al.,

1981, 1982).

In ovariectomized rats pretreated with gcnadal steroids

the intraventricular administration of NE stimulates IH

release (Krieg and Sawyer, 1976; Vijayan and McCann, 1978;









Gallo and Drouva, 1979). It would appear that in an

ovariectcaized animal, the inhibitory effects of NE on 1H

release predominate, while in the presence of gonadal

steroids, stimulatory nodes of BE cn LH release are

primarily operative.

Despite many studies, the nature of the coupling of the

noradrenergic neuron to the IBH-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 BE infusion (Gallo, 1982). 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 IHBB

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-containing neurons. On a

molar basis EPI is more potent than either NE or DA in

eliciting H release when injected into the ventricles of

gonadal steroid treated ovariectomized rats (Vijayan and







38

McCann, 1978). Epinephrine might be iaFortant 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 sale rats (Begro-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 H1 and FSB release in sale and

female rats. In vitro studies suggest that DA stimulates

LHRH release from hypothalamic tissue fragments (Schneider

and McCann 1969; Botsztejn 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 HBB of female rats (Simpkins et al., 1979, 1980). While

this might argue for a stimulatory role for EA neurons on LH

release, DA itself does not consistently induce LE secretion

when injected into the ventricles (Drouva 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








LH release depending on the dose employed (Kamberi et al.,

1970; Cramer and Porter, 1973). Studies with the neurotozin,

5,7-dihydroxytryptamine, suggest that serotcnergic neurcns

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 Babii, 1983; Walker and

Wilson, 1983; Chen et al., 1984). Since many serctcnergic

drugs also act upon catecholamine neurons or their

receptors, more information must accumulate on this subject

before a more definitive assessment can be made.



Endoqenous Opioid Peptides and the cntrcl cf Gonadotrovin
Secretion

The first experimental evidence that cpicids 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 Froestrous gcnadctrcin surge (Pang

et al., 1977). At this time Cicero et al. (1975a, b)

demonstrated that chronic treatment with acrphine induced

changes in the reproductive system of the male rat similar

to the effects of narcotic abuse in men, i.e. depressed

serum I and diminished secondary sex organ function. These

studies indicated that the effects cf opiates cn








reproductive function could be assessed in a laboratory

setting.



Reproductive Pharmacologq of Opioids

The antigonadotropic effects of opiate administration are

ultimately exerted at the level of the hypothalamus through

the inhibition of LBBS release. Many careful studies have

eliminated other possible sites of action (Cicero 1980a).

Opiate administration has no effect cn the metabclisa 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 I synthesis or the

response of the leydig cell to gonadotropins. At the

pituitary gland, morphine does not alter the release cr

synthesis of LH or the response of the gonadotrope to LHBH.

In one study however, leucine-enkephalin acutely inhibited

the LH secretary response to IHEB (Bay et al., 1979).

Several lines of evidence suggest that opiates exert

their antigonadotropic actions by inhibiting LHRB release.

The increased LHEH concentrations in hypophyseal portal

plasma which accompanies the proestrous gonadotropin surge

are prevented by morphine treatment (Ching, 1983). Alsc,

opiate agonists and antagonists can modulate the release of

LHBH from in vitro hypothalamic incutations (Rotsztejn 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 LHBR 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 LH-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 LHRH output. The

acute effects of opioids on LH secretion satisfy the

criteria for mediation ty 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 intraventricularly

(Bruni et al., 1977; Cicero, 1980b; Johnson and

Rosencrans, 1981; Kinoshita et al., 1981; Kato et

al., 1982; Bhanot and Wilkinson, 1983; Leadem and

Kalra, 1983; Marko and Romer, 1983;

2. The relative potency of opiates in suppressing 1H

release parallels their pharmacological efficacy in

other preparations such as the displacement of

tritiated opiates, the ability to inhibit the

contraction cf guinea pig illeum, and analgesia

(Cicero et al., 1976; Cicero, 1980a).






42
3. The effects of opiates on LH secreticn are reversed

by opiate antagonists like nalcxone 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 dextrorctatcry

isomers (Cicero et al., 1976).



Physiological Inhibition of Gonadotropin Secretion bv
Opioids

Although the existence of opiate receptors in the train

and hypothalamus and the presence of a pharmacologic

response to stimulation of these receptors suggest opicid

pathway which effects IH secretion, this does not, per se,

verify that EOP normally act to inhitit LH release. If

physiologically released EOP do act to inhibit 1H 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 frequently verified evidence favoring

an EOP inhibition of LB 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 secreticn, then sequestering IfE with

an appropriate antibody might produce the same effect.

