CENTRES. MECHAMSMS {N'WLVEB IN RELEASE
€35 PBGLACT’EH.. ANE' CGHECWERGRE EN
RESPONSE TO RESTBAENT STRESS.

T319843 {our fine Degree of M. S.
MICBEGAN STATE UNIVERSE”

Mary S. Vomachka
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ABSTRACT

CENTRAL MECHANISMS INVOLVED IN
RELEASE or PROLACTIN AND CORTICOSTERONE
IN RESPONSE TO RESTRAINT STRESS

BY

Mary S. Vomachka

The purpose of these experiments was to investigate the mechanisms
controlling the stress—induced rise in serum prolactin and plasma corti-
costerone.

l. The first study examined the relationship between time and the
release of prolactin and corticosterone after 3—minute supine immobiliza-
tion stress in male rats. Serum prolactin and plasma corticosterone con-
centrations were observed before and O, 5, 10, 15, 30, and 60 minutes
after this stress. Prolactin rose rapidly after immobilization stress
and reached a maximum level by 5 minutes of the onset of stress. By con—
trast, the concentration of plasma corticosterone rose gradually in re—
sponse to restraint stress and did not achieve a maximum until 15 minutes
after the initiation of stress. The elevation in the levels of these hor-
mones after acute stress was only transient. The levels of both prolactin
and corticosterone declined shortly after their initial rise and returned
to pre-stress values within 60 minutes after stress. The hypothalamus is
believed to be the site where prolactin and corticosterone release in re—

Sponse to stress is regulated.

2. The second study indicated that central monoamines were intimately

dary S. Vomachka

involved in regulating prolactin and corticosterone release in response

to restraint stress. Pre-treatment of male rats with drugs, which alter-
ed the levels of dopamine, norepinephrine, serotonin, and acetylcholine,
produced marked changes in the pattern of prolactin and corticosterone
release observed before and 15, 30, and 60 minutes after immobilization
stress. Alteration in amine activity changed peak levels, the time of
the peak, and the rate of decline of these hormones after stress. In
addition, the reaction of each hormone to these drugs was different. En-
hancement of catecholamine activity by l—dopa lowered the maximum stress-
induced increases of both prolactin and corticosterone. Depletion of
catecholamine levels by a-methyl dopa, however, elevated the resting level
of prolactin but inhibited the release of prolactin after the application
of restraint stress. By contrast, a—methyl dopa did not increase the non-
stress level of corticosterone but delayed the peak rise of corticosterone
consequent to restraint stress.

When both catecholamine and serotonin activities were increased by
the MAO inhibitor, iproniazid, this potentiated the release of corticost-
erone in reSponse to stress but had the Opposite effect on prolactin re-
lease. Specific depletion of serotonin stores by parachloroamphetamine
partially inhibited the stress-induced rise of prolactin 15 minutes after
stress. However, this drug did not adversely affect the rise of corti-
costerone in reSponse to stress but did accelerate the rate at which this
hormone returned to pre—stress levels.

The cholinomimetic agent, pilocarpine, not only elevated the non‘stress
level of corticosterone, but also potentiated its release in response to

immobilization stress. Both pilocarpine and its antagonist, atropine

Mary S. Vomachka

sulfate, inhibited the stress—induced rise of prolactin. Atropine sul-
fate, on the other hand, did not adversely affect the response of corti-
costerone to stress.

These findings suggest that the neurotransmitters, norepinephrine and
serotonin may play a role in mediating the response of prolactin and
corticosterone to acute stress. What appears to be important in the
stress-induced secretion of these hormones is not the action of one mono-
amine but an interaction between norepinephrine, dopamine, and serotonin,
all of which are increased during acute stress.

3. The possible interaction of corticosterone and prolactin in re-
sponse to restraint stress was examined in the third study. A single in-
jection of either corticosterone or hydrocortisone acetate reduced the
basal secretion of prolactin in male rats, but did not adversely affect
the pattern of prolactin release 15, 30, and 60 minutes after immobiliza-
tion stress. In contrast, adrenalectomy did not affect the resting level
of prolactin secretion, but did result in an increased release in response
to restraint stress over that observed in sham—Operated controls and in
adrenalectomized animals given corticosteroid replacement treatment. It
was demonstrated that the effect of either removal or administration of
glucocorticoids on prolactin release is mediated by the hypothalamus, since
there was no difference in pituitary prolactin content under either con-
dition. Both the non-stress and stress—induced changes in prolactin sec-
retion were releated to alterations in turnover of catecholamines and ser-

otonin after corticosteroid administration and adrenalectomy.

CENTRAL MECHANISMS INVOLVED IN RELEASE OF PROLACTIN AND

CORTICOSTERONE IN RESPONSE TO RESTRAINT STRESS.

By

Mary S. Vomachka

A thesis submitted to
Michigan State University
in partial fulfillment of
the requirements for the
degree of Master of Science,
Department of Physiology,
1974

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kid—b IL

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TABLE OF CONTENTS

Page
INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . 1
MATERIAL AND METHODS. . . . . . . . . . . . . . . . . . . . . . 3

EXPERIMENTAL

I. Relation of time to the secretion of prolactin and
corticosterone in reSponse to restraint stress. . 5

Introduction .

Procedure.

Results. . . . . . . . . . . .
Discussion . . . . . . . . . . . . . . . . . . . l

Ji-‘OOOO‘

II. Relation of biogenic amines to the stress response
of prolactin and corticosterone . . . . . . . . . 21

Introduction . . . . . . . . . . . . . . . . . . 22
Procedure. . . . . . . . . . . . . . . . . . . . 31
Results. . . . . . . . . . . . . . . . . . . . . 32
1) Catecholamine System. . . . . . . . . . 32
2) Serotonergic System . . . . . . . . . . 38
3) Cholinergic System. . . . . . . . . . . 41
Discussion . . . . . . . . . . . . . . . . . . . 46

III. Possible role of corticosterone in the response
of prolactin to stress. . . . . . . . . . . . . . 55

Introduction . . . . . . . . . . . . . . . . . . 56
Procedure. . . . . . . . . . . . . . . . . . . . 58
Results. . . . . . . . . . . . . . . . . . . . . 60
l) Intact Male Rats. . . . . . . . . . . . 60
2) Adrenalectomized Male Rats. . . . . . . 64

Discussion . . . . . . . . . . . . . . . . . . . 71

GENERAL DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . 78

REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . . . 91

iii

Figures

1.

10.

11.

LIST OF FIGURES

Page

Changes in the levels of serum prolactin in male
rats subjected to 3—minute supine immobilization. . 10

Changes in the levels of plasma corticosterone in
male rats subjected to 3-minute supine immobili-
zation. . . . . . . . . . . . . . . . . . . . . . . 13

Effects of drugs which alter catecholamine neuro-
transmission on the stress-induced changes in serum
prolactin of male rats. . . 35

Effects of drugs which alter catecholamine neuro-
transmission on the stress-induced changes in plas-
ma corticosterone of male rats. . . . . . . . . . . 36

Effects of agent which affect serotonergic trans-
mission on the stress-induced changes in serum pro-
lactin of male rats . . . . . . ... . . . . . . . . 39

Effects of agents which affect serotonergic trans—
mission on the stress-induced changes in plasma
corticosterone of male rats . . . . . . . . . . . . 40

Effects of drugs which alter cholinergic activity

on the stress-induced changes in serum prolactin
of male rats. . . . . . . . . . . . . . . . . . . . 42

Effects of drugs which alter cholinergic activity
on the stress-induced changes in plasma corticoster-
one of male rats. . . . . . . . . . . . . . . . . 43

Effect of corticosteroid administration on serum

prolactin before and after 3-minute restraint stress
in male rats. . . . . . . . . . . . . . . . . . . . 62

Effect of adrenalectomy, adrenalectomy plus corti—
costeroid replacement or sham—adrenalectomy on
serum prolactin in response to stress . . . . . . . 66

Hypothetical model for a communication system employ—

ed by the brain that could modify the release of ACTH
and prolactin in response to stress . . . . . ... . 80

iv

Table

k)

(A

LIST OF TABLES

Page
Effect of 3-minute supine immobilization on pit-
uitary prolactin content. . . . . . . . . . . . . . 12
Procedure Table: drugs, doses, and schedule of
administration. . . . . . . . . . . . . . . . . . . 31
Effects of drugs which alter central monoamine
neurotransmission on the stress—induced changes
in serum prolactin of male rats . 33
Effects of drugs which alter central monaomine
neurotransmission on the stress-induced changes
in plasma corticosterone of male rats . . 34

Summary of the effect of drugs on monoamine neuro-
transmission and the secretion of prolactin and
corticosterone in reSponse to restraint stress. . . 45

Effect of corticosteroid administration on serum
prolactin before and after 3—minute restraint stress
in male rats. . . . . . . . . . . . . . . . . . . 61

Effect of corticosteroid administration on pituitary
prolactin content before and after 3-minute restraint

stress. . . . . . . . . . . . . . . . . . . . . . . 63
Effect of adrenalectomy, adrenalectomy plus corti-
costeroid replacement, and sham—adrenalectomy on

serum prolactin in response to stress . . . . . . . 65
Effect of adrenalectomy and adrenalectomy plus
corticosteroid replacement on pituitary prolactin

in response to stress . . . . . . . . . . . . . . . 69

INTRODUCTION

One of the prominent themes of scientific interest for many years
has been how the body participates in what Seyle has termed the ”general
stress reaction”. It has been shown that virtually every organ and
chemical constituent of the body are involved in this reaction. The
nervous and endocrine systems play an important part in maintaining re-
sistance during stress. Both systems help to keep the structure and func-
tion of the body at homeostasis despite exposure to stress producing ag-
ents or stimuli. During stress the release of 4 of the 6 hormones secre-
ted by the anterior pituitary gland may be increased. The concentration
of ACTH and prolactin in the blood rise rapidly after application of such
stressful stimuli as surgical trauma, cold, ether anesthesia, or re-
straint. Both Dunn et al. [1972) and Krulich et a1. (1973) demonstrated
that the levels of plasma LH are increased after application of 2 minute
ether stress. A similar but smaller rise was also observed for FSH. In
addition, the secretion of TSH was shown to be enhanced in response to
ether stress. However, other workers have found that the secretion of
TSH may decline after such stresses as surgical trauma, introduction to
novel stimuli, and injection of saline or nembutal (Duncommun et al., 1966
and Kraicer et al., 1963). The secretion of CH appears to be inhibited by
stress. Stresses such as etherization and cardiac puncture, cold, hypo-
gylcemia, and intense exercise all have been shown to depress the levels

of GH in rats (Schalch et al., 1968 and Collu et al., 1973). What is

significant is that all hormones secreted by the anterior pituitary are
changed simultaneously during the stress raction. Other stimuli may also
induce alteration in the release of all anterior pituitary hormones.

There is evidence that estrOgen, thyroid hormones, suckling, and under-
feeding also can modify the secretion of all anterior pituitary hormones.
Since stress initiates a change in all anterior pituitary hormones simul-
taneously, perhaps there is a common mechanism involved in controlling
their secretion in response to stress. The studies reported here were
designed to determine if there were common factors involved in initiating
the stress-induced rise of ACTH and prolactin, two hormones known to be
elevated in reSponse to acute stress. The first experiment examined the
relationship of time to levels of serum prolactin and plasma corticoster-
one after 3 minutes of restraint stress. The second experiment determined
whether the pattern of release of these hormones after this stress could
be changed by altering the turnover of biogenic amines. The third study
investigated the interaction between adrenal steroid secretion and release

of prolactin in response to stress.

