Relationship of hypothalamic catecholamine alterations and reproductive function in aging female rats

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Relationship of hypothalamic catecholamine alterations and reproductive function in aging female rats
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Hypothalamic catecholamine alterations and reproductive function in aging female rats
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xiii, 170 leaves : ill. ; 29 cm.
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Estes, Kerry S., 1952-
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Catecholamines   ( mesh )
Gonadotropins   ( mesh )
Hypothalamic Hormones   ( mesh )
Pharmacy thesis Ph.D   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida.
Bibliography:
Bibliography: leaves 149-169.
Statement of Responsibility:
by Kerry S. Estes.
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Photocopy of typescript.
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Vita.

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RELATIONSHIP OF HYPOTHALAMIC CATECHOLAMINE ALTERATIONS
AND REPRODUCTIVE FUNCTION IN AGING FEMALE RATS






BY

KERRY S. ESTES


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1982














ACKNOWLEDGEMENTS

I express my deepest appreciation to Dr. James W. Simpkins for his

thoughtful and gentle guidance of these studies. His scientific insights

and enthusiastic approach to neuroendocrine research truly have been inspi-

rational to me. I am most grateful to Dr. Satye Kalra for his efforts

toward my scientific maturation. Dr. William Thatcher is thanked for his

advice, concern and kindness.

Many thanks are also due to Murl Casey and Dave Fritz for their tech-

nical assistance. I thank Ray Naslund especially for faithfully smearing

the rats and his help in the maintenance of the aging colony.

I appreciate the financial support provided by the College of Pharmacy.

Finally, I thank my family. Through their love and support, my par-

ents have encouraged me to pursue my goals. My husband, Hartmut Derendorf,

has been most patient during the first month of our marriage. I reserve

very special thanks for him.














TABLE OF CONTENTS

CHAPTER PAGE

ACKNOWLEDGEMENTS........................................ 11

LIST OF TABLES.......................................... vii

LIST OF FIGURES.......................................... ix

KEY OF ABBREVIATIONS.................................... x

ABSTRACT............................................ xii

INTRODUCTION.............................................. 1

LITERATURE REVIEW....................................... 4

Hypothalamic Control of Anterior Pituitary Function.... 4
Hypophysiotropic Hormones........................... 4
Hypothalamic Anatomy................................. 6
General... .. .............................. 6
Vascular Supply.................................. 7
Hypothalamic Nuclei................................ 8

Innervation of the Hypothalamus........................ 9
Afferent and Efferent Pathways....................... 9
Noradrenergic Pathways.............................* 10
Dopaminergic Pathways............................... 12
Other Putative Neurotransmitter Pathways.............. 14

Catecholamine Regulation of Luteinizing Hormone........ 15
Initial Evidence.. ....................... ........... 15
Anatomical Evidence. ................................ 15
Biochemical Evidence. ............................... 16
Pharmacological Evidence............................. 16

Dopamine-Prolactin Regulation......................... 18
Dopamine Regulation of Prolactin...................... 18
Prolactin Regulation of Dopamine.................... 19

Age-Related Alterations in Reproductive Function....... 19
Estrous Cycles in Young Mature Animals............... 19
Patterns of Reproductive Senescence.................. 20
Luteinizing Hormone Secretion........................ 23
Prolactin Secretion................................. 26









CHAPTER PAGE

Age-Related Alterations in the Central Nervous System.... 27
Dopamine......................................... ..... 27
Norepinephrine.......... ........................ 30
Luteinizing Hormone Releasing Hormone................. 32
Other Neurosecretory Changes.......................... 33

GENERAL RATIONALE ....................................... 34

GENERAL MATERIALS AND METHODS.............................. 35

Animals............................... ......... 35
Selection of Animal Model............................ 35
Establishment of Rat Colony........................... 37
Monitoring Cycle Status................................ 38
Monitoring Health Status............................... 38

Surgical Treatment and Blood Collection.................. 39
Hormone Radioimmunoassays............................... 41
Microdissection of Brain Areas.......... ............ 42
Determination of Catecholamine Neuron Activity........... 44
Catecholamine Radioenzymatic Assay....................... 47

EXPERIMENTAL............................................... 49

Evaluation of Age-Related Alterations in Catecholamine
and Luteinizing Hormone Releasing Neuronal Activity
Within Microdissected Brain Areas....................... 49
Changes in Catecholamine Concentration in Microdis-
sected Brain Regions of Aged Male Rats................ 49
Objectives................................... 49
Materials and Methods.............................. 49
Results........................... ... ........ 50
Discussion............... ....................... 50

Changes in Catechol amide Activity in Microdissected Brain
Regions of Aging Ovariectomized Fischer 344 Rats....... 52
Objectives.................. .................... 52
Materials and Methods............................... 53
Results....................... .. ............. 55
Discussion............. ...... .............. ... 61

Response of Luteinizing Hormone Releasing Hormone Neurons
to Ovariectomy in Microdissected Brain Regions of Aging
Fischer 344 Rats..................................... 63
Objectives.............. ................... .... 63
Materials and Methods .... ........................... 63
Results.................................. ...... 65
Discussion................................. .. ...... 67










CHAPTER PAGE

Changes in Catecholamine Activity in Microdissected
Brain Regions of Aging Ovariectomized Long-Evans Rats.. 67
Objectives............................................ 67
Materials and Methods................................ 68
Results.................................... 68
Discussion............ .......................... 70

Response of Luteinizing Hormone Releasing Hormone Neurons
to Ovariectomy within Microdissected Brain Regions of
Aging Long-Evans Rats.................................. 77
Objectives. .......................................... 77
Materials and Methods ............................... 77
Results .............................................. 78
Discussion............................................ 81
Age-Related Alterations in Luteinizing Hormone and Prolactin
Response to Ovariectomy and a-Methylparatyrosine......... 82
Changes with Age in Serum Luteinizing Hormone and Prol-
lactin Levels in Fischer 344 Rats...................... 82
Objectives............................................ 82
Materials and Methods................................ 83
Results................................... 83
Discussion....................................... 86

Changes with Age of Serum Luteinizing Hormone and Pro-
lactin Levels in Long-Evans Rats...................... 90
Objectives ........................................ ........ 90
Materials and Methods ................................ 90
Results ............................................... 91
Discussion ........................... ................ 94

Age-Related Alterations in the Regulation of Pulsatile
Luteinizing Hormone Release in Ovariectomized Constant
Estrous Rats .......................................... 96
Changes of Pulsatile Luteinizing Hormone Secretion Pro-
files in Ovariectomized Rats During Advanced Age....... 96
Objectives ............................................ 96
Materials and Methods................................. 96
Results ..................................... 97
Discussion................................... 02

Restoration of Pulsatile Luteinizing Hormone Release by
Clonidine in Young Ovariectomized Rats with Acute
Norepinephrine Depletion............................... 103
Objectives .......................... .................. 103
Materials and Methods .............................. 104
Results. ................................... ...... 107
Discussion ....................................114
DiS US 100 .. .. *** ** ** *** ** *** ** ** *** ** **









CHAPTER PAGE

Restoration of Pulsatile Luteinizing Hormone Release
by Clonidine in Aged Ovariectomized Long-Evans Rats...118
Objectives .................. ...................... 118
Materials and Methods............................... 119
Results ............................................ 133
Discussion........................................... 130

Reinitiation of Estrous Cycles in Old Pseudopregnant
Rats with a Dopamine Agonist......................... 132
Objectives........................ ............132
Materials and Methods...............................133
Results......................................133
Discussion........... ...................................... 135

GENERAL DISCUSSION....................................... 137

REFERENCES................. ....................... 149

BIOGRAPHICAL SKETCH. ....................................170














LIST OF TABLES


TABLE PAGE

I Reproductive Status of Fischer 344 Rats at Various Ages.... 36

II Parameters Used in Microdissection of Brain Regions.........43

III Age-Related Alterations in Dopamine (DA) and Norepi-
nephrine (NE) Concentrations in Microdissected Brain
Regions from Male Rats..................................... 51

IV Body and Organ Weights and Health Status of Fischer 344
Rats Employed in Catecholamine Studies...................... 54

V Age-Related Change in Dopamine Activity Within Microdis-
sected Brain Regions of Ovariectomized Fischer 344 Rats.....56

VI Age-Related Change in Norepinephrine Activity Within
Microdissected Brain Regions of Ovariectomized Fischer
344 Rats................... ........................... 59

VII Body and Organ Weights and Health Status of Fischer 344
Rats Employed in Luteinizing Hormone Releasing Hormone
Studies.................................................. 64

VIII Effects of Age and Ovariectomy on Luteinizing Hormone
Releasing Hormone Concentrations Within Microdissected
Brain Regions of Fischer 344 Rats.......................... 66

IX Body and Organ Weights and Health Status of Long-Evans
Rats Employed in Catecholamine Studies...................... 69

X Age-Related Change in Dopamine Activity Within Microdis-
sected Brain Regions of Ovariectomized Long-Evans Rats...... 71

XI Age-Related Change in Norepinephrine Activity Within
Microdissected Brain Regions of Ovariectomized Long-Evans
Rats ........................................................ 73

XII Body and Organ Weights and Health Status of Long-Evans Rats
Employed in Luteinizing Hormone Releasing Hormone Study..... 79

XIII Effects of Age and Ovariectomy on Luteinizing Hormone
Releasing Hormone Concentrations Within Microdissected
Brain Regions of Long-Evans Rats........................... 80











XIV Effects of Ovariectomy on Serum Luteinizing Hormone Concen-
trations in Aging Fischer 344 Rats ........................ 87

XV Effects of Ovariectomy on Serum Prolactin Concentrations in
Aging Fischer 344 Rats ................................. 88

XVI Effects of a-Methylparatyrosine on Serum Luteinizing Hormone
and Prolactin Levels in Aging Ovariectomized Long-Evans Rats92

XVII Effects of Ovariectomy on Serum Luteinizing Hormone and Pro-
lactin Levels in Aging Long-Evans Rats**********.................93

XVIII Luteinizing Hormone Pulse Amplitude in Ovariectomized Rats
of Several Ages and Reproductive States................******************100

XIX Age-Related Changes in Mean Plasma Luteinizing Hormone Con-
centrations in Ovariectomized Rats ...*......*..........***.101

XX Effects of Various Doses of Clonidine (CLON) on Pulsatile
Luteinizing Hormone Release in Ovariectomized Rats Pre-
treated with Diethyldithiocarbamate (DDC)...................108

XXI Luteinizing Hormone Release for Two Hours After Clonidine
Injection in Ovariectomized Rats Pretreated with FLA-63....111

XXII Luteinizing Hormone Release between Two to Four Hours After
Clonidine Administration in Ovariectomized Rats Pretreated
with FLA-63....................... ........................113

XXIII Characterization of Luteinizing Hormone Release for Three
Hours After Clonidine Administration in Ovariectomized Rats
Pretreated with FLA-63..................................... 115

XXIV Effects of Diethyldithiocarbamate (DDC) and FLA-63 on Cate-
cholamine Concentrations in the Preoptic Area and Medial
Basal Hypothalamus...................... .. .116

XXV Parameters of the Luteinizing Hormone Assay Employed in
Evaluation of Pulsatile Luteinizing Hormone Secretion....... 21

XXVI Body and Organ Weights of Ovariectomized Long-Evans Rats
Employed in Study of Clonidine Effects on Pulsatile Lutein-
izing Hormone Secretion ...............******................123

XXVII Age-Related Changes in Luteinizing Hormone Secretory Para-
meters of Ovariectomized Long-Evans Rats .......*........... 126

XXVIII Effects of Clonidine on Luteinizing Hormone Secretory Para-
meters in Ovariectomized Middle-Aged and Old Long-Evans Rats128


viii


PAGE


TABLE














LIST OF FIGURES


FIGURE PAGE

1 Age-Related Changes in Dopamine (DA) Turnover Rates in Micro-
dissected Brain Regions of Ovariectomized Fischer 344 Rats ... 57

2 Age-Related Changes in Norepinephrine (NE) Turnover Rates in
Microdissected Brain Regions of Ovariectomized Fischer 344
Rats ................................................... 60

3 Age-Related Changes in Serum Luteinizing Hormone (LH) Concen-
trations After a-Methylparatyrosine (aMPT) in Ovariectomized
Fischer 344 Rats....... ................................ 84

4 Age-Related Changes in Serum Prolactin (PRL) Concentrations
After a-Methylparatyrosine (aMPT) in Ovariectomized Fischer
344 Rats ..................................................... 85

5 Representative Luteinizing Hormone (LH) Secretion Profiles
in Aging Sprague-Dawley Rats Ovariectomized at Various Repro-
ductive States .............................. ............... 99

6 Representative Luteinizing Hormone (LH) Secretion Profiles of
the Effects of Clonidine (CLON, 0.03 and 0.3 mg/kg) and Saline
in Ovariectomized Rats Pretreated with Diethyldithiocarbamate
(DDC) ......................................................... 109

7 Representative Luteinizing Hormone (LH) Secretion Profiles of
the Effects of Clonidine (CLON, 0.3 mg/kg) or Saline in
Ovariectomized Rats Pretreated with FLA-63 ................... 112

8 Representative Luteinizing Hormone (LH) Secretion Profiles in
Ovariectomized Long-Evans Rats of Three Ages ................. 125

9 Representative Luteinizing Hormone (LH) Secretion Profiles for
Six of Nine Individual 11-12 Month Old Ovariectomized Long-
Evans Rats Treated with Clonidine (CLON)...................... 127

10 Representative Luteinizing Hormone (LH) Secretion Profiles for
Five of Eight Individual 21-23 Month Old Ovariectomized Long-
Evans Rats Treated with Clonidine (CLON)...................... 129

11 Estrous Cycle Reinitiation in Old Fischer 344 Repeated Pseudo-
pregnant (PP) Rats Treated Daily with CB-154 (3 mg/kg)........ 134














KEY OF ABBREVIATIONS

AP anterior pituitary

ARC area retrochiasmatica

CA catecholamine

CE constant estrous

CLON clonidine

CV coefficient of variation

DA dopamine

DBH dopamine-a-hydroxylase

DDC diethyldithiocarbamate

EDTA ethylenediamine tetraacetic acid

F344 Fischer 344

FLA-63 bis(4-methyl-1-homopiperanzinyl thiocarbonyl)disulfide

IC irregularly cycling

K rate constant of amine loss

L-DOPA dihydroxyphenylalanine

LH luteinizing hormone

LHRH luteinizing hormone releasing hormone

LSD least significant difference

MBH medial basal hypothalamus

ME median eminence

MFB medial forebrain bundle

caMPT a-methylparatyrosine









NA nucleus arcuate

NAc nucleus accumbens

NC normally cycling

NE norepinephrine

NHA anterior hypothalamic nucleus

NSC nucleus suprachiasmatica

NSO nucleus supraoptica

NVM nucleus ventromedialis

OVLT organum vasculosum of the lamina terminalis

POA preoptic area

POAm preoptic area medialis

POAs preoptic area suprachiasmatica

PP repeated pseudopregnant

SCN suprachiasmatic nucleus

SON supraoptic nucleus

RIA radioimmunoassay

TIDA tuberinfundibular dopamine

TRH thyrotropin releasing hormone

VMN ventromedial nucleus










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


RELATIONSHIP OF HYPOTHALAMIC CATECHOLAMINE ALTERATIONS AND
REPRODUCTIVE FUNCTION IN AGING FEMALE RATS


By

Kerry S. Estes

December, 1982

Chairman: James W. Simpkins
Major Department: Pharmacy

This work has examined the relationship between age-related changes

in hypothalamic catecholamine (CA) neurons and impaired gonadotropin re-

gulation in female rats which exhibit two patterns of reproductive senes-

cence. Long-Evans and Fischer 344 (F344) rats which exhibit the constant

estrous (CE) and repeated pseudopregnant (PP) states respectively, during

advanced age were studied. Norepinephrine (NE) and dopamine (DA) turnover

rates were examined in microdissected hypothalamic regions in young,

middle-aged and old ovariectomized Long-Evans and F344 rats to character-

ize the pattern, extent and loci of age-related changes in CA neuronal

function. Endocrine and pharmacological manipulations were employed to

evaluate the contribution of the described changes in CA activities to

altered regulation of luteinizing hormone (LH), prolactin and LH releasing

hormone (LHRH) during advanced age.

Results of these studies show that no widespread progressive decreases

in DA or NE neuronal function accompany the aging process. Age-related

changes in magnitude and direction within and between F344 and Long-Evans
xii










rats. While CA concentrations generally decrease during advanced age,

CA turnover rates are maintained or augmented in several hypothalamic

regions of older compared to younger animals, particularly in long-lived

F344 rats. Hyperprolactinemia appears to maintain the PP state in old

F344 rats and may be responsible for increased DA turnover in several hypo-

thalamic regions during aging. Decreased postcastration LH response in

CE rats results from impaired LHRH neuronal function as reflected by de-

creased frequency and amplitude of LH pulses. An a-adrenergic agonist

can restore pulsatile LH release in both ovariectomized young rats with

induced NE depletion and in middle-aged CE rats in which decreased NE acti-

vity was observed, but not in old CE rats. Thus, adrenergic dysfunction

appears to mediate the onset of the CE state, while subsequent alterations

may maintain the CE state. These results show that complex patterns of

CA neuronal change contribute to impaired hormone regulation during ad-

vanced age and suggest that pharmacological agents may be employed to

improve age-related neuroendocrine dysfunction.


xiii














INTRODUCTION

Advanced postmaturational age is associated with a generalized decline

of many physiological functions. Although several hypotheses have been

proposed to explain age-related impairments in regulatory processes, the

mechanisms responsible for these alterations remain poorly understood.

Due to the central role of the neuroendocrine system in maintaining homeo-

stasis through regulation of the hormonal milieu, changes in this system

with age could potentially be responsible for many age-related impairments.

This concept predicts that changes in hormone regulation occur during ad-

vancing age and further suggests that age-related impairments might be

corrected by restoring or maintaining neuroendocrine regulatory function.

