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an. '—

_ Lfig'flA IIY
Michigan State
Graven“?

FHESlI

 
  

This is to certify that the

dissertation entitled

CHOLINERGIC REGULATION
OF FEMALE SEXUAL BEHAVIOR

presented by
GAR Y PETER D OHAN I CH

has been accepted towards fulfillment
of the requirements for

PhoDo degree in ZOOlogy

 

 

 

/ Major professor

/"
\ 4

Date #5} uéuc/a/i/l, W/g/

 

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

OVERDUE FINES:
25¢ per day per item

RETURNING LIBRARY MATERIALS:

Place in book neturn to remove
charge from circulation records

 

 

CHOLINERGIC REGULATION OF FEMALE SEXUAL BEHAVIOR

By

Gary Peter Dohanich

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology
1981

 

G- I /S‘ ‘75“ 3

 

ABSTRACT

CHOLINERGIC REGULATION OF
FEMALE SEXUAL BEHAVIOR

By

Gary Peter Dohanich

The role of cholinergic activity in the regulation of
hormone-dependent sexual behavior in female rats was
examined. In two experiments, stimulation of cholinergic
receptors in specific areas of the brain facilitated the
occurrence of sexual behavior. Specifically, carbachol, a
cholinergic receptor agonist, increased the incidence of
lordosis in estrogen-primed female rats following bilateral
microinfusion (0.5 ug/side) into the medial preoptic area
or ventromedial hypothalamus. The behavioral response was
normally highest 15 min after carbachol infusion and had
returned to control levels by 90 min. Intracerebral carb-
achol facilitated lordosis at lower levels of estrogen
priming than did systemic progesterone which may indicate
that carbachol and progesterone achieve their effects by
different mechanisms. The ineffectiveness of exogenous
progesterone at these low levels of estrogen priming also
suggests that carbachol infusion did not facilitate lordosis
by inducing the release of adrenal progestins. Carbachol
failed to activate lordosis following infusion into the
mesencephalic reticular formation or frontal cortex. It
appears that the potential of a brain area to respond to

cholinergic stimulation may be related to the sensitivity

 

 

 

 

 

Gary Peter Dohanich
of that area to estrogen.

In two other experiments, an agent which interferes
with central cholinergic activity was found to inhibit
sexual behavior displayed by female rats. Bilateral fore-
brain infusion of hemicholinium-3 (HO-3, 1.25, 5, or 7.5
ug/side), an acetylcholine synthesis blocker, reduced the
incidence of lordosis in females receiving behaviorally-
active combinations of estrogen and progesterone. The
inhibition of lordosis by HC-3 was prevented when choline
chloride (120 ug/side) or carbachol (0.5 ug/side) was
simultaneously infused with HC-3. There was no evidence
of behavioral inhibition following infusion of HC—3 into
the frontal cortex of sexually receptive females.

In the final experiment, the ability of estrogen to
alter in X2332 binding of a radioactive cholinergic mus-
carinic antagonist within certain areas of the brain was
demonstrated. However, these data did not consistently
support behavioral findings since an expected increase in
muscarinic binding was not detected at the estrogen-priming
dose (0.125 ug) utilized in behavioral experiments.
Furthermore, higher doses of estrogen (1 or 10 ug) increased
the number of muscarinic binding sites in the medial basal
hypothalamus but decreased the number of muscarinic bindig
sites in the medial preoptic area.

These experiments suggest that lordosis in female

rats is influenced by an estrogen-dependent cholinergic

 

Gary Peter Dohanich

mechanism within the brain. The facilitation of lordosis by
cholinergic stimulation of specific brain regions does not
appear to be correlated with an estrogen—induced increase

in cholinergic binding as measured by in vitro uptake of a

muscarinic antagonist.

 

 

 

 

 

"I never been nobody's idol

But at least I got a title."

- Merle Haggard

ii

ACKNOWLEDGEMENTS

The contributions of various individuals were indis-
pensable to the completion of this work. The result is
dedicated to those individuals: Dr. Lynwood Clemens for
his insight, enthusiasm, and confidence; Jeffrey Witcher
for his invaluable collaboration; David Brigham for his
capable technical assistance; Cheryl Rapp for her skillful
histology and illustrations; and Jo Ann Dohanich for her
proficient typing and patience.

The advice and guidance of Drs. Edward Convey,

Richard Rech, and John King are gratefully acknowledged.

iii

 

TABLE OF CONTENTS
Page
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . vi
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . vii
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1
GENERAL METHOD (Experiments 1-4) . . . . . . . . . . . 21
EXPERIMENT 1. Facilitation of Lordosis in Hormone-

treated Female Rats Following Intra-
cerebral Infusion of Carbachol . . . . 25

 

MTHOD I I I I I I I I I I I I I I I I I I I I I 26
RESULTS . . . . . . . . . . . . . . . . . . . . . 28
SUMMARY I I I I I I I I I I I I I I I I I I I I I 32

EXPERIMENT 2. Brain Regions Implicated in Cho-
linergic Mediation of Lordosis . . . . 33