MATERIALS AND METHODS

A pilot study examining the response of serum prolactin and plasma
corticosterone to a variety of non-specific stresses demonstrated the
great sensitivity of both hormones to the influence of environmental stim-
uli. It was found that noise produced by Opening and closing cage doors
and introduction to novel stimuli, such as transferring animals from their
animal quarters to a strange room greatly elevated the level of serum pro-
lactin and plasma corticosterone over control animals (E=13.06, P<.002).
Further, non-handled rats displayed an increased concentration of prolac-
tin and corticosterone over handled rats (Duncan's P<.05). The results
of this study demonstrated the need to avoid non—specific stresses and to
standardize the conditions for each experiment. Therefore, 24 hours be-
fore each experiment, all experimental animals were placed in individual
cages and isolated in a separate animal room, which was not entered for
at least 18 hours before the start of the experiment. Before placing the
animals in separate animal quarters, the animals were kept in groups of
3-5 rats per cage for a 5—9 day period of acclimatization under constant
temperature (25:20C) and controlled lighting (fluorescent illumination
5 A.M.-7 P.M.). Wayne Lab Blox (Allied Mills, Chicago, Illinois) and

tap water were made available ad libitum. Experimental studies were

 

carried out between 9 A.M. and 3 P.M. to avoid the naturnal diurnal rise
in both serum prolactin and plasma corticosterone that occurs in the late

afternoon. All stress and collection procedures were preformed outside

(/1

the animal quarters in a separate laboratory adjacent to the animal quar-
ters. A second pilot study investigating the variation between different
stress and blood collecting technics revealed that 1 minute etherization
and cardiac puncture produced the greatest variation among the different
methods tested. Etherization and orbital sinus puncture plus etherization
and decapitation exhibited less but still a high degree of variation.
Decapitation alone produced the least variation of all methods compared.
For this reason, decapitation was chosen as the blood collecting method
and 3—minute supine immobilization was selected as the stress. The ex—
perimental procedure employed for each study is outlined in each experi-

mental section.

RELATION OF TIME TO THE SECRETION OF PROLACTIN AND

CORTICOSTERONE IN RESPONSE TO RESTRAINT STRESS

INTRODUCTION

Stimulation of the pituitary-adrenal axis by stressful stimuli has
long been recognized. The interaction of this axis with a variety of
stresses has been reviewed by several authors (Ganong, 1963, Mangili et
al., 1966, and Sayers and Sayers, 1947). One criterion for a stressful
stimulus has been a rise in the level of adrenal corticoids.

Although the rapid reaction of ACTH to stress has been well establish-
ed, the quick response of prolactin to stress has only recently been re-
cognized. Nicoll and Meites (1960) were the first to demonstrate that
stress might play a role in the release of prolactin. They found that
five days of continuous exposure to a variety of stresses such as cold,
restraint, starvation, or an injection of formaldehyde, induced lactation.
in estrogen-primed female rats. Since then, several acute stress condi-
tions have been found to stimulate prolactin secretion in a variety of
species. Grosvenor et al. (1965) demonstrated that laparotomy, bleeding,
and cervical stunning depleted pituitary prolactin stores in lactating
rats. Subsequently, Neil (1970) found that these acute stresses were
accompanied by an elevation in plasma prolactin in the rat. Stress in-
duced by prolonged ether inhalation or soon after pentobarbital admin-
istration elevated serum prolactin in both male and female rats (Wuttke
and Meites, 1970, Wakabayshi et al., 1971). In addition, Dunn et a1.
(1972) observed that ether stress markedly increased serum prolactin in

male rats throughout a 24 hour test period. He noted that both ether

stressed and non—stressed rats exhibited a circadian periodicity in pro-
lactin levels. In contrast, stress abolished the rhythmic secretion of
LH and dampened the daily peaks found in non-stress corticosterone secre-
tion. Stress has also been found to be a stimulus for prolactin release
in cows, goats, and humans (Meites and Clemens, 1972). Increased levels
of serum prolactin were observed in cows after 10 mi.utes of noise and
restraint stress (Raud et al., 1971). Both emotional anxiety and surgi-
cal trauma have been shown to promote prolactin release in human patients.
Less traumatic situations, such as intense exercise, also were found to
increase plasma prolactin in normal men and women (Noel et al., 1972).
Since the previous studies did not investigate the relation of
time to the release of both prolactin and corticosterone after acute
stress, the objective of the present study was to examine this relation-
ship. The experiment was designed to test the rapidity and duration of
the release of these hormones in response to 3 minutes of supine immobil-

ization.

PROCEDURE

Animals used in this experiment were male Sprague—Dawley rats (250-
300g) purchased from Spartan Research Animals, Haslett, Michigan. One
hour before the start of the experiment, the rats were given an intraperi-
toneal injection of .5 ml. of phosphate buffer saline in .1% gelatin sus-
pension. Non-stress blood samples were obtained by rapid decapitation
after animals were removed from their animal quarters to an adjacent lab-
oratory (time<20 sec.). Animals were stressed by subjecting them to sup-
pine immobilization in a plastic rat restrainer. following supine immob-
ilization, blood samples were removed at 0, 5, 10, 15, 30, and 60 minutes
by decapitation. Half of the trunk blood was collected in glass tubes
for serum samples to be used for prolactin assays and half in heparin-
ized tubes for plasma to be used for corticosterone assays. At the term-
ination of the experiment, blood samples were centrifuged and serum and
plasma samples were stored at —200C. until assayed. In addition, each
anterior pituitary was removed and weighed following decapitation. At
the end of the experiment the pituitaries were homogenized, diluted, and
stored frozen until assayed. Both serum and pituitary prolactin were
measured by a double antibody radioimmunoassay (Niswender et al., 1969)
and an average of four dilutions was expressed in terms of a purified
rat prolatin reference standard (NIAMD-RAT-PROLACTIN—RPl). Plasma corti-
costerone concentrations were measured by the fluormetric procedure of-

DeMoor and Steeno (1963).

RESULTS

The release of prolactin in response to restraint stress occurs with
great rapidity. Figure 1 shows the changes in the levels of serum pro-
lactin in male rats subjected to 3-minute supine immobilization. Pro-
lactin levels started to rise immediately after stress, although no sig-
nificant difference was found between levels at time 0 and non-stress
levels (P>.OS). When the concentration of serum prolactin was compared
to both non-stress and stress levels at the other time intervals, a max-
imum level of serum prolactin was reached between 5-10 minutes after the
beginning of stress (Duncan's P<.05). However, this may not represent
the actual peak. Terkel et al. (1972) noted that 6 minute ether stress
enhanced prolactin secretion in non-suckling lactating female rats. Pro-
lactin levels started to rise between 1-2 minutes and reached a maximum
3—4 minutes after exposure to ether anesthesia. The increased levels of
prolactin release produced by restraint stress in the present study were
not sustained. Prolactin levels started to decline 15 minutes after
stress (Duncan's P<.OS), even though at this time the levels were still
statistically greater than non-stress levels (Duncan's P<.05). Prolactin
levels continued to fall at 30 and 60 minutes after stress. At 60 min-
utes, the concentration of serum prolactin was not statistically different
from the non-stress state (P>.OS). These results extend and confirm those
of Krulich et a1. (1973) who noted a consistent "biphasic change” in the

concentration of plasma prolactin in rats decapitated 0, 10, 30, 60, and

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120 minutes after acute stress. The stresses used in their experiment
included 2 minutes of ether inhalation, repeated ether inhalation, or ex-
posure to a novel situation. In their study prolactin levels rose within
10 minutes of application of stress, followed by a decline to slightly be-
low initial levels 2 hours after stress.

The fall in pituitary prolactin content seen in Table 1 appeared to
reflect the rise in serum prolactin following three minute immobilization
stress. However, analysis of variance indicated no significant difference
(P>.025) in the concentration of prolactin when all time periods were ex-
amined. This may be because the amount of prolactin released into the
general circulation in response to acute stress is relatively small com—
pared to the pituitary stores of prolactin.

Figure 2 shows the changes in the levels of plasma corticosterone in
male rats subjected to 3 minutes of supine immobilization. Stimulation
of the adrenals after acute stress is slow relative to prolactin. Dun-
can's New Multiple Range test showed that there was no significant ele-
vation in the levels of plasma corticosterone at either 0, 5, or 10 min-
utes after stress. However, the concentration of corticosterone appeared
to gradually increase following stress until it reached a maximum level
15 minutes after stress. At this time there was a 3-fold increase in
plasma corticosterone levels above non—stress levels (P<.05). Further
analysis revealed there was no significant difference between the levels
of corticosterone at 15 and 30 minutes following restraint stress (P>.05),
even though at 30 minutes the levels appeared to decline. The levels of
plasma corticosterone 60 minutes after stress were not statistically dif-

ferent from non-stress levels (P .03).

Table 1.

Effect of 3-Minute Supine Immobilization Stress on Pituitary Prolactin
Content.

 

Group n Pituitary Prolactin Content
ng./Anterior Pituitary

Non-Stress S 21,509.5t 3079.6

Minutes After Stress

o s 20,499.53 4082
s 6 17,399.0_+_ 1320.8
10 6 18,777.0i 2440
15 6 lS,l82:llS.S
so 6 , 15,214.835401
60 s 15,145.517259

 

* Standard Error of Mean

n represents the number of samples taken

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DISSCUSION

The secretion of prolactin and corticosterone in response to acute
stress appears to display a definite pattern. Prolactin levels rose rap—
idly after 3-minute immobilization stress and achieved a maximum level
within 5 minutes of the onset of stress. By contrast, the concentration
of plasma corticosterone rose gradually in response to this stress and
did not reach a maximum level until 15 minutes after the initiation of
stress. Following this rise, the stress-induced release of each hormone
declined. Both prolactin and corticosterone at 60 minutes after stress
had returned to levels which were not statistically different from pre-
stress values. Activation of the adrenal in response to stress is slow
when compared to the rapid release of ACTH after stress. Hodges (1971)
followed the concentration of ACTH in the blood of rats at various time
intervals after laparotomy under ether anesthesia. He observed a sig-
nificant rise in ACTH by one minute after stress. A maximum level was
reached 2.5 minutes after the beginning of anesthesia. The concentration
of ACTH started to decline 5 minutes after stress and continued to fall
at 10, 20, and 40 minutes after stress. At 40 minutes after stress the
concentration of ACTH was barely detectable. One criticism of the exper-
iment is that the concentration of pituitary ACTH after the application
of stress was not compared to the concentration prior to stress. A low
initial concentration could have resulted in a smaller release of ACTH

in response to stress. It is thus difficult to assess the true

significance of the rapid elevation of serum ACTH after ether anesthesia
and laparotomy. However, the findings of Hodges (1971) demonstrate the
rapidity with which stress promotes the release of ACTH. His study also
shows that the release of ACTH in response to stress exhibits a pattern
similar to that outlined for prolactin release after restraint stress.
When this pattern is examined, there are two main events which must

be explained in terms of existing mechanisms regulating the function of
the anterior pituitary. One event involves the rapid rise of both blood
prolactin and ACTH after the initiation of stress. The rapidity with
which the release of ACTH and prolactin occurs after acute stress is in
agreement with the existence in the hypothalamus of neural or neuro—
humoral mechanisms controlling the function of the pituitary gland. The
hypothalamus appears to be the principal mediator of the stress response.
Disruption of the functional integrity of the hypothalamus by lesions
placed in the medial basal hypothalamus or median eminence, by pituitary
stalk section or by pituitary transplantation has been shown to prevent
stress-induced release of ACTH (Mangili et al., 1966). It seems likely
that the hypothalamic influence on pituitary ACTH secretion in response
to stress is mediated by CRF. Venikos—Danellis (1964) demonstrated in
female rats that ether or ether and surgical stress caused a rapid and
marked increase in CRF activity in the median eminence approximately one
minute after stress. The enhanced CRF activity one minute after stress
correlates with the elevated levels of ACTH found by Hodges (1971) 2.5
minutes after laparotomy under ether anesthesia.

The influence of the hypothalamus on prolactin secretion is different

‘16

from ACTH The predominant action of the hypothalamus on prolactin secre-
tion is inhibitory. Suppression or removal of this tonic inhibition by
either disruption of the hypothalamo-pituitary connection or by appropri-
ate drug administration has been shown to increase the release of prolac-
tin from the anterior pituitary (Meites et al., 1972). These.findings
have led some workers to interpret the rapid rise of prolactin following
stress as a result of acute inhibition of PIF release (Wakabayshi et al.,
1971, Meites et al., 1972). In addition to a PIF, recent evidence sug—
gests that prolactin secretion may also be influenced by a releasing fac—
tor (PRF) (Meites and Clemens, 1972). Valverde et a1. (1973) found that
ether stress further elevated the levels of prolactin in rats pre-treated
with reserpine (500ug/100g). Since reserpine has been shown to lower PIF
activity and enhance prolactin release (Meites, 1970), they proposed that
:flmastress augmented rise in plasma prolactin seen in reserpine-treated
animals was due to stimulation of PRF rather than to acute inhibition of
PIF. Whether acute suppression of PIF or stimulation of PRF is the mech-
‘anism governing the prolactin response to stress is not known. Only by
directly measuring the respective PIF and PRF activities after stress

can this question be answered.