Among the most recognized age-related changes in physiological func-

tions is the decline of reproductive capacity and cessation of regular

ovulatory cycles. While the menopause in middle-aged women appears to re-

sult primarily from ovarian oocyte exhaustion, altered gonadotropin regu-

lation appears primarily responsible for demise of reproductive function

in female rats. Similarly, altered gonadotropin levels have been associated

with impaired reproductive activity in aged men and male rats. Aging fe-

male rats exhibit various patterns of reproductive senescence which appear

to result from alterations in neuroendocrine regulatory functions. Most

rats have irregular and lengthened ovarian cycles in middle-age prior to

establishment of noncyclic reproductive states. Following a variable

period of irregular cycles many rats enter a constant estrous (CE) non-

cyclic state. Constant estrous rats have well developed follicles which










secrete moderate levels of estrogen, but fail to secrete sufficient lutein-

izing hormone (LH) to stimulate ovulation. A hypothalamic alteration ap-

pears to prevent the normal steroid induced release of LH in these CE

animals. Other rats enter a repeated pseudopregnant (PP) state in later

life. In this PP state animals ovulate at 8 to 20 day intervals with cor-

pora lutea maintained and elevated progesterone secretion between ovula-

tory events. Old PP rats appear to have relatively normal LH response to

a stimulatory regimen of gonadal steroids. Some old rats become anestrous

with atretic ovaries and have chronically elevated prolactin levels. This

state appears to result from a pathological condition at the level of the

anterior pituitary (AP) characterized by a relative absence of functional

gonadotrophs and proliferation of mammotrophs.

Interestingly, the PP state appears to be associated with longer lived

rats compared to the CE state. This observation together with the demon-

strated difference in ability of the neuroendocrine system of these two

animal models to respond to stimulatory regimens of gonadal steroids with

LH secretion is consistent with the concept that alterations in the neuro-

endocrine system may be responsible for many age-related declines in

physiological function. However, the nature of alterations which result

in the onset of the CE or PP state in aging rats is largely unknown. Elu-

cidation of changes in hormone regulation which occur in these animal

models would provide a better understanding of mechanisms responsible for

age-related alterations in homeostasis and perhaps suggest approaches to

treat these impairments.

Neuroendocrine systems can broadly be described as transducer sys-

tems which convert neural signals into endocrine output. The importance

of neuroendocrine regulation of reproductive function has been identified









by numerous investigators through a variety of experimental approaches.

The secretion of AP hormones is regulated by neurohormones produced in

the hypothalamus and transported to the AP via the hypophysial portal

system. Inputs which modulate activity of neurohormone secretion of hy-

pothalamic neurons have been extensively studied. These inputs consist

of synaptic transmission signals from putative neurotransmitters. Although

age-related alterations in neurotransmitter activities have been reported

in several brain regions, the extent and rate of these changes on repro-

ductive function in rats are not well characterized. Experiments described

in this thesis examine the activity of luteinizing hormone releasing hor-

mone (LHRH) neurons and activity of the neurotransmitters, dopamine (DA)

and norepinephrine (NE), within hypothalami of aging rats which enter the

CE or PP state. These studies are designed to evaluate the extent and

identify the locus of neuroendocrine alterations which occur in the CE

and PP states. In addition to identifying the locus of age-associated

alterations in the neuroendocrine system of CE and PP animals, studies

are described which evaluate the effects of manipulation of neurotrans-

mitter systems on LH secretion in old animals.

It is hoped that the results of studies described in this thesis will

improve our understanding of the mechanisms responsible for age-associated

declines in hormone regulation by (i) more clearly identifying central

loci responsible for altered gonadotropin secretion (ii) permitting quan-

tification of the consequences of altered neuron function on gonadotropin

secretion and, (iii) evaluating the potential for pharmacological correc-

tion of age-related alterations in neuroendocrine function.














LITERATURE REVIEW

Hypothalamic Control of Anterior Pituitary Function

Hypophysiotropic Hormones

The concept that neurohormones regulate AP secretary function evolved

from a variety of experimental observations and several experimental ap-

proaches conducted by numerous investigators. McCann and Porter1 reviewed

early studies which led to this concept.

The close anatomical proximity of the AP and hypothalamus suggested

to Popa and Fielding that the portal vasculature connecting these tissues

served to transport hormones from the AP to the hypothalamus.2 A few

years later, the direction of blood flow was shown to course from the

brain to the AP by following the movement of dyes in the portal blood.3

The Scharrers first proposed and later demonstrated that hypothalamic

neurons secreted hormones.4'5 This concept led Harris to propose the

"chemotransmitter hypothesis" which essentially stated that neurosecretory

products from hypothalamic neurons traveled via the hypophysial portal

system to regulate hormone secretary activity of the AP.6

Early evidence supporting this hypothesis came from studies which

lesioned the hypothalamus or severed the connections between the hypo-

thalamus and pituitary. These procedures resulted in atrophy of the

gonads, thyroid and adrenal glands (see 1). Similarly, removing the pi-

tuitary from the sella turcica and transplanting it to the anterior cham-

ber of the eye or under the kidney capsule resulted in a variety of meta-

bolic changes including atrophy of endocrine glands stimulated by hypo-

physiotropic hormones. However, ectopic pituitaries maintained corpora
4









lutea7 and mammary gland function.8 These experiments indicated the cen-

tral nervous system (CNS) exerted both a stimulatory and an inhibitory

effect on AP hormone secretions.

Another approach used in early investigations to confirm the "chemo-

transmitter hypothesis" used electrical stimulation of hypothalamic regions.

Harris and coworkers showed in a series of studies that hypothalamic stim-

ulation could induce ovulation in rabbits, increase adrenal activity,10

and increase thyroid weight."

A classic approach used by endocrinologists when attempting to iden-

tify the origin and characteristics of a putative hormone is to study the

effects of various types of extracts made from the tissue suspected of

secreting the substance. Results of studies utilizing this approach

during the late 1950's and 1960's demonstrated hypothalamic releasing hor-

mone activity for each of the AP hormones and hypothalamic inhibiting ac-

tivity for prolactin and growth hormone (see 1, 12). The effects of hypo-

thalamic extracts were generally negated when extracts were treated with

trypsin which indicated the putative neurohormones were peptides or pro-

teins.

The structural elucidation of neurohormones was and continues to be

of major interest to investigators who examine CNS regulation of AP func-

tion. Successful identification and synthesis of four neurohormones

during the last decade have vastly improved understanding of neuroendocrine

regulatory mechanisms. Synthesis of these hormones permitted development

of both radioimmunoassay (RIA) and immunohistochemical techniques to quan-

tify these hormones as well as to facilitate studies designed to character-

ize the physiological function of these substances. The first releasing

factor to be identified was thyrotropin releasing hormone (TRH).13'14









The tripeptide has identical structure and releases thyrotropin in all

species which have been examined. Thyrotropin releasing hormone also

stimulates prolactin secretion in rats, cows and humans.15-17 Two years

after the identification of TRH, the ten amino acid sequence of LHRH was

elucidated in bovine18 and porcine19 hypothalamic extracts. Extensive

studies of LHRH have shown it has identical structure and similar ability

to release both LH and follicle stimulating hormone in all species tested.

Growth hormone inhibiting hormone somatostatinn) was identified and syn-

thesized in 1973.20 Recently, a 41 amino acid sequence with corticotropin

releasing activity has been characterized.21

Extensive study of these four peptide structures confirmed their

roles as neurohormones through meeting the criteria proposed by McCann and

Porter to prove neurohormone function. These criteria include

1. demonstration of secretary elements in the brain which produce the

putative neurohormone within diffusable distance from the portal

vessels,

2. identification and characterization with biological, physical and

chemical methods of the putative neurohormone within portal vessels,

3. demonstration that the putative substance alters AP hormone release

after entering the portal vessels.'

Conclusive identification of other hypothalamic substances as neurohor-

mones with these criteria awaits further study.

Hypothalamic Anatomy

General. The hypothalamus is the most ventral portion of the dien-

cephalon and is partially exposed on the ventral surface of the brain.

Hypothalamic structures evident on the ventral surface (rostral to caudal)

are the optic chiasm, infundibulum, tuber cinerium and mammillary bodies.









The base of the infundibulum together with the rostral portion of the

tuber cinerium is the median eminence (ME). Anatomically, the hypothalamus

is defined dorsally by a groove in the wall of the third ventricle called

the hypothalamic sulcus; rostrolaterally by the first segment of the optic

tract; caudolaterally by the groove formed by the cerebral peduncles;

and, caudally by the limiting border of the mammillary bodies.22 The ros-

tral border is indistinctly separated from the preoptic area (POA) and the

POA is sometimes included as part of the hypothalamus.23 The optic chiasm

and lamina terminalis are generally considered to form the rostral most

hypothalamic border.22 The hypothalamus is often divided into four gray

areas: the POA, the anterior hypothalamic region, the medial hypothalamic

area and the posterior hypothalamus. It should be noted that these regions

are arbitrarily defined since several hypothalamic nuclei occupy more than

one region. The ME is often considered as a separate region.

Vascular supply. While all of the hypothalamus is vascularized from

the ventral surface via branches from the Circle of Willis, the five hy-

pothalamic regions each receive blood from separate arteries of this sys-

tem and are generally thought to have relatively separate venous drainage.

The vascularization of each region has been discussed in detail by Ambach

and Palkovits.23 Of special interest is the vascularization of the ME

region. The ME and nucleus arcuate (NA), which lies dorsal to the ME, are

richly and exclusively supplied by hypophysial arteries. These arteries

form a capillary network in the ME by multiple anastomoses called the

primary plexus of the ME or palisadic zone. The ME arterial supply con-

tains fenestrated capillaries characteristic of the circumventricular

organs and therefore the ME is outside of the blood-brain barrier. Veins

collect in several directions from these capillaries to form a portal










circuit of the AP. Portal veins of the AP collect blood from the rostral,

dorsal and lateral portions of the ME and branch into a secondary capil-

lary plexus in the pituitary.

Hypothalamic nuclei. Anatomically, the nuclei within the hypothalamus

are often less distinct compared to other brain regions such as the thal-

amus. Since a brain nucleus is defined as an area where the density of

cells is higher than in surrounding areas, boundaries between nuclei are

usually identified on the basis of relative position of cellular densities.24

Hypothalamic nuclei are bilaterally distributed along either side of the

third ventricle with the exception of the ME and organum vasculosum of the

lamina terminalis (OVLT). The OVLT, like the ME, is a circumventricular

organ.

Although nuclear regions within the hypothalamus were identified by

early anatomists, Palkovits most extensively characterized the location

and size of more than 20 specific hypothalamic nuclei in the rat.24 Con-

comitant with his anatomical description, he developed a microdissection

or "punch" technique which has been used extensively to identify the po-

tential neuroendocrine function of many hypothalamic nuclei.24

The importance of the hypothalamus in maintaining homeostasis was

recognized from numerous early studies which employed both lesion and

stimulation techniques. In addition to regulating AP secretary activity,

the hypothalamus regulates appetite, water balance, body temperature,

blood pressure, the sleep-wake cycle and behavior. Although the known

functions of hypothalamic regions have been reviewed extensively,25 the

identification of the anatomical loci associated with each of these func-

tions has been, and continues to constitute, a major aspect of neuroscience

research.










The medial base hypothalamus (MBH) was first identified as the region

of primary importance in regulating AP hormone secretion by Halasz and co-

workers.26,27 They noted that AP tissue was able to maintain integrity of

the thyroid, adrenals and gonads when it was transplanted to the MBH of

rats but the AP and its target tissues degenerated when it was implanted

in other brain regions.26 Halasz and Pupp severed the "hypophysiotropic"

region from other brain regions in rats and observed that the AP histology

and thyroid and testicular weights were maintained, but the ovaries and

uterus were atrophied.27 Further studies showed that the neural connec-

tion between the POA and MBH is required for cyclic release of gonadotro-

pins in rodents, but does not appear essential in primates (see 28).

The majority of evidence now indicates that hypothalamic regulation

of basal gonadotropin secretion originates in nuclei of the MBH while the

ovulatory hypersecretion of LH or "cyclic release" is mediated by more

rostral hypothalamic regions in rodents (see 29). Of the nuclei located

between the POA and MBH, the suprachiasmatic nucleus (SCN) appears impor-

tant in timing LH hypersecretion with diurnal photic cycles.30

Innervation of the Hypothalamus

Afferent and Efferent Pathways

The hypothalamus receives afferent and sends efferent projections

through a variety of pathways to communicate with many brain regions as

might be expected from its diverse regulatory functions. Evidence for

seven major fiber tracts integrating the hypothalamus with other brain

regions has been reviewed by Palkovits and Zaborszky.31 The medial fore-

brain bundle (MFB) is a diffuse fiber tract which courses through the lateral

hypothalamus. Descending afferents from the MFB to the hypothalamus origi-

nate in the olfactory bulbs, septum, pyriform cortex and nucleus









accumbens (NAc). Projections from the raphe nuclei and dorsal and ven-

tral tegmental nuclei ascend via the MFB. Both ascending and descending

MFB projections exchange afferent and efferent fibers most densely in the

medial portion of the hypothalamus as well as innervating the POA regions.

The fornix provides afferents to the mamnmillary bodies from the hippocam-

pus. The medial corticohypothalamic tract diverges from the fornix to

innervate the periventricular, SCN and MBH regions of the hypothalamus

with hippocampus projections. The stria terminalis originates in the

amygdala and projects to the POA and MBH regions. Three nerve tracts

course primarily through the mammillary bodies: the mammillothalamic tract,

mammillotegmental tract and the mammillary peduncles. The first two do

not involve major connections to other hypothalamic regions, while the

last connects the posterior and lateral hypothalamus with the ventral teg-

mental area.

Noradrenergic Pathways

The role of NE as a putative neurotransmitter was first suggested by

Vogt32 who observed high concentrations of the catecholamine (CA) in the

brain and its differential distribution among brain regions. Subsequent

development of histofluorescent and radioenzymatic techniques in the late

1960's and early 1970's demonstrated that the hypothalamic neuronal net-

work is rich in CAs. Experimental evidence leading to elucidation of the

CA containing pathways to the hypothalamus and detailed descriptions of

these pathways have been extensively reviewed.25,31,33-35

Norepinephrine pathways in the brain originate in cell bodies located

in the lower brain stem. No NE-containing perikarya have been detected

in the hypothalamus. The cell bodies are located in five major clusters:

the lateral reticular nucleus (Al cell group), solitary tract nucleus









(A2 cell group), ventrolateral pontine nucleus (A5 cell group), locus

ceruleus (A6 cell group) and mesencephalic reticular formation (A7 cell

group). All NE cell groups participate in hypothalamic innervation through

three fiber tracts: the ventral NE bundle and the ventral and dorsal peri-

ventricularNE bundles. The dorsal NE bundle which originates primarily

in the A6 cell group sends projections to the dorsal hypothalamic, peri-

ventricular and paraventricular nuclei via branches to the central NE and

dorsal periventricular NE pathways. The majority of NE fiber projections

to the hypothalamus course through the ventral NE bundle which contains

axons from cell bodies located primarily in the Al cell groups, although

each NE cell group contributes to the bundle. At the rostral mesence-

phalic region, the ventral NE bundle receives fibers from the dorsal NE

bundle and then joins the MFB. The ventral periventricular NE bundle is

formed from medial projections of MFB fibers at its entrance to the hypo-

thalamus. Other fibers from the MFB diverge at the lateral hypothalamus

and terminate in the MBH while the majority of fibers course further ros-

trally. At the posterior border of the optic chiasm, fibers from the MFB

branch through the lateral retrochiasmatic region and enter the MBH from

the rostral and lateral aspects.

The external source of hypothalamic NE pathways has been confirmed

by studies which severed the regions from other brain areas. Deafferen-

tiation of the medial hypothalamus resulted in a 70-90% NE depletion in

this region.36'37 The residual 10-30% of NE may be present in neuroglial

cells and thus be unresponsive to lesion of NE fiber tracts.38 Similar

approaches which lesioned specific NE cell groups in the brain stem re-

sulted in general NE reductions in all hypothalamic areas; however, the

rate of NE depletion within hypothalamic regions varied depending on which









brain stem regions were lesioned.39 The depletion in each case was not

as great as complete hypothalamic deafferentiation. The dense innervation

network in the hypothalamus and results of lesion studies of brain stem

cell groups suggests axon collaterals originating from other NE cell groups

may compensate to innervate all hypothalamic regions when the primary

source of NE to a particular region is lost.35 Thus, the specific pri-

mary source of NE input to each hypothalamic area has not been clearly
established.

Dopaminergic Pathways

Unlike NE, DA innervation of the hypothalamus originates from both

extrahypothalamic and intrahypothalamic pathways. Intrahypothalamic

sites of DA perikarya include the NA (A12 cell group), hypothalamic and

preoptic periventricular nuclei (A14 cell group) and zona incerta area of

the dorsomedialis subthalamus which extends to the adjacent ventral peri-

ventricular nuclei (A13 cell group). Extrahypothalamic sources of DA

originate in cells located in the pars compact of the substantial nigra

and the ventral tegmental area. Catecholamine-containing perikaryia with-

in these regions are often designated as A8, A9 and A10 cell groups, al-

though the anatomical distinction between DA containing cell groups is

not clear.34 Projections from these mesencephalic regions form two major

DA systems. The nigrostriatal DA system arises primarily from the pars

compact of the substantial nigra and projects rostrally via a pathway

which includes the lateral hypothalamus to terminate in the neostriatum

caudatee putamen) and glabus pallidus. The mesocorticol-mesolimbic DA

system arises primarily from the ventral tegmental region and ascends

rostrally close to the nigrostriatal fibers. Four major branches from

the ascending mesocorticol pathway form projection routes which terminate









in the exocortex, and allocortex. The allocortex includes the olfactory,

bulb and tubercle, perform cortex, NAc and amygdala or amygdaloid areas.