METHOD . . . . . . . . . . . . . . . . . . . . . 34
RESULTS I I I I I I I I I I I I I I I I I I I I I 314’
SUNHVIARY I I I I I I I I I I I I I I I I I I I I I 36

EXPERIMENT 3. Inhibition of Lordosis in Hormone-
treated Female Rats Following
Intracerebral Infusion of
Hemicholinium-3 . . . . . . . . . . . . 40

EXPER INENT 38- I I I I I I I I I I I I I I I I I I LPO
METHOD . . . . . . . . . . . . . . . . . . . 40
RESULTS I I I I I I I I I I I I I I I I I I 41

EXPER INENT 3b I I I I I I I I I I I I I I I I I I Li’s
NETH OD I I I I I I I I I I I I I I I I I I I 45

RESULTS I I I I I I I I I I I I I I I I I I 46
EXPER INENT 3 C I I I I I I I I I I I I I I I I I I 48
NETHOD I I I I I I I I I I I I I I O I I I I L"9
RESULTS I I I I I I I I I I I I I I I I I I 50
SUNHVIARY I I I I I I I I I I I I I I I I I I 50

iv

 

Table of Contents cont'd
Page

EXPERIMENT 4. Reversal of Hemicholinium-3 In-
hibition of Lordosis by Carbachol . . . 53

NETHOD e o o e e e e e e o e o o o o o e e e o o 53
RESULTS . . . . . . . . . . . . . . . . . . . . . 55
SUNHVIARY o o e o o e o e o e e o e o e o e e o e o 58

EXPERIMENT 5. Alterations in Cholinergic Binding
Sites within Specific Brain Areas

Following Estrogen Treatment . . . . . 59

NETHOD o e o e e e e e e e e e e o e e e o e o 60
RESULTS 0 e e e o e e e e o o e o e e o 61
SUNHVIARY a o I o 0 o o o o o o o o e e e e e e 0 64
DISCUSSION 0 e e o e e e e e e o o e e e o e o e e o e 68
LIST OF REFERENCES . . . . . . . . . . . . . . . . . . 84

 

 

Table

LIST OF TABLES
Page

Proportion of Female Rats Responding

Following Intracerebral Infusion of Carb—

achol or Artificial Cerebrospinal Fluid

(CSF) Vehicle . . . . . . . . . . . . . . . . 39

Mean Lordosis Quotients Recorded from Female
Rats Following Bilateral Infusion of
Hemicholinium—3 into the Frontal Cortex . . . 52

Musoarinic Binding in the Parietal Cortex
and Caudate Putamen Following Estradiol
Benzoate (EB) Treatment . . . . . . . . . . . 63

 

Vi

LIST OF FIGURES

Figure

1

Distribution of cannula tips as verified histo-
logically in female rats from Experiment 1 . . .

Mean lordosis quotients recorded from female
rats followin bilateral infusion of carb-

achol (0.5 u cannula) or artificial cerebro-
spinal fluid (CSF) vehicle into the medial
preoptic area . . . . . . . . . . . . . . . . . .

Mean lordosis quotients recorded from female
rats following bilateral infusion of carbachol
(0.5 ug/cannula) or artificial cerebrospinal
fluid (CSF) vehicle into various brain areas . .

Carbachol infusion sites in the medial preoptic
area (upper left), ventromedial hypothalamus
(upper right), frontal cortex (lower left), and
mesencephalic reticular formation (lower right) .

Distribution of cannula tips as verified histo-
logically in female rats from Experiments 3 . .

Mean lordosis quotients (LQ) recorded from
female rats following bilateral infusion of
hemicholinium-3 (HG-3, 5 or 7.5 ug/cannula) or
artificial cerebrospinal fluid (CSF) vehicle
into the bed nucleus of the stria terminalis . .

Mean lordosis quotients (LQ) recorded from
female rats following bilateral infusion of
hemicholinium-3 (HG-3, 5 ug/cannula) or
artificial cerebrospinal fluid (CSF) vehicle
into the medial preoptic area . . . . . . . .

Mean lordosis quotients (LQ) recorded from
female rats following bilateral infusion of
hemicholinium—3 (HO-3, 5 ug/cannula) or HC—3

(5 ug/cannula) plus choline chloride (120 ug/
cannula) into the medial preoptic area . . . . .