Since catecholamines (dOpamine and norepinephrine) and serotonin have
been shown to have a profound influence on the release of the hypothala-
mic hypophysiotropic hormones and the anterior pituitary hormones, it is
relevant to determine how stress affects these neurotransmitters. A rap-
id elevation in the levels of these 3 amines has generally been observed

within only a few minutes of the inception of stress (Kato et al., 1967,

l7

Breitner et al., 1963). Welch and Welch (1968a) also found that re-
straint stress produced a marked increase in the levels of norepineph-
rine, dopamine, and serotonin in various parts of the mouse brain with—
in one to five minutes. The rise in monoamines during stress appears to
proceed and parallel the maximal stress—induced release of ACTH at 2.5
minutes and prolactin at 5 minutes. No direct correlation has been made
between the changes in brain monoamines and the release of the anterior
pituitary hormones after a stressful stimulus. However, from this in-
direct evidence it would appear that changes in brain monoamines in re-
Sponse to stress might play a role in mediating the stress—induced re-
lease of ACTH and prolactin.

The second event is the decline of ACTH and prolactin following their
stress—induced rise. One possible explanation for this event involves
“the changes in brain monoamines following acute stress. The effects of
physical stress on brain catecholamines and serotonin are "biphasic".

The initial tendency at the onset of stress is for the levels of these
amines to be elevated. Bliss et al. (1971) found that the levels of homo-
vanillic acid, the principal dopamine metabolite, increased in the brains
of mice during electric foot shock. At the termination of one hour of
electric foot shock the levels of homovanillic acid remained elevated for
30 minutes and then began to return to normal levels. Depending upon the
type, intensity, and duration of the stress, the levels of brain amines
may be decreased. The concentration of brain norepinephrine and dopamine
was shown to decline in mice as a result of intense fighting (Welch and

Welch, 1969) and in rats by electric foot shock (Bliss et al., 1968,

l8

/

Thierry et al., 1968). Whereas low frequency stimulation of the mid—
brain raphe with implanted electrodes tended to elevate the levels of
serotonin, stimulation at higher frequencies lowered it (Sheard and
Aghajanian, 1968). Brain norepinephrine also has been reported to de-
crease as a result of intense exercise for one hour (Gordon et al., 1966
and four and eight hours of restraint stress (Corrodi et al., 1968).
Thus, it would appear that the utilization of catecholamines and sero—
tonin in response to stress follows a pattern similar to that found for
the release of ACTH and prolactin. The evidence indicates that both
brain monoamines and the anterior pituitary hormones ACTH and prolactin
rise after the inception of acute stress. This stress-induced rise is
then followed by a decline in brain amines and hormones to normal levels
or lower than normal levels depending upon the type, intensity, or dura-
‘Cion of the stress. The rise and subsequent fall of brain monoamines
after stress therefore, might explain the increase and decline or pro-
lactin and ACTH levels after acute stress. However, it has been reported
that hypothalamic catecholamines inhibit both ACTH (Canong, 1971) and
prolactin (Meites et al., 1972). Therefore, the relation of hypothalamic
biogenic amines to stress-induced increases in blood prolactin and ACTH
is not entirely clear.

Another possible explanation for the decline of ACTH and prolactin
may involve the ”short loop” feedback mechanism. It has been found that
the hypophysiotrophic area of the hypothalamus is sensitive to feedback
from anterior pituitary hormones. Thus, these hormones may influence

their own secretion through what has been termed the ”short loop"

l9

feedback mechanism. Prolactin implants in the median eminence region
resulted in decreased levels of serum and pituitary prolactin in intact

and ovariectomized female rats. The reduction in prolactin concentration
was linked to an increase in hypothalamic PIP activity (Clemens and Meites,
1968). A similar autoregulatory mechanism exists for ACTH (Mangili et al.,
1966). ACTH or anterior pituitary tissue implanted into the hypophysio-
tropic area inhibited ACTH release (Halasz and Szentagothai, 1960).
Therefore, the decline in ACTH and prolactin seen following a stress pro-
moted increase of these hormones might be the result of feedback inhibi-
tion. Both ACTH and prolactin released after stress might possibly feed
back on neurons in the hypophysiotropic area of the hypothalamus to sup-
press the release of CRF or enhance the release of PIF. Consequent to

the action of these hormones on their respective hypophysiotropic factors,
the secretion of ACTH and prolactin would be reduced. This mechanism, how-
ever, does not appear to be important since prolactin release is maintain-
ed as long as a stressful stimulus is continued.

A third possible explanation involves the role of adrenal corticoids
in suppressing the secretion of ACTH by feedback inhibition. Corticoids
implanted into the medial basal hypothalamus resulted in a decrease in
hypothalamic CRF and pituitary ACTH content (Chowers et al., 1967). In-
hibition of ACTH secretion has also been reported following injection or
implantation of corticoids into other brain areas, such as the midbrain,
septal region, and amygdaloid nuclei. In addition, the pituitary he
also been established as another probable site of feedback inhibition of

ACTH by adrenal corticoids (Canong, 1970). Thus, corticosterone, which

20

has also been shown to rise in response to restraint stress, may be in-
volved in suppressing ACTH secretion. This feedback inhibition of ACTH
by adrenal corticoids does not appear to operate under acute stress con-
ditions, since the concentration of ACTH starts to fall before the levels

of plasma corticosterone has increased significantly.

RELATION OF BIOCENIC AMINES TO THE STRESS RESPONSE

OF PROLACTIN AND CORTICOSTERONE

INTRODUCTION

Since several pharmacological agents, which either mimic central mono-
amine neurotransmission or interfere with synaptic transmission, have been
shown to enhance both ACTH and prolactin secretion, a question arises as
to whether both hormones are controlled by the same monoamine regulatory
mechanism. The major emphasis of existing evidence has suggested a stim—
ulatory role for the neurotransmitter, serotonin, in the release of both
prolactin and ACTH. A single injection of serotonin into the third ven-
tricle of male rats produced a marked increase in serum prolactin levels
(Kamberi et al., 1971a). Although systemic administration of serotonin
has no effect on serum prolactin concentrations, an injection of 5-hydroxy-
tryptophan (5-HTP), the immediate precursor of serotonin, did prove stim-
ulatory to the release of prolactin in both proestrous female rats and
hypOphysectomized rats with an anterior pituitary graft (Lu et al., 1970).
5—HTP also has been found to be capable of stimulating the pituitary-ad-
renal axis (Fiore-Donati et al., 1959). Oral administration of 5-HTP (150
mg) resulted in increased levels of both ACTH and cortisol in human male
subjects (Imura et al., 1973). Direct application of serotonin to the
median eminence or different areas of the hypothalamus, midbrain, and fore-
brain has produced significant elevations in the tonic secretion of corti-
costeroids (Krieger et al., 1970, Naumenko et al., 1968). It has also
been shown that serotonin may play a role in mediating the circadian per-

iodicity of the pituitary—adrenal axis (Krieger et al., 1969). Scapagnini

. PO
Ix)

et al. (1971) observed that there was a direct correlation between the
variations in serotonin content of the amygdala and hippocampus and plas-
ma corticosterone levels. Both values were low at 8 A.M. and high at 8
P.M. Roch et al. (1971) and Dunn et al. (1972) independently showed that
the concentration of prolactin is higher during the late afternoon than in
the morning. Thus, serotoneric neurons may play a part in mediating the
diurnal changes in both prolactin and ACTH levels. The important role
played by the serotonergic system in ACTH regulation has been indicated by
the findings of Naumenko et a1. (1968) and Popova et a1 (1972). Inter-
ruption of neural afferents to the hypothalamus either by midbrain trans-
section in guinea pigs or complete deafferentation of the medial basal
hypothalamus in rats did not prevent the stimulation of the pituitary-ad-
renal axis by 5-HTP or serotonin administration. Direct action of S-HTP
on the adrenal cortex was ruled out by finding that hypophysectomy compl-
etely abolished the stimulatory effect of S-HTP on corticosterone secre-
tion. In contrast, while regional injections of carbacol or norepinephrine
into the hypothalamus or midbrain significantly.increased the levels of
plasma corticosteroids in intact guinea pigs, both of these compounds were
ineffective in midbrain transected animals. These results strongly sug-
gest that the terminal neurons in the hypophysiotrophic area of the hypo-
thalamus stimulating the release of CRF are serotonergic in nature.

The main difference between the regulation of ACTH and prolactin re-
lease is that prolactin secretion is controlled principally by an inhibi-
tory factor and perhaps a releasing factor (Meites et al., 1972). ACTH

secretion is governed mainly by a releasing factor (CRF) (Mangili et al.,

’

1966). From the evidence presented it appears that serotonin overcomes
the dominant inhibitory influence of the hypothalamus on prolactin secre-
tion by either suppression of PIF or release of PRF, although neither hypo-
thesis has been proven conclusively. CRF, on the other hand appears to
be unique among the hypothalamic hypophysiotrOphic hormones in that sero-
tonin has been shown to be stimulatory to its release. In contrast, this
neurotransmitter has been suggested to inhibitory to the releasing fac-
tors controlling gonadotrophin secretion (Kamberi et al., 1979 and 1971a).
The dominant inhibitory influence of the hypothalamus on prolactin
secretion appears to be maintained by dopaminergic neurons present in the
hypothalamus and median eminence. The correlation made between the dop-
aminergic system and inhibition of prolactin secretion is based upon sev-
eral lines of evidence. CA single injection of dopamine into the third
ventricle was reported by Kamberi et al. (l971b)to depress the concentra-
tion of serum prolactin in male rats. This decrease in serum prolactin
levels was accompanied by an increase in PIF levels in pituitary stalk por-
tal blood. Increasing the concentrations of catecholamines, particularly
dopamine, either by enhanced synthesis or reduced metabolism has resulted
in the inhibition of prolactin release. L-dopa, the immediate precursor
of depamine, was shown to depress serum prolactin and increase hypothala-
mic PIF content in hypophysectomized female rats with an anterior pitui-
tary graft (Lu and Meites, 1972). The monoamine oxidase inhibitors, par-
gyline, iproniazid, and also the catechol-o—methyl transferase inhibitor,
pyrogallol, all caused a reduction in serum prolactin (Lu and Meites, 1971,
Clemens and Meites, 1972). In contrast, pharmacological agents which re—

duce hypothalamic catecholamine activity by interfering with synthesis,

25

storage, or receptor interaction of these amines, have been shown to en-
hance prolactin secretion. Chlorpromazine, pimozide, and halperidol, drugs
which block central dopamine and norepinephrine receptors all have been
shown to stimulate prolactin secretion (Meites and Clemens, 1972). In ad-
dition, reserpine, which decreases monoamine transmission by inhibiting the
uptake and storage mechanism of amine granules, was reported to enhance
prolactin secretion by suppressing PIF (Ratner et al., 1965). Inhibition
of catecholamine biosynthesis by a-methyl-p-tyrosine (AMPT), a—methyl-m-
tyrosine (AMMT), or e-methyl dopa has proved stimulatory to prolactin re-
lease. A single injection of these drugs caused a marked increase in ser-
um prolactin levels within 30 minutes of administration and all resulted
in reduced pituitary prolactin content except AMMT (Lu and Meites, 1970).
From this evidence it would thus appear that dOpaminergic neurons tonical-
1y stimulate PIF secretion and thereby mediate the inhibitory influence of
the hypothalamus on prolactin secretion.

The exact neuroendocrine role of the neurotransmitter, norepinephrine,
on prolactin and ACTH is difficult to define. Meites and Clemens (1972)
found that disulfram, an agent which inhibits norepinephrine biosynthesis,
caused a significant reduction in the level of serum prolactin in ovari-
ectomized rats. Likewise, Donoso et al. (1972) showed that a single in-
jection of DL-DOPS (DL-threo-3,4,hydroxyphenylserine 200mg/kg) which sel—
ectively increases norepinephrine biosynthesis, caused a significant ele-
vation in plasma prolactin in ovariectomized rats. These results suggest
that while norepinephrine probably has no role in controlling tonic prolac-
tin secretion, it may be stimulatory to prolactin release under special

conditions.