Two intrahypothalamic and one extrahypothalamic DA pathways have

been described. The tuberoinfundibular DA (TIDA) pathway consists of

short axons which course from DA cell bodies in the NA (A12 cell group)

and project to the ME. The majority of DA in the ME originates from cells

located in the NA. Axons from the TIDA system have been shown to ter-

minate from the ME to the caudal end of the infundibulum, although some

evidence indicates cells from the rostral portions of the TIDA system

send axons caudally to innervate the posterior pituitary gland.34 Addi-

tionally, projections from the A12 cell group are thought to terminate in

the NA, ventromedial nucleus (VMN) and premammillary nuclei since deaffer-

entiation of the MBH which includes these areas fails to alter DA concen-

trations in these nuclei.36 Axon collaterals from fibers running into

the ME are thought to innervate these three nuclei.35 The incerto-hypo-

thalamic DA system originates primarily in the A13 cell group but also

receives contributions from some axons originating in the A10 and All cell

groups. Fibers from this system run periventricularly in the dorsal hypo-

thalamus and have been detected to course rostrally to the POA-hypothalamic

border. The incerto-hypothalamic DA system has been shown to innervate

the dorsomedial and paraventricular nuclei;40'41 however, its role in AP

hormone regulation has not been demonstrated.42 Axons originating from

cell bodies in the A14 cell group innervate the medial preoptic nucleus

(POAm) and periventricular nucleus. Dopamine sources from extrahypothal-

amic regions enterthe hypothalamus via the mesocortical DA pathway. De-

afferentiation of the hypothalamus fails to substantially alter intrahypo-

thalamic DA levels indicating this pathway does not constitute the primary









DA innervation to the hypothalamus. However, lesion of A8, A9 and A10

cell groups decreases ME levels of DA and results in degenerated nerve

terminals in the external layer of the ME.43'44 The origins of DA innerva-

tion of the supraoptic nucleus (SON), anterior hypothalamic nucleus

(NHA), SCN, and posterior hypothalamic nuclei are not yet conclusively

identified.35

Other Putative Neurotransmitter Pathways

In addition to the high concentrations of CA found in the hypothal-

amus relative to other brain regions, several other putative neurotrans-

mitters are localized in this region. Pathways of these substances have

been reviewed.31,35 Epinephrine is found in axons terminating in the

dorsomedial, paraventricular, periventricular, NA and SON hypothalamic

nuclei. Cell bodies for epinephrine axons are located in the C1 and C2

regions of the medulla oblongata; however, the pathways by which these

fibers reach the hypothalamus have not been demonstrated.35 Serotonin

innervation to the hypothalamus arises from the narrow band of midbrain

raphe nuclei and projects via the MFB to terminate in the SCN as well as

other hypothalamic and POA regions.31 Although most serotonin appears to

arise from extrahypothalamic origins, some evidence indicates the dorsome-

dial nucleus may contain serotonin perikarya. 45 Histamine is present in

all hypothalamic regions and hypothalamic deafferentation does not great-

ly reduce histamine levels in the MBH.31 These results have been inter-

preted to indicate histamine may be located in nonneuronal cells.46

Similarly, deafferentation does not change MBH concentrations of acetyl-

choline nor choline acetyltransferase.31









Catecholamine Regulation of Luteinizing Hormone

Initial Evidence

A role for CA in the regulation of LH release was initially proposed

by Sawyer and colleagues who observed intraventricular injection of NE

resulted in ovulation in estrous rabbits.47'48 Since NE did not alter

LH release in vitro, a central mechanism for NE action was proposed.49

The resulting hypothesis that noradrenergic neurons exert a stimulatory

influence on LH release has been supported by a variety of experimental

approaches which have been extensively reviewed.49,50

Anatomical Evidence

Anatomical evidence is presently insufficient to state the precise

nature of CA and LHRH neuron connections; however, high densities of CA

nerve terminals and LHRH containing cell bodies have been histologically

identified in close synaptic-like proximity in the ME.51 Early immunohis-

tochemical studies failed to conclusively identify LHRH containing peri-

karya (see 50); however, more recent studies showed positive staining

LHRH perikarya in the POA and SCN regions as well as the NA-ME areas.52

This evidence, together with the observation that LHRH in the MBH is de-

pleted following anterior deafferentation,53 supports the existence of a

preopticoinfundibular LHRH pathway proposed by Everett as a result of

electrophysiological studies.54

Electrophysiological studies demonstrated electrical stimulation of

the POA, results in altered multiple unit activity in the NA and ME in

rats and increased LH release.54,55 Further, the magnitude and duration

of the stimulus were somewhat proportional to the amount of LH released.56

Since intraventricular injection of NE resulted in similar alteration in

multiple unit activity in the ME57 and augmented LH release in a dose









dependent manner,58 correlative evidence implicated that NE stimulates the

preopticoinfundibular LHRH pathway.

Biochemical Evidence

A biochemical approach has been to examine CA metabolism during

physiological states of increased LH secretion. Following ovariectomy,

anterior hypothalamic NE concentrations increase,59 the rate of tritiated-

NE depletion increases in whole brain,60 hypothalamic NE turnover is aug-

mented,61 and NE synthesis from tritiated-tyrosine increases.62 Ovariec-

tomy also augments the activity of the rate limiting enzyme in CA syn-

thesis, tyrosine hydroxylase.63 Additionally, the preovulatory discharge

of LH as well as the gonadal steroid induced surge of LH in ovariectomized

rats is preceded by increased NE turnover which reflects augmented activity

in hypothalamic neurons.64'66

Pharmacological Evidence

Probably the most compelling evidence for the involvement of CA in

LH secretion comes from studies using pharmacological techniques. Intra-

ventricular injection of NE has been shown to induce ovulation67 and in-

creased LH serum concentrations58 in rats. Acute blockade of NE synthesis

with dopamine-B-hydroxylase (DBH) inhibitors dampens episodic LH release,68*69

mean serum LH concentrations70 and the surges of LH induced by electrical

stimulation as well as by gonadal steroids.71'72 Further, the effects of

DBH inhibition of LH release could be overcome with dihydroxyphenylserine

but not L-DOPA.72 Similarly, a-adrenergic receptor antagonists blocked

ovulation,73 reduced LH response to castration,68 and blocked the gonadal

steroid induced LH release in ovariectomized rats.72

Destruction of the ventral NE pathway by injection of the neurotoxic

agent, 6-hydroxydopamine, early on proestrus blocked the LH surge.74








Studies designed to identify sites of LHRH-NE interaction microimplanted

6-hydroxydopamine into the POA, SCN and the NMBH regions in ovariectomized

rats treated with estrogen and progesterone. Implants in the POA and

SCN but not the MBH blocked the gonadal steroid induced LH surge and de-

pleted NE in the POA-anterior hypothalamic areas.75 These results support

the concept that NE-LHRH neuronal interactions involve hypothalamic regions

rostral to the ME in the ovulatory hypersecretion of LH in rats. Inter-

estingly, after chronic depletion of hypothalamic NE by lesion of the ven-

tral NE bundle, cyclic activity returns after several weeks.76 These re-

sults were interpreted as evidence that NE normally is facilitory for LHRH

neuron function, but with prolonged absence of NE influence, the LHRH

neurons of young mature animals are able to regain normal function.

Although the majority of studies support a facilitory role for NE

in LH secretion, recently an inhibitory role of NE in regulation of LH

secretion has been proposed in view of the findings that intraventricular

NE infusion77 and electrical stimulation of the ventral NE bundle78 can

suppress the pulsatile mode of LH secretion in ovariectomized, nonsteroid

treated rats. Ciceres and Taleisnik have proposed the inhibitory effects

of NE on LH release are mediated through e-adrenergic receptor activation

on LHRH neurons, while the facilitory effects involve primarily a-adrenergic

receptors.79 Confirmation of this hypothesis requires further study.

The role of DA in LH regulation is less clearly understood than that

of NE in the regulation of LH secretion. Early reports suggested DA

exerts a stimulatory influence on LH secretion.80 However, administration

of both DA agonists and antagonists have been reported to inhibit or have

no effect on LH secretion while intraventricular injection of DA stimulated

or had no effect on LH secretion in the rat (see 49). The observation









that DA antagonists generally have no acute effect on LH release indi-

cates the role of DA is not essential for this phase of LHRH neuron func-

tion.81 Whether DA has a role in the synthesis of LHRH or in modulating

the input of other neuronal systems remains to be critically evaluated.

Dopamine-Prolactin Regulation

Dopamine Regulation of Prolactin

The inhibitory effect of DA on prolactin secretion is probably the

best characterized neurotransmitter-AP functional relationship. Unlike

the other AP hormones, prolactin secretion is normally tonically inhibited

by hypothalamic factors. The evidence indicating DA is the primary hypo-
thalamic factor responsible for prolactin inhibition has been reviewed.49

This evidence includes several studies which indicate DA originating in

the TIDA neurons enters the portal vasculature in concentrations which

effectively dampen prolactin secretion.8284 Further, alterations in TIDA

neuron activity and altered DA concentrations in portal blood have been

associated with altered states of prolactin secretion.85'86

In contrast to the above evidence, several studies suggest DA may

not be the sole hypothalamic substance which inhibits prolactin secretion.

Purified hypothalamic extracts contained significant prolactin inhibiting

activity after all CAs were removed from the extracts.87"89 Similarly,

pretreating hypothalamic-AP coincubation preparations with DA antagonists

did not eliminate hypothalamic inhibition of prolactin secretion.90 In

vivo studies showed that while injected hypothalamic extracts decreased

prolactin secretion induced by suckling, the dose of DA required to inhibit

prolactin release was more than 30-fold the amount of DA contained in

hypothalamic extracts.91 Conclusive identification of hypothalamic pro-

lactin inhibiting substances other than DA awaits further study.









Prolactin Regulation of Dopamine

Shortly after the demonstration of DA inhibitory effects on prolac-

tin secretion, Hikfelt and Fuxe showed exogenous prolactin augmented TIDA

neuron activity.92 Studies which extended and confirmed the negative feed-

back role of prolactin on TIDA neuron activity have been reviewed.93

More recent studies have also indicated prolactin augments extrahypothal-

amic DA systems.94-97 These findings suggest prolactin may exert some

of its effects on behavior via CNS pathways outside the hypothalamus.

The negative feedback relationship of DA and prolactin is altered by es-

trogen.98 The ability of estrogen to augment prolactin secretion99 ap-

pears to result from alterations in the intracellular response of the

mammotroph to DA.100,101 Thus, alterations in portal blood DA levels

may not correlate with prolactin secretion in an inverse relationship.

Age-Related Alterations in Reproductive Function

Estrous Cycles in Young Mature Animals

The changing hormone milieu which occurs during the reproductive

cycle in young mature females ensures that ova are released under optimal

conditions for successful fertilization and implantation. The coordina-

tion of cyclic physiological and behavioral changes required for success-

ful reproduction is closely regulated by dynamic fluctuations in hormone

secretion and involves interplay of the CNS, AP and ovary. These hormonal

interactions have been reviewed.29',102',103 Briefly, ovulation is trig-

gered by a hypersecretion or surge of LH. The interval between LH surges

is characteristic of the length of the ovarian cycle in different species

(i.e., 4-5 days in rats and 28 days in women). A surge of FSH is coin-

cident with the LH surge and prolactin is also elevated at this time in

rats. Estrogen secretion gradually increases with follicular growth and

increases more rapidly prior to the LH surge. Progesterone secretion









is also increased before the LH surge and appears important in inducing

estrous behavior in rats (see 103). Progresterone secretion is highest

during the luteal phase of the cycle.

The LH surge is triggered by increasing circulating estrogen levels

which is the positive feedback effect of estrogen on LH secretion. This

effect of estrogen is exerted at both the AP and hypothalamic level since

both augmented AP response to LHRH and hypersecretion of LHRH are asso-

ciated with these positive feedback effects (see 29). In contrast to

positive feedback effects of estrogen responsible for the LH surge, tonic

basal LH secretion is modulated by the inhibitory feedback actions of

estradiol and progesterone. Thus, ovariectomy results in elevated LH

levels which occur from secretary bursts or pulses of LH from the AP.

Pulsatile LH secretion appears to be the result of pulsatile LHRH release

into the portal vasculature (see 29 and 81).

Elucidation of the CNS regulatory mechanisms involved in both posi-

tive and negative feedback effects of estradiol used intact cycling

animals as well as ovariectomized animals treated with differing regimens

of estrogen and/or progesterone (see 103). Results of studies employing

these animal models indicate that NE facilitates LHRH neuron secretary

activity, while DA neuron activity is more closely associated with prolac-

tin secretion (see 49, 50 and 104).

Patterns of Reproductive Senescence

Although advanced postmaturational age is accompanied by a decline

in reproductive function in all species, the patterns of reproductive

senescence differ. Patterns of reproductive decline with increasing age

have been studied most extensively in women, rats, mice and hamsters.

Each of these species shows a decrease in fertility as measured by viable









offspring during the middle third of their normal expected life span (see

105). Accompanying this decrease in fertility is a change from normal,

regular ovarian cycles to irregular cycles. Premenopausal women experi-

ence shortened-irregular menstrual cycles while rats and mice have length-

ened, irregular cycles in middle-age.106'108 The hamster generally main-

tains regular estrous cycles through old age (19 months); however, 10-20%

of old hamsters have lengthened irregular cycles.109 The decreased fer-

tility of ovulating middle-aged women and rodents has been associated with

altered uterine function (see 105).

Following variable periods of irregular ovarian cycles and diminished

fertility, most animals enter states of noncyclic ovarian function or re-

productive senescence. In postmenopausal women, ovaries atrophy and be-

come fibrotic. Ovaries from old mice often have well developed follicles

which secrete sufficient estrogen to maintain persistent cornified vaginal

epithelium.110 These senescent mice are often classified as "persistent

vaginal cornified state" or CE. Other old mice have persistent diestrous

vaginal lavages and are acyclic.111

Extensive longitudinal studies in Long-Evans and Sprague-Dawley

strains of rats revealed the incidence of irregular cycles diminishes and

the frequency of CE rats begins to increase between 10 and 12 months of

age.112"114 By 19 months of age most rats experience the CE state. In

later life PP animals predominate.114 Everett characterized the now ex-

tinct line of short-lived inbred rats from the Duke Anatomy Department

which entered CE between 6 and 8 months of age.115 In contrast, the

long-lived Fisher 344 rat has predominantly irregular cycles in late

middle-age and then enters the PP state in late life (20-27 months).116









The CE rat has large, well developed follicles, no corpora lutea and

moderately elevated levels of serum estrogen compared to diestrous young

rats.'17 The old PP rat ovulates at 8 to 20 day intervals and maintains

functional corpora lutea between ovulatory events.117 These ovarian

changes are reflected in the vaginal cytology of PP rats which typically

have one or two days of cornified epithelial cells intermittent with sev-

eral consecutive days of predominantly leukocytic smears, while the CE

rat shows consistent cornified vaginal epithelium.

The locus of impairment responsible for the diverse patterns of re-

productive senescence differs between species. In the mouse and woman

ovarian oocyte exhaustion appears primarily responsible for loss of ovarian

cyclicity as indicated from two lines of evidence. Hemiovariectomy de-

creases reproductive life-span by 50% in both mice"18 and women;119 how-

ever,no decrease was observed in hemiovariectomized rats.120 Conversely,

hypophysectomy which retards oocyte loss prolonged ovarian function in

mice.121 When young mice received ovary transplants from old hypophysec-

tomized donor mice, normal cycles resulted.122 Prior to menopause, ovaries

in women have a reduced response to gonadotropin and produce decreased

levels of gonadal steroids.123 This decrease in gonadal steroids may re-

sult in shortened menstrual cycles. Postmenopausal women have little or

no ovarian response to gonadotropins.

A CNS impairment has been implicated in the noncyclic reproductive

states of rats.24 Indirect evidence includes the results of ovarian trans-

plant studies which showed ovaries from old CE and PP rats regain function

upon transplant to young recipients.125 In contrast to results in human

ovaries, gonadotropin receptor responses are not diminished with age in

rats.126 Second, multiple injections of LHRH induced LH secretary









responses in old CE rats which were similar in magnitude to LH response

in young animals.'27

More direct evidence implicating a central nervous system (CNS) dys-

function includes several studies in which successful reinitiation of

estrous cycles in old rats has been accomplished with a variety of CNS

mediated stimuli (see 105). These stimuli include electrical stimulation

of the POA, chronic ether stress, and administration of CNS acting drugs

such as dihydroxyphenylalanine (L-DOPA), iproniazid, epinephrine, proges-

terone and corticotropin.128-134

While reproductive decline in aging males is well known, the patterns

of alteration are not as well characterized compared to females. Aging

is generally associated with decreased libido, decreased sperm production,

and declines in function or size of testosterone dependent tissues.135

The majority of studies indicate that testosterone production generally

decreases with age in men and rats (see 135), an apparent result of a de-

cline in the diurnal phase of hypersecretion of testosterone.136'137

Testicular alterations may-be in part responsible for diminished gonadal

steroid production.138 Recent studies which carefully monitored health

status of old men and animals suggest that the age-related decline in

testosterone may be more closely related to age-related pathologies.139,140

A central locus for age-related reproductive alterations in men and rats

has recently been suggested from studies which examined the diurnal pat-

terns of testosterone secretion in rats136 and men.'37

Luteinizing Hormone Secretion

Basal LH secretion changes in different directions in humans compared

to laboratory rodents with increasing age. Basal LH secretion in women

gradually increases prior to menopause and more rapidly increases after









menopause.123 Serum LH concentrations during the later decades are simi-

lar to levels measured in young ovariectomized women until the ninth dec-

ade when LH decreases.l41 Similar LH increases occur with age in men,

although the increase is gradual and maintained through the ninth decade.142

Elevated LH levels appear to result from diminished gonadal steroid se-

cretion since treatment with appropriate gonadal steroids decreases gonado-

tropin levels to those of young control subjects.143 In contrast, basal

LH secretion in healthy old male laboratory rodents is modestly decreased

or unchanged,'44-147, while old female rats114,148,149 and mice150'151

have LH levels which are slightly elevated or unchanged compared to young

diestrous rodents.

Hypersecretory LH responses appear to be generally reduced with age

in laboratory rodents. Several studies have shown the ovulatory surge of

LH on proestrus is dampened in middle-aged normal cycling and irregular

cycling rats'52-155 and mice.151 Similarly, the positive feedback LH

response to stimulatory regimens of gonadal steroids is impaired in CE

rats.156-159 Interestingly, stimulatory regimens of gonadal steroids were

less effective in augmenting LH in postmenopausal women compared to pre-

menopausal women.160 However, no age-related decreases were shown in

similar studies which examined positive LH feedback in hamsters109 and

both normal and decreased148 LH responses were found in old PP rats.