Distribution of cannula tips as verified histo-
logically in female rats from Experiment 4 . . .

vii

Page

27

30

35

38

43

A4

47

51

54

 

 

List of Figures cont'd
Figure Page

10 Upper panel. Mean lordosis quotients recorded
from female rats following bilateral infusion
of hemicholinium-3 (HO-3, 1.25 ug/cannula) or
HC-3 (1.25 ug/cannula) plus carbachol (0.5 ug/
cannula) into the medial preoptic area. Lower
panel. Linear regression lines of lordosis quo-
tient on log1o transformed EB doses for pretests
and 45 min tests . . . . . . . . . . . . . . . . 57

11 Specific binding of 3H quinuclidinyl benzilate
(QNB) in synaptosomal preparations of tissue
from the medial basal hypothalamus (MBH; left)
and medial preoptic area (POA: right) of
female rats . . . . . . . . . . . . . . . . . . . 62

12 Scatchard plot of specific 3H quinuclidinyl
benzilate (QNB) binding in the medial basal
hypothalamus (MBH; upper) and medial preoptic
area (POA; lower) of female rats treated with
estradiol benzoate (EB, 10 ug) or 0.1 ml sesame
seed oil 72, 48, and 24 hr before sacrifice . . . 66

 

viii

INTRODUCTION

Various behaviors which increase the probability of
successful mating have evolved in vertebrates. Lordosis
is a distinctive posture assumed by females of several
mammalian species during the period of sexual receptivity.
It is characterized by flexion of the epaxial muscles
which causes a ventral arching of the spine and elevation
of the perineum (Pfaff, 1980). In species such as the rat,
in which the male achieves multiple intromissions before
ejaculating, lordosis promotes genital stimulation of both
sexes and facilitates sperm deposition during ejaculation.
The lordosis reSponse is elicited in the female rat by the
mount of a male and is dependent on somatosensory stimu-
lation to the flanks, rump,and tail base (Pfaff, 1980).

Steroid hormones secreted by the ovary are clearly
the principle hormonal factors controlling lordosis
(Young, 1961). Accordingly, removal of the ovaries
abolishes the capacity for sexual behavior in female rats.
Sexual behavior is restored in ovariectomized females by
systemic administration of estrogen (estradiol) followed
by progesterone (Boling and Blandau, 1939). This treatment
regimen mimics the natural hormonal profile of the intact
cyclic female such that maximum receptivity is preceded

by several days of rising estrogen titers followed by a

1

 

 

 

2

brief increase in progesterone levels (Clemens and
Christensen, 1975). However, when given in sufficient
quantities, estradiol alone will activate a high incidence
of lordosis in ovariectomized female rats (Pfaff, 1970).

Although lordosis has been described as a stereotyped
reflex (Pfaff, 1980), the neuroendocrine mechanisms which
regulate the occurrence of this behavior are complex. The
selective uptake of estradiol from the circulation by
specific areas of the brain appears to be prerequisite.
Visualization of tritiated estradiol uptake by autoradio-
graphy has identified the medial preoptic area (POA) and
the ventromedial hypothalamus (VMH) as two forebrain areas
with dense distributions of neurons which concentrate
estradiol (Pfaff and Keiner, 1973). The ability of
preoptic and hypothalamic neurons to take up estradiol
suggests that these particular cells may direct the perfor-
mance of an estrogen-dependent behavior such as lordosis.

Appropriately, results obtained with a variety of
techniques have implicated both the POA and the VMH in the
control of estrogen-activated lordosis. For example,
estradiol benzoate (EB) has been reported to activate
sexual receptivity in ovariectomized rats within several
days after direct implantation in either the POA or the VMH
(Lisk, 1962; Lisk and Barfield, 1975; Yanase and Gorski,
1976; Barfield and Chen, 1977). The VMH appears to be
particularly sensitive to estrogen stimulation since a

higher incidence of lordosis was observed in female rats

 

 

3
following implantation of EB in the VMH than was observed
following implantation of EB in the POA (Barfield and Chen,
1977). Furthermore, dilute estradiol implants confined to
the VMH activated lordosis in female rats when combined
with systemic administration of progesterone (Rubin and
Barfield, 1980). However, this same treatment was ineffec-
tive when dilute estradiol implants were placed in other
brain areas including the POA.

In addition to displaying different sensitivities to
estrogen, evidence suggests that the POA and VMH may exert
different actions in mediating female sexual behavior.
Although lesions of the VMH have been demonstrated to
inhibit lordosis in female rats treated with estrogen
(Mathews and Edwards, 1977), lesions of the POA increased
the potential of estrogen—treated females to display lor-
dosis (Powers and Valenstein, 1972)o Further, electrical
stimulation of the VMH facilitated lordosis (Pfaff and
Sakuma, 1979) while stimulation of the POA has been found
to inhibit lordosis (Malsbury and Pfaff, 1973). Finally,
EB has been shown to increase the number of cells in the
VMH displaying detectable spontaneous activity while
decreasing their number in the POA (Bueno and Pfaff, 1976).
These results seem to suggest that the VMH exerts an
excitatory influence over estrogen—activated lordosis and
the POA exerts a tonic inhibitory influence.

In contrast to the POA and VMH, implantation of BB in

the mesencephalic reticular formation (MRF) failed to

 

 

Ll,
facilitate lordosis in female rats (Yanase and Gorski,
1976). However, combinations of EB implants in the MRF
with systemically injected progesterone or progesterone
implants in the MRF with systemically injected EB activated
a high incidence of lordosis (Ross, Claybaugh, Clemens, and
Gorski, 1971; Yanase and Gorski, 1976). Furthermore,

local exposure to estrogen was necessary for MRF implants
of progesterone to be effective (Yanase and Gorski, 1976).
Although the MRF does not concentrate tritiated estradiol
as proficiently as basal forebrain areas (Pfaff and Keiner,
1973) mesencephalic control of female sexual behavior
nevertheless may be mediated by an interaction between
estrogen and progesterone.