26

With regard to ACTH secretion, it has been found that both reserpine
and chlorpromazine are stimulatory to ACTH secretion. Bhattacharya and
Marks (1969) found that a single injection of either agent into female
rats produced a marked rise in plasma and adrenal corticosterone concen-
trations and was accompanied by a dramatic fall in CRF content in the med-
ian eminence. If these effects are related to blockade of central cate—
cholamine neurotransmission, it would mean that norepinephrine or dOpamine
released from their various terminal systems of the brain would act to in~
hibit the secretion of ACTH. This view is also supported by the observa-
tion that an intaventricular injection of l-dopa (20mg), dopamine (4mg),
or l-norepinephrine (5mg) suppressed the 17-hydroxyg1ucocorticoid response
to laporatomy stress in dogs. In addition, an intraventricular injection
of agents which have been shown to release catecholamines, such as tyr-
amine (20mg) and a—ethyltyramine (8mg), reduced the concentration of 17-
hydroxyglucocorticoids after surgical stress (Van Loon et al., 1971).
However, since the doses needed for ACTH inhibition were large relative
to the levels of amines normally present in the hypothalamus, it was sug-
gested that suppression of ACTH release by catecholamines might be second-
ary to vasoconstriction of the portal blood vessels. This possiblity was
ruled out by the finding that an intraventricular injection of angiotensin
II, a proven vasoconstrictive agent, failed to prevent the adrenocortical
response to laparotomy stress (Canong, 1971). In addition, stimulation of
the hypothalamus in animals pretreated with e-ethyltyramine overcame this
agent's inhibition of ACTH secretion (Ganong et al., 1965). Thus, a cate-
cholamine precursor, catecholamines, and two drugs that are capable of re-

leasing catecholamines were shown to inhibit the release of ACTH when

IN
\I

injected into the third ventricle of dogs. Further evidence for a poss-
ible adrenergic inhibition of ACTH secretion is based on experiments using
pharmacological agents which inhibit catecholamine synthesis and release.
Van Leon et a1. (1971) found that either a systemic injection (100mg/kg)
or an intraventricular injection (20mg) of AMPT, which depletes brain nor-
epinephrine and dOpamine, increased plasma corticosterone levels in male
rats. When rats were given l—dopa along with AMPT, the depletion of cate-
cholamines was partially prevented and the mean increase in plasma corti-
costerone was less than in those rats receiving AMPT alone. These authors
also found a negative correlation between plasma corticosterone and the
hypothalamic content of norepinephrine and dopamine after the administra-
tion of AMPT and l-dopa. Vhen brain catecholamines were depleted, there
was a marked increase in plasma corticosterone, but when catecholamine
levels were high, a significant decrease in plasma corticosterone was
found. Intraventricular administration of guanethidine (lmg/kg), which pre-
vents catecholamine release from adrenergic neurons, caused a marked ele-
vation in plasma corticosterone in male rats and was accompanied by a de-
pletion in hypothalamic catecholamines (Scapagnini et al., 1972). Use of
an inhibitor of dopamine—B-hydroxylase, which would prevent the conversion
of depamine to norepinephrine and thereby reduce the levels of brain nor-
epinephrine, suggested that norepinephrine rather than dOpamine might be
the neurotransmitter responsible for inhibition of ACTH secretion. When
the dopamine-B-hydroxylase inhibitor, FLA-63 was administered intraperiton-
eally to male rats, the hypothalamic concentration of norepinephrine de-
clined, while that of dopamine remained unchanged. A marked rise in plas-

ma corticosterone following ELA-63 administration was attributed to this

reduction in brain norepinephrine (Scapagnini et al., 1972). An inhib-
itory role for the norepinephrine terminal system on ACTH secretion is al—
so indicated by results from lesion studies. Fuxe and Hokfelt (1971) re-
ported that 1esioning the ascending norepinephrine pathways produced an
increase in the tonic secretion of corticosterone. All these pharmacolo-
gical experiments would favor the view that increased release of norepi-
nephrine from its various terminals in the brain acts to inhibit ACTH
secretion.

The results obtained with psychoactive drugs have to be interpreted
with caution, particularly since many workers report different results.
Reserpine implants in the median eminence failed to alter ACTH secretion
in response to various stimuli, including reserpine and chlorpromazine
(Smelik, 1967). It has also been found that a stress-induced elevation
in ACTH secretion was not changed by combine treatment with reserpine and
a-methyltyrosine (Carr and Moore, 1968), :hich would block central cate-
cholamine transmission. Furthermore, data recently have been obtained
suggesting that the increase in plasma corticosterone observed after a-
methyltyrosine administration was due to a non-specific stress (Kaplanski
et al., 1972). Nembutal administration 30 minutes prior to e-methyltyro-
sine administration prevented the subsequent rise in plasma corticosterone
found by Scapagnini et a1. (1970) and Van Loon et a1. (1971), even though
catecholamine depletion was still present. In addition, repeated adminis-
tration of 50 mg/kg a-methyltyrosine to male rats resulted in a decrease
in brain catecholamine levels without any effect on ACTH secretion. These
findings question the inhibitory role of norepinephrine neurons on ACTH

secretion and demonstrate that intact function of hypothalamic

29

norepinephrine nerve terminals is not crucial in the regulation of ACTH

In addition to serotonin, dopamine, and norepinephrine, cholinergic
neurons have also been shown to be capable of influencing ACTH and prolac-
tin secretion. Krieger and Krieger (1970) and Endrocrzi et al. (1963) in-
dependently found that cholinergic chemical stimulation of the median em-
inence and different areas of the hypothalamus, midbrain and forebrain re-
sulted in increased ACTH secretion in cats. On the other hand, injection
or implantation of cholinomimetic drugs into other areas of the hypothala-‘
mus, midbrain, or forebrain was found to inhibit pituitary-adrenal activ-
ity. Naumenko et a1. (1968) further demonstrated that carbacol injected
into the hypothalamus of midbrain-transected guinea pigs failed to enhance
or inhibit ACTH secretion. Thus, while cholinergic neurons are capable
of relating information to the neurons controlling the production of CRF,
this system is not essential to the regulation of ACTH secretion. The ef—
fects produced by cholinergic stimulation are probably the consequence of
reflex mechanisms. This means that cholinergic stimulation serves as a
source of efferent nervous impluses and activates the hypothalamic-pitui-
tary—adrenal system through corresponding secondary mechanisms. With re—
gard to prolactin secretion, Meites and Clemens, (1972) found that both
acetylcholine and its antagonist, atropine sulfate, induced lactation in
estrogen-primed rats and rabbits. However, the mechanism by which cholin—
ergic neurons affect PlF and prolactin release has not yet been investi-
gated.

The purpose of the present study was two fold: (l) to correlate the
presumed rise and decline in brain amines in various stresses with the

rise and fall of prolactin and corticosterone levels observed after stress.

SO

(2) to investigate how dopamine, serotonin, norepinephrine, and acetyl-
choline, wnich can influence the secretion of these hormones, might af-
fect their release in response to a stressful stimulus. Experiments were
designed using psychoactive drugs to determine which monoamines are import-
ant in the release of ACTH and prolactin during the stress reaction.

Three minute supine immobilization was chosen as the stress stimulus.

Serum prolactin and plasma corticosterone concentrations were examined

in relation to changes in catecholamines, serotonin, and acetylcholine,

produced by specific drugs at O, 15, 30, and 60 minutes after stress.

PROCEDURE

Animals used in the present study were adult male Sprague-Dawley rats
obtained from Spartan Research Farms, Haslett, Michigan. The same proced-
ures were used as outlined in the previous experiment for stressing, coll—
ecting blood samples, and assaying serum prolactin and plasma corticost-
erone. Drugs were dissolved or suspended in phosphate buffered saline
.1% gelatin and given to groups of 5-6 rats. All drugs were administered
intraperitoneally in .5 cc of the vehicle. The drugs, their doses, and

schedule of administration employed in the present study are listed below

 

 

 

in Table 2.
Table 2.
Schedule of Adminis-
Drugs Doses tration
L-dopa (L-3,4,dihydroxyphenyl- 20 mg/rat 1 hour before stress

alamine) (Hoffmann-LaRoche Inc.)

Iproniazid phosphate 40 mg/rat “
(Hoffmann—LaRoche Inc.)

a-methyldopa (L-3,4,hydroxy- 80 mg/rat "
phenyl-Z—metylalanine (Merk
Sharp and Dohme)

P-Chloroamphetamine-HCl (Com— 0.83 mg/rat daily for 3 days be-

pound 511600) (Regis Chemical total dose 2.5 mg/ fore stress (1 hr.)

Co.) rat

Atropine Sulfate 0.8 mg/rat daily for 2 days be-

(Sigma Chemical Co.) total dose 1.6 mg/ fore stress (1 hr. )
rat

Pilocarpine nitrate (Nutri— 0.25 mg/rat 15 minutes before

tional Biochemical Corp.) stress

 

 

A

 

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LA

 

RESULTS

1) EFFECTS ON CATECHOLAHINE ACTIVITY

The drug d—methyldOpa, was used as an example of a drug causing de-
pletion of brain catcholamine levels through synthesis inhibition. L-dopa
was chosen for the opposite effect, since this drug enhances catecholamine
neurotransmission through increased synthesis. Only the effects produced
by 20 mg. of l-dOpa are presented since further studies using higher doses
of l—dopa did not change the response of prolactin or corticosterone.
Iproniazid was selected as an example of a monoamine oxidase inhibitor.
Use of such an agent would, therefore, enhance catecholamine and serotonin
neurotransmission as a result of inhibition of interneuronal catabolism.

The stress—induced changes in serum prolactin and plasma corticoster—
one of male rats subsequent to treatment with these drugs are shown in
Figures 3 and 4 and in Tables 3 and 4. Analysis of variance indicated a
significant difference in the non-stress levels of prolactin and corticos-
terone among treatment groups (F=11.83, P<.002;,F=4.74, P<.002 for prolac-
tin and corticosterone respectively). Treatment of rats with a-methyldopa
one hour prior to immobilization stress increased the resting levels of
prolactin approximately 50% above the control level. In contrast, the
Same drug produced no significant change in the non—stress concentration
0f plasma corticosterone. Further, iproniazid administration, which fail-
ed to alter the levels of prolactin before stress, tripled the concentra-

tion of plasma corticosterone. Animals treated with I—dopa showed no

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36

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significant change in the non-stress levels of either prolactin or corti-
costerone from control animals.

The previous study showed that 15 minutes post—stress represented the
maximum response of plasma corticosterone to immobilization stress and al-
so a time when prolactin levels were still increased over non-stress lev-
els. Figures 3 and 4 demonstrate how either suppressing or enhancing cate-
cholamine neurotransmission influences the peak response of plasma corti-
costerone and serum prolactin to acute stress. All three drugs, l-dopa,
a-methyldopa, and iproniazid, blocked the elevation in prolactin levels
seen 15 minutes after immobilization stress (Duncan's P<.05). In contrast,
neither a-methyldopa nor iproniazid prevented the peak plasma corticoster-
one response to restraint stress. There was no significant difference
(P>.OS) in the concentration of plasma corticosterone between animals
treated with these drugs and control animals. However, l—dopa did decrease
the maximum levels of plasma corticosterone induced by acute stress (Dun—
can's P<.05).