Antibodies to both beta-endorphin and dynorphin have been

found to stimulate LH secretion (Schulz et al., 1981; Forman

et al., 1983)








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 froc 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.



ulti ple_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, termed 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 au-opioid







44
receptor appears to mediate the effects cf cmrphine and its

cogeners. The delta-opioid receptor appears to be acre

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 subtype may mediate the

effects of opioids on LH secretion. Opioid inhibition of LH

secretion appears to involve both a nu and kappa-opicid

receptor component (Cicero et al., 1983c; Gabriel and

Siapkins, 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-Honoaminergic Interactions

The interaction between opioid and monoamine-containing

neurons has been investigated in several systems (Schwartz,

1979; Kuchinsky, 1977). Several recent studies have

characterized a possible adrenergic interaction with opicids

in the control of gonadotropin secretion. The stimulatcry

effects of naloxone on LH secretion are prevented by

adrenergic antagonists, DBH inhibitors and EPI synthesis


I







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 LH secretion without an

intermediary opioid interaction since the inhibition cf LH

secretion seen following morphine is reversed ty subsequent

treatment with clonidine or the intraventricular EEI

injection (S. Kalra and Siapkins, 1981; S. Kalra and Gallo,

1983).

In addition to both NE and EPI-containing neurons, ECP

have teen shown to interact with dcpaminergic 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 LB secreticn are influenced ty each cf

these monoaminergic neurcnal systems (Botsztejn et al.,

1978; leiri et al., 1980k).



Opioid-Gonadal Steroid Interacticns

While it is apparent that BOP-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 gonadal

steroids in the brain. Morphine, like 1, can prevent the







46
post-castration rise in serum LH, while naloxone and I are

mutually antagonistic on LH secretion in orchidectcmized

rats. Similarly estrogens can inhibit naloxone's stimulation

of LH secretion in ovariectomized 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 ECE concentrations or their

release under varying steroid milleus Peptide release wculd

be the most preferable estimate of neuronal activity. To

date, only beta-endorphin has been evaluated in the








hypoihyseal portal plasma of non-human primates. Cf

interest, beta-endorphin levels decline precipitcusly

following ovariectomy and during menstruation (Ferin et al.,

1984), This would imply that the activity of hypothalamic

beta-endorphin-containing neurons depends cn ovarian

factors, particularly P.

Tissue levels of EOP show changes which may be relevant

to gonadotropin secretion. Both beta-endorphin in the septum

and medial preoptic area and sethionize-enkephalin in the

MBH 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). Orchidectomy dces

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 antericr

pituitary following castration (Lee et al., 1980; Bong 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).








Bationale

From the literature presented it appears that EOP say

function as one of several neurotransmitters that regulate

the release of the gonadotropins, LH and FSH. Based on the

pharmacological effects of opicid agonists and antagonists

during various reproductive states, and the ability of

gonadal steroid alterations to modify EOP levels in the

brain and pituitary it appears that ECP-containing neurons

respond to changes in the steroid milleu. These changes may

reflect alterations in EOF neuronal activity which mediate

the feedback effects of gonadal steroids on gonadotrcpin

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 Icrphine. In the

female rat, the extent of EOP inhibition of LE secretion has

not been fully assessed. Naloxone was used to evaluate the

potential EOP inhibition of LB secretion present during the

estrous cycle and following gonadal steroid treatment to

ovariectomized rats. Finally, the feedback effects of

gonadal steroid treatment on gcnadotropin secretion in

females was evaluated in the presence of continuous opiate

receptor stimulation with morphine.














CHAPTER III
GENERAL MATERIALS ADD METHODS



Animals

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

Wilmingtcn, 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 i 10 C.) controlled room with

food and water provided ad lititum.

Reproductive status cf female rats was verified by

microscopic examination of vaginal lavages (Ingram, 1956).

Rats which displayed two consecutive 4-day estrous cycles

were chosen for studies employing gonadal intact female

rats. The normal sequence of cell morphclogy in the vaginal

smear consists of lavages containing cornified epithelial

cells (estrus), followed by two days of predominately

leukocytic smears diestruss I and diestrus II), which is

then followed by a day in which the lavages contain

nucleated epithelial cells (proestrus). The cornified

epithelium and leukocytic smears are characteristic of a








gonadal steroid milieu dominated by estrogens and

progestins, respectively.

Surgical procedures consisted of subcutaneous

implantation of drugs or steroids and bilateral gonadectomy

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 acst

experiments, trunk blood was collected by decapitation.