The sequence of events initiated by intracellular
uptake of estradiol and culminating in the lordosis
response is presently speculative. However, evidence
(O'Malley and Means, 1974; Woo and O'Malley, 1975) that
ovarian hormones regulate the synthesis of specific
proteins within the chick oviduct by interaction with the
cellular genome may have important implications for the
hormonal control of sexual behavior. A hormone, such as
estrogen, is believed to diffuse passively into the cyto-
plasm of a target cell from the general circulation and
bind to high-affinity receptor proteins. These hormone-
receptor complexes are subsequently translocated into the
cell nucleus where they interact with nonhistone chromo-

somal proteins. This interaction facilitates transcription

5

of new messenger RNA, with the location of the nonhistone
protein on the DNA molecule determining the code sequence
of the messenger RNA synthesized. Consequently, these
events may result in ribosomal formation of various
proteins which regulate cellular function and composition.

The process by which ovarian hormones influence syn—
thesis of proteins in the chick oviduct is clearly applic-
able to estrogen target cells in the brain. It has been
suggested that exposure of appropriate brain areas to
estrogen may alter the activity of various putative neuro-
transmitter systems, thereby activating the neural circui—
try which mediates female sexual behavior (McEwen, Davis,
Parsons, and Pfaff, 1979). Conceivably, the synthesis of
a wide variety of transmitter-related proteins may be
influenced by the presence of estrogen within a cell. For
instance, estrogen might effect an alteration in neuro-
transmitter activity by regulating the production of
enzymes which are responsible for the synthesis or
degradation of a particular neurotransmitter. Alterna-
tively, estrogen might control the synthesis or degra-
dation of membrane-bound neurotransmitter receptor proteins,
modify the formation of enzymes involved in secondary
cyclase systems, or determine the level of various intra-
cellular transport proteins.

Although the specific mechanism or mechanisms have
yet to be identified, evidence supports the conclusion

that the activation of lordosis by estrogen is dependent

 

6
upon protein synthesis. Actinomycin D, an inhibitor
of DNA-dependent RNA synthesis, blocked estrogen-
progesterone-induced lordosis in female rats when applied
to the POA, VMH, or third ventricle within 12 hr after
estrogen treatment (Quadagno, Shryne, and Gorski, 1971;
Terkel, Shryne, and Gorski, 1973; Ho, Quadagno, Cooke,
and Gorski, 1973). Cyclohexamide, an inhibitor of protein
synthesis, produced an identical effect on lordosis
following infusion into the POA or third ventricle
(Quadagno and Ho, 1975). More recently, systemic admini—
stration of anisomycin, a protein synthesis inhibitor with
low toxicity, was reported to block estrogen-progesterone—
activated lordosis, as well as induction of a specific
protein, the progestin receptor, in the POA and VMH
(Rainbow, Davis, and McEwen, 1980). These results indicate
that some form of estrogen—induced protein synthesis within
preoptic and hypothalamic neurons is critical to the
occurrence of lordosis.

If estrogen activates lordosis in female rats by
altering the activity of certain neurotransmitters within
the brain, then pharmacological manipulations which produce
similar alterations in neurotransmitter activity should
also activate lordosis. Meyerson (1964) first demonstrated
that systemic administration of agents which depleted the
level of whole brain serotonin facilitated lordosis in
female rats. However, these antiserotonergic compounds

were only effective in ovariectomized females that had been

 

7
pretreated with low doses of estrogen. Since the original
work of Meyerson, every known major neurotransmitter system
has been shown to influence the display of sexual behavior
in female rodents in some manner. Consistently, the
ability of any pharmacological agent to increase the inci-
dence of lordosis in female rodents has been contingent
upon estrogen pretreatment. The importance of neuro—
transmitter functions to sexual behavior now appears

certain and the potential of estrogen to regulate the

 

activity of these neurotransmitter systems is a logical

mechanism of hormone action. However, at present, it is >
not possible to completely circumvent the role of estrogen

by pharmacologically altering the activity of neuro-

transmitters which mediate lordosis. This observation

suggests that estrogen probably influences a number of

cellular functions which are critical to lordosis.