Studies designed to evaluate negative feedback responses consistently

showed LH response to castration is slower and lower in aged male and

female rats,144,148,156,'158161-163 female hamsters'09 and male mice.164

Similarly, the LH response to ether stress was decreased in old male rats.133

Both in vivo and in vitro studies suggest that alterations in the

pituitary response to LHRH may contribute to dampened hypersecretory LH









responses in old animals. Luteinizing hormone secretion following a single

injection of LHRH is reduced in old compared to young male and female

ratsl27,1333,1,62,63,165-167 and female hamsters.109 Studies using

incubated pituitaries from male and female rats showed a large decrease

with age in both LH response to LHRH and pituitary LH content.117,135,168'169

However, more recent studies which monitored AP morphology rigorously found

no age-related difference in LH response to LHRH of AP cell cultures between

young cycling and old PP rats170 or in vivo LH response of young and old

male mice to LHRH.105 Age-related differences have been shown in the prop-

erties of secreted LH both in vitro170 and in vivol71 indicating the bio-

logical activity of LH measured with RIA may increase in old rats.

The majority of evidence does not implicate pituitary dysfunction in

the initiation or maintenance of noncyclic reproductive states. The mag-

nitude of LH required to stimulate ovulation of graffian follicles is 10

to 20% of the LH hypersecretion measured on proestrus in young rats.172

Since the degree of decreased LH response to LHRH has never been shown to

approach 80%, impaired ability of old pituitaries to secrete LH is unlikely

to result in the CE or PP state. Further, multiple injections of LHRH in

both male165 and female CE rats,127 stimulate LH levels which are compar-

able to levels assayed in young rats indicating no drastic age-related

impairment in LH secretary capacity exists when the pituitary is suffi-

ciently stimulated. The differential diminished response to a single in-

jection of LHRH and near normal response to multiple LHRH injections of

old rats suggests that old AP's may require augmented LHRH priming. Al-

ternatively, the diminished response to a single LHRH challenge might

result from diminished endogenous LHRH input of old compared to young









rat AP's. The self-priming of LHRH on its own secretion is well described

in the rat173 and appears to participate in the generation of the proes-

trous LH surge.29 The latter possibility implies a CNS alteration may

change the secretion modes of hypothalamic LHRH input to the AP in old

rats.

Prolactin Secretion

The best documented age-related hormone alteration is the increase in

basal prolactin levels during advanced age. In man, serum prolactin con-

centrations are generally stable through the seventh decade and then in-

crease,174 although prolactin decreases are seen in postmenopausal women

as estrogen declines.174 Elevated prolactin concentrations have been reported

consistently in old male and female rats.113,114,133,144,159,161,175-180

The magnitude and time of age-related increases in prolactin differs with

the reproductive status of old rats. Old CE females typically have serum

prolactin levels which are more than twice those seen in old male and PP

rats.113,144 Further, prolactin appears to increase during middle-age in

strains of rats which predominantly show the CE state with advanced age,

while levels are stable through middle-age in strains which enter the PP

state late in life.176 The mouse appears to be exceptional in that serum

prolactin levels remain stable throughout the life span164 or decrease

during middle-age.150,176

Mechanisms responsible for prolactin increases with age are not clear.

Ovariectomy of old CE rats reduces prolactin to levels comparable to young

animals159,161,175 while little postcastration decrease is apparent in

PP rats.161,175 Thus, the elevated estrogen levels associated with the

CE state appear to cause prolactin increases in old CE rats. Whether

age-related increases in prolactin in PP and male rats result from AP









alterations, diminished hypothalamic inhibition or augmented hypothalamic

stimulation of prolactin secretion is unclear. Elevations in human serum

prolactin levels during late decades have been associated with a high

incidence of pituitary microadenomas compared to young controls.181

Further, AP adenomas from subjects greater than eighty years of age had

positive immunostaining for prolactin, while young hypophysectomized

patients predominantly had growth hormone secreting adenomas.181 The

difference in basal prolactin secretion alterations with age in rodents

may also be partially attributed to the increased incidence of AP adenomas

in rats182 compared to the low incidence of AP pathology in C57/6J mice.105

Since the majority of studies which examined AP hormone secretion dif-

ferences with age have not rigorously monitored health status or AP mor-

phology, the relative contribution of AP pathological conditions to reported

age-related differences in neuroendocrine function is difficult to eval-

uate.

Age-Related Alterations in the Central Nervous System

Dopami ne

The well documented increased incidence of movement disorders in

older individuals precipitated investigation of age-related alterations

in CNS function. Results of these studies have been reviewed.'83 Although

the majority of studies examined extrahypothalamic DA systems, the pre-

viously discussed changes in AP hormone regulation also stimulated inves-

tigation of hypothalamic alterations during advancing age.

In general, there are no massive overall changes of neuronal function

in healthy old humans or animals.105,184 Rather,in the absence of

pathological conditions, alterations appear limited to quite focalized

brain regions. 105,183,184 Thus, studies whichexamined whole brain tissues










rarely reported age differences. Studies which do report age changes

within brain regions usually report the direction of change is toward

impaired DA function. Decreases in DA activity have been reported most

consistently in the nigrostriatal DA system. In this region decreased

DA concentrations,185-188 impaired enzymatic synthesis,189-193 dampened

DA turnover187'188,192,194 and diminished receptor recognition195-203

have been shown in rodents, rabbits and humans. An initial glance at

these alterations suggests their cumulative effects might result in se-

vere functional impairment of this DA system. However, more recent studies

indicate the complex mechanisms which regulate neurotransmitter function

may at least partially compensate for reported changes. In contrast to

several studies which suggest DA binding affinities remain constant while

the number of binding sites decrease in striatal and other brain regions

(see 204,205), Marquis and coworkers recently reported an increase in the

numberofstriatum-DA binding sites in old mice and rats.203 These diver-

gent reports may be partially explained by different methods employed to

estimate DA binding characteristics since some evidence suggests not all

classes of DA receptors are altered with age.206

The significance of reported alterations in DA binding sites with age

is presently difficult to evaluate. In vitro studies have demonstrated

consistently that DA-stimulated adenyl cyclase in the rodent striatum

decreases with age.201,207,208 However, in vivo studies suggest age-

related impairments do not involve primarily receptor sites. A series of

studies by Joseph and coworkers showed that rotation behavior induced by

amphetamines in rats with unilateral lesions of the substantial nigra is

attenuated with age.208-210 However, no age differences were seen in

rotation after administering the DA agonist, apomorphine.208 Further,









treating rats with the DA precursor, L-DOPA, does not restore amphetamine

induced rotation deficits in old compared to young lesioned rats.209 Another

study by these investigators showed the extent of DA depletion in substan-

tia nigra after injection of the neurotoxin, 6-hydroxydopamine, was damp-

ened in old compared to young rats.210 In contrast to these results, two

reports from Finch's laboratory did not observe induction of DA receptor

proliferation (supersensitivity) in old mice after haloperidol treatment.202'211

An explanation for these divergent results may lie in species difference or

in the methods employed to induce receptor augmentation following drug-

induced impairment of DA transmission. Another in vivo approach to examine

DA receptor alterations was used by Marshall and Berrios who examined the

mechanisms of impaired swimming efficiency in old rats to levels similar

to young animals.212 Collectively, these in vivo data indicate that al-

terations in the substantial nigra DA system with age appear to primarily

involve the ability of neurons to release DA rather than receptor altera-

tions. Anatomical evidence supporting alterations in DA release mechanisms

in old mice comes from a study that showed synaptic varicosities associ-

ated with stored rather than releasable pools of DA were more prevalent

in old mice.213

Intrahypothalamic DA systems have not been studied as extensively as

the nigrostriatal system. Earlier investigations reported little if any

effect of age on DA concentrations in whole hypothalamic tissues.188,192'214-216

However, examination of DA concentrations within hypothalamic regions re-

vealed age-related variations. Doapmine levels were substantially decreased

in the ME of old rats and mice217-220 and in the MBH of old rats.215,216

Few studies have examined age-related changes in enzymatic synthesis of

hypothalamic DA. Tyrosine hydroxylase activity in hypothalami was increased









in 24 to 26 months old Fischer 344 rats221 and unchanged in 29 months old

Sprague-Dawley rats.193 Aromatic amino acid decarboxylase was unchanged

in the old mouse221 and decreased in older men.191

The activity of hypothalamic DA neurons in old rodents has been ex-

amined with two methods. Turnover rates of DA estimated by steady state

and nonsteady state methods was decreased in whole hypothalami of male

mice188 and rats192 and MBH of male rats,216 but was unchanged in the

anterior hypothalamic region of old female Long Evans rats.216 The

latter study saw no augmentation of DA turnover in response to ovariectomy

in the anterior hypothalamus which was observed in young rats. However,

Finch's laboratory recently reported that turnover was maintained in old

male mice ME tissues in spite of a 25% decrease in DA concentration.218

Another approach examined TIDA neuron activity by measuring DA concen-

trations in portal blood. These studies found age-related decreases in

portal DA blood levels, supporting data which found decreased TIDA turn-

over during advanced age.222.223 Histological studies which support de-

creased TIDA function reported DA was decreased in the ME of old male

Fischer 344 rats and increased in NA perikarya217 and evidence of degraded

TIDA neurons was found in old male mice.224 Collectively, these data in-

dicate that, of the intrahypothalamic DA systems, the TIDA system appears

to be more severely affected by increasing age than other DA systems.

Norepinephrine

The majority of studies which examined changes in NE neuron function

indicate activity decreases with age, although age-related alterations in

NE activity are not massive and also appear to be confined to localized

brain regions. Norepinephrine concentrations have been reported to decrease

in human and monkey hindbrain and hypothalamic regions225',226 as well








as in rat hypothalami,214 216 and MBH tissues.215,216 The mouse appears

exceptional in that NE concentrations remain stable in all regions examined

through postmaturational life.218 However, the rate of NE turnover is de-

creased in hypothalami from both old mice and rats.188,192,215 Similarly,

the normally observed increase of NE hypothalamic turnover in response

to ovariectomy is dampened in old CE rats.216 Enzyme activity of DBH was

shown decreased with age in rats but not in mice.221 However, a recent

study indicates that the augmentation of DBH on proestrus is reduced in

old mice.227

Alterations in the number or affinity of hypothalamic NE receptors

withage have not been characterized. Investigations of extrahypothalamic

brain areas indicate both a- and a-adrenergic binding sites decrease in

cortex, cerebellum and pineal regions of old rats.199'228 These regions

as well as the hippocampus have decreased adenyl cyclase activity in re-

sponse of NE stimulation.208 Further, the proliferation of adrenergic

binding sites in response to decreased adrenergic stimulation is dampened

in old rats.229',230 The majority of studies which employed Scatchard

analysis suggest that the affinity of binding sites is not altered but

rather the number of binding sites diminishes with age.199,228-230

Although decreases in both a- and a-receptors have been indicated, one

study suggests B, binding sites may increase with age in the cerebellum

of rats.231 A paucity of information is available on the nature of changes

in adrenergic receptors in all brain regions throughout advancing age as

most studies compared only young and old rats. The limited availability

of tissues from old animals has precluded rigorous examination of binding

characteristics within specific brain regions.









Luteinizing Hormone Releasing Hormone

The relationship between altered gonadotropin secretion and LHRH

neuron activity in old rats is not clear. Luteinizing hormone releasing

hormone concentrations in the hypothalamus of intact female rats were

reported to increase,232 decrease,220,233,234 or remain stable235 with

age, while LHRH in the MBH of male rats was decreased with age.217 Ex-

planation for these discrepant results may include the type of assay em-

ployed or stage of reproductive senescence of rats examined. Reports

employing bioassay methods for LHRH activity found little change with age

in whole hypothalamic extracts.135,169,235 In contrast to studies which

found decreased LHRH concentrations with RIA methods, Barnea et al.

reported LHRH content increased in synaptosome preparations from whole

hypothalami of old rats.232 These investigators suggested age-related

alterations in physiochemical cell properties and subneuronal LHRH dis-

tribution may result in impaired ability of old rats to release LHRH.

Studies designed to evaluate LHRH neuronal function examined the magnitude

of LHRH depletion response following ovariectomy. Wise and Ratner examined

this response in young cycling and middle-aged noncycling rats.234 They

found LHRH concentrations were reduced in intact noncycling compared to

young rats; however, the extent of LHRH depletion in response to ovariec-

tomy depended on the type of noncyclic reproductive state in older rats.

Constant estrous rats had little LHRH depletion response while PP rats

had near normal magnitudes of LHRH decrease after ovariectomy.234 How-

ever, Wilkes and Yen reported no diminished LHRH response to ovariectomy

in CE rats.220 These various results indicate the extent of LHRH neuronal

alteration with age is not characterized conclusively. Studies which suc-

cessfully induced ovulation in old noncycling rats (see 105) indicate LHRH









neurons can function sufficiently to release ovulatory quotas of LH.

However, the dampened LH surges associated with middle-aged rodents152-155

may result from mild impairments in LHRH neuron function.

Other Neurosecretory Changes

Age-associated physiological and behavioral impairments have stimu-

lated investigation of other CNS components for alteration during aging

in addition to the CA's. Of particular interest to students of neuroen-

docrine function are the changes in the serotonin system during aging.

In man and rhesus monkeys, serotonin was decreased in hypothalamic

regions226'236 but stable in hindbrain areas from postmortem patients.237

In rodents, hypothalamic concentrations were stable188,215 or decreased225

and raphe and hippocampus levels were decreased.238 Walker et al. have

recently suggested that dampened serotonin activity may contribute to im-

paired cyclic release of LH in old rats.239 However, changes in the activity

of serotonic neurons during age are unclear. Tryptophan hydroxylase has

been reported to decrease238 or remain stable221 with age in serotonin

perikarya of rats. Although the serotonin metabolite, 5-hydroxyindole-

acetic acid, has been shown to be increased in both old rat215 and human240

tissues, evidence suggests clearance of the metabolite may be decreased

with age.215

Alterations in cholinergic systems with age have recently been re-

viewed.241 Extensive investigation suggests that this systemmay be impaired

in patients who show memory loss; however, the extent of alteration in

normal aging is equivocal.241













GENERAL RATIONALE

The evidence reviewed in the preceding section indicates that a cor-

relation may exist between changes in central CA metabolism and reproduc-

tive dysfunction. These CNS changes may be responsible for decreased LH

and elevated prolactin secretion frequently seen in reproductively senes-

cent rats. Two approaches were selected to test this hypothesis. First,

studies were undertaken to systemically characterize the pattern of changes

in CA metabolism and LHRH levels and to identify the loci of these changes

during the postmaturational aging process. Second, this hypothesis was

tested directly by examining the effects of pharmacological manipulation

of CA systems on AP secretary function in reproductively senescent rats.

It is hoped that these approaches will enhance our understanding of the

mechanisms which are responsible for sub-optimal neuroendocrine regulation

in aging rats. Further, these studies may identify avenues for rational

treatment of these neuroendocrine disorders.













GENERAL MATERIALS AND METHODS

Animals

Selection of Animal Model

The laboratory rat was chosen as the experimental animal in the pres-

ently described studies for several reasons. First, neuroendocrine inter-

actions between the brain and AP are more thoroughly known for the

rat than any other species. Second, the neuroanatomy of both CA and LHRH-

containing pathways has been most extensively characterized in the rat.

Third, the rat life span is relatively short compared to many other mammals

and sufficient numbers of uniformly aged rats could be obtained to investi-

gate neuroendocrine alterations in regulation of LH secretion in this ani-

mal model. Additional considerations included the ready access to rat and

prolactin RIA systems, size of the animal for surgical manipulation, blood

and tissue collection, and the availability of sufficient cages and facil-

ities for long-term housing.

The Long-Evans rat was chosen as the model for studying the CE state

because it has a relatively early onset of persistent vaginal cornifica-

tion,113'114 appears more resistant to respiratory infection than the

Sprague-Dawley rat, and has been the most commonly used animal model to

examine age-related alterations in hypothalamic-AP-ovarian regulation.

The F344 rat was chosen to examine age-related neuroendocrine changes in

the PP state because healthy, barrier-reared old rats of uniform age could

be regularly supplied. Additionally, the results of our initial studies

showed that the majority of F344 rats (80%) maintain normal estrous cycles

until 15 to 18 months of age and then enter the PP state without experiencing
the CE state (Table I).









TABLE 1. Reproductive Status of Fischer 344 Rats at Various Ages


Age (months)


9-12


Number of Animals

Reproductive Status


13-20

44


21-27

53


Percentage


Normally Cycling (NC)

Irregularly Cycling (IC)

Constant Estrous (CE)

Repeated Pseudopregnant (PP)


aNearly 100% of animals less tha
to five day estrous cycles. Al
monitored for at least 30 days.


n nine months of age showed normal four
1 animals included in this Table were









Establishment of Rat Colony

Studies which employ aged animals are subject to some considerations

which are usually not aspects of experiments that use only young mature

animals. Of primary consideration is the source of old animals. Animals

for most of these studies came from two sources. Barrier-reared Fischer

344 rats were purchased at 2-3, 9-10 and 19-21 months of age from Charles

Rivers Laboratories (Wilmington, MA) through an arrangement with the

National Institute on Aging. Because old Long-Evans rats are not commer-

cially available, 150 Long-Evans retired breeder females were purchased

from Blue Spruce Farms (Altamont, NY) at 8-10 months of age and 50 to 75

animals at 8-10 months of age were purchased at 3-4 month intervals there-

after. This "stocking" purchase scheme of retired breeders combined with

purchase of 2-3 month old animals resulted in sufficient numbers of animals

of the same cohort and reproductive status to ensure completion of these

experiments.

Animals were housed two per cage in one of three animal rooms upon

arrival at the animal facility. Rats were provided free access to Purina

Rat Chow (Ralston Purina Co., St. Louis, MO) and tap water. Rooms are

temperature (230 20C) and light (lights on 0500 to 1900 h) controlled

and each room houses animals from a single source. In order to minimize

the incidence of murine pneumonia in the colony, two precautions were taken.

First, animal rooms were kept locked to minimize traffic. Second, animal

technicians fed, watered and cleaned the senescent colony at the beginning

of their work day prior to entering other animal rooms. Only one episode

of respiratory distress arose in the colony in three years. In this in-

stance, tetracycline was added to the drinking water for a seven day

period and thereafter animals showed no signs of pneumonia.