Decapitations were completed within 30 seconds of remcval of

each rat from its home cage. In studies employing LBRH

injection, blood samples were collected by cardiac puncture

under light ether anesthesia. All blcod samples were

collected in a room separate from the animal quarters. Sera

was separated from trunk blood by centrifugation (1000 1 g)

for 15 minutes while jugular and cardiac blood samples were

centrifuged in a microcentrifuge for two minutes. All sera

were stored at -20o C. for later hormone analysis ty

radioimmunoassay (RIA).








Dissection of Brain Tissue

Brains were rapidly removed and placed with their dorsal

surface on ice. Tissue sections containing the MHB and POA-

AH were removed using fine iris scissors. Cuts for the MBH

fragment were made at the posterior order of the optic

chiass, then caudally at the level of the maamillary bodies,

2 an laterally at the hippocampal sulcus, and 2 as 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 pcsterior border of the critic chiasa.

Additional cuts were placed 2 an lateral to the midline and

approximately 2 an from the dorsal surface of the PCA-AH at

the level of the anterior commissure.



Gonadal Steroid Treatment

Gonadal steroids were obtained from Steraloids Inc.,

Hilton, N.J. and administered as subcutaneous implants or as

injections. Implants consisted of Silastic tubing (1.57 am

i.d., 3.17 as o.d.) of lengths ranging from 2.5 am tc 30 am.

Capsules were filled with either crystalline foras 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 blcod levels cf






52

gonadal steroids for several weeks. In the sale rat these

implants reversed Eost-castraticn LH hypersecretica at

physiologically relevant dosages (P. Kalra and Kalra, 1980).

The imalantation of crystalline E2 in ovariectomized rats

provided sustained E2 levels which immediately reduced 18

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

microliters of oil at 1000 h. This treatment produced a fall

in LH secretion followed by a sidaftereccn LH surge two days

later. If 5 ag 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. Cther

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 ag morphine (free

base, Merk, St. Louis, NO), 37.5 ag micrccrystalline

cellulose (Avisil, FMC Corporation, Philadelphia, PA.), 0.56

ag Cab-o-sil (Cabot Corporation, Bostcn, 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 (Gibson and

Tingstad, 1970; Simpkins et al., 1983b). Control animals

received placebo pellets which were formulated with an

additional 75 ag. Avisil rather than morphine free base.

Naloxone HC1 (Dupont Pharmaceuticals, Garden City, N.J.)

was dissolved in normal saline and administered

subcutaneously.



Measurement of Catecholamines Indolamines and Metabolites

Concentrations of NE, DA, 5HT, the NE metabolite

normetanephrine (N E), the DA metabolites dihydroxyphenyl-

acetic acid (DOPAC) and honovanillic acid (HVA) and the 5HT

metabolite 5-hydroxy-indolacetic acid (SHIAA) were measured

by amperometric methods following their separation by high-

pressure liquid chromatography using a modifaction of the

procedure described by Michaud et al. (1981). The

separation was accomplished using reverse phase

chromatography across an IBM LC-18 (15 ca X 4.6 an, 5

micrometer particle size) with a mobile phase composed of 81

methanol, 0.2 aM octyl sodium sulfate, 0.1 M. NaB2r04 and

0.1 aM EDTA at pH 2.9. The flow rate was varied from 0.5

al/min for the first 7.5 minutes of separation, 0.7 al/min








from 7.5 through 23 minutes, 2.0 ml/ain from 23 to 39

minutes and 3.0 mi/min thereafter, through 57 minutes. This

procedure allowed the elution of catecholamines, indolamines

and their aetabolites in standards and samples within a one

hour period. The times for eluticn (in order) for BE, NE,

DA, DOPAC, 5HIAA and BVA were 5.5, 13, 17.5, 21, 28.5, 33.5

and 36 minutes respectively. The detection of amines,

indolamines and their metabolites was accoarlished with an

electrochemical detector (IBB,model LC 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 EBB and EOA-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 mg% EDTA at a weight:volume

ratio of 1 ag/10 microliters. Average weights of these

tissues were 19.0 1 0.5 ag for BBH and 20.2 0.6 mg for

POA-AH.

To each 20 microliter of sample of EBB and POA-AB tissue

homogenate was added 2 ng dibydrcxybenzo-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 NE, DA, 5HT, and their

metabclites.