The initial finding that pharmacological depletion of
brain serotonin facilitated lordosis in female rats primed
with estrogen (Meyerson, 1964) was later confirmed and
extended by several investigators. Increases in the
frequency of lordosis were reported in estrogen—treated
female rats following administration of a serotonin syn-
thesis inhibitor, parachlorophenylalanine (PCPA); serotonin
receptor blockers, methysergide and cinanserin; or
serotonin release and reuptake blockers, reserpine and
tetrabenazine (Meyerson and Lewander, 1970; Zemlan, Ward,

Crowley, and Margules, 1973; Everitt, Fuxe, and Hokfelt,

 

 

 

 

8
1974; Everitt, Fuxe, Hokfelt, and Jonsson, 1975;
Ward, Crowley, Zemlan, and Margules, 1975; Eliasson and
Meyerson, 1977; Foreman and Moss, 1978a). In addition,
reductions in the incidence of lordosis were observed in
sexually receptive female rats following treatment with
agents which enhance central serotonin activity such as
monoamine oxidase inhibitors, lysergic acid diethylamide,
5-hydroxytryptophan, fenfluramine, or serotonin (Meyerson,
1966; Eliasson, Michanek, and Meyerson, 1972; Everitt et
al., 1974, 1975; Meyerson, Carrer, and Eliasson, 1974;
Eliasson, 1976; Eliasson and Meyerson, 1976, 1977;
Foreman and Moss, 1978a). Furthermore, the POA, VMH,
anterior hypothalamus, posterior hypothalamus, and medial
forebrain bundle have been identified as active sites in
this inhibitory serotonergic system (Ward et al., 1975;
Foreman and Moss, 1978a).

Despite this impressive body of evidence, reservations
have been raised concerning the validity of serotonergic
inhibition of lordosis. It has been suggested that
increases in the incidence of lordosis following treatment
with antiserotonergic agents may be due to a drug-induced
release of adrenal steroids, particularly progesterone
(reviewed by Clemens, 1978). In support of this con-
tention, PCPA failed to facilitate lordosis in adrenalec-
tomized, estrogen-primed rats (Eriksson and Sodersten,
1973). Reserpine was similarly ineffective in adrenalec-

tomized, estrogen-primed mice (Uphouse, Wilson, and

 

9
Schlesinger, 1970). Furthermore, an increased concen-
tration of plasma progesterone was detected in female rats
following reserpine treatment (Paris, Resko, and Goy,
1971). When the rise in plasma progesterone was prevented
by pretreatment with a corticotropin suppressor, reserpine
failed to facilitate lordosis in estrogen-primed females.
In contrast, other reports have demonstrated that the acti-
vation of lordosis by PCPA or methysergide treatment per-
sisted in absence of the adrenal glands (Zemlan et al.,
1973) although the magnitude of response may be reduced
(Everitt et al., 1975).

Since PCPA was found to deplete brain catecholamines,
as well as brain serotonin, the roles of dopamine and
norepinephrine in the mediation of lordosis have also
received considerable attention. Indeed, the increase in
the frequency of lordosis observed following PCPA treatment
was reported to be temporally correlated with a reduction
in brain catecholamine content rather than with a reduction
in brain serotonin level (Ahlenius, Engel, Eriksson, Modigh,
and Sodersten, 1972). However, much of the subsequent
research on the effects of the catecholamines on female
sexual behavior has proven to be ambiguous and contra-
dictory. Some of this confusion appears to stem from the
multiple receptor types which are believed to mediate
catecholamine transmission.

Support for an inhibitory dopaminergic system was

provided by a series of pharmacological experiments.

 

10

Systemic administration of alpha methylparatyrosine, a
catecholamine synthesis inhibitor, or pimozide, a dopamine
receptor blocker, facilitated lordosis in female rats primed
with estrogen (Ahlenius et al., 1972; Everitt et al., 1974,
1975; Davis and Kohl, 1977; Fuxe, Everitt, and Hokfelt,
1977). Adrenalectomy failed to reduce this response to
dopamine antagonists (Everitt et al., 1974, 1975). In
addition, dopamine receptor agonists, such as apomorphine
and ET 495, were found to reduce the frequency of lordosis
in females brought into receptivity by administration of
estrogen and progesterone (Everitt et al., 1974, 1975;
Eliasson, 1976). Pretreatment with pimozide prevented the
reduction in lordosis induced by apomorphine, further
implicating a dopaminergic system (Meyerson, Carrer, and
Eliasson, 1974; Eliasson and Meyerson, 1976). An inhib-
ition of lordosis following d-amphetamine treatment was
also reported to be blocked by pimozide pretreatment
(Michanek and Meyerson, 1977). Furthermore, selective
depletion of brain catecholamines by intraventricular
administration of a neurotoxin, 6-hydroxydopamine, increased
the incidence and duration of lordosis in female rats primed
with estrogen (Herndon, Caggiula, Sharp, Ellis, and
Redgate, 1978; Caggiula, Herndon, Scanlon, Greenstone,
Bradshaw, and Sharp, 1979).

Although these results suggest a significant inhib-
itory role for a central dopamine system, several exper-

iments directly contradict this conclusion. For example,

 

 

11
in contrast to the purported inhibitory effects of dopa—
mine receptor agonists, a facilitation of lordosis has
also been reported in hormone-primed female rats following
systemic administration of apomorphine or ET 495
(Hamburger-Bar and Rigter, 1975; Everitt and Fuxe, 1977).
This facilitation was not mediated by release of adrenal
steroids (Hamburger—Bar and Rigter, 1975). These contra-
dictory effects of dopamine receptor agonists on lordosis
may be related to drug dose. While high doses of agonists
inhibit lordosis in estrogen-progesterone-treated females,
low doses appear to facilitate lordosis in estrogen-primed
females. Everitt and Fuxe (1977) have attempted to recon-
cile these effects with the concept of an inhibitory dopa—
minergic system. They suggest that, at high doses, dopa-
mine agonists act at postsynaptic receptors to inhibit
lordosis; whereas, at low doses, these agonists act at
presynaptic receptors to reduce endogenous dopamine release
and thereby facilitate lordosis. Although appealing, this
hypothesis has yet to be substantiated.