Monitoring Cycle Status

Animal reproductive status was monitored by histological examination

of vaginal lavages.108 Each animal was "smeared" for ten consecutive days

each month and classified for that month according to the following cri-

teria:

-- Normally cycling (NC) rats lavages showed two estrous cycles each,

four to five days in length;

-- Constant estrus (CE) rats lavages showed ten consecutive days with

cornified epithelium;

-- Repeated pseudopregnant (PP) rats lavages showed ten consecutive

days of predominant leukocytes or one or two days of cornified

epithelium;

-- Irregular cycling (IC) rats lavages showed several consecutive days

of cornified epithelium or several days of leukocytes.

One month prior to the experimental day, animals were selected on the

basis of cycle history and assigned to treatment groups. Vaginal lavages

were then examined daily through the end of the experimental period. This

monitoring procedure ensured animals with similar reproductive histories

could be assigned to experimental groups. Although the ten day evaluation

period alone may not have been sufficient to always distinguish PP and IC

rats, constant cycle evaluation is extremely tedious, time consuming and

costly. Daily monitoring of animals assigned to treatment groups 30 days

prior to the experimental period ensured reproductive status was accurately

evaluated.

Monitoring Health Status

Separating normal age-related physiological alterations from patho-

logical conditions that occur with increased frequency in advanced age is









a major consideration in gerontological studies.242 Three routine pro-

cedures were employed to minimize the contributions of possible patho-

logical effects to experimental results in these experiments. First,

general animal robustness was monitored during the period of reproductive

cycle evaluation. Daily handling ensured that animals with observable

tumors, skin lesions, abnormal urinary tract discharges, weight loss or

behavior alterations were identified and could be removed from the colony.

Second, animals were necropsied upon completion of the experiment for

visible abnormalities. Viscera were examined for gross pathology and kid-

ney, heart, adrenal, and pituitary weights were recorded. Although this

procedure could not detect many pathological conditions, extensive histo-

logical necropsy of each animal was not feasible. Third, because the in-

cidence of prolactin secreting adenomas is relatively high in Long-Evans

old rats,180,182 AP tissues were assayed for prolactin and LH content.

Animals with disproportionately high ratios of prolactin versus LH were

omitted from the experiments.

The monitoring procedure 30 days prior to the experiment and at the

termination of the experiment routinely results in elimination of some

old animals from each treatment group. Therefore, group sizes were aug-

mented 10 to 20% at the time old animals were assigned to treatment groups.

Surgical Treatment and Blood Collection

Animals in experiments using castrated rats were ovariectomized under

deep ether anesthesia two weeks prior to the experimental day. A bilateral

dorsal surgical approach was used for ovariectomies. Following surgery,

animals were monitored for wound healing.

Blood was obtained by one of two methods. Several experiments examined

trunk blood collected at decapitation. Decapitations were completed within









30 seconds of removal of each rat from its home cage. Since decapitation

experiments were designed to characterize brain CA and LHRH neuron func-

tion, trunk blood was collected while brains were rapidly exteriorized

and frozen on dry ice. These procedures were completed within 30 seconds

of decapitation. Posterior pituitaries were dissected and homogenized

in 40 pl of 0.4 N perchloric acid for CA assay. Anterior pituitaries

were dissected, weighed and homogenized in phosphate buffered saline for

later LH and prolactin assay. Animals were then necropsied as described

above.

Experiments designed to characterize the pulsatile nature of hormone

secretion employed cannulated rats. Silastic catheters (id. 0.025 in,

od 0.047 in, Dow Corning, Midland, MI) were implanted into the atrium via

the right jugular vein243 while rats were under deep anesthesia induced

by chloral hydrate (400 mg/Kg body weight, ip) or pentobarbital (40 mg/Kg

body weight, sc). Catheters were exteriorized on the dorsal aspect of the

neck, filled with heparin (1000 units/ml) and stoppered with stainless

steel plugs. Animals were then individually housed. The following day,

silastic extension tubes (30 cm long, id 0.025 in, od 0.047 in) were con-

nected to the catheters and draped outside of the animal's home cage.

One milliliter syringes connected to the extension tubes permitted repeated

blood sampling from freely moving animals. The syringe weight prevented

the extension tubes from tangling without hindering rat movement. Thirty

or 60 min prior to blood sampling, rats received 200 units of heparin

(200 pl) via the catheter.

Serial blood samples were then obtained at 10 or 15 min intervals for

a 3 h period. Saline filling the cathether (about 100 pl) was removed and

discarded. For studies which examined LH profiles at 15 min intervals,









300 pl of blood was removed for assay of plasma LH and an equal volume

of 0.9% saline was returned to the animal via the catheter. During the

3 h period, hematocrits decreased 10 to 25%. In studies which examined

LH fluctuations in samples collected at 10 min intervals, 500 p1 of blood

was removed via the catheter. Blood samples were centrifuged (Beckman

Microfuge B, Palo Alto, CA) for 30 sec, and 200 ul of plasma was separated

for assay of LH. Cells were resuspended in 200 pl of heparinized saline

(5 units/ml) and returned to the same animal following the next blood

sample. This procedure resulted in a 0 to 10% decrease in the hematocrit

during the 3 h sampling period. Animals were then necropsied as described

above.

Hormone Radioimmunoassays

Plasma and serum samples were assayed for LH and prolactin in dupli-

cate using routine RIA methods described in assay kits provided by the

National Institutes of Arthritic, Metabolic and Digestive Diseases (NIAMDD).

Pituitaries were homogenized and diluted in phosphate buffered saline con-

taining 1% gelatin. Diluted homogenates were assayed for hormone levels

in quadruplicate. Hormone concentrations are expressed in terms of the

standard reference preparations NIAMDD rat LH-RP-1 for LH and NIAMDD rat

PRL-RP-1 or PRL-RP-2 for prolactin. These reference standards have respec-

tive biological potencies of 0.03 x NIH-LH-S1 (ovarian ascorbic acid de-

pletion test), 11 international units of prolactin (mouse deciduoma test)

and 30 international units of prolactin (pigeon local crop sac assay).

Minimum assay sensitivities were 1 ng for LH and 0.05 ng for prolactin

per assay tube.

Levels of LHRH in supernatants of hypothalamic tissues homogenized

in 0.1 N hydrochloric acid were determined using previously described RIA









methods.244 Acid supernatants were neutralized with 0.1 N sodium hydrox-

ide during the assay procedure. Synthetic LHRH obtained from Beckman Co.

(Palo Alto, CA) was used as reference standard and for radioiodination.

Monoiodo-LHRH was employed in the assay.245 Rabbit antibodies against

LHRH were generously provided by Dr. Nett (R-42, Colorado State Univer-

sity, Fort Collins, CO) or purchased from Miles Laboratories (Elkhart, IN).

Minimum LHRH assay sensitivity was established as 2 pg/tube at which 10%

of total labelled binding was displaced for both LHRH antibodies. Con-

centrations of LHRH were expressed in terms of protein measured with the

dye binding method of Bradford246 in pellets formed after centrifuging

tissue homogenates.

Samples from individual experiments were evaluated in a single assay

to avoid interassay variability. Hormone concentrations were determined

only from serum or homogenate assay volumes which resulted in values on

the linear portion of the standard curves.
Microdissection of Brain Areas

Five experiments designed to examine regional brain CA and LHRH al-

terations with age utilized the microdissection method first described by

Palkovits.24 This "punch" technique has been used extensively to map the

distribution of neurotransmitters,24,35,247 neuropeptides,24,50'248 and

neurotransmitter synthetic enzymes63,249 within the rat brain.

Brains which were collected during the morning were trans-

ferred from dry ice to a cryostat chamber (IEC model CTD-Harris Cryostat,

Needham Heights, MA) held at knife temperature of -10C. Serial frontal

sections (300 pm thick) were then cut beginning rostral to the NAc or OVLT

and extended through the mammillary bodies. Regions were identified in

frozen sections under a stereomicroscope with the aid of K6nig and Klippel's









TABLE II. Parameters Used in Microdissection of Brain Regions


Region
Dissecteda


OVLT


Striatum


POAm

POAs


Approximate
Coordinated


A 9410

A 7770

A 7470

A 7200

A 7020

A 6360

A 6360

A 6360

A 6060

A 5660

A 4950

A 4950

A 4950


Punches/
Brainc


Needles
I.D. (mm)


0.5

1.0


0.75


0.75

0.75

0.75

0.75

0.75


6-8

6-8


0.5

0.5


aAbbreviations used are as follows: nucleus accumbens (NAc); organum
vasculosum of the lamini terminalis (OVLT); preoptic area medialis
(POAm); preoptic area suprachiasmatica (POAs); anterior hypothalamic
nucleus (NHA); medial forebrain bundle (MFB); nucleus supraoptica (NSO);
nucleus suprachiasmatica (NSC); area retrochiasmatica (ARC); nucleus
ventromedialis (NVM); nucleus arcuate (NA); median eminence (ME).
bThe coordinates listed utilized the orientation of KOnig and Klippel250
and represent the first slice from which nuclei were punched. For sev-
eral regions punches were taken from subsequent slices and pooled.
cExcept for the OVLT, regions were dissected bilaterally. For the ME,
overlapping bilateral punches were taken to include the DA rich lateral
palisades zones.251









stereotaxic atlas.250 Table II presents details of the microdissection

parameters employed in these studies. Microdissected tissues were imme-

diately homogenized in 40 ul of 0.1 N perchloric acid containing 10

mg EDTA/100 ml and frozen for later CA assay. Microdissection was com-

pleted with 8 h of decapitation and acid homogenates were stored at -200C

or -800C until assay.

The internal diameter of needles used for microdissection in these

studies was 1.5 to 2.5 times the diameter of punches used in the original

description of this technique.24 Previous studies reported the need to

pool tissue obtained from two to three animals to estimate CA concentra-

tions in some hypothalamic regions.24 The present studies anticipated

lower CA levels in old animals and animals treated with ac-methyl paratyro-

sine (aMPT) compared to these other reports. Since the high cost and

limited availability of old rats precluded the option of pooling tissues

from several animals, larger punch sizes were examined. The internal

diameter of needles employed in these studies dissected the entire region

of interest and in several areas, some surrounding tissue. All regions

except the OVLT were dissected bilaterally. The ME region dissected con-

tained only anatomically distinct ME tissue.25'

Hypothalamic nuclei examined for LHRH and CA concentrations in these

studies were selected on the basis of their suspected roles in the regu-

lation of LH secretion24'50 or because they were terminal beds for various

CA neuronal systems.

Determination of Catecholamine Neuron Activity

Although altered CA concentrations during various physiological or

experimentally induced states suggest that functional changes in the re-

lease of neurotransmitters might be present, this approach does not resolve









whether concentration differences result from altered release or synthe-

sis. Additionally, several reports have shown that CA concentrations

remain remarkably stable in spite of large differences in neuron activ-

ity.252 Because direct measurements of CA release from presynaptic ter-

minals is presently not feasible, several approaches have been used to es-

timate the neuronal activity of CA systems. Each method used thus far

has some limitations which have been reviewed.252

The present studies used the nonsteady state method of Brodie et al.253

to estimate CA activity within microdissected brain regions. This method

measures the rate of CA depletion following blockade of CA synthesis with

the tyrosine hydroxylase inhibitor, aMPT. It assumes that the rate of CA

depletion is equivalent to the rate of CA release. An advantage of this

method over other nonsteady state methods which measure accumulation of

DA, NE or their metabolites lies in evidence that feedback inhibition of

these accumulated products may decrease the synthesis of CA's (see 50).

It has also been shown that aMPT treatment does not alter the uptake or

storage capacity of CA neurons.254

Steady state methods to monitor activity of CA neurons inject trace

doses of labelled CA or precursors which do not disturb the steady state

CA levels. The rate of accumulated labelled CA after injection of precur-

sors or the rate of labelled CA disappearance after injection of radio-

active amines reflects neuron activity. These methods assume a uniform

distribution of injected trace material into the CA neuronal pool and

further assume that the administered amounts do not disturb the ongoing

neuron activity. While these assumptions are subject to some controversy252

the major disadvantage of this approach is the large amount of tissue

required to measure activities.









The major advantage of the aMPT method lies in the ability to detect

changes in CA neuron activity within small tissue regions. Two cautions

must be kept in mind using this approach. First, since the rate of CA

depletion after synthesis inhibition exhibits first order kinetics,253

the times after drug treatment chosen to examine CA concentrations should

be selected over periods that have detectable CA levels. Second, the

method assumes that indirect effects of aMPT on hormone secretion (such

as the increase in prolactin with DA decreases) do not result in feedback

alterations on activity of CA neurons. It should also be stressed that

this method reflects relative neuron activities which should be compared

to different experimental groups examined under the same conditions.

Thus, the direction of changes have been shown to be similar in physio-

logical states tested under different experimental conditions, but the

values reported for CA activity vary among experiments.

The experimental procedure for estimating CA turnover by this non-

steady state method is as follows. On the morning of experiment, 30 animals

from each age group were divided into three subgroups. Two of the sub-

groups were treated with aMPT (250 mg/kg ip, Sigma Chemical Co., St. Louis,

MO) and were killed by decapitation either 30 and 60 or 45 and 90 min later.

As a control for possible stress-related effects of ip injection, the third

subgroup received saline in place of aMPT and was killed 30 or 45 min after

injection. Tissues were then collected and processed as previously described.

Turnover rates of NE and DA (ng/mg protein/h) were calculated for each

brain region from the product of the rate constant of amine loss and the

steady state CA concentration (ng/mg protein) as described by Brodie etal.253

The rate constant was determined from least squares fit analysis of the

aMPT induced depletion for DA and NE within each area. The rate constant









of amine loss reflects the activity of CA neurons while the turnover rates

indicate the relative amounts of CA released for postsynaptic recognition

in the tissue region examined.253 Age-related differences in turnover

rates were tested by LSD tests after determining the variance of the turn-

over rate from application of the Taylor expansion formula.255 Since

treatment with aMPT reduced CA levels to below the detectable limits of

the assay in some regions, turnover rates were determined only for regions

which had measurable CA levels throughout the experimental period.
Catecholamine Radioenzymatic Assay

Acid homogenates of microdissected tissues were thawed and centrifuged

(Beckman Microfuge B, Palo Alto, CA) for 90 sec immediately prior to evalua-

uation for DA and NE content. Supernatant samples were assayed in volumes

of 5 or 10 pl for DA and NE using a modification of the radioenzymatic

method of Cuello et al.256 as previously described.42 This method employs

catechol-0-methyl transferase, isolated from rat livers,257 to catalyze

the 0-methylation with tritiated S-adenosyl methionine (New England Nuclear,

Boston, MA) of DA and NE to methoxytyramine and normetanephrine, respec-

tively. Tritiated metabolites were separated by thin layer chromatography,

identified under ultraviolet light and fluorescent spots were scraped for

quantification of tritiated activity with liquid scintillation.258 The

separation procedures employed resulted in complete separation of the NE

metabolite and less than 2% normetanephrine contamination in the DA meta-

bolite fraction.42

Each microdissected region was evaluated in a separate assay to elim-

inate interassay variability within a region. Minimum assay sensitivities,

calculated from twice the tritium activity obtained from blank standards,

were approximately 20 pg for DA and 50 pg for NE. Protein content of the






48


pellet obtained after centrifugation was measured with the dye binding

method of Bradford.246 Bovine serum albumin was the protein standard

and CA values are expressed in terms of protein concentrations.














EXPERIMENTAL

Evaluation of Age-Related Alterations in Catecholamine
and Luteinizing Hormone Releasing Hormone Neuronal Activity
Within Microdissected Brain Regions

Changes in Catecholamine Concentrations in Microdissected Brain Regions
of Aged Male Rats

Objectives. A large body of evidence has implicated a role for cen-

trol CA neurons in the secretion of LH and prolactin from the AP of the

rat.25,49,50,104 Although the distribution of DA and NE among discrete

regions of the brains of young mature animals has been well characterized,24

little is known about the distribution of these putative neurotransmitters

in the aged rat. Since the secretion of LH and prolactin appear to be de-

pendent upon CA activity in discrete regions along the preopticotuberal

pathway and hormone secretary patterns are altered in old rats (see 113,

148, 182), we compared the concentrations of DA and NE in discrete regions

of the brain structures in young and old male rats.

This study was undertaken to establish both the feasibility of measur-

ing CA from microdissected brain regions in our laboratory and to demon-

strate that differential CA alterations occur between neural regions of

aged rats. Male rats were initially used in this study because they do not

have variation in hormone levels and CA fluctuation due to cyclic changes

in ovarian cycles.

Materials and Methods. Male Wistar rats (Harlan Industries, Indiana-

polis, IN) 3-4 months (n=8) and 24-25 months (n=7) of age were housed in

our animal facilities for one week. On the morning of experiment animals

were decapitated, brains were removed and frozen on dry ice and trunk blood









was collected for later assay of serum hormones. Within 2 h following

decapitation, 11 POA and hypothalamic nuclear regions were microdissected,

homogenized in perchloric acid (containing EDTA 10/mg/100 ml) and frozen

for CA assay as described above. The CA assay was sensitive to 31 pg of

DA and 30 pg of NE.

Results. Table III presents the changes detected in CA concentration

with age for each region examined. Abbreviations used for each dissected

region are shown on Table II. Concentrations of NE were significantly

decreased in the NA, POAm and OVLT of aged,compared to young, mature male

rats. As anticipated, DA concentrations were decreased significantly in

some areas. Concentrations of DA were decreased nearly 50% in the ME, ARC

and NA. In contrast, DA concentrations were increased 35% in the NHA, 290%

in the POAm and 220% in the POAs of old versus young rats.

Mean serum prolactin concentrations were increased significantly in

the 23-24 month old rats (165 47 ng/ml) compared to 3-4 month old males

(37 4 ng/ml), while serum LH concentrations were not different in young

(14 4 ng/ml) and old (16 8 ng/ml) rats. It was noted that three of

seven aged rats had AP adenomas which were identified from weights greater

than 30 mg. However, neither brain CA concentrations nor serum hormone

levels differed consistently among old tumor and nontumor bearing rats.

Discussion. These results demonstrate that differential alterations

are present and can be detected with methods employed in our laboratory in

CA concentrations among microdissected POA and hypothalamic regions of old

rats. The decreased DA in the MBH region and elevated serum prolactin

measured in old rats indicates that mammotrophs of old animals may augment

prolactin secretion as a result of diminished activity of the TIDA system.