Hormone Badioimsunoassays

Luteinizigi Hormone and Follicle Stimulating Eormone

Serum and medium samples were assayed for LH and PSR

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 NIADDK-anti-rFSB-S-11 for the

FSH assay. Radioiodinations were performed in our laboratory

using standard procedures for a chloramine-T iodination with

gel filtration to separate free iodine from hcraone-bound

iodine.

Because these studies were performed cver 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, tH-BR-2, was 61 times more potent

than the original LH-RP-1. To aid in the comparison cf IH

values across experiments, all 1H values were expressed

relative to the original LH-BP-1 standard. The figure cr

table legends will note when this conversion was made. ISH

values were expressed in relation to the reference standard,

FSH-BP-2. The intra-assay variation for the LB and FSE

assays, determined by the coefficient of variation for 10

replicates of pooled castrate serum which inhibited the






56

binding of the radiolabeled hormone 40X to 60% was 6.8% 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 LH and FSH, respectively.

The sensitivity of these assays, defined as a the account of

standard hormone required to inhibit the binding of the

radiolabeled hormone by 20% was C.90 mg (LH-BP-1) for LH and

0.40 ng for FSH.



Luteinizinq Hormone Releasinq Hcroone

Tissue sections containing the MHE and POA-AH were

homogenized in 2 al of 0.1 N. BCl and supernates were

analyzed for LHRH using RIA methods described previously (S.

Kalra, 1976). Acid supernates were neutralized with 2 N.

NaOH during the assay procedure. Synthetic LHRH obtained

from Beckman Co. (Palo Alto, CA.) was used as the reference

standard and for iodinaticn. Mcnoradioiodinated LHBH was

employed as described previously (Bett and Adams, 1977).

Rabbit antibodies against LBRH 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 LHBH were expressed in

terms of tissue sample (i.e. ng per HBH or POA-AH).








Testosterone

Serum T levels were analyzed according to procedures

described previously (P. Kalra and Kalra, 1982).



Statistical Analysis

For most experiments, analysis of variance with Student

Neuman Keuls tests were 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 to determine the

significant effects of LHBH 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.














CHAPTER IV
THE EFFECTS OF CHBCNIC BCRPHINI TBEATHENT ON TESTCSIEBONE
NEGATIVE FEEDBACK IN CASTRATED MALE RAIS



Introduction

The feedback effects of gonadal steroids on 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 EOP can acutely suppress LB release in

intact and gonadectcmized rats (Heites et al., 1979; Cicero,

1980; Kinoshita et al., 1981; leaden and Kalra, 1983).

Interestingly, gonadal steroids have also been found to

modify EOP levels in various sites within the hypcthalamus

and the secretion of beta-endorphin in the hypophyseal

portal system (Barden et al., 1981a; lardlaw et al., 1982;

Wehrenberg et al., 1982). Further, acute blockade of central

opiate receptors with narcotic antagonists transiently

reverses the inhibitory feedback effects of T cn LH release

(Cicero et al., 1980). Since gonadal steroid treatment and

opiate receptor stimulaticn suppress LH release and EOP-

producing neurons are found in the vicinity of LHBH neurons,

it is logical to suspect that ECE-containing neurons may

either mediate the feedback effects cf gonadal steroids cr






59
that they may act through similar hypothalamic mechanisms to

decrease LH release (P. Kalra and Kalra, 1980; Matson et

al., 1980; Sar and Stumpf, 1975; Shivers et al., 1983b). The

following study compares continuous opiate receptor

stimulation with morphine to T 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 (twc 15 mm tubes) cr

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 1, while the

brains were rapidly removed and dissected for analysis of

BBH and AH-POA LHBH concentrations.








Experiment 2

In the second study, rats were castrated and two weeks

later received either control pellets or implants of

morphine, T, or morphine plus 1. Testcstercne-containing

tubes were either 2.5, 5.0, or 10.0 am 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

Rats which had been castrated two weeks previously

received either control, 5 mm T, or morphine plus 5 am T

implants. After 4 days, rats were killed by decapitation,

anterior pituitaries were removed, hemisectioned, and

preincubated in control medium (minimal essential medium +

25 mm Hepes, pH 7.2, Gibco Inc., Grand Island, NY) for one

hour at 370 C. Fresh control medium or medium containing 1

X 10-7 M. LHRH 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.