A facilitative role for dopamine has also been indi-
cated by infusion of dopaminergic agents into various
brain areas. Microinfusion of dopamine or apomorphine
into the POA or VMH was found to increase the incidence of
lordosis in hormone—primed female rats (Foreman and Moss,
1979; Dohanich and Clemens, 1980). Conversely, the fre-
quency of lordosis was reduced in females treated with

high doses of estrogen following preoptic or hypothalamic

 

12

infusion of dopamine receptor blockers, haloperidol and
alpha flupenthixol (Foreman and Moss, 1979). However,
since the maximum behavioral reSponse to dopamine infusion
usually was observed 1-2 hr after infusion, it is difficult
to interpret this effect as a direct neurotransmitter
action. The contribution of adrenal steroids, although
not assessed, may represent a significant factor in these
intracerebral infusion experiments.

,As in the case of dopamine, the role of norepinephrine

in the control of hormone-dependent lordosis has not been

 

well defined. A facilitative influence of the noradrener-
gic system was initially indicated by the observation that
d-amphetamine potentiated the stimulatory effect of a
dopamine receptor blocker, pimozide, on lordosis (Everitt
et al., 1975). It was suggested that this enhancement was
due to an amphetamine-induced release of norepinephrine.
More recently, infusion of norepinephrine into the POA or
VMH was reported to increase the incidence of lordosis in

estrogen-primed female rats (Foreman and Moss, 1978b).

 

Selective stimulation of beta noradrenergic receptors in
the POA or VMH by infusion of isoproterenol similarly
facilitated lordosis while blocking beta receptors by
infusion of propranolol inhibited lordosis (Foreman and
Moss, 1978bL However, the possible dependence of
noradrenergic facilitation of lordosis on adrenal steroid
release has not been determined.

Several experiments have also indicated an inhibitory

 

13
role for norepinephrine. Generally, systemic or intra-
cerebral administration of alpha noradrenergic receptor
blockers such as piperoxane, yohimbine, phentolamine, or
phenoxybenzamine has been found to increase the frequency
of lordosis in estrogen-primed females (Everitt et al.,
1975; Foreman and Moss, 1978b%. In addition, lordosis was
inhibited in receptive females following treatment with
alpha noradrenergic receptor agonists, clonidine and
methoxamine (Davis and Kohl, 1977; Foreman and Moss, 1978b).
The inhibition of lordosis by clonidine was prevented by
pretreatment with the alpha receptor antagonist, yohimbine,
but not by the alpha receptor antagonist, phenoxybenzamine.
Davis and Kohl (1977) suggested, therefore, that noradre-
nergic alpha receptors may be present in two forms with
unequal potential to inhibit lordosis.

Consequently, in the rat, available data seem to indi-
cate a facilitative role for beta noradrenergic receptors
and an inhibitory role for alpha noradrenergic receptors
in the mediation of hormone-induced lordosis. Alterna-
tively, norepinephrine appears to exert a major facili-
tative action on lordosis in the guinea pig based on
observations that agents which promote noradrenergic
activity, such as clonidine, increased lordosis duration
while agents which reduce noradrenergic activity, such as
phenoxybenzamine or a norepinephrine synthesis blocker,
inhibited the display of lordosis (Crowley, Feder, and

Morin, 1976; Crowley, Neck, and Feder, 1978; Neck and

 

 

14
Feder, 1979).

Only one report presently available documents the role
of the gamma-aminobutyric acid (GABA) system in the control
of lordosis (McGinnis, Gordon, and Gorski, 1980). Picro-
toxin, a GABA receptor blocker, was found to decrease the
incidence of lordosis in estrogen-progesterone—treated
female rats following bilateral infusion into the sub-
stantia nigra. In addition, the frequency of lordosis was
increased in estrogen-primed females by bilateral nigral
infusion of hydrazinopropionic acid (HPA), an agent which
reduces GABA degradation. The maximum behavioral reSponse
occurred 2 hr after HPA infusion. The authors suggest that
increased nigral GABA facilitated lordosis by inhibiting
dopamine activity in the striatum and nucleus accumbens.
However, the delayed onset of an HPA effect and the failure
to control for HPA-induced release of adrenal steroids
weaken their conclusion. Furthermore, although evidence
is presented that dopamine activity was modified in the
striatum and nucleus accumbens following nigral infusion of
GABAminergic agents, a proposed inhibitory effect of dopa-
mine on lordosis is itself tenuous.