The previous experiment demonstrated that 30 and 60 minutes post-stress
represented the time interval when the concentration of corticosterone and
prolactin returned to normal. Figures 3 and 4 Show the effect of these
psychoactive drugs upon the rate of decline of these hormones to pre-stress
levels. The concentration of serum prolactin in animals treated with ipron-
iazid and l-dopa was still lower than in control animals at both 30 and 60
minutes after immobilization stress (Duncan's P<.05). In contrast, these
drugs did not cause a significant change (P>.OS) in the levels of corti-
costerone 30 and 60 minutes after stress. However, a-methyldopa enhanced

the concentration of plasma corticosterone 30 minutes after stress (Duncan's

P<.OS), even though the levels of serum prolactin in animals treated with
this agent were not statistically different from controls (P>.OS). At 60
minutes after stress, there was no significant difference (P>.OS) in the‘

concentration of either prolactin or corticosterone between animals treat—

ed with a—methyldopa and control animals.
2) EFFECTS ON SEROTONERGIC ACTIVITY

Figures 5 and 6 reveal the stress promoted changes in serum prolactin
and plasma corticosterone of male rats after treatment with pharmacologi-
cal agents which affect serotonergic neurotransmission. Parachloroamphet-
amine (PCA) was chosen as an example of a drug which selectively reduces
serotonergic activity. This pharmacological agent lowers the concentra-
tion of serotonin in the brain subsequent to inhibition of the rate limi-
ting enzyme of serotonin biosynthesis, tryptophan hydroxylase. The action
of PCA on the secretion of prolactin and corticosterone before and after
stress was compared to that of iproniazid, since this monoamine oxidase
inhibitor increases serotonergic activity in addition to catecholamine
neurotransmission. Duncan's New Multiple Range test indicated that there
was no significant difference (P>.OS) in the resting concentration of
either prolactin or corticosterone between thrice daily PCA (2.5 mg/rat
total dose) treated animals and control animals. However, reduction in
serotonergic activity in animals treated with PCA significantly reduced the
non-stress levels of plasma corticosterone when compared to animals treated
with iproniazid (Duncan's P<.05). In addition, rats which received PCA
exhibited a lower maximal prolactin response to immobilization stress at

15 minutes than did control rats (Duncan's P<.05). However, this

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41

inhibition of peak prolactin levels was not as great as seen with iproni—
azid (Duncan‘s P<.05). In contrast, specific epletion of serotonin stores
by PCA had no effect on the maximum levels of plasma corticosterone ob-
served 15 minutes after restraint stress. In addition, there was no sig-
nificant difference (P>.05) in the concentration of corticosterone 15 min-
utes .fter stress when iproniazid and PCA treatments were compared. The
rate of decline of both serum prolactin and plasma corticosterone to pre-
stress levels was not significantly affected by PCA. At 30 and 60 minutes

after immobilization stress, the concentration of corticosterone and pro-

lactin in animals given PCA was not statistically different from control

anim'ls.
3) EFFECTS ON CHOLINERGIC ACTIVITY

Figures 7 and 8 show the stress—induced changes in serum prolactin
and plasma corticosterone of male rats after treatment with pilocarpine
and atropine sulfate. Pilocarpine was chosen as an example of a choline-
mimetic agent. Atropine sulfate was selected as its antagonist. This ag-
ent blocks the action of acetylcholine at its receptor by competitive in—
hibition. The administration of either pilocarpine or atropine sulfate
to male rats prior to stress produced no significant change (P>.OS) in the
resting concentration of serum prolactin from that found in controls. By
contrast, while the administration of atr0pine sulfate produced no signi-
ficant change (P>.05) in the non-stress concentration of plasma corticosta
erone, treatment of animals with pilocarpine did elevate the resting levels
of corticosterone over control animals (Duncan's P<.05). Further, there

was a significant difference in the levels of plasma corticosterone when

 

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44

pilocarpine treatment was compared to atrOpine sulfate treatment (Duncan's
P<.05). Both pilocarpine and its antagonist, atropine sulfate, prevented
the stress-induced rise of prolactin seen 15 minutes after stress (Dun-
can's P<.05). Atropine sulfate was equally as effective as l-dOpa in sup-
pressing the elevated levels of prolactin observed in response to stress.
In addition, there was no significant difference (P>.OS) between the res-
ponse of atropine sulfate and pilocarpine treated animals 15 minutes after
stress. In contrast, pilocarpine increased the maximum corticosterone
stress-induced response (Duncan's P<.05). Atropine sulfate, however, pro-
duced no significant change (P>.OS) in the concentration of plasma corti-
costerone 15 minutes after stress. At this time the levels of corticost-
erone of pilocarpine treated animals were elevated over those of atropine
sulfate treated animals (Duncan's P<.05). The concentration of serum pro-
lactin in animals given atropine sulfate and pilocarpine was still lower
than controls at 30 minutes after stress (Duncan's P<.05). The levels of
prolactin in atropine sulfate treated rats were also significantly decreas-
ed 60 minutes after stress (Duncan's P<.05). In contrast, there was no
significant difference in the concentration of corticosterone when atro-
pine sulfate treated animals were compared to control animals 30 and 60
minutes after stress (P>.OS). However, pilocarpine did cause a marked
rise in the levels of corticosterone 30 minutes after stress. The levels
of corticosterone in pilocarpine treated animals were significantly greater
than those observed in control animals (Duncan's P<.05) and in atropine

sulfate treated animals (Duncan's P<.05).

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DISCUSSION

It is difficult to define which particular central monoamine neuro-
transmitter is responsible for the rise and fall of prolactin and ACTH
subsequent to a stressful stimulus. Part of the problem lies in our lack
of understanding of the relationship between the liberation of amines at
Specific synapses by nerve impluses and the occurrence of specific post-
synaptic events in the brain. The difficulty is further compounded by
the many uncertainties that remain concerning the action of psychotrophic
drugs on synthesis, release, uptake, and metabolism of monoamines or upon
the interaction of amines with postynaptic receptors. Despite these pro-
blems, the results of this study do demonstrate that a change in the bal-
ance of central monoamine neurotransmission has a profound effect on the
secretion of ACTH and prolactin in response to stress.

An example of the way in which alteration of central amine neurotrans-
mission affects the stress-induced release of prolactin and ACTH is illus-
trated by l-dopa administration. While the administration of l-dopa one
hour prior to stress had no effect on the pre-stress concentration of
either prolactin or corticosterone, this drug did suppress the stress—pro-
moted rise of these hormones. Furthermore, l—dopa appeared to be more in-
hibitory to prolactin secretion after stress than to corticosterone.
There are two possible explanations for the inhibitory effect of l—dopa
on the release of these hormones after stress. Glowinski and Baldessarini

(1966) reported that the administration of l—dopa increased dopamine levels

46

throughout the brain, but had only a small effect on norepinephrine con—
centrations. The correlation between elevation of brain dopamine content
subsequent to l—dopa administration and inhibition of ACTH and prolactin
secretion is in aggreement with the findings of other workers (Canong,

1970 and Meites et al., 1972). Another possible explanation is that the
administration of l-dopa has recently been found to interfere with seroton—
ergic transmission, which is stimulatory to both ACTH and prolactin (Ng et
al., 1970 and 1971). These workers found that l-dopa rapidly reduces cen-
tral serotonin stores from brain slices incubated in vitro. They also
found that l—dopa may enter serotonergic neurons, undergo decarboxylation
to dopamine, and subsequently be liberated in response to electrical stim-
ulation. Therefore, dopamine formed in serotonergic neurons, as a conseq-
uence of l—dopa administration, may act as a false serotonergic transmit-
ter. The suppression of the stress-induced rise of prolactin and corti-
costerone 15 minutes after stress by l-dopa may thus be explained in terms
of enhanced brain dopamine levels or to interference of serotonergic neuro-

transmission subsequent to administration of this drug.

Monoamine oxidase (MAO) inhibitors have effects upon brain amines that
are similar to the effects induced by stress. Bliss et al. (1968) found
that the MAO inhibitor, iproniazid, increased the concentration of cate-
cholamines and serotonin throughout the brain and prevented the depletion
of these amines following electric foot shock stress. Various stressors,
such as restraint, fighting, and d-amphetamine, also may elevate mouse
brain norepinephrine, dopamine, and serotonin, within S-lO minutes (Welch

and helch, 1968b). Likewise, MAO inhibitors may also enhance brain amine

48

levels with great rapidity. Welch and Nelch (1968b) found that brain cate-
cholamines and serotonin were significantly increased within ten minutes
after the administration of pargyline, another monoamine oxidase inhibitor.
Still other workers have found elevated brain monoamine levels when meas-
ured 30 and 60 minutes after pargyline (Spector et al., 1963, Everett and
Wiegand, 1962). These worker also found that the increase of serotonin
concentration was 20% greater than that of norepinephrine. MAO inhibitors
mimic the effects of stress on brain monoamines and prevent their deple-
tion after stress. Therefore, the administration of the MAO inhibitor,
iproniazid, should enhance the response of prolactin and ACTH to stress
and delay the return of these hormones to resting levels. Treatment of
rats with iproniazid one hour before immobilization did appear to potenti-
ate the release of corticosterone and partially delayed the decline of
this hormone to pre-stress levels. However, the exact opposite was found
for prolactin. The administration of iproniazid prevented the stress-in-
duced rise of serum prolactin and accelerated its return to normal levels
30 and 60 minutes after stress. Although MAO inhibitors do elevate the
concentration of brain amines and prevent their depletion after stress,
they also have other effects. MAO inhibitors also have been shown to de-
press the spontaneous release of HS-norepinephrine from sympathetic nerve
endings and to prevent or diminish the release of this amine by catechol-
amine releasing agents such as reserpine, histamine, and nicotine (Bliss
et al., 1968). Therefore, the reduction in prolactin secretion in response
to stress observed after the administration of iproniazid might be the re-

sult of decreased norepinephrine release. If this assumption is correct,

49

it would implicate norepinephrine as playing a part in mediating the re—
lease of prolactin in response to stress.

The results obtained with e-methyldopa administration illustrates the
distinction that must be made between the effects induced by this drug be-
fore stress and after a stressful stimulus. a-methyldopa administered to
male rats one hour prior to stress did elevate the non-stress concentra-
tion of prolactin 50% above that found in controls. However, after immobi-
lization stress, prolactin secretion was not further enhanced by o-methyl-
dOpa treatment rather it declined. Administration of this catecholamine
depleting agent prevented the stress-induced rise in prolactin levels 15
minutes after stress. Prolactin levels remained low 30 minutes after
stress, then started to rise above those found in control animals 60 min-
utes after stress. These results are in partial agreement with those of
other workers (Lu and Meites, 1971), who found that a single injection of
a-methyldopa into female rats increased serum prolactin over pre—treatment
levels within 30, 60, and 120 minutes of injection. However, these workers
did not observe that a-methyldopa decreased prolactin secretion after
stress probably as a result of non-specific stresses inherent in their ex-
perimental design. They used serial bleeding by cardiac puncture under
repeated etherization as their method for determining whether a—methyldopa
enhanced prolactin secretion. Etherization alone has been shown to cause
a 2—4 fold rise in prolactin secretion in l-4 minutes of application (Terkel
et al., 1972). Elevated prolactin levels induced by ether inhalation do
not return to normal until 1-2 hours after stress (Krulich et al., 1973).

Thus, the stress of injection, repeated etherization, and bleeding might

have masked the inhibition of prolactin release by a—methyldopa found
after immobilization stress and decapitation in male rats. e-methyldopa
caused transient reduction in the concentrations of depamine and serotonin
and also induced a prolonged decrease in brain norepinephrine (Glowinski
and Baldessarini, 1966). If suppression of norepinephrine neurotransmiss-
ion proves inhibitory to prolactin release under other specific stressful
conditions, such as exercise, cold, electric foot shock, or d-amphetamine,
it would further implicate norepinephrine as having a stimulatory role in
the release of prolactin during stress.

Although a—methyldopa administration did not alter the pre-stress
levels of plasma corticosterone, this drug did appear to delay the peak
response of this hormone to immobilization stress. These results would
suggest that while catecholamines, particularly norepinephrine, may not.
be an important factor in the regulation of tonic ACTH secretion, they may
be involved in mediating the rise in ACTH during stress. This view is
supported by the results of Lippa et al. (1973). These workers found that
an intraventricular injection of 6-hydroxydopamine, which destroys adren-
ergic neurons, resulted in a significant though transient decrease in the
resting levels of plasma corticosterone. However, chronic depletion of
catecholamines by 6-hydroxydopamine impaired the ability of the pituitary-
adrenal system to respond to a ketamine stressor 28 days after initial'
treatment.

The idea that norepinephrine may participate in the release of ACTH
and prolactin in stress is based on the additional evidence that the syn-

thesis and utilization of norepinephrine is increased after various types

51

of stresses: electric foot shock (Thierry et al., 1968a), cold and exer-
cise (Gordon et al., 1966), and restraint (Corrodi et al., 1967). In ad-
dition, Corrodi et al. (1971) found that minor tranquilizers such as
chlordiazepoxide (Librium) blocked the stress—induced activation of cen—
tral norepinephrine neurons in immobilization stress. These drugs have
also been shown to inhibit ACTH secretion (Gold and Ganong, 1967). It is
not definietly known if the increase in norepinephrine turnover during
various types of stress is related to the increased ACTH and prolactin
secretion after stress. However, the evidence presented would suggest
such a relationship. An experiment to determine if drugs such as chlor-
diazepoxide, which blocks the activation of central norepinephrine neurons,
in immobilization stress would influence the increase in prolactin and
corticosterone secretion found during this type of stress, might answer
this question.