Results
Effects of Time After Castration on the Serum LH and
Hypothalamic LHRH Responses to T and morphine

Figure 1 illustrates serum 1H concentrations in rats

treated with either T or morphine immediately after or two

weeks after castration. Serum T levels attained by the two

15 an implants were 2161 120 pg/al in rats treated

immediately after castration and 2439 t 129 pg/ml in rats

receiving T two weeks after castration. Ihese 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 1 (p < 0.05). However, when the

initiation of treatment was delayed for twc weeks after

castration (long-term castrate), unlike T, morphine was no

longer effective in suppressing LB 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. Bcth T and morphine

prevented the post-castration decline in MBB LEBE

concentrations if the treatments commenced at the time of

castration (p < 0.05). Two weeks after castration, however,

only T was effective in stimulating LBBH accumulation in the

MBH.

Levels of LHRH in the AH-POA of castrated rats treated

with I or morphine were unaffected by any experimental

treatment (data not shown).













1 Control
Testosterone


Morphine


Short Term
Castrate


I_


Long Term
Castrate


Figure 1: Serum LB 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 orchidectcay. Lcng-terz
castrate = treatment started two weeks post-
castraticn. p denotes < 0.05 vs. control. IH
concentrations among the control groups were not
significantly different and were therefore
pooled.


IL


C 600-


r 400-
-j
E
L
* 200-


i 1i~s I












E Control


M Testosterone


Short Term
Castration


Long Term
Castration


Figure 2: MBH LHRH concentrations in rats treated with
morphine or T at the time of castration or two
weeks after castration
Short-tern castration = treatment started
immediately after orchidectcay. Icng-term
castration = treatment started two weeks post-
castration. p denotes < 0.05 vs. control. lHH
LBBH concentrations among the control groups were
not significantly different and were therefore
pooled.


T


I
m

C



I
-.
=2c











The Effects of Chronic Morphine Treatment on the LH
Secretor_ and MBH LBRHResponses to Graded Doses of I.

Figure 3 illustrates serum T levels attained from various

sized implants in rats castrated for two weeks treated

additionally with either morphine or placebo pellets. Low

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 LB concentrations in rats treated with mcrphine or

placebo pellets in combination with graded doses of I are

also shown in Figure 3. In placebo-treated rats, low

circulating levels of T (355 t 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 1 (10 ma implant). As

observed in Experiment 1, morphine treatment alone did not

significantly change serum LH levels in castrated male rats.

However, it influenced the LH response to I treatment. In

rats treated with bcth morphine and T, the LE response curve

to T shifted to the left such that a 501 suppression of






65
serum LH was observed with the 2.5 am T implant and nearly

complete suppression of serum LB was seen with 5 am 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/al, 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 IB or IHBH concentrations;

3. between 500 and 999 pg/al, representing I levels

which stimulate LHBH accuaulaticn in the MEH but have

little effect on LH secretion; and

4. greater than 1000 pg/ml, representing T levels which

consistently suppress LH secretion (P. 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 LB 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 inhibition

occurring at 622 44 pg/al. In some of these morphine-

treated rats, near baseline LH levels were found with I

concentrations as low as 420 pg/ml serum. In contrast. tut








in agreement with previous studies (P. Kalra and Kalra,

1982), significant depression of serum LH concentrations was

only apparent with T concentrations cf greater than 600

pg/ml. Serum T concentrations necessary tc elicit a 501

reduction in LH secretion were 300 pg/ml in morphine-treated

rats and 960 pg/ml in placebo-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 (F <

0.C01) as were the drug treatment (placebo or morphine ), T

treatment, and the interaction of drug with I (p < 0.01, for

each).

The effects of simultaneous T plus chronic morphine

treatment on LHRH concentrations in the MBH are shown in

Figure 5. In placebo-implanted rats, I exposure for 4 days

resulted in an accumulation of LHRH in the BBH (i < 0.05).

As has been noted previously, this accumulation of LBBB in

the MBH occurred at T levels lower than that required to

inhibit LH secretion (5 mm T implants, Figure 5 vs. 10 am

implants, Figure 4; P. Kalra and Kalra, 1982). It can also

been seen from Figure 5 that in the presence of morphine, T

was unable to cause any significant increases in LBRE stores

in the MEH.















state W2.5mm T
levels I implant s


r3-n-s1 I:mTh


,-Morphine
,5 -
T T


I I I I I I
0 200 400 600 800 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 LB in
morphine-treated and placebo-treated sale rats
castrated for two weeks

The vertical and hcrizcntal bars represent sean i
standard error for serum LB 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.