The role of each of the major neurotransmitter systems
reviewed thus far may be summarized. A substantial amount
of evidence strongly indicates that serotonin exerts an
inhibitory action on lordosis. The facilitation of
lordosis following treatment with certain agents which

reduce serotonergic activity may be the result of adrenal

 

 

 

 

15
steroid secretion. However, the ability of several anti-
serotonergic compounds to activate lordosis in adrenalec-
tomized females clearly implies a central action of sero-
tonin. The contribution of other neurotransmitter systems
to the control of lordosis is not as well characterized.
Dopamine activity has been reported to exert either facili-
tative or inhibitory actions on lordosis depending on the
experimental contingency. Agonists, as well as antago-
nists, increased the incidence of lordosis in estrogen-
primed female rats even following adrenalectomy. The
suggestion that these contradictory effects may be mediated
by two types of dopamine receptors, presynaptic and post-
synaptic, has yet to be confirmed experimentally.
Similarly, both facilitative and inhibitory actions have
been described for the norepinephrine system.. It has been
hypothesized that stimulation of beta receptors activates
lordosis, whereas, stimulation of alpha receptors inhibits
lordosis. However, the available evidence is weak, parti-
cularly since a facilitation of lordosis by beta agonists
has not been demonstrated in adrenalectomized females.
Finally, recent data indicate a facilitative role of brain
GABA in the control of lordosis. Again, the possible
contribution of adrenal steroids has not been determined.
Furthermore, the proposed mediation of the facilitative
effect of GABA by the dopamine system must be regarded as
speculative.

The remaining major neurotransmitter, acetylcholine,

 

 

16

may represent another central influence on hormone-
activated lordosis. Lindstrom and Meyerson (1967) reported
that systemic administration of cholinergic receptor
agonists such as pilocarpine, oxotremorine, or arecoline
reduced the incidence of lordosis in female rats primed
with estrogen and progesterone. This inhibition was
observed within 30 min after injection and was prevented by
pretreatment with atropine, a cholinergic receptor blocker.
Systemic pretreatment with methylatropine, an agent which
only blocks cholinergic receptors in the periphery, failed
to prevent the reduction in lordosis by cholinergic ago-
nists, implying a central action of these compounds.

Lindstrom (1970) later found that the inhibition of
lordosis following pilocarpine treatment was magnified when
females were pretreated with monoamine oxidase inhibitors,
pargyline or nialamide. Monoamine oxidase inhibitors were
previously shown to inhibit lordosis in receptive females,
presumably by increasing central monoaminergic, princi—
pally serotonergic activity (Meyerson, 1964). Lindstrom
suggested, therefore, that elevations in cholinergic and
monoaminergic activity may inhibit lordosis by related
mechanisms. Subsequently, it was demonstrated that pre-
treatment with the monoamine synthesis inhibitor PCPA
prevented the inhibition of lordosis by pilocarpine
(Lindstrom, 1971). Furthermore, pretreatment with a
catecholamine synthesis inhibitor failed to block the

inhibitory effects of pilocarpine. Lindstrom concluded

 

17
that pilocarpine inhibited lordosis in estrogen-progester—
one—treated females via a serotonergic mechanism.

Pilocarpine and oxotremorine were also reported to
increase the incidence of lordosis in female rats primed
with estrogen (Lindstrom, 1973). In contrast to the short
latency of the inhibitory effects previously observed (30
min), the facilitation of lordosis by cholinergic agonists
occurred 4 hrs after systemic injection. The increase was
blocked by atropine pretreatment, reinforcing a cholinergic
interpretation. However, cholinergic agonists failed to
facilitate lordosis in estrogen-primed females that had
been adrenalectomized, hypophysectomized, or pretreated
with a corticotropin suppressor. Consequently, it appears
that the enhancement of lordosis following pilocarpine or
oxotremorine treatment was dependent on a drug—induced
release of adrenal steroids.

The work of Lindstrom suggests that the inhibitory
effect of cholinergic agonists is mediated by a sero—
tonergic mechanism while the facilitative effect of cho—
linergic agonists is mediated by adrenal steroids. The
cholinergic compounds used by Lindstrom act primarily at
cholinergic muscarinic receptors. A more recent study
(Fuxe et al., 1977), indicated that stimulation of cho~
linergic nicotinic receptors following systemic admini—
stration of nicotine increased the incidence of lordosis
in estrogen—primed female rats. This facilitation was

observed within 5 min after injection and was blocked by

 

 

18
pretreatment with a nicotinic receptor blocker but not by
pretreatment with catecholaminergic agents. Although the
effect of nicotine treatment on lordosis was not evaluated
in adrenalectomized females, it is doubtful that adrenal
steroids would activate lordosis within 5 min after
release.