Since serotonin has been shown to be stimulatory to both the release
of ACTH and prolactin, the increase in serotonin turnover found during
various types of stress (Thierry et al., 1968b and Welch and Welch, 1968c)
might be an important factor in the stress-induced rise of ACTH and pro-
lactin. If this neurotransmitter participates in mediating the release of
ACTH and prolactin in response to stress, then depletion of this amine by
parachloroamphetamine (PCA) should prevent the rise in prolactin and corti—
costerone observed after immobilization stress. The administration of PCA
for three days prior to stress did not affect the resting levels of either
prolactin or plasma corticosterone. Likewise, Donso et al. (1972) found

that blockade of serotonin biosynthesis by para-chlorophenylalanine did

_52

pet modify plasma prolactin levels. However, treatment of rats with PCA
did partially inhibit the rise in serum prolactin induced by restraint
stress. In contrast, PCA did not affect the stress-induced rise of plas-
ma corticosterone, but did appear to accelerate the decline of this hor-
mone to pro-stress levels 30 and 60 minutes after stress. Prezoisi et al.
(1968) and De Schaepduver et a1. (1969) also found that depletion of brain
serotonin content subsequent to para-chlorophenylalanine administration
failed to affect the ability of ACTH to be secreted in response to various
stresses and in response to reserpine administration. However, these find-
ings do not exclude a role for serotonin in mediating the increased secre-
tion of ACTH and prolactin in response to stress. Fuller et al. (1973)
found that either a single injection or multiple injections of parachloro-
amphetamine (20.6 mg/kg/rat) resulted in only a 50% reduction in brain ser-
Otonin content. His results and those of others (Deguchi et al., 1972)
suggest that some serotonin neurons in the brain are not susceptible to
depletion by parachloroamphetamine. Since serotonin levels are not totally
depleted after parachloroamphetamine treatment, it is possible that a small
amount of residual or newly formed serotonin could maintain effective act-
ivity in the serotonergic system. This could explain why PCA administra-
tion produced only partial inhibition of the stress-induced rise of pro-
lactin and failed to alter plasma corticosterone levels in response to im-
mobilization stress.

The exact role of acetylcholine during stress is not known. Maynert
and Levi (1963) reported that electric foot shock stress failed to alter

the levels of acetylcholine in rats. However, administration of

pilocarpine and its antagonist, atrOpine sulfate, did modify the release
of ACTH and prolactin in response to immobilization stress. While pilo-
carpine and atropine sulfate administration had no effect on the non-
stress levels of prolactin in the present study, both agents prevented

the rise in prolactin after immobilization stress. The opposite effect
was observed for corticosterone secretion. Pilocarpine not only increased
the resting level of corticosterone secretion, but also potentiated the
release of this hormone in response to stress. Atropine sulfate, on the
other hand, did not impair the ability of the pituitary-adrenal system to
respond to stress. In contrast, Hedge and Smelik (1968) found that im-
plants of atropine sulfate in the hypothalamus did prove inhibitory to the
release of CRF. Since both atrOpine sulfate and pilocarpine have been
shown to principally affect peripheral cholinergic systems (Goodman and
Gillman, 1970), the effect of these agents on the secretion of anterior
pituitary hormones is probably secondary to stimulation of perpherial re-
flex mechanisms. Animals subsequent to pilocarpine administration exhib-
ited sweating, salivation, watering eyes, and diarrhea. Naumenko et al.
(1968) showed that central cholinergic stimulation of ACTH secretion was
secondary to activation of peripheral reflex mechanisms. Whether inhib-
ition of prolactin secretion is also the result of such a mechanism is not
known. Perhaps by implanting more specific centrally acting cholinomime-
tic agents like physostigmine or its antagonist, scopolamine, into the
hypothalamus and other areas of the brain, a clearer picture of the influ-

ence of the cholinergic system on the secretion of prolactin and ACTH

might be obtained.

54

In summary, the results of the present study reveal several points.
First, while pharmacological agents which alter central monoamine neuro-
transmission do not necessarily affect the tonic secretion of ACTH and
prolactin, they do produce marked changes in the secretion pattern of
these hormones in response to a stressful stimulus. Differences were ob-
served between the acute stress response of ACTH and prolactin after treat-
ment with drugs which altered the activity of adrenergic and serotonergic
systems. Second, from the evidence presented, it would also appear that
the secretion of ACTH and prolactin is closely regulated in response to
acute stress. All drugs used in this study altered the individual patterns
of prolactin and corticosterone secretion after initiation of immobiliza-
tion stress. Whereas these agents did not always impair the ability of
ACTH and prolactin to be secreted in response to restraint stress, they
did alter the peak levels, the time of the peak, and the rate of decline
of these hormones after stress. Third, the results of this study also
implicate the neurotransmitters, norepinephrine and serotonin, as being
important in mediating the release of prolactin and ACTH after immobiliza-
tion stress. Furthermore, it would appear that what is important in the
stress-induced secretion of ACTH and prolactin is not the action of one
monoamine, but an interaction between norepinephrine, dopamine, and sero-
tonin, all of which are increased during acute stress. This raises the
interesting question as to how these neurotransmitters interact with hypo—
thalamic neurons that contain CRF and PIF, and thus control the release of

ACTH and prolactin.

POSSIBLE ROLE OF CORTICOSTERONE IN

THE RESPONSE OF PROLACTIN TO STRESS

55

_INTRODUCTION

Many physiological conditions and drugs which elicit ACTH release have
also been shown to influence prolactin secretion. Elevated levels of
ACTH have been reported after adrenalectomy, suckling and various stress-
es (Sydnor et al., 1954, Turner and Bagnara, 1971, and Ganong, 1963). In
addition, the injection of different drugs which alter the balance of
biogenic amines in the brain, such as chlorpromazine and reserpine, have
been shown to produce marked changes in ACTH secretion.

All these factors also affect the release of prolactin from the an-
terior pituitary. It is known that different types of stresses cause
elevated serum prolactin in both man and animals. Fseudopregnancy, a
physiological consequence of increased prolactin levels has been induced
in rats by adrenalectomy, surgical trauma, and also by the administration
of reserpine and chlorpromazine (Swingle et al., 1951a and 1951b, and
Baraclough and Sawyer, 1959). Lactation, another manifestation of ele-
vated prolactin and ACTH levels, may be initiated by acute and chronic
stress, suckling, and injections of morphine and serotonin (Nicoll et al.,
1960 and Meites et al., 1959). Although it appears that many stimuli
which elicit ACTH also cause the release of prolactin, the interrelation
between the pituitary-adrenal axis and prolactin secretion remains unclear.

The objective of the present set of experiments was to study the in-
fluence of adrenal steroids on prolactin secretion in response to re-

straint stress, a condition shown to promote both ACTH and prolactin

57

release. The study was divided into two parts. The first part was de—
signed to assess the effect of glucocorticoid administration on pituitary
synthesis of prolactin and prolactin secretion. Experimental animals
were normal male Sprague-Dawley rats purchased from Spartan Research
Animals, Haslett, Michigan. The second part involves the influence of
adrenalectomy and subsequent replacement with adrenal steroids on the

secretion of prolactin.

PROCEDURE

The effect of glucocorticoid administration and removal of glucocorti-
coids on prolactin secretion was studied before and after restraint stress.
Non—stress blood samples were obtained by rapid decapitation (time < 20
sec.) after animals were removed individually from their animal quarters
to an adjacent laboratory. tress blood samples were taken at 15, 30,
and 60 minutes after 3 minutes of supine immobilization in a rat re-
strainer. Trunk blood was collected, centrifuged, and serum samples were
frozen at -2OOC. until assayed. Pituitaries were removed immediately fol-
lowing decapitation a d at the end of the experiment were weighed, homo-
genized, diluted, and stored frozen until time for assay. Both serum
and pituitary prolactin were measured by a double antibody radioimmuno—
assay (Niswender et al., 1969) and an average of four dilutions of each
sample assayed were expressed in terms of purified rat prolactin referen-
ce standard (NIAMD-RAT-PROLACTIN-API).

All experimental substances used in the study were prepared on the
day of the experiment in a phosphate buffer saline .1% gelatin suspension.
Each animal received .5cc of their reSpective treatment substance intra-
peritoneally 4 hours before the start of the experiment. Intact male rats
used in the first part of the study received a single injection of either
hydrocortisone acetate (lmg/rat) or corticosterone (3mg/rat or lmg/rat).
Control animals received only the vehicle. Animals in the second part

of the study were bilaterally or sham adrenalectomized under ether

58

anesthesia 9 days before the experiment. Adrenalectomized animals were
maintained on 0.9% saline in the interim. Animals in this part of the
study_were divided into the following three groups: sham—operated con-
trols, adrenalectom zed controls, and adrenalectomized animals given a
single injection of either hydrocortisone acetate (lmg/rat) or corticost-
erone (3mg/rat or lmg/rat). These constituted the replacement steroids
for three separate groups of adrenalectomized rats. Sham-Operated and

adrenalectomized controls received phosphate buffered saline .1% gelatin.

RESULTS

1) EFFECT OF CLUCOCORTICOID ADMINISTRATION ON PROLACTIN SECRETION IN

THE INTACT MALE RAT BEFORE AND AFTER STRESS.

The effect of glucocorticoid administration on serum prolactin before
and after 3-minute restraint stress in male rats is summarized in Table 6
and Figure 9. Analysis of variance indicated that there was a significant
difference in the non—stress levels of prolactin between animals treated
with glucocorticoids and control animals (F=8.95, P<.003). Both hydro—
cortisone acetate and corticosterone suppressed the resting levels of pro—
lactin by 50%. Further analysis revealed that the prolactin levels in an-
imals treated with glucocorticoids did not statistically differ from con-
trol animals at 15, 30, or 60 minutes after immobilization stress. Neither
adrenal steroid inhibited the rise in serum prolactin after stress, nor
was the pattern of prolactin release after acute stress changed by the ad-
ministration of corticosteroids.

The pituitary prolactin content of control animals and animals treated
with either hydrocortisone acetate (1 mg/rat) or corticosterone (3 mg/rat),
measured before and after stress is presented in Table 7. Analysis of var-
iance revealed that the pituitary prolactin content in control animals did
not differ statistically from that of treated animals before and 15 minutes
after restraint stress (P>.025). Additional analysis showed that there
was no difference between the levels of pituitary prolactin in the control

and hydrocortisone acetate group at either 30 or 60 minutes after

60

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64

immobilization stress. However, there was a significant difference in pit-
uitary prolactin content between animals receiving 3 mg of corticosterone
and control animals in this time interval (Duncan‘s P<.05). The amount of
pituitary prolactin in these animals was increased over that of animals re-
ceiving hydrocortisone acetate or the vehicle. Several factors could ac-
count for the apparent difference between the corticosterone group and the
other treatment groups. One is that this group demonstrated a large stand-
ard error between samples. The mean of pituitary prolactin in each group
represented an average among the individual animals tested. It was notic-
ed that individual animals showed a variable response to stress. In ad-
dition, the weight and the amount of pituitary prolactin has been seen to
'vary with different shipments of rats and among rats of the same shipment.
Consequently, the group given 1 mg of corticosterone was not included in
the statistical analysis. These results are similar to the findings of
Nicoll and Meites (1965). They observed that a high dose of cortisone or
cortisol over a 10 day period elicited only a slight rise in the pituitary
prolactin content of female rats. They also found that corticosterone

had no effect on the amount of prolactin released from pituitary explants

when cultured in vitro (Nicoll and Meites, 1964).

2) EFFECT OF ADRENALECTOMY, ADRENALECTOMY PLUS CORTICOSTEROID REPLACEMENT,

AND SHAM-ADRENALECTOHY BEFORE AND AFTER STRESS.

The effect of adrenalectomy, adrenalectomy plus corticosteroid replace-
ment, or sham-adrenalectomy on serum prolactin in response to stress is

summarized in Table 8 and Figure 10. Analysis of variance showed that

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63

there was no difference in prolactin levels prior to stress when all five
groups were compared (P>.025). however, there was a significant differ-
ence in the amount of prolactin released among these groups 15, 30, and

60 minutes after restraint stress (F=3.10, F=6.54, F=4.93; P<.OS, P<.OOl,
P<.OOS reSpectively). Further analysis revealed that at all three time
periods following stress, prolactin levels in adrenalectomized animals
were increased over those in sham-operated controls (Duncan's P<.05).
Duncan's New Multiple Range test indicated that serum prolactin in the
adrenalectomized was also increased above those animals receiving replace-
ment therapy 15, 30, and 60 minutes after acute stress (P<.05). However,
there was no difference in prolactin release among sham-operated control
animals and adrenalectomized animals receiving either hydrocortisone acet-
ate or corticosterone. The latter results are similar to the observations
made in the normal rat after the administration of corticosteroids.