The somewhat surprising increases of DA measured in POA regions from old









TABLE III. Age-Related Alterations in the Dopamine (DA) and Norepine-
phrine (NE) Concentrations in Microdissected Brain Regions
from Male Rats


DA (ng/mg Protein)
Age (Months)
3^ ----424-25


NE (ng/mg Protein)
Age (Months)
3-4 24-----25


69.4 9.8a


15.2

4.1

3.9

2.3

2.7

4.8

2.3

14.1

3.9

28.0


2.5

0.5

0.5

0.3

0.6

1 .1

0.3

5.9

0.5

6.2


37.0 4.3**


9.7

2.1

3.7

2.1

2.8

2.6

3.1

55.2

12.4

20.2


1.2*

S0.4***

0.9

0.5

1.5

0.5

0.3*

18.0*

4.3*

8.3


" = p < 0.05;


Area


NA

ARC

NVM

NSC

NSO

MFB

NHA

POAm

POAs

OVLT


30.8

19.0

35.7

28.6

25.7

18.8

14.8

22.5

72.3

30.8

35.3


+ 5.8

+ 1.7

3.6

1.4

+ 1.9

+ 1.9

1.7

+ 1.3

6.0

2.9

+ 6.9


21.2

15.3

29.0

21.4

27.4

17.4

14.9

24.7

52.8

22.5

18.3


2.6

1.0*

4.9

5.1

3.2

1.4

2.1

1.6

7.1*

3.7

3.4*


-- ----


means SEM; w = p < 0.1;


"" = p < 0.01










rats further suggests that DA neuronal systems may respond different to

the effects of advanced age.

While NE concentrations were significantly decreased in several POA

and hypothalamic regions, basal LH concentrations were not different in

young and old male rats. Thus, NE neuronal function in old male rats ap-

pears to be adequate to maintain basal secretion of LH. However, old male

and female rats are less able to secrete LH in response to several cen-

trally mediated stimuli.133,148,156-159,161,162 This may indicate that a

functional deficiency in NE may become apparent when challenged to augment

LH secretion.

Therefore, these results clearly point out the need to examine more

closely the activities of CA neurons within these hypothalamic regions and

to characterize the extent of alterations in these systems during advanced

age.

Changes in Catecholamine Activity in Microdissected Brain Regions of
Aging Ovariectomized Fischer 344 Rats

Objectives. Several patterns of reproductive senescence have been

described in the female rat.113'114 While the ovaries and pituitary of

the old CE rat can be induced to function relatively normally (see 105),

a hypothalamic deficiency appears to prevent the steroid-induced cyclic

release of LH in these old animals.182 This deficiency appears to involve

CA neurons, since hypothalamic NE concentrations have been reported to be

decreased in old ovariectomized CE rats113 and several drugs which enhance

CA activities can reinitiate estrous cycles in animals experiencing this

reproductive state (see 105).

The PP state occurs late in life in a small proportion of Long-Evans

and Sprague-Dawley rats114 and in a high proportion of F344 rats116 (Table

I). In the PP state, animals ovulate at 8 to 20 day intervals with maintained









functional corpora lutea between ovulatory events.113 The LH secretary

capacity of PP rats appears to be relatively normal in response to a stimu-

latory regimen of gonadal steroids162 which is in contrast to the response

reported in old CE rats.156-159,162

In view of the paucity of information on regional alterations in brain

CA metabolism during reproductive senescence, this study was undertaken to

determine NE and DA concentrations and neuron activity in aging F344 rats

which enter the PP state in advanced age. Regions selected for analysis

were primarily those along the preopticotuberal pathway, and also regions

which contain extensive DA nerve terminal fields from several DA systems.

Animals were examined at two ages prior to the PP state and after estab-

lishment of the PP state. Animals were examined two weeks after ovariec-

tomy. This experimental design was employed to eliminate the confounding

effects of cyclic variation in gonadal hormones on CA activity. Although ex-

perimentally induced pseudopregnancy may have served as a control in young

animals, it is unclear whether similar mechanisms control the PP state of

old rats. Further, the ovariectomized rat model has the advantage of ex-

amining CA neuron activity during a state in which hormone response dif-

ferences have been demonstrated to occur with age.144,148'156,161-163

Materials and Methods. Barrier-reared F344 female rats were employed

in these studies. Animals were purchased at 3-4, 8-9 and 20-21 months of

age and housed in our animal facility. After 30 days of daily monitoring

of vaginal cytology, NC young (4-5 months) and middle-aged (9-10 months)

and PP old (21-22 months) old rats were ovariectomized. Two weeks later,

30 animals of each age group were divided into three subgroups. Rats in

the two subgroups receiving aMPT were killed 45 and 90 min following drug

administration while the third saline subgroup was killed 45 min after









Body and Organ Weights and Health Status
Employed in Catecholamine Studies


of Fischer 344 Rats


Age (Months)


Number of Rats


Body Weight
(g, B.W.)
Anterior
Pituitary


Weight
mg/100


(mg)
g B.W.


Weight (mg)
mg/100 g B.W.


Adrenal


Kidney


Weight
mg/l 00

Weight
mg/100


(g)
g B.W.

(mg)
g B.W.


210 2.4


9.85 0.19
4.7 0.1

45.6 + 0.8
21.7 + 0.4

1.57 0.03
745 + 14

505 7
240 2


218 2.5


10.10 0.20
4.6 0.1


294 5.7


15.1 3.4
3.9 0.2


44.4 0.9 52.9 1.4
20.5 0.5 18.1 0.5

1.70 0.03 2.29 0.06
780 13 784 + 21


527 6
245 2


730 11
240 4


Abnormalities


Number per group


Pituitary
Lung
Liver


TABLE IV.


4-5


9-10


21-22


Heart









injection. Tissues were collected as described above and animals were

necropsied. Minimum sensitivity of the CA assays was approximately 20 pg

for DA and 50 pg for NE.

Results. The evaluation of health status of the animals employed in

this experiment is shown in Table IV. Both body and organ weights increased

with age. When organ weights were expressed as proportions of body weight,

no age-related changes were apparent. The incidence of pituitary and liver

abnormalities increased in old animals. However, these lesions involved

only a small portion of the organ and did not affect organ weight (Table IV).

Further, no consistent trends in hormone or CA levels were apparent between

lesioned and nonlesioned animals.

Steady state DA concentrations which were measured in every region

are shown in Table V for the three age groups. Concentrations of DA de-

creased in old versus young rats in several areas. These decreases were

most dramatic (42 to 78%) in the POAs, ARC, ME and NIL. In the NSC, DA con-

centrations were reduced 78% to levels below the limits of sensitivity of

the assay in the old age group.

Age-related changes in DA turnover rates are shown in Figure 1 for

those areas in which DA was still detectable after aMPT treatment. In con-

trast to the general decreases found in DA concentrations, DA turnover rates

were stable, decreased or increased with age in various regions. Turnover

rates of DA decreased significantly in the OVLT (81%), POAs (49%) and NA

(63%) of old compared with young rats. In contrast, DA turnover increased

significantly by 100% in the NVM, 42% in the ME and 22% in the striatum.

No significant alteration in DA turnover with age was detected in the POAm

or the NIL.








TABLE V. Age-Related Change in Dopamine Activity Within Microdissected Brain
Regions of Ovariectomized Fischer 344 Rats


Area


OVLT


S


POAm


POAs


Rate Constant
(K)_


Age
(Months)

4-5
9-10
21-22
4-5
9-10
21-22
4-5
9-10
21-22
4-5
9-10
21-22
4-5
9-10
21-22
4-5
9-10
21-22
4-5
9-10
21-22
4-5
9-10
21-22
4-5
9-10
21-22
4-5
9-10
21-22
4-5
9-10
21-22
4-5
9-10
21-22


Dopami ne
Concentrations
(ng/mg protein)
16.7 1.8
13.3 0.7
13.7 1.2
61.0 7.1
54.2 6.6
54.3 6.4
2.5 0.5
3.6 1.0
2.4 0.3
4.0 0.3
3.3 0.6
2.1 0.2*
27.5 + 2.0
25.9 + 5.5
19.6 + 4.2
1.5 + 0.3
2.3 0.2
1.9 0.2
98.7 10.3
109.2 2.0
56.8 11.8 t
6.5 0.8
6.3 0.6
3.4 0.3*t
3.0 0.5
2.8 0.5
1.8 0.8
2.2 1.2
3.2 0.7
ND
4.4 0.9
2.1 0.3
1.3 0.3
1.4 0.4
1.9 0.3
1.2 + 0.3


Turnover Rate
(ng/mg protein/hr)


0.37
0.07
0.09
0.26
0.26
0.36
0.43
0.48
0.60
0.77
0.53
0.75
0.35
0.31
0.18
0.49
0.38
0.77
0.68
0.73
1.67
0.46
0.55
0.84


0.07
0.03*
0.05
0.06
0.07
0.08
0.07
0.13
0.11
0.07
0.10
0.09
0.06
0.11
0.11
0.10
0.10
0.07t
0.09
0.12
0.17*t
0.06
0.08
0.08*t


6.2
0.9
1.2
16.1
14.0
19.6
1.1
1.7
1.4
3.1
1.7
1.6
9.6
8.1
3.5
0.7
0.9
1.5
67.5
79.3
95.1
3.0
3.5
2.9


0.4
S0.1*
0.1
1.3
1.3
1.6t
0.1
0.3
0.1
0.1
0.2
0.1*
0.5
1.1
0.6*t
+ 0.1
+ 0.1
0.1*t
4.4
2.6
12.5*
0.2
0.2
0.2
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND


NVM


ME


NIL


MFB


NSC


ARC


NHA


*p < 0.05 vs 4-5 mo; t p < 0.05 vs 9-10 mo.; ND = Rate Constant and Turnover
Rate could not be accurately determined (see text)
























i 4-5mo
O 9-IOmo
S21-22 mo

* p<.05 vs 4-5mo
t p<.05 vs 9-IOmo


S OVLT POAm PQAs NVM NA ME NIL


Age-Related Changes in Dopamine (DA) Turnover Rates in
Microdissected Brain Regions of Ovariectomized Fischer
344 Rats. Each column represents mean turnover rate as
determined by a non-steady state method employing 24-30
rats. Only those regions for which turnover rats could
be accurately determined are included. Abbreviations
used are the same as in Table II.


80


, 60
a.


C

c 15
o
a

I 10
o
c

'5


Figure 1.









The decline in DA turnover rates with age in the OVLT appears to be

a consequence of a decreased activity in DA neurons since, while DA con-

centrations were not changed, the K of DA loss was substantially decreased.

The acceleration in the DA turnover observed in the NVM and the ME of old

rats was the result of a substantial increase in the K of DA loss. Inter-

estingly, in both the NIL and ME the activity of DA neurons increased co-

incident with a 48 and 42% decline, respectively, in DA concentrations in

these two brain regions.

Table VI illustrates differences in steady state NE concentrations be-

tween age groups for each area examined. As found for DA, NE concentrations

were decreased in old compared to younger animals in several areas. Rela-

tively large decreases in NE concentration between 4-5 and 21-22 month old

animals were observed in the POAs (56%), ARC (56%), MFB (48%), NHA (46%),

NA (42%) and NSC (38%). No significant differences in NE concentration

were detected between young and middle-aged rats in any area examined.

Turnover rate changes of NE with age present a strikingly different

picture from the generalized decreases in NE concentration detected in old

rats. As shown in Figure 2, significant increases in NE turnover rates were

found in middle age compared to 4-5 month old rats in the POAm (31%), NHA

(3.0-fold), MFB (2.9-fold), NSC (2.6-fold) and NA (11.6-fold) regions.

Interestingly, NE steady state concentrations did not change with age in

the ME but the NE turnover rate increased 5-fold in old animals in this

region (Table VI and Figure 2). With the exception of the POAs, the pat-

tern which emerges from these data suggests that increases in NE turnover

rates precede the decreases in NE concentrations within discrete hypothal-

amic areas during the aging process. The increase in turnover rates observed

in several regions in middle-aged rats was due primarily to increased K of









TABLE VI. Age-Related Change in Norepinephrine Activity Within
Brain Regions of Ovariectomized Fischer 344 Rats


Microdissected


Norepinephrine
Concentrations
(ng/mg protein)


Area



POAm



POAs



MFB



NHA


1.6
1.4
1.4


2.4
3.2*
1.1*t

1.0
1.9
0.8*t


4-5
9-10
21-22

4-5
9-10
21-22

4-5
9-10
21-22

4-5
9-10
21-22

4-5
9-10
21-22

4-5
9-10
21-22

4-5
9-10
21-22

4-5
9-10
21-22

4-5
9-10
21-22

4-5
9-10
21-22


10.6
11.0
8.5

25.2
20.2
11.1

11.6
14.6
6.0

12.0
13.6
6.5

10.3
9.6
6.4

19.2
15.7
8.4

9.0
12.1
5.2

11.6
14.8
10.7

16.3
17.3
15.5

11.7
16.1
11.9


Rate Constant
i)n


0.24
0.33
0.32

0.35
0.28
0.53

0.30
0.69
0.19

0.29
0.72
0.42

0.29
0.80
0.49

0.41
0.55
0.22


Turnover Rate
(ng/mg protein/br)


2.5
3.6 +
2.7 +

8.9 +
5.7 +
5.9 +


0.05
0.08
0.07

0.06
0.10
0.09t

0.09,
0.08
0.15

0.17
0.13
0.21

0.10
0.09*
0.09t1

0.09
0.08
0.10+


3.5
10.2
1 .1

3.5
10.3
2.7


3.0 +
7.7
3.1

7.8
8.7
1.9 +


0.08 0.14
0.67 0.34*


0.9
2.4
1.0

+ 0.9
+ 2.2
1.7

+ 2.4
2.2
+ 3.3

1.4
+ 1.5
1.9


0.18
0.27
0.98


0.08
0.08*
0.10 t


0.2
0.3*
0.3t

0.6
0.6*
0.4*t

0.3
0.8*
0.2*t

0.7
1.2*
0.4t

0.5
0.8*
0.2t

0.8
0.7
0.2*t


0.7 0.3
8.1 + 1.3
ND


2.1
4.1
10.5


0.2
0.4*
0.4*t


"p < 0.05 vs 4-5 mo.; t p < 0.05 vs 9-10 mo.; ND =
Rates could not be accurately determined (see text)


Rate Constants and Turnover


Age
(months)


3.9
2.6
1.3

2.9
1.7
0.6


NSC


ARC


2.9
2.1
1.6*t


















4-5mo
9-10mo
21- 22 mo


* p<.05vs 4-5mo
t p<.05 9-10vs 21-22mo


POAm POAs HA MFB NSC ARC NA ME


Figure 2. Age-Related Changes in Norephinephrine (NE) Turnover Rates
in Microdissected Brain Regions of Ovariectomized Fischer
344 Rats. Each column.represents mean turnover rate as
determined by a non-steady state method employing 24-30
rats. Only those regions for which turnover rates could
be accurately determined are included. Abbreviations
used are the same as in Table II.









NE loss since initial concentrations of NE changed little between 4-5 and

9-10 months of age (Table VI). In contrast, the decline in turnover rates

seen in the POAs, MFB, NHA, and ARC was due either to a decline in initial

NE concentrations (POAs and NHA) or to a decline in both components of

turnover rate (MFB and ARC).

Discussion. The results of this study clearly demonstrate that with

increasing postmaturational age, regional alterations in concentrations,

activities and turnover rates of DA and NE are observed in the ovariec-

tomized F344 rat. Other studies, which have examined larger brain regions,

have demonstrated differential responses of DA and NE neurons to advancing

age (see 105, 184). The present study indicates that both DA and NE neurons

show disparate responses to age within microdissected regions of the ven-

tral diencephalon. Thus the heterogeneity of CA neuronal aging previously

observed in large brain region is apparently existent even within these

regions. These results further suggest that ubiquitous loss of CA neurons

or a generalized decline in CA activity does not occur in the F344 rat.

The responses ofdopaminergic neurons to advancing age are of particular

interest since most studies have reported an age-related decline in DA con-

centration and/or DA turnover rates in the hypothalamus.215'220 In male

rats,215 as well as female Long-Evans rats,216 the age-related decline in

medial basal hypothalamic (MBH) DA concentrations is associated with a de-

crease in MBH DA turnover215,216 as well as a decline in DA levels in pi-

tuitary stalk blood.222,223 The present study demonstrates that in the

F344 rat, a decline in DA concentrations in the ME and NIL of the pituitary

is associated with an increase in DA neuronal activity in these two regions.

Thus an age-related decline in DA levels is not obligatively associated

with a decline in the turnover rates of DA in these neurons.









The response of noradrenergic neurons to advancing age appears to be

considerably more uniform than the alterations observed in dopaminergic

neurons. Of the six regions which showed a substantial decline in NE con-

centration in the 21-22 month old group, five of these areas showed enhanced

NE turnover in the 9-10 month old group. These data suggest that the age-

related decline in NE concentration may be caused by a preceding state of

hyperactivity of these noradrenergic neurons. In the ME, NE turnover was

accelerated in both middle-aged and old rats and NE concentrations were

stable with age. In the POAs, while a decline in NE concentration in the

old group of rats occurred without an acceleration in NE activity in middle-

aged animals, it is interesting to note that NE turnover was extremely high

in the POAs of young animals. Collectively, these data suggest that a

period of chronic hyperactivity of NE neurons may lead to subsequent deple-

tion of NE stores with advancing age.

The PP state can be induced in young rats by electrolytic destruction

of the POAm259 or the OVLT.260 While we were unable to demonstrate any

alteration in CA concentration or turnover in the POAm in old rats, a sub-

stantial decline in both DA and NE metabolism was observed in the POAs, and

a decline in DA turnover rate was seen in the OVLT. Since large electro-

lytic lesions of the POAm are likely to include the POAs, it is possible

that the CA deficiencies observed in the OVLT and/or POAs may contribute

to PP state. This idea is consistent with the observation that local im-

plantation of the CA precursor L-dopa, into the preoptic area,131 or sys-

temic administration of the dopamine agonist, lergotrile mesylatel79,259

reinitiates normal estrous cycles in old PP rats. However, it is not cer-

tain whether the ability of these drugs to reinitiate estrous cycles in

PP rats is due to an action in the central nervous system or to their sup-
pression of PRL secretion.