400-


S300-


_200-
E

100-


0-
















(8)


400- -


E 300- T3 (9

S \ (7
I 200- Placebo
-J 200
E
:3 ,-Morphine T
U 100-

(7)

I I I I
0o-- -- ----- -- ---I (9)*T

200 400 600 800 1000 1200
Serum Testosterone (pg/ml)





Figure 4: Relationship between LH and I levels in acrEhine-
treated and placebo-treated sale rats castrated
for two weeks

In the interest of clarity in presenting the
relationship between the 1H response and I
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/al, or > 1000
pg/ml. Number in parentheses represent the number
of rats in each T level. The vertical and
horizontal error tars represent mean i standard
error for the IH and T concentrations,
respectively. denotes p < 0.01 vs. I < 199
pg/al group; the dagger symbol denotes p < 0.05
vs. placebo-iamlanted group at the same T level.



















E
Ca
CO
n1
o



oa
0
0.


Implant Size
O 0.0 mm (Sham)
12.5 mm
S5.0 mm
W10.0 mm


Placebo Morphine


Figure 5: The effects of various sized T implants on MBE
LHRH concentrations in morFhine-treated and
placebo-treated male rats castrated for two
weeks.
LHRH concentrations were determined per NBH
tissue section and expressed relative to shaa-
implanted MBH 1HBH (control) concentrations. *
denotes p < 0.05 vs. sham-implanted grcup.











Effects of T and morphine Treatment cn the Pituitary
Resocnsiveness to LHRH

Testosterone levels achieved by 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 (Table 1,

legend, p < 0.05)

The effects of the in vivo T or morphine plus T treatment

on the in vitro release of LH frcm pituitary incubations are

also shown in Table 1. Incubation of pituitaries for cne

hour with 1 X 10-7 N. LHRH significantly increased IH

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.


I









TABLE 1

Effects of In Vivo Morphine and I Pretreatment on In Vitro
LH Release from Hemisectioned Pituitaries (LB release rate:
ng/mg pituitary tissue/ hour)



In vitro In vivo treatment


treatment Control T Morphine + T



Control 1,583 313 957 51 1,393 1 166

1 X 10-7 M. LHRH 3,930 555 3,682 275 4,588 285

delta-LH 2,346 f 666 2,612 362 3,194 t 425


Serum I levels achieved by the 5 mm T implants were 683 89
ng/ml for morphine-implanted and 636 73 pg/ml for
placebo-treated rats. Serum LH concentrations were
225 33 ng/al in castrated (placebo plus sham treated) rats,
227 61 ng/al in 5 mmT implanted rats, and 16 1 ng/nl in
5 amm plus morphine treated rats.



Discussion

These studies reveal a putative underlying interaction

between opiates and T on LH release. As in the case of I

implants, placement of morphine pellets immediately after

castration prevented the post-castration rise in serum IH

and the decline in LHBH 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 of LEBE

concentrations in the MBH after T implantation, LH and its








releasing factor were unaffected by morphine treatment

initiated two weeks after castration. Apparently, cpiate

receptor stimulation does not mimic the actions of I 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 LH release

in rats which had been castrated for two weeks is surprising

in view of the observation that administration of mcrphine

or opioids systemically or ECE intraventricularly promptly

suppressed LH release in gonadectcaized rats (Cicero et al.,

1980; Kinoshita et al., 1981; leaden 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 LB suppression must

be transient because with sustained supply of morphine these

rats appear to overcome the inhibition and LH secreticn

seemingly occurred unabated 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., 1982a; Bhanot and Wilkinson, 1983). The

ability of testosterone to inhibit LH release diminishes






73

with time after castration (Cicero et al., 1982a). 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. Ihis is shown by the

observation that low concentrations of T, while failing to

exert any impact on LB release nc their own, were highly

effective in suppressing LH release in morphine-treated

rats. The LH response curve to graded doses of 7 was shifted

to the left (Figures 3 and 4) in morphine-treated rats with

maximal inhibition occurring at T concentrations cf 622 t 44

pg/ml serum. In some morphine-treated rats, near baseline LH

levels were seen with T concentrations as low as 420 pg/ml.

Furthermore, it appears that serum I levels needed to

achieve a 50% reduction in serum La levels were three times

lower in morphine-treated rats than iv control rats.

Evidently morphine treatment concurrently with I rendered

rats more responsive towards T feedback action. T has been

shown to decrease pituitary rescnsiveness to LHRH (Drouin

and Labrie, 1976; P. Kalra and lalra, 1980). It is possible

that morphine may interact synergistically with T at the

level of gonadotropes to suppress pituitary responsiveness

to endogenous LBRH 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 (Cicerc et

al., 1977; Wiesner et al., 1814), there was no evidence of

modification by morphine of LBBH 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 LfBH-

producing neurons and opiate receptors overlap (Sarr and

Stumpf, 1975; Watson et al., 1980; S. Kalra, 1981; Shivers

et al., 1983b).