The pituitary prolactin content of adrenalectomized rats and adrenal-
ectomized rats receiving either hydrocortisone acetate (1mg) or corticost-
erone (3mg and 1mg) in response to 3 minute immobilization stress is
shown in Table 9. Comparison of the pituitary prolactin levels among
these different treatment groups proved variable, yet there was a consis-
tent trend. There was no statistically significant difference between the
amount of pituitary prolactin of adrenalectomized animals and animals re-
ceiving corticosterone or hydrocortisone acetate as replacement three of
the four time periods analyzed. Analysis of variance revealed that there
was no difference between the levels of pituitary prolactin of all four

gTOUps at either 15 or 30 minutes after stress (P>.025]. However, a

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significant difference did appear between pituitary prolactin content of
adrenalectomized animals and adrenalectomized animals given replacement
treatment before and 60 minutes after restraint (F=4.30, F=S.-9; P<.OS,
P<.OOl respectively). Duncan's New Multiple Range test indicated that
prior to stress there was no significant increase in the level of pitui-
tary prolactin of adrenalectomized rats over adrenalectomized rats given
corticosterone (1mg and 3mg) (P>.OS). There was, however, a significant
difference between adrenalectomized animals and animals receiving hydro-
cortisone acetate replacement (Duncan's P<.05). At 60 minutes after
stress, further analysis showed that there was no difference (P>.OS) in
pituitary prolactin between adrenalectomy and adrenalectomy-hydrocortisone
acetate treatment groups, even though there was a significant difference
when the former group was compared to the adrenalectomy—corticosterone
(1mg and 3mg) treatment groups (Duncan's P<.05). Some of the reasons
which could account for the variation among the treatment groups at the
different times before and after stress include the large standard error
observed between samples, the variable response of individual animals to
stress, and the difference in pituitary weight and prolactin content among
individual animals. Despite this variation, there was no statistically
significant difference in pituitary prolactin between adrenalectomized
animals and animals receiving replacement treatment for the majority of
times analyzed. These results are similar to those obtained by Ben-David
et al. (1970). These workers observed that adrenalectomy alone increased
serum prolactin levels 56% above intact controls, even though removing
the influence of glucocorticoidsadid not significantly affect pituitary

prolactin content.

DISCUSSION

The results of the present study indicate that both the removal or
administration of glucocorticoids greatly influenced prolactin secretion.
Administration of corticosteroids to male rats depressed the resting lev-
el of serum prolactin, but did not adversely affect this hormone's re-
lease in response to a stressful stimulus. In contrast, adrenalectomy
greatly increased prolactin levels following restraint stress over sham-
operated control animals and animals given replacement treatment. The
effect of glucocorticoids or their removal on prolactin release does not
appear to be the result of a direct action on the pituitary, since there
was no change in pituitary prolactin content under either condition. Ra-
ther the influence of the pituitarycadrenal axis on prolactin secretion
seems to be mediated via the hypothalamus. Therefore, the interpretation
of these results lies in the effect adrenalectomy and corticosteroid ad-
ministration may have on the balance of the biogenic amine systems in the
hypothalamus. It has been shown that ch nges in brain monoamine neuro-
transmission produce marked changes in prolactin secretion (Meites et al.,
1972).

Recent reports have presented evidence indicating that norepinephrine
is capable of stimulating prolactin release. Workers using drugs which
selectively alter norepinephrine levels have found significant changes in
prolactin release. DL-DOPS, a drug which promotes norepinephrine syn-

thesis, caused a significant rise in serum prolactin in ovarectomized rats

(Donoso et al., 1971). Heites and Clemens (1972) found similar results

in that di

U‘a

ulfram, a drug which blocks the synthesis of norepinephrine,
led to a significant inhibition of prolactin release. Further, Koch et
al. (1970) found small doses of norepinephrine increased the release of
prolactin from pituitaries in vitro: These results support the idea that
although the main influence of the adrenergic system on prolactin secre-
tion is inhibitory, norepinephrine may stimulate its secretion under cer-
tain conditions. It is thus possible that an increased utilization of
norepinephrine seen after adrenalectomy and stress could result in a fac-
ilitation of prolactin release in adrenalectom'zed animals subjected to
restraint stress. Adrenalectomy has been associated with an increased
synthesis of norepinephrine in brain tissue. Javoy et al. (1968) found
that brain norepinephrine turnover was significantly increased 6 days
after adrenalectomy, even though this increase was not present in animals
adrenalectomized for 2 er 3 days. Similar results were obtained by Fuxe
et al. (1970). These workers also found that an injection of cortisol
(2.5mg/100g) partially blocked the increase in norepinephrine turnover
found after adrenalectomy. Thus, there is an increase in the utilization
of norepinephrine not only after various stresses, but also after adrenal-
ectomy.

It is know that dopamine inhibits prolactin secretion both in vitro
(Mac Leod, 1969 and Birge et al., l9?0) and in vivo and acts partly by
stimulating the release of PIP (Kamberi et al., 1970). Agents such as
iproniazid and pargyline, which inhibit monoamine oxidase activity and

thereby suppress the metabolism of dOpamine, have been shown to elevate

73

PIF and reduce prolactin secretion (Lu and Meites, 1971). It is possible
that a rise in monoamine oxidase activity, which would accelerate the
degradation of dopamine, would lower the level of PIP and increase the
amount of prolactin released in response to stress. Recent experiments
have shown that adrenal steroids play a role in the degradation of cate-
cholamines. monoamine oxidase activity is significantly increased in the

a
heart, brain, vas deferens, and kidney after adrenalectomy (Avakian and
Callingham, 1968, Sampath and Clarke, 197“, and Parvez and Parvez, 1972).
Parvez and Parvez (1973] showed that adrenalectomy produced a marked in-
crease in monoamine oxidase activity in the hypophysis and a less marked
but still significant rise in the hypothalamus. In addition, the inhib-
ition of glucocorticoidgenesis by metopirone, a drug which blocks 118-
hydroxylation, was followed by a significant rise in monoamine oxidase
(MAO) activity in the hypophysis, hypothalamus, and the rest of the brain.
This increase in enzyme activity returned to normal following administra-
tion of hydrocortisone. These observations suggest that the presence of
adrenal steroids might be a rate limiting factor for catecholamine degra-
dation in normal rats. The absence of these hormones removes this limi-
tation, resulting in higher levels of monoamine oxidase. Therefore, an
increase in dopamine metabolism, the consequence of increased monoamine
oxidase activity, would decrease the availability of dopamine, suppress
stimulation of PIF and result in higher levels of prolactin.

It is not known which of these two effects predominates or whether it
is a combination of these alterations in the balance of catecholamine
neurotransmission that is responsible for the rise in prolactin seen after

adrenalectomy and stress. Most likely, it is the combination of these two

74

effects, since a lowering of dopamine and thereby of the inhibitory in—
fluence of PIF would facilitate the action of a stimulatory neurotrans-
mitter such as norepinephrine, which is elevated during adrenalectomy and
stress.

It has also been shown that serotonin or the precursors of serotonin,
tryptOphan or S-hydroxytryptophan elevate serum prolactin levels. There
have been reports on the effects of adrenalectomy and administration of
corticosteroids on the serotonergic system. The evidence is confusing
and it is questionable whether changes in this amine system following ad-
renalectomy could account for the rise in prolactin of adrenalectomized
animals after stress. Reports have shown that adrenalectomy can decrease
(DeMaio, 1959), increase (Pleifer et al., 1963), or leave unchanged
(Garattini et al., 1961) whole brain serotonin content. More recently,
evidence has been presented indicating that serotonin turnover is reduced
following adrenalectomy [Fuxe et al., 1970). However, this decrease in
serotonin turnover varied in intensity depending on which area of the
brain was studied. Thus, the brain stem appeared to have a greater de-
crease than did the telecephalon and diencephalon regions (Azimitia et al.
1970). As was seen in connection with the catecholamine system, administra-
tion of corticosteroids restored serotonin turnover to normal or even
super-normal rates. Thus, alterations in the serotonergic system caused
by the removal of adrenal steroids does not offer an explanation for the
rise in prolactin found following adrenalectomy and stress. If the sero-
tonin system were involved, there would be a decrease in prolactin secretion
after adrenalectomy and stress, since a decrease in the utilization of

serotonin was found following adrenalectomy. It is possible that the

7S

effect of corticosteroid administration on the serotonergic system would
have a greater effect in the normal rat,

ln intact rats administration of corticosteroids appears to produce
changes in the serotonergic system which are dissimilar from those found
in the catecholamine system. Opposing alterations in these two systems
of neurotransmission could affect prolactin secretion in different ways.
The decrease in the resting level of prolactin following corticosteroid
administration in male rats might be explained by the effect that adrenal
steroids have on the degradation of catecholamines. It has been shown
that hydrocortisone inhibits both MAO and CONT activities in vitro and
that this inhibition is dose dependent (Parvez and Parvez, 1972). Thus,
inhibition of these two enzymes, resulting in suppression of catecholamine
degracation, could enhance PIF stimulation through dopamine and thereby
cause a decrease in prolactin secretion. H wever, this inhibition of pro-
lactin secretion was only transitory, since restraint stress was capable
of stimulating prolac in release to levels comparable with those of con-
trol animals. Furthermore, my results and the findings of Krulich et al.
(1973) indicate that after acute stress there is only a transient rise in
serum prolactin fellowed by a gradual decline which continues until the
levels return to normal 60 minutes after the initiation of stress. A pos-
sible explanation for the decline in prolactin seen at 30 minutes after
stress is that adrenal steroids which also rise shortly after the initia-
tion of stress might act indirectly to suppress prolactin secretion. The
lack of inhibition of prolactin found after stress in animals receiving
corticosteroid treatment argues against this explanation.

Reports concerning the effects of corticosteroid treatment on the

(J\

serotonergic system have shown that prolonged treatment with glucocorti-
coids caused an increase in serotonin turnover (Fuxe et al., 1970). In
addition, administration of large doses of corticosterone or ACTH was
found to enhance the conversion of radiolabeled tryptOphan to serotonin

30 minutes after its administration, possibly by stimulating the enzyme
tryptophan-S-hydroxylase (Millard et al., 1972). Thus in contrast, to
the catecholamine system an increase in the utilization of serotonin could
lead to an increase in the level of prolactin since it has been shown that
serotonin stimulates prolactin secretion. This might explain why a small
increase in the level of catecholamines caused by the inhibition of MAO
and COMT was not successful in suppressing the stress-induced release of
prolactin. However, it could also argue against finding a decrease in
the non-stress level of prolactin. Some reasons for this seeming discre-
pance might lie in the different dose levels, duration of treatment, and
experimental measures used by different workers. Thus, different para-
meters would cause different changes in monoamine neurotransmission de-
pending upon the various experimental conditions used.

The consequence of adrenalectomy and corticosteroid administration on
brain monoamine neurotransmission and their subsequent effects on prolac-
tin secretion are both numerous and complex. The presence or absence of
glucocorticoids alters the transport, synthesis, and degradation of cate-
cholamines. In addition, adrenal steroids also effect the rate limiting
enzyme on serotonin biosynthesis. Thus, shifts in the balance of biOgenic
amine neurotransmission brought on by alterations in the concentration of
glucocorticoids have been shown to influence prolactin secretion. However,

more work needs to be done to find out how the interaction between the

77

pituitary-acrenal axis and the biogenic amine systems affect the release
of prolactin. It would be of interest to further determine the physiolo-
gical significance of these interactions in states wnere both prolactin
and ACTH are elevated, such as parturition and lactation. Perhaps by
measuring PIE activity in the hypothalamus after adrenalectomy and corti-
costeroid administration a more definitive answer could be obtained to

these questions.

GENERAL DISCUSSION

Each of the preceding studies demonstrated that central monoamines
were intimately involved in regulating the patterns of ACTH and prolactin
responses to restraint stress. The first study suggested that the rise
and subsequent decline of norepinephrine, dopamine, and serotonin might
be related to the fluctuations in prolactin and corticosterone levels
after stress. The next study showed that modification of central amine
transmission profoundly changed the pattern of hormone release in response
to stress. Alterations in central neurotransmission produced by drugs,
which modified the concentration of catecholamines, serotonin, or acetyl-
choline, changed peak levels, the time of the peak, and the rate of de-
cline of these hormones after stress. The final study indicated that
either the administration or removal of adrenal steroids not only modified
the non-stress levels of prolactin, but also changed the secretion of pro-
lactin in response to restraint stress by altering the turnover of cate—
cholamines and serotonin. The question that remains unanswered is how
central amines are involved in eliciting the release of ACTH and prolactin
in response to stress.

The literature on the regulation of ACTH and prolactin indicated that
there are excitatory and inhibitory amines controlling the secretion of
each hormone. Based upon the results obtained from intraventricular in—
jections of dOpamine, this transmitter has been preposed to be inhibitory

to prolactin release by stimulating PIF (Kamberi et al., 1971b).