Response of LHRH Neurons to Ovariectomy in Microdissected Brain Regions
of Aging Fischer 344 Rats

Objectives. The results of previous reports which examined the effects

of age on LHRH concentrations in intact rats are equivocal in that increased,232

decreased,220,233,234 or stable235 values were reported in old versus young

animals. The present study was undertaken to characterize LHRH concentra-

tions within regions of the preopticotuberal pathway and to examine the

ability of LHRH neurons within these regions to respond to the stimulatory

effects of ovariectomy in young, middle-aged and old F344 rats.

Materials and Methods. Barrier-reared F344 rats were purchased at 2-3,

8-9 and 20-21 months of age. Daily vaginal lavages were obtained for 30

days to assess the reproductive state of each animal. Normally cycling

young (4-5 months old) and middle-aged (10-11 months old) and PP old (22-

23 months old) rats were selected for study on the day of diestrus or two

weeks following ovariectomy. Animals were killed by decapitation, trunk

blood was collected and necropsies were performed as described above.

Eight brain regions were microdissected and tissues were homogenized

in 150 il of 0.1 N HC1 for subsequent LHRH assay. The intraassay coef-

ficient of variation was 6.9% as determined from 12 replicate standard

tubes which displaced about 50% of labelled hormone. Minimum LHRH assay

sensitivity was established as 2 pg/tube at which 10% of total labelled

binding was displaced. Luteinizing hormone releasing hormone was detect-

able in all samples with the exception of 8% of the OVLT samples. These

samples were assigned LHRH levels at minimum assay sensitivity for statis-

tical analysis. Concentrations of LHRH are expressed in terms of pellet

protein measured by the dye binding method of Bradford.246









TABLE VII.


Body and Organ Weights and Health Status of Fischer 344 Rats
Employed in Luteinizing Hormone Releasing Hormone Studies


4-5


22-23


Number of Rats 20

Body Weight 185.3 2.2
(g, B.W.)


Anterior
Pituitary


Adrenal


Weight
mg/100

Weight
mg/100


(mg)
g, B.W.

(mg)
g, B.W.


10.9 0.5
5.9 0.3

48.5 1.3
26.3 0.7


20

207.5 3.2


12.1 0.6
5.9 0.3

52.6 1.6
25.4 0.8


20

277.5 5.3*t


12.1 0.5
4.4 0.2

56.1 1.6*
20.3 0.6*


Abnormal cities


Number per group


Pi tui tary
Lung
Liver


*p < 0.05 vs 4-5 month;


p < 0.05 vs 4-5 month; t p < 0.05 vs 10-11 month


4-5


Age (months)
10-11


t p < 0.05 vs 10-11 month









Data were analyzed for age effects with ANOVA followed by Student-

Neuman-Kuels tests and for response to ovariectomy with t-tests. Proba-

bility values of less than 0.05 were considered statistically significant.

Results. Although the F344 rat has been shown to have a lower inci-

dence of pathologies in advanced age and to maintain general robustness

compared to other varieties of inbred rats,261 inclusion of the occasional

abnormal or lesioned animal often confounds the results of studies of se-

nescence.242 For the present experiments, therefore, only animals with

robust appearance which maintained body weight throughout the course of

the experiment were chosen. As shown in Table VII, body and adrenal weights

increased significantly with age, while anterior pituitary weights did not

change. However, when organ weights were expressed as proportions of body

weight, a significant decrease was noted for both the adrenal and anterior

pituitary glands. While the incidence of visible anterior pituitary and

liver abnormalities increased in 22-23 month old rats, these alterations

were point-like lesions which did not affect organ weight and no differences

in serum hormone or brain LHRH concentrations were observed between lesioned

and nonlesioned rats.

Luteinizing hormone releasing hormone concentrations were detectable

in all eight of the microdissected areas and the expected regional distri-

bution of LHRH was observed (Table VIII).262 The highest LHRH concentra-

tions were observed in the median eminence (ME) and this area alone showed

a significant decrease in LHRH levels in response to ovariectomy. This

decrease in LHRH concentration within the ME of ovariectomized rats was con-

sistently about 20% in all three age groups. In all eight regions examined,

no age-related differences in LHRH concentrations were detected.









TABLE VIII.


Effects of Age and Ovariectomy on Luteinizing Hormone
Releasing Hormone (LHRH) Concentrations Within Microdissected
Brain Regions of Fischer 344 Rats


LHRH (pg/pg
Diestrous


Area


OVLT


0.09a
0.05
0.09

0.03
0.02
0.08


4-5
10-11
22-23

4-5
10-11
22-23

4-5
10-11
22-23

4-5
10-11
22-23

4-5
10-11
22-23

4-5
10-11
22-23

4-5
10-11
22-23

4-5
10-11
22-23


months
months
months

months
months
months

months
months
months

months
months
months

months
months
months

months
months
months

months
months
months

months
months
months


Protein)
Ovariectomized


0.31
0.50
0.34

0.21
0.28
0.26

1.97
1.64
1.28

0.08
0.08
0.13

1.05
1.00
0.81

2.73
1.77
1.96

1.53
2.08
1.41

112.8
135.0
113.4


0.50
0.47
0.79

0.30
0.20
0.20

0.71
1.55
1.44

0.09
0.08
0.08

0.81
0.92
0.94

2.28
1.75
2.13

0.95
0.99
1.53

87.8
102.9
92.0


aMean SEM; *Significantly less than diestrous p <0.05


0.44
0.26
0.17

0.01
0.01
0.04

0.15
0.08
0.99

0.77
0.29
0.48

0.30
0.39
0.33


Age


POAm


POAs


NHA


4.7
8.8
7.0


0.20
0.12
0.19

0.08
0.03
0.02

0.39
0.28
0.21

0.01
0.01
0.01

0.10
0.10
0.13

0.78
0.72
0.69

0.18
0.35
0.43

8.3*
5.5*
4.8


NSC



ARC









Discussion. The major finding in this study is the observation that

the PP state in aged F344 rats occurs concomitantly with the maintenance

of LHRH concentration along the preopticotuberal pathway and a normal post-

castration LHRH decline in the ME. In view of the capacity of these PP

rats to ovulate, albeit less frequently than NC younger animals, it is

apparent that the two year old F344 rat can respond relatively normally to

signals for LH hypersecretion. These data are consistent with the observed

normal LH secretion response to a stimulatory regimen of gonadal steroids

in the aged PP rat of other strains162 and the postcastration LHRH deple-

tion response reported in the MBH of middle-aged PP Sprague-Dawley rats.234

The results of this study, in conjunction with the previous study which

showed that the old ovariectomized F344 maintains NE turnover at levels

equal to or greater than those observed in young rats in several regions

of the preopticotuberal pathway including the POAm, NHA and ME, indicate

that the primary locus of alteration leading to the PP state in old F344

rats is not an impaired responsiveness of LHRH neurons. It is further sug-

gested that a primary defect does not occur in the ability of NE neurons

to modulate LHRH neuronal function.

Changes in Catecholamine Activity in Microdissected Brain Regions of
Aging Ovariectomized Long-Evans Rats

Objectives. At least two lines of evidence suggest changes in central

CA neuronal function may be causally related to altered gonadotropin secre-

tory responses in old rats. First, several stimuli which augment central

CA systems are able to restore near normal ovarian cyclic activity in old

CE rats (see 105). Second, studies which compared hypothalamic CA concen-

trations113.194 and metabolism145,216 in old male and CE rats reported de-

creases with age. These decreases in CA neuronal function have been asso-

ciated with dampened LH response to centrally mediated stimulil56-159,161-163
and persistent hyperprolactinemial13'223 in CE rats.









The present study was designed to characterize age-associated altera-

tions in CA neuronal activity within microdissected brain regions in ovar-

iectomized Long-Evans rats. Regions selected for evaluation were primarily

those along the preopticotuberal pathway and secondly, regions which con-

tain extensive DA nerve terminal fields from various DA neuron systems.

Emphasis was placed upon comparison of young and middle-aged animals prior

to establishment of the CE state with old CE rats.

Materials and Methods. Daily vaginal lavages were examined for 30

days in three different cohort groups of Long-Evans rats. Young NC (3-4

months old), middle-aged NC (10 months old) and old (20-22 months old)

rats which had been in the CE state for six to ten months were ovariecto-

mized. Two weeks later, each cohort age group was divided into three sub-

groups. Two subgroups of each cohort were treated with aMPT and killed by

decapitation 30 and 60 min after drug treatment. The third subgroups re-

ceived saline injections and were killed 30 min later. Trunk blood was col-

lected for later evaluation of serum LH and prolactin concentrations.

Pituitary tissues were removed and homogenized as previously outlined and

animals were necropsied for visible abnormalities. Ten brain areas were

microdissected and homogenized as described above. Neural tissue homo-

genates were then assayed for DA and NE concentrations. Minimum sensitivi-

ties of the DA assays were approximately 20 pg for DA and 50 pg for NE.

Results. In an attempt to separate the effects of age from effects

which might be attributed to pathology, only animals with robust appearance

and maintained body weight were selected for study. Additionally, animals

were necropsied for gross visible lesions and weights of several organs

were recorded. Of the 32 old rats selected for study, five were eliminated

from data analysis on the basis of putuitary tumors noted at necropsy.









Body and Organ Weights and Health
Employed in Catecholamine Studies


Status of Long-Evans Rats


Age (months)
10


Number of Rats


Body Weight
(g, B.W.)

Anterior
Pituitary


Adrenal


Ki dney


Heart


Weight (mg)
mg/100 g B.W.


Weight
mg/100

Weight
mg/100


(mg)
g B.W.

(g)
g B.W.


Weight (mg)
mg/100 g B.W.


31

285.3 3.8a


9.2 0.3
3.2 0.1

61.5 2.6
21.5 0.9

2.11 0.04
742.8 15.9

726.7 13.6
255.0 4.2


31

338.9 + 5.8*


10.5 0.4
3.1 0.1

58.0 2.6
17.2 + 0.8


27

411.3 9.2*t


13.1 + 0.6*
3.2 0.1

66.5 3.01
16.2 0.7


2.46 0.07* 3.13 0.08*t
728.2 18.6 768.3 20.2

790.1 19.2 966.8 20.6*t
233.5 4.7* 236.3 4.5*


Abnormal i ties


Pituitary
Lung
Kidney
Liver


Number per group

0
15
1
3


mean SEM; *p < 0.05 versus 3-4 months; t 0.05 versus 10 months


TABLE IX.


20-22









Results recorded at necropsy are summarized on Table IX for animals used

in this study. Both body and organ weights tended to increase with age;

however, values were stable or decreased with age when expressed as pro-

portions of body weight. Decreased ratios of adrenal and heart weights

may be attributed to increased proportions of body fat with age; however,

these ratios did not change between 10 and 20-22 month old rats. The in-

cidence of pituitary, liver and kidney abnormalities increased with age.

These abnormalities consisted primarily of small point-like lesions and

no consistent trends in hormone or CA levels were apparent between lesioned

and nonlesioned animals included in data analysis.

Steady state DA concentrations (measured in saline treated rats)

tended to decrease with age in seven of nine brain regions examined for

this amine (Table X). Decreased DA concentrations in regions from old

versus young rats were most dramatic in the NAc (34%), NHA (54%), NIL

(51%), and ME (74%). Concentrations of DA decreased significantly between

3-4 and 10 months of age only in the NHA (31%) and ME (40%).

Steady state NE concentrations similarly tended to decrease from

23 to 59% in old versus young rats in the regions examined (Table XI).

Decreases were significant in the POAs (54%), MFB (44%), NSC (49%), and

ME (59%). Concentrations of NE in 10 month old rats were significantly

diminished compared to young rats only in the NSC (46%) and ME (40%).

Turnover rates of DA were decreased substantially in five regions from

old versus young animals (Table X). These regions were the POAm (45%),

POAs (59%), NHA (81%), ME (63%) and NIL (48%). Interestingly, DA turnover

rates increased in old compared to middle-aged and/or young rats in the

NAc, striatum and NA. The increases in DA turnover in these three regions

were attributed to augmented neuronal activity as reflected by DA rate






71


TABLE X. Age-Related Change in Dopamine Activity Within Microdissected
Brain Regions of Ovariectomized Long-Evans Rats


[DA] initial
(ng/mg protein)


Rate Constant
iK --


Turnover Rate
(ng/mg protein/hr)


233.9
180.9
154.7

227.0
254.0
226.0

14.4
14.3
9.3

24.3
24.6
24.7

10.8
7.4
5.0


13.4
18.8.
11.1

15.4
12.0
15.7

2.6
1.8
0.6

3.2
5.1
2.8

1.2*
0.8*
0.9*


3-4
10
20-22

3-4
10
20-22

3-4
10
20-22

3-4
10
20-22

3-4
10
20-22

3-4
10
20-22

3-4
10
20-22

3-4
10
20-22

3-4
10
20-22


43.7
33.6
29.9

210.9
125.4
54.4

12.5
10.0
6.1


5.4
5.7
4.1


22.4
13.8*
7.6*t

1.7
0.9*
0.6*


0.53
0.48
1.13

0.30
0.11
0.36

0.48
0.59
0.41

1.03
0.92
0.41

0.53
0.40
0.21

0.33
0.38
0.50

1.00
0.77
1.32

0.83
0.81
1.18

0.80
0.33
0.91


0.09
0.09
0.08*+t

0.08
0.07
0.11

0.13
0.12
0.12

0.17
0.14
0.13*


0.16
0.10
0.15

0.19
0.16
0.14


0.14
0.14
0.15t

0.11
0.10
0.15

0.12
0.22
0.16t


123.5
87.5
174.8

68.5
29.2
81.7


6.9
8.5 +
3.8


24.9
22.8
10.1


5.8
2.9
1.1


5.7
6.0
7.8 t

4.3
3.3*
5.8t

0.8
0.7
0.3*t

2.1
2.9
0.9*t

0.5
0.2
0.2*t


0.8 0.1
0.7 0.3
0.9 0.1


43.9
26.0
39.6

175.9
102.1
64.4

10.0
3.3
5.2


1.2
2.7
3.it

11.
7.0
5.2*t

0.9
0.5*
0.4


*p < 0.05 versus 3-4 month old group; t p < 0.05 versus 10


Age
(months)


Area


POAm



POAs



NHA


2.4 + 0.6
1.7 + 0.2
1.8 + 0.2


NVM


month old group









constants (K). Although rate constants were not significantly different

in any region between young and middle-aged rats, they increased signifi-

cantly in the NAc (135%), striatum (139%), NIL (175%), and NA (71%) and

were moderately elevated in the NVM (36%), and ME (45%) between middle-

and old-age. Only the POAs region had a significant decrease in DA rate

constant in old versus young rats.

As shown in Table XI, turnover rates of NE in microdissected tissues

from 20-22 month old rats were significantly decreased compared to 3-4

month old rats in the POAs (98%), NSC (21%), NVM (51%) and ME (38%), while

turnover rates were augmented in the POAm (44%) and NHA (85%). Interest-

ingly, for four regions in which decreased NE turnover was observed in

old animals, a significant decline in NE turnover was seen in middle-aged

rats. Rate constants of NE remained stable although they tended to increase

in old compared to younger rats in all regions except the POAs. Thus, as

in the case for DA, decreases in NE turnover rates observed in several

brain regions of old animals were primarily the result of decreased NE

concentrations rather than neuron activity. In contrast, regions which

showed a decreased NE turnover in middle-aged animals, both a decline in

NE concentration and K,appeared to contribute to the turnover decreases.

We noted that regions with relatively high NE rate constants in young

rats were those regions which showed the greatest NE concentration deple-

tion in old animals. When this relationship was analyzed, a strong posi-

tive correlation was found between neuronal activity (K) measured in regions

from young animals and the magnitude of the NE depletion measured in corre-

sponding regions from old animals (r = 0.72). Similarly, the relationship

between NE activity in young animals and percent NE depletion calculated

between 4-5 and 10 month old rats was positively correlated (r = 0.84).









TABLE XI. Age-Related Change in Norepinephrine Activity Within Micro-
dissected Brain Regions of Ovariectomized Long-Evans Rats


[NE] initial
(ng/mg protein)


Area

POAm



POAs


2.9
3.3
1.9


Rate Constant
-M


Age
(months)

3-4
10
20-22

3-4
10
20-22


7.9
6.4*
3.3


1.1
1.0
1.0

1.8
1.3
4.4


28.1
24.0
21.7

50.0
33.8
22.9

10.2
11.2
7.7

18.8
14.4
10.6

18.9
10.2
9.6

11.0
7.4
6.9

37.0
29.2
29.0

29.8
18.0
12.2


2.1
0.8
0.8

2.5
7.9
4.2


3-4
10
20-22

3-4
10
20-22

3-4
10
20-22

3-4
10
20-22

3-4
10
20-22

3-4
10
20-22


0.27
0.39
0.51

0.63
0.38
0.03

0.13
0.53
0.31

0.49
0.86
0.57

0.59
0.00
0.91

0.41
0.16
0.32

0.48
0.58
0.42

0.50
0.26
0.76


Turnover Rate
(ng/mg protein/hr)


7.7
9.4
11.1


31.5 +
12.8
0.6

1.3 +
5.9
2.4


0.08
0.11
0.11

0.13
0.15k
0.17

0.12
0.10
0.14

0.11
0.15
0.18

0.13
0.14*
0.15t

0.19
0.15
0.14

0.13
0.16
0.14

0.14
0.12
0.23


0.6
0.9
0.8*

3.1
1 .7*
0.8*t

0.2
0.4*
0.3*t

0.7
0.8
1.5t


11.1 0.7
0
8.8 0.6*


4.5 +
1.2 +
2.2 +


17.7
16.9
12.3

15.0
4.8
9.3


0.6
0.2*
0.2*

1.1
2.8
1.3

1 .5
0.5*
0.9*t


*p < 0.05


vs 3-4 month old group; t p < 0.05 vs 10 month old group


9.2
12.4
6.1


1 .6*
1.2*
1.1*


4.4*
2.1*
1.7*


NVM









In contrast, no correlation was detected when a similar comparison of DA

concentration and neuronal activities was evaluated (r = 0.05).