Precisely how T and chronic morphine interact to inhitit

Lf secretion is not known. The apparent inability of

morphine to reduce 1H 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 chronic

morphine to block the accumulation of LHBB in the MBB

following exposure, it is possible that chronic morphine

suppressed the activity of the IBRH neuron at several steps

in the secretary process.

While the LHRH neuron may be a likely site for the

interaction between T and morphine, cther 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 requires 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 Slapkins, 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 CHBROIC MORPHINE TREATMENT CH THE NEGATIVE
FEEDBACK REGULATION OF GONADOTBCPIN SECRETION BY GCNADAI
STEECIDS



Introduction

The negative feedback effects of testicular hormones on

gonadotropin secretion appear to te exerted at the level of

the hypothalamus and the pituitary (Drouin and Labrie, 1976;

Franchimont et al., 1979; S. Kalra and Kalra, 1983). In the

male rat, three majcr gonadal steroids, I, DHT and E2 have

been implicated in the feedback regulation of LB secretion,

and to a lesser extent, FSH secretion (P. Kalra and Kalra,

1980; D'Agata et al., 1981; Sherins et al., 1982; acCann et

al., 1983; Nishihara and Takahashi, 1983). While E2 and EHT

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 (Hassa et al., 1972; laftolin 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 anatcmical relaticnshiF

exists between steroid concentrating, LHBB and EOP-

containing neurons (McEwen et al., 1979; Watson et al.,







77
1980; Shivers et al., 1983b). EOP neuronal systems have been

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 own, enhanced by

3-fold the negative feedback effects cf T. The present study

extends these observations by comparing the effects on LH

and PSH secretion of T, DHT and E2 in male rats treated

chronically with morphine.



gxperjimental
Chronic morphine and Gonadal Steroid Treatments

Groups of placebo or mcrphine-treated rats were

simultaneously exposed to either sham implants or one of the

three gonadal steroids at various dosages. All treatments

lasted for 4 days, after which animals were sacrificed by

decapitation between 1100 h and 1300 h. Serum from trunk

blood was stored at -20o C. for subsequent analysis of IH

and FSH by RIA. The steroid treatments were:

1. 5 am tubes packed with crystalline T;

2. 7.5 mm tubes packed with crystalline EBT or HDT which

had been diluted with cholesterol on a weight:weight

ratio of 1:1 or 1:3; and






78

3. E2 dissolved into sesame seed cil at a concentration

of 300 micrograms/ml and filled into 7.5 am tubes or

E2 diluted in oil to concentrations of 150 or 75

micrograas/ml and then placed into tubes of 5.0 am in

len th.



Evaluation of Pituitary _gesponsiveness to LHEB

Based on the results of the first series of experiments,

groups of placebo or chronic mcrphine treated rats were

simultaneously exposed to either sham, 5 am T, 7.5 ma DET or

7.5 maa 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.R., s.c.). Blood samples were obtained

by cardiac puncture under light ether anesthesia, pricr to,

and 30 minutes after LHRH injection. This dose of LBBH was

based on earlier cwrk (Lu et al., 1980). Serum was

separated by centrifugation and stored at -20o C. for

analysis of LH by BIA.



Results

The effects of simultaneous morphine plus I

administration are shown in Figure 6. While 5 mm T implants

alone reduced serum LB concentrations by greater than 40%,

this effect was not significant. Chronic morphine treatment

alone did not affect serum LH levels in castrated rats.

However, as was noted in Chapter IV, the coatination of 5 am







79
T plus morphine treatment reduced serum LH concentrations by

90% to levels seen in intact male rats (p < 0.05; P. Kalra

and Kalra, 1977b).

The consequences of chronic morphine exposure on serum IH

and FSH levels in castrated rats concurrently exposed tc

various dosages of E2 are shown in Figures 7 and 8,

respectively. Chronic morphine did not alter serum LE

concentrations in animals receiving sham implants.

Similarly, E2 treatment alone failed to significantly reduce

LH levels. In contrast, the ccnbinaticn cf morphine

treatment plus 5 mm E2 (150 micrograms/al) or 7.5 am E2 (300

micrograms/al oil) reduced serum LH concentrations (p <

0.05). The highest E2 dosage (7.5 as at 300 micrograms/al)

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/al) plus mcrphine

significantly reduced FSH levels by 301 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 dcsages of DHT in
















D Placebo

M Morphine


I


I.