78

Alterations in catecholamine activity produced by reserpine, chlorproma-
zine, dOpa, and dopamine-B—hydroxylase inhibitors indicated that norepi-
nephrine may have an inhibitory role in the regulation of ACTH (Ganong,
1971). The neurotransmitter, serotonin, has been shown to be stimulatory
to the release of both prolactin and ACTH (Kamberi et al., 1971a and
Naumenko, 1968). however, all three present studies failed to find a con-
sistent correlation between the prOposed excitatory and inhibitory amines
for ACTH and prolactin and the release of these hormones in response to
stress. The evidence obtained from these studies suggested that the rise
of these hormones subsequent to stress was the result of interaction be-
tween various amine systems and the hypothalamic neurons regulating PIP
and CRF secretion. This raises the question as to how such interactions
might occur.

Figure 11 illustrates a hypothetical model for a communication system
employed by the brain that could modulate the release of prolactin and
ACTH from the anterior pituitary. Various transmitter systems make up
the components of this interneuronal system of communication (refer to l
of the model). A list of these substances would include acetylcholine,
norepinephrine, amino acids, peptides, and other biogenic amines such as
depamine, serotonin, and histamine. Whether the message conveyed by a
transmitter is excitatory or inhibitory depends in part upon the chemical
nature of the transmitter (see Control Boxes). For example, (control box
A,6) dicarboxylic amino acids such as glutamate or aspartate excite, while
monocarboxylic omega amino acids such as glycine and GABA generally inhi-

bit. The transmitters norepinephrine, dOpamine, and serotonin are

80

Figure 11.

Hypothetical model for a communication system in the brain that could
modify the release of ACTH and prolactin in response to restraint stress.

+ excitatory - - - hypothesized
- inhibitory

S—HT=serotonin

DA=d0pamine

NE=norepinephrine
ACh=acetylcholine

PIF=prolactin inhibiting factor
PRF=prolactin releasing factor
CRF=corticotr0phin releasing factor

 

 

 

 

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83

generally capable of being either excitatory or inhibitory (Bloom, 1973).
Discrimination between interneuronal transmitters may occur at synaptic
receptors (control box A, 2; B; C). For example, the terminal system of
sympathetic neurons contain cholinergic receotors which respond to acetyl-
choline in a variety of ways. Acetylcholine can excite rapidly via nico-
tinic receptors, excite more slowly and with longer duration at muscar-
inic receptors, and inhibit slowly and for longer duration at still other
receptors (Smith, 1972). Thus, it becomes apparent that receptors in ad-
dition to differentiating between different types of transmi ters also
have the property of amplifying transmitter messages. Receptors for trans-
mitter substances are an integral part of the synaptic membrane (control
box A,4). Therefore, the coupl ng between the receptor site and its part-
icular transmitter could change the electrophysiological properties of the
synaptic membrane in favor of excitation or inhibition (control box A,7).
Another mechanism whereby transmitter messages may be amp ified invol-
ves the activation of secondary messengers (control box A,3). Recent
evidence reveals that the transmitters dopamine, serotonin, and norepi-
nephrine all can activate adenyl cyclase. For example, application of
norepinephrine or activation of noradrenergic pathways has been shown to
lead to an increased content of cyclic AMP in Purkinje neurons of the
cerebellar cortex (Siggins et al., 1973). In addition, while the mechan-
isms of peptide receptors in the pituitary are still unknown, action in
the pituitary may involve activation of adenyl cyclase (control box C, 1,
2, 3). Borgeat et al. (1972) demonstrated that synthetic luteinizing hor—

mone releasing factor (LRF) stimulated the accumulation of cyclic AMP in

84

the pituitary gland in vitro. Cyclic AMP and its dibutyryl derivative
also have been shown to enhance the release of TSH, CH, ACTH, FSH, and

LH from the pituitary gland (Zor et al., 1972). The activation of adenyl
cyclase would therefore combine the precise stereochemical information of
a transmitter with the unique biochemical consequences which attend ini-
tiation of cyclic AMP synthesis in the postsynaptic cells. Whether acti-
vation of adenyl cyclase reflects special properties of the receptor,
transmitter molecule, or the postsynaptic nucleus is not known. lowever,
mediation of synaptic messages by secondary messengers offers the possibi-
lity for longer terms changes in the neuronal membranes and in carbohydr-
ate and protein metabolism. Thus, activation of secondary messengers may
also represent a type of filtering device that distinguishes between sig-
nificant and nonsignificant messages.

The activation of another group of secondary messengers, prostagland-
ins, (control box A,3&9) by the interaction of norepinephrine at its ef—
fector cell has been shown to inhibit the further release of this trans-
mitter from its terminals (Smith, 1972). Such an action might represent
a participant in the mechanism for interneuronal feedback control.

Another property of the actions of various transmitter substances is
the duration of the rCSponse (control box A, B, C). As can be seen, this
is an integrated function of the chemical nature of the transmitters, re-
ceptors, and activation of secondary messengers. Therefore, as revealed
by electrophysiological and behavioral observations, certain nicotinic
cholinergic actions and amino acis produce effects with rapid onset. Mono-

amines and muscarinic cholinergic actions proceed more slowly and for a

longer duration. By contrast, hypothalamic releasing factors and other
substances with central action act over periods of hours-to—days in dura-
tion.

In summary, the transmitter systems comprising the CNS communication's
network could be represented as control boxes where modulation within
transmitter systems and between different transmitter systems may occur
as functions of the properties discussed.

The interaction between different transmitter systems can take many
forms (2). At the level of the membrane, the reaction between one trans-
mitter and another could be translated into permeability changes for pot-
assium—, sodium—, or chloride- ions that result in electric currents which
either depolarize or hyperpolarize the neuronal membrane. The amino acid,
CABA, has been shown to produce inhibition not only by post-synaptic in-
hibition through hyperpolarization, but also presynaptic inhibition via
a depolarizi g action (Bloom, 1973). Schade and Wilgenburg (1970) found
that iontOphorectic application of acetylcholine and dopamine to two dif-
ferent types of neurons of the snail (Helix ponatia) changed the firing
pattern of these neurons. Whereas application of acetylcholine led to de-
polarization in one type of neuron, its application resulted in hyper-
polarization of the other type of neuron tested. The same observation
was made for dopamine. Thus, interaction between transmitter systems may
be different depending upon which transmitter system is effected. Like-
wise, each transmitter system may employ different means fer generating
inhibition or excitation.

Reaction between transmitter systems may produce longer changes in

86

that the action of different transmitter systems may involve modifications
in enzymatic profiles. Depamine neurons have been shown to have a stimu-
latory influence on norepinephrine neurons. Depletion of depamine at its
synapses resulted in a decreased rate of norepinephrine disappearance as
assessed through the use of dopamine and norepinephrine synthesis inhib-
itors and receptor stimulating agents (Persson and Waldeck, 1970). Fur-
thermore, elevated levels of brain norepinephrine, dopamine, and serotonin
have been found to cause.a significant increase in brain choline acetyl-
ase activity, the enzyme mediating acetylcholine biosynthesis. By con-
trast, reduction of dopamine and norepinephrine induced by 6-OH—d0pamine
resulted in a significant though transient decrease in enzyme activity
(Ho and Loh, 1972). Thus, the biosynthesis of the transmitter acetyl-
choline and cholinergic mechanisms may be modified by catecholamines and
serotonin. Finally, destruction of the medial forebrain bundle has been
shown to produce a marked reduction in the concentration of brain seroto-
nin and norepinephrine secondary to a loss of enzymatic activities essent-
ial for the biosynthesis of these amines (Heller, 1972).

The third part of the model (3) represents an example of how differ-
ent transmitter systems might react with either dopamine or serotonin,
two transmitter systems in the hypothalamus hypothesized to be directly
involved in regulating the secretion of ACTH and prolactin. The compon-
ents that comprise part 4 are the peptide releasing factors which either
elicit or inhibit the release of ACTH and prolactin. This transmitter
system is located in the medial basal hypothalamus and functions similarly

to the other transmitter systems, except that its postsynaptic cell is not

\ Cf)
\]

another neuron but the portal blood vessels and the anterior pituitary.
As other transmitter systems possess multiple receptor sites for various
chemical transmitters, so may this peptide transmitter system. This would
allow this system to respond either to a sum of different transmitter in-
teractions conveyed by a single transmitter or to integrate over multiple
transmitter system input. Thus, the peptide transmitter system is the
final integrator of messages conveyed by other transmitters from various
parts of the CNS, ie. it decides upon what message should be sent to the
pituitary. Part 5 of the model renresents the final output of all the
preceding interactions in terms of hormone release from the anterior pit-
uitary gland.

Let us now consider in detail how interactions between various trans;
mitter systems could modify the release of ACTH and prolactin. Our analy-
sis will be limited to two transmitter systems in the hypothalamus, dopa-
mine and serotonin, and the interaction of each system with other trans-
mitter systems. One assumption will be made for simplication, ie. that
both dopamine and serotonin are excitatory to its respective releasing
factor. Norepinephrine has been shown to be both an excitatory and inhib—
itory transmitter (Bloom, 1973). Thus, inhibition of the serotonin system
could be produced by an inhibitory action of norepinephrine. he result-
ing CECTCQSC in serotonin release from its terminals would be translated
by CRY neurosecretory cells into insufficient membrane permeability changes
to elicit the release of this peptide. Failure of CRF secretion would
ultimately lead to inhibition of ACTH release. he exact opposite would

occur if norepinephrine was stimulatory to the serotonin system. In this

88

case, an excitatory interaction between norepinephrine and the serotonin
system would cause release of CRF and stimulate ACTH secretion. Further
inhibition of the serotonin system could resu t from this system reacting
with an inhibitory dopamine, acetylcholine, or even another serotonin
transmitter molecule. Such an inhibition of serotonin, in turn, would
cause inhibition of CRF release and suppression of ACTH secretion. Excit-
ation of the serotonin system and finally stimulation of ACTH could be
produced by an orcitatory coupling of the serotonin system with an excit-
atory dopamine, acetylcholine, or serotonin molecule. Krieger and Krieger
(1970) found that implantation of various transmitter substances into dif-
ferent areas of the cat brain resulted in a differential plasma cortico-
steroid response. For example, implantation f carbacol into the median
eminence, posterior hypothalamus, and amygdala (anterior and central) re-
sulted in stimulation of corticosteroid release. However, inhibition of
corticosteroid secretion was obtained from lateral amygdalar, hippocampal,
and septal implantations. Similar differential corticosteroid responses
were seen with implantation of norepinephrine, serotonin, and GABA. How-
~ev=r, the pattern of response for each transmitter substance was different.
These findings provide indirect evidence that various transmitter systems
in different areas of the brain do modify the secretion of ACTH possibly

by a mechanism similar to that proposed.

A similar pattern of transmitter interaction might be preposed for the
modification of prolactin secretion. In this case, reactions may occur

for example between depamine and the other transmitter systems. These

C0
l O

interactions would be interpreted further by PIP neurosecretory cells to
either elicit or suppress the secretion of prolactin. Serotonin has been
shown to be stimulatory to prolactin release. One possible explanation
for this finding is the inhibition of the dopamine system via an inhibi-
tory reaction with serotonin. The resulting decrease release of dopamine
from its terminals would suppress the secretion of PIF and allow for stimu-
lation of prolactin release. The transmitter norepinephrine has been
found to both stimulatory and inhibitory to prolactin release. The seem-
inly dual role for norepinephrine might be explained in terms of trans-
mitter systems interaction. The coupling of the dopamine system with an
inhibitory norepinephrine transmitter would result in suppression of dopa-
mine release from its terminals, depression of PIF secretion, and stimu-
lation of prolactin release. The opposite effect would result if an ex-
citatory interaction occurred between the depamine system and norepineph—
rine. Further inhibition of the dopamine system and therefore stimulation
of prolactin secretion could be produced by this system interacting with
an inhibitory acetylcholine or another dopamine system. On the other
hand, excitation of the depamine system and thus inhibition of prolactin
release could also be the result of the depamine system reacting with an
excitatory acetylcholine or serotonin system.

This hypothetical model therefore answers the question as to how in—
teractions between various tansmitter systems might occur. It also offers
a possible explanation whereby the fluctuations in the concentrations of
depamine, norepinephrine, and serotonin could cause the rise and subsequ-

ent decline of prolactin and corticesterone levels in response to

90

restraint stress. Thus, the common factor involved in the release of
ACTH and prolactin and other anterior pituitary hormones in reSponse to

stress is the interaction of various transmitter systems during stress.

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