Discussion. The results of this study clearly demonstrate that CA

concentrations and neuron activities within microdissected regions along

the preopticotuberal pathway in ovariectomized Long-Evans rats are differ-

entially changed with age. Collectively, this study and our results dis-

cussed above from male Wistar and ovariectomized F344 rats extend and con-

firm earlier reports which examined the effects of age on CA function in

large brain regions from rodents and humans.184-190 Thus, age-associated

changes in central CA function are not uniform in their magnitude or direc-

tion within brain areas. Age-associated alterations are relatively focal-

ized and appear to be maximal within specific nerve terminal fields. The

importance of this study and the previous study in ovariectomized F344 rats

lies in the observation that CA neuron activities are not generally decreased

with age but, rather, are augmented in some brain regions from old compared

to younger animals.

A rather progressive age-related decline in NE concentrations was ob-

served in the eight brain regions examined in the present study. Since NE

neuronal activity (K) decreased significantly in only the NSC and POAs of

middle-aged and old animals, a progressive decline in the activity of NE

neurons cannot account for the loss of stores of this CA. Rather, the age-

related decline of NE concentrations appears to be the primary contributor

to the substantial decline in NE turnover observed in the NSC, NVM, and the

ME. Interestingly, an increase in NE turnover was observed in the POAm

and the NHA in old ovariectomized rats. This increase resulted from enhanced

neuronal activity (K). Thus, the age-related loss of NE stores rather than

a decline in the activity of neurons appears to be the primary contributor









to the decline in NE turnover observed in several hypothalamic regions.

A partial explanation for the divergent effects of age on NE turnover may

be the activity of the NE neurons. An interesting positive correlation

between NE neuronal activity in young animals and the magnitude of the

age-related loss of NE stores was observed. Thus, initial high neuronal

activity was associated with a loss of NE and a decline in NE turnover.

In contrast, initial low NE activity in regions from young animals was

associated with the maintenance of NE concentration and an increase in

NE turnover in corresponding areas from old rats. In view of the evidence

that stimuli which acutely accelerate NE turnover cause moderate depletion

of NE stores,263 it is not unreasonable to suggest that persistent hyper-

activity of NE neurons may contribute to the age-related loss of NE stores.

In this regard, in the F344 rat, the loss of NE stores between middle-aged

and old rats was associated by an increase in NE neuron activity in younger

animals.

The observation that in five of six regions in which DA concentrations

were reduced in old rats, DA turnover rates were also reduced indicates

that, as for NE neurons, reduced DA stores are a primary contributor to

the age-related decline in DA turnover. Neuronal activity of DA was reduced

with age only in the POAs and was significantly increased with age in the

NAc, striatumand the NA. The enhanced neuronal activity in these latter

three DA regions indicates that some DA systems retain into late life the

capacity to respond to DA loss with enhanced activity, a capacity clearly

established for CA neurons in young animals.93 However, for most brain

regions, DA neuronal hyperactivity is not sufficient to compensate for the

dramatic loss of DA stores as this is reflected in a decline of DA turnover.









The roles of CA neurons in the regulation of AP hormone secretion

have been studied extensively. The significant decreases in NE turnover

detected in five of eight regions examined along the preopticotuberal path-

way from old versus young ovariectomized Long-Evans rats support the con-

cept that NE neuronal dysfunction with age is related to impaired LH hyper-

secretion responses in old CE rats previously proposed.113 However, ex-

amination of NE turnover from ovariectomized previously NC middle-aged

rats indicates that a progressive age-related decrease in NE turnover does

not occur in most hypothalamic regions. Whether decreases in NE turnover

detected in some regions from middle-aged ovariectomized rats may be related

to the diminished preovulatory surge of LH reported in these animals152-'55

is unclear. Interestingly, a reduced NE response to stimulatory regimens

of gonadal steroids has been reported in middle-aged animals prior to the

onset of CE.264

Decreased DA concentrations and DA turnover in MBH tissues of old male

and CE rats appear to be associated with hyperprolactinemia.113,145,216

Although DA turnover rates were progressively decreased with age in ovariec-

tomized Long-Evans rats in the ME, serum prolactin levels were not elevated

in old ovariectomized rats.159,'161,175 Thus, TIDA neuron activity, although

reduced in old ovariectomized Long-Evans rats, appears sufficient to prevent

prolactin increases. The results of the present study support the concept

that hyperprolactinemia in the CE rat is the result of chronically increased

estrogen levels.161 In contrast to the preceding similar study in F344 rats,

TIDA neurons terminating in the ME of old Long-Evans rats do not maintain

DA turnover rates in the presence of decreased DA concentrations. Since

hyperprolactinemia has been shown to reduce DA concentrations in MBH regions

of young rats,92 elevated prolactin levels, previously maintained in these









intact CE rats, may have significantly contributed to the decreased DA

concentrations.

Response of LHRH Neurons to Ovariectomy within Microdissected Brain
Regions of Aging Long-Evans Rats
Objectives. Results of several studies have shown that the post-

castration rise in LH and the LH response to stimulatory regimens of

gonadal steroids, both CNS mediated events, are attenuated in old CE

rats.144'149,156,157-159,161-163 Because similar LH secretion patterns

are observed in young and old CE rats after appropriate treatment with

LHRH, a CNS defect appears primarily responsible for dampened LH responses

in these CE rats.127 Results of previous studies which examined LHRH

concentrations in aging rats are equivocal in that increased,232

decreased,220,233,239 or unchanged235 levels were reported in CE old versus

young rats. Although the preceding experiments suggested diminished NE

turnover in several areas of old ovariectomized rats may contribute to im-

paired LH secretion, the extent of decreased NE turnover was similar in some

regions of middle-aged rats which exhibited normal estrous cycles at the

time of ovariectomy. Age-related alterations in LHRH neuronal function

might clarify the locus of impairment leading to the CE state. The pur-

pose of the present study was first to characterize the effects of age on

LHRH concentrations within regions along the preopticotuberal pathway and

second, to examine the ability of these neurons to respond to the stimula-

tory effects of ovariectomy. Because of the lack of methods to more direct-

ly measure LHRH neuron activity, LHRH concentration depletion after ovariec-

tomy was used as an index of LHRH neuron function.
Materials and Methods. After 30 days, during which reproductive cycles

were monitored, NC young (3-4 month old), IC middle-aged (7-8 month old)

and CE old (20-24 month old) Long-Evans rats were selected for study.









Middle-aged rats were classified as IC if they consistently had 3-5 con-

secutive days of cornified vaginal epithelium with two or three days of

leukocytic smears during the evaluation period. A subgroup of rats from

each cohort group was ovariectomized. Rats were killed by decapitation

on the morning of estrus or two weeks following ovariectomy. Another sub-

group of 3-4 month old rats was killed on the morning of diestrus. Brains

were removed and frozen, trunk blood was collected and animals were necrop-

sied as described above. Seven areas along the preopticotuberal pathway

were microdissected, homogenized in 100 pl of 0.1 N hydrochloric acid and

assayed for LHRH content. The intraassay coefficient of variation was

11.5% as determined from 11 replicate standard tubes which displaced about

50% of labelled hormone. Minimum assay sensitivity at which 10% of labelled

hormone was displaced was 3.7 pg. Supernatants from NA and ME tissues were

assayed in duplicate. Data were analyzed for age effects with analysis of

variance followed by Student Neuman-Kuels tests and for response to ovariec-

tomy with t-tests. Probability values of less than 0.05 were considered

statistically significant.

Results. Table XII summarizes the health status of the animals used

in this study. As previously found in Long-Evans rats in our colony, both

body and organ weights tended to increase with age. Organ weights remained

stable or decreased in old animals when expressed as proportions of body

weight. Although the incidence of visible abnormalities increased with

age, no consistent trends in hormone levels were apparent between lesioned

and nonlesioned rats.

The expected regional distribution of LHRH was observed among the

seven microdissected regions,262 as shown in Table XIII. Although LHRH

levels tended to decrease from 13 to 27% in ME tissues from ovariectomized









TABLE XII.


Body and Organ Weights and Health Status of Long-Evans Rats
Employed in Luteinizing Hormone Releasing Hormone Study


Age (months)


Number of Rats


Body Weight 272.5 3.8
(g, B.W.)


Anterior
Pituitary


Weight
mg/100


(mg)
g B.W.


Weight (mg)
mg/100 g B.W.

Weight (g)
mg/100 g B.W.


Weight
mg/100


(mg)
g B.W.


10.4 0.3
3.8 0.1

56.8 1.2
20.9 0.5

1.89 0.03
696.4 13.2

758.8 11.1
279.1 4.1


335.0 7.9


11.8 0.7
3.7 0.2

54.0 1.7
16.2 0.6

2.13 0.07
643.4 18.6

855.5 16.8
255.8 5.6


430.8 12.8


14.3 0.9
3.4 0.3

67.6 3.2
15.8 0.6

3.04 0.08
718.2 27.9

1145.9 23.2
268.3 5.5


Abnormal i ties


Number per group


Pituitary
Lung
Kidney
Liver


20-24


Adrenal


Kidney


Heart









TABLE XIII.


Effect of Age and Ovariectomy on Luteinizing Hormone
Releasing Hormone Concentrations Within Microdissected
Brain Regions of Long-Evans Rats


LHRH (pg/pg protein)
Diestrous


0.82 0.17


0.96 0.24


0.03 0.00


0.40 0.06


Ovariectomized


Area

OVLT



POAs


3-4
7-8
20-24

3-4
7-8
20-24

3-4
7-8
20-24

3-4
7-8
20-24

3-4
7-8
20-24

3-4
7-8
20-24

3-4
7-8
20-24


0.68
1.74
0.62

0.75
0.93
1.22

0.03
0.03
0.05

0.32
0.26
0.38

3.37
1.36
0.86

1.80
1.10
1.97

47.3
64.3
57.3


+ 0.20
0.76
0.72

0.45
0.37
0.35

0.01
0.01
0.01

+ 0.06
0.03
0.06

+ 0.44
0.78
0.54

0.20
+ 0.17
0.77

+ 3.6
7.1
4.4


*significantly different from 3-4 month old groups p < 0.05


Age
(months)


0.88
1.42
1.48

1.36
1.40
1.33

0.07
0.05
0.06

0.62
0.34
0.38

1.08
2.14
2.47

1.12
0.85
2.29

41.1
46.6
43.0


Estrous


POAm


0.11
0.74
0.19

0.30
0.24
0.25

0.00
0.00
0.01

0.05
0.03
0.08

1.38
0.45
0.21

0.42
0.29
0.67

4.7*
5.9
4.8


1.62 + 0.48



47.0 4.8


2.31 0.53









compared to intact rats of each age group, these differences were not sig-

nificant at the 95% confidence level. Only one significant effect of age

was detected in any area of intact or ovariectomized rats examined. Con-

centrations of LHRH in the ME were increased in middle-aged compared to

young estrous rats. No significant age-related changes in LHRH concentra-

tions were observed among brain areas in either intact or ovariectomized

rats.

Discussion. The results of the present study show that LHRH concen-

trations along the preopticotuberal pathway in both intact and ovariecto-

mized Long-Evans rats are not significantly changed during advanced age.

Thus, the severely impaired LH response of CE rats to ovariectomy156'158

and to stimulatory regimens of gonadal steroids161-163 cannot be explained

by inadequate amounts of hypothalamic LHRH. Either impaired LHRH neuro-

secretion and/or altered AP response to LHRH might contribute to dampened

LH secretion response in these animals. This latter possibility was sup-

ported by studies which observed decreased LH response after a single in-

jection127 or during continuous infusion166 of LHRH in CE rats. However,

the similar LH secretion profiles observed in both old CE and young rats

after repeated LHRH administration indicates AP response to LHRH is not

severely altered during advanced age.127

Although the LHRH depletion response to ovariectomy has been used as

an index of neurohormone secretion262 and the present study found no effects

of age on LHRH response to castration, these results must be interpreted

with caution. The primary limitation of this method is that it does not

reflect the mode of LHRH secretion. Since several studies have shown that

the pattern of LHRH stimulation affects the LH secretion response,28'29

diminished LH release observed in old CE rats may result from altered modes









of LHRH secretion. Specifically, either the amplitude or frequency of

LHRH release may be altered in the old animal. Additionally, this method

cannot distinguish between altered synthesis and release rates of LHRH.

Depletion response measured 2-3 weeks after castration suggests that syn-

thesis of the peptide is insufficient to compensate for increased release

rates in the young animal.262 Although the magnitude of LHRH depletion

response was similar in young and old animals in the present study, changes

in the regulatory mechanisms which maintain LHRH synthesis and release

rates could be altered with age. While results of this study clearly show

hypothalamic LHRH concentrations are not altered in old CE rats, further

studies are required to clarify whether LHRH release mechanisms are im-

paired in these animals.

Age-Related Alterations in Luteinizing Hormone and Prolactin Response
to Ovariectomy and a-Methylparatyrosine

Changes with Age in Serum Luteinizing Hormone and Prolactin Levels in
Fischer 344 Rats

Objectives. Basal levels of LH have generally been reported to remain

stable with age in several species.1 '.148'149 In contrast, the response

of LH to stimulatory regimens of gonadal steroid in old rats appears to

depend upon the stage of reproductive senescence. Steroid-induced hyper-

secretion of LH is consistently decreased in old CE rats,156-159 while in

old PP rats it is reported to be normal162 or dampened.161 The castration-

induced hypersecretion of LH is consistently dampened in old rats.144,'156,161163

Thus, the mechanisms regulating LH hypersecretion appear to be affected to

varying extents in the CE and PP old rat.
Increases in serum prolactin levels during advanced age are probably

the best characterized age-associated changes in AP hormones (see 113).

However, the mechanisms responsible for prolactin elevations also appear to









differ with reproductive status of old rats. Ovariectomy decreases pro-

lactin in CE rats to levels similar to young animals,159,161,175 but has

little effect on prolactin levels in old PP rats.161,175

The present studies examined serum LH and prolactin levels and moni-

tored pituitary concentrations of these hormones from F344 rats used in

experiments designed primarily to evaluate CA and LHRH neuronal alterations

with age. Serum hormone levels were evaluated in an attempt to charac-

terize the magnitude of age-associated effects on AP secretion in the ani-

mals employed in these studies. Characterization of these concentrations

also provided a basis for comparison with previously reported results.

Materials and Methods. Prolactin and LH concentrations were evaluated

by standard RIA methods in sera and AP tissues from F344 rats employed in

the previous studies of CA metabolism in ovariectomized rats and LHRH con-

centration response to ovariectomy.

Results. Serum LH concentrations measured in each of three age groups

of ovariectomized F344 rats killed 0, 45 and 90 min after treatment with

acMPT are shown in Figure 3. There were no differences in LH concentrations

between age groups in saline treated rats (0 min after aMPT). Levels of

LH tended to increase in both young and middle aged rats after drug treat-

ment, but no significant differences were detected between saline and aMPT

treated rats of these two age groups. In marked contrast, 21-22 month old

rats had significantly decreased LH levels after drug administration.

These decreases were significant compared to both younger groups of drug

treated animals and old saline treated rats.

Serum prolactin levels were elevated about four-fold in old compared

to younger saline treated ovariectomized rats as shown in Figure 4. Animals

in all age groups responded to aMPT with increased serum prolactin






































4-5 mo


Omin aMPT
45 min aMPT
90min aMPT


9-10 mo


* p<.05 vs 4-5 mo
t p<.01 vs Omin










21-22 mo


Age-Related Changes in Serum Luteinizing Hormone (LH) Con-
centrations Aftera-Methylparatyrosine (aMPT) in Ovariecto-
mized Fischer 344 Rats. Columns represent mean hormone
concentrations of 9-10 animals at 0, 45 or 90 min after
aMPT (250 mg/Kg, ip) while bars represent SEM.


400-


300-


200-


Figure 3.






















600-


500-


400-


300.


200-


0 min CMPT
45 min QMPT
90 min aMPT


* p<.01vs 4-5 mo
t p<.01vs 45min


*w





t


t t


4-5mo


9-10 mo


21-22 mo


Age-Related Changes in Serum Prolactin (PRL) Concentrations
After a-Methylparatyrosine (aMPT) in Ovariectomized Fischer
344 Rats. See Figure 3 for further explanation.


Figure 4.









concentrations; however, the hormone response to drug treatment was delayed

in 21-22 month old rats.

The effects of ovariectomy in the three cohort groups of F344 rats

studied on serum LH and prolactin levels are shown in Tables XIV and XV,

respectively. Although basal LH concentrations in intact animals tended

to increase in middle-aged compared to young diestrous and old PP rats,

these differences were not significant. Values for intact 4-5 and 22-23

month old animals were often below sensitivity limits of the assay. Con-

sistent with the data on Figure 3, no significant differences between age

groups were detected in ovariectomized rats. Prolactin levels were sig-

nificantly increased by more than three-fold in all PP rats compared to

younger diestrous animals (Table XV). Serum prolactin levels did not de-

crease in response to ovariectomy in old rats, but were decreasedin younger

ovariectomized rats compared to their diestrous cohorts.

Discussion. The results of these studies clearly show that the F344

rat maintains a normal postcastration LH response to ovariectomy through

advanced age. Together with the results of the preceding study which showed

these animals maintain normal LHRH concentrations and depletion response to

ovariectomy, these data indicate that LHRH neurons are able to function in

a relatively normal manner. This conclusion is supported by the unimpaired

LH response to stimulatory regimens of gonadal steroids previously reported

in old PP rats.162 Although LHRH depletion response to ovariectomy has been

shown in the MBH of PP Sprague-Dawley rats,234 a diminished LH postcastra-

tion response has been described in Long-Evans, Wistar and Sprague-Dawley

strains of rats following establishment of the PP state.'61'179'234 These

apparent discrepancies in LH secretion response between PP rats may possibly

be attributed to the strain of rat or the possible presence of the CE state









TABLE XIV.


Effects of Ovariectomy on Serum Luteinizing Hormone Concen-
trations in Aging Fischer 344 Rats


Serum LH (ng/ml)
Age Diestrous Ovariectomized % Increase

4-5 months 21.8 6.7(10)a 370.8 63.2(11) 1600

10-11 months 55.5 16.3(10) 340.5 50.0(10) 514

22-23 months 8.1 1.8(10)*b 244.3 32.5(10) 2916


aMean SEM (no./group); bsix of 10 animals < detectable at the
volume used. These six animals were therefore assigned values
the lower limits of sensitivity of the LH assay at the volumes
(200 vl serum); *p < 0.05 versus 10-11 months.


serum
of 5 ng/ml,
employed




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