*

*.os


-------------L-I------___X <_____________________ _._________________________


SHAM
IMPLANT


5mmT


Figure 6: The effects of simultaneous morphine and 5 an T
implants on LH secreticn in rats which had been
orchidectomized two weeks previously

denotes p <0.05 compared to shai-implanted
rats; the dagger symbol denotes p < 0.05 when
compared to placebo-irmlanted rats.


600-

E

c



Cr
- 400-


-J
U,
2(
200-
















D Placebo

D Morphine
2_1


5mm E2
(150,ug/ml)


+j


7.5mm E2
(300pg/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. HB was determined using the
LH-RP-2 reference standard and expressed relative
to LH-BP-1 (LH-RP-1 = 61 X LH-BP-2). denotes p
< 0.05 vs. sham-iamlanted rats; the dagger symbol
denotes p < 0.05 vs. placebo-implanted rats at
the same E2 dose.


600-




C 400-

"-


W 200-
0-


SHAM
IMPLANT


5mmE2
(75jug/ml)


I


------ --;----






82








1f Placebo

SMorphine

30 -


CE
cr

I 20-


IV

Io-





SHAM 5mm E2 5mm E2 7.5mmE2
IMPLANT (75,ug/ml) (150ug/mi) (300pg/ml)





Figure 8: The effects of simultaneous treatment with
morphine and various dcses cf E2 cn PSH secretion
in rats which had been orchidectonized twc weeks
previously

E2 was dissolved into oil and filled into tutes
of lengths as noted in the figure. denotes F <
0.05 vs. sham-implanted rats; the dagger systol
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 DET

crystals) significantly reduced serum I1 concentrations by

63%, while the coatination of chronic morphine plus DHT (at

1:1) or 7.5 mm DBH reduced 1H levels 39g and 83%. The

combination of morphine plus DHT was not significantly sore

effective in inhibiting LH levels than DHT alone at any DHT

dosage, however. Chronic morphine and BHT 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 shown 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 (91, p < 0.05). Morphine

treatment with 7.5 mm DHT reduced serum PSH levels 43%. This

reduction in serum FSH concentrations was significant

relative to sham-implanted rats and to placebo-iapanted rats

at the same DHT dosage.

The effects of combinations of chronic morphine plus

steroid treatments on the in vive LH secretary response to

LHRH injection are shown in Table 2. Prior to 1HBH

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














SPlacebo


SMorphine
600-




I 400-
_J


Cn
200 -





SHAM 7.5mm DHT 7.5mm DHT 7.5mm DHT
IMPLANT (1:3) (1 1)




Figure 9: The effects of simultaneous treatment with
morphine and various doses cf DHT cn LB secretion
in rats which had been orchidectomized two weeks
previously

Crystalline DHT or DHI which had been diluted cn
a weight:weight basis with cholesterol was packed
into tubes 7.5 an in lengths as ncted in the
figure. Serum 1H was determined using the LE-BP-2
reference standard and expressed relative tc IH-
RP-1. (LH-RP-1 = 61 X IH-BP-2). denotes p <
0.05 vs. sham-implanted rats; the dagger symbcl
denotes p < 0.05 vs. placebo-implanted rats at
the same DHT dose.






















45


0E

C
" 30
I
LL



- 15-
Cr)
W:













Figure 10:


SHAM 75mm DHT 7.5mm DHT 75mm DHT
APLANT (1:3) (1:1)


The effects of simultaneous treatment with
morphine and various doses cf DHI on FSH
secretion in rats which had been castrated two
weeks previously.

Crystalline DHT or DBT which had been diluted on
a weight:weight ratic with cholesterol was
packed into tubes 7.5 mm in length as noted in
the figure. denotes p < 0.05 vs. sham-
implanted rats; the dagger symbol denotes p <
0.05 vs. placebo-implanted rats at the same UBT
dose.








on serum LH levels in sham-implanted rats prior tc LHBB

injection. When compared to sham-implanted rats, 5 mm T was

without effect, while 7.5 am E2 (300 micrograms/ml oil) and

7.5 mm DHT reduced serum LH levels by 57% and 69X,

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 an DBT 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 I reduced LH

concentrations before LHBH injection, this combination did

not alter the pituitary response to LHRH. In rats treated

with E2 alone, pituitary responsiveness to LHBBE was

increased significantly. Despite a reduction in LH

concentrations prior to IHRH injection in morphine plus

E2-implanted rats, the LH secretary response to 1HBH was

further enhanced. Finally, DHT alone diminished the

responsiveness of the pituitary to LBBH and this reduction

in sensitivity was not modified by morphine exposure.