Rodgers and Law (1968) implanted a muscarinic receptor
agonist, carbachol, or a muscarinic receptor blocker,
atropine, in various brain areas and reported a facili-
tation of lordosis in ovariectomized female rats. The
failure to prime females with estrogen and the negligible
level of reSponse to intracerebral treatments make the
significance of their results difficult to interpret.
However, evidence has recently been presented which
strongly suggests that stimulation of central cholinergic
muscarinic receptors facilitates lordosis independently of
adrenal secretions, contradicting the early reports of
Lindstrom. Implantation of muscarinic receptor agonists
carbachol and bethanechol in the POA or MRF consistently
activated lordosis in female rats primed with estrogen
(Clemens, Humphrys, and Dohanich, 1980). This facilitation
was usually observed within 30 min of intracerebral treat-
ment, persisted in the absence of the adrenal glands, and
was blocked by pretreatment with the muscarinic receptor
blocker atropine. In support of a facilitative role of
muscarinic receptors, Singer (1968) earlier reported that

systemic administration of a high dose of atropine alone

 

 

 

 

19
reduced the incidence of lordosis in female rats treated
with estrogen and progesterone.

The experiments comprising this dissertation were
designed to extend the analysis of cholinergic facili-
tation of sexual receptivity in female rats. Several
design features were introduced in order to more fully
characterize this phenomenon. The contradictory behavioral
effects often observed following treatment with various
pharmacological agents may arise partly from the mode of
administration. The systemic injection technique often
exposes the entire brain, if not the entire body of an
organism, to the drug. Nonphysiological reSponses under
these experimental conditions may be expected. Conse-
quently, in order to limit drug actions to specific areas
of the brain, cholinergic agents were applied directly to
brain sites implicated in the control of female sexual
behavior. In addition, compounds were administered in
solution form via a microinfusion technique which delivers
low doses of a drug and provides transient stimulation of
specific brain areas not possible with traditional intra-
cerebral implants of crystalline compounds. The effects of
a cholinergic agonist, carbachol, as well as a cholinergic
antagonist, hemicholinium-3, were investigated. Since
cholinergic mediation of lordosis appears to be a hormone-
dependent phenomenon, the behavioral responses of female
rats following intracerebral cholinergic treatment were

examined under a variety of estrogen and estrogen-

 

 

 

 

20
progesterone regimens. Finally, the ability of manipu-
lations of the cholinergic system to influence sexual
behavior may indicate that ovarian homones alter cho—
linergic processes within the brain. In the last experi-
ment, the effect of estrogen treatment on a cholinergic
parameter, specifically muscarinic receptor binding, was

evaluated in specific areas of the female rat brain.

 

 

 

 

GENERAL METHOD
(Experiments 1-4)

Sherman female rats, 60—70 days of age, were obtained
from Camm Research Company, Wayne, New Jersey. The animals
were housed singly with free access to food and water. A
reversed light-dark cycle was maintained in the Vivarium
(14 hr light: 10 hr dark, light off at 1100 hr).

Females were placed under ether anesthesia
'(Mallinckrodt, Inc.) or Ketamine anesthesia (Parke, Davis,
and Co.) and bilaterally ovariectomized at 75-85 days of
age. To verify a normal behavioral response to exogenous
hormone treatment, all ovariectomized females were
pretested for lordosis prior to stereotaxic surgery.
Females were injected intramuscularly with 0.5 ug estra—
diol benzoate (EB, Schering Corp.) 72, 48, and 24 hr
before behavioral testing. Approximately 5 hr before
testing 0.5 mg progesterone (Sigma Chemical Co.) was
injected intramuscularly. EB and progesterone were
administered in 0.1 ml volumes of sesame seed oil (Sigma
Chemical 00.). Hormone doses always represent the total
dose administered to each animal per day.

Behavioral tests were conducted in a Plexiglas arena
(45 x 50 x 58 cm) occupied by a Long-Evans stimulus male

(Charles River Laboratories). Each female was mounted a

21

 

 

22
total of 10 times during a single test. Any female failing
to receive 10 mounts within 10 min was transferred to
another arena with a different male where the test was
completed. The number of lordosis responses observed per
10 mounts was recorded and a lordosis quotient was
computed for each female: L0 = (number of lordoses/10
mounts) x 100.

Females displaying an LQ of 70 or higher on the
pretest underwent stereotaxic surgery under ether or
Ketamine anesthesia approximately 1 wk later. Cho—
linergic drugs were infused intracerebrally via a double-
barrel cannula assembly constructed from stainless—steel
hypodermic tubing (Small Parts, Inc.). Each assembly
consisted of a chronic 23 gauge outer barrel (guide) and
a removable 27 gauge inner barrel (insert). Guides were
implanted bilaterally 1 mm dorsal to the target brain
sites and anchored to the skull with machine screws and
dental acrylic. Each insert extended 1 mm beyond the end
of the guide into the brain site. Stereotaxic coordinates
were provided by Albe-Fessard, Stutinsky, and Libouban
(1966) for five different sites as follows: POA
(anterior 7.9, lateral 1.0, horizontal 4.0); VMH
(anterior 5.8, lateral 0.8, horizontal 2.0); MRF
(anterior 3.2, lateral 1.0, horizontal 2.0); stria
terminalis (anterior 7.9, lateral 1.0, horizontal 5.5);
frontal cortex (anterior 10.0, lateral 1.0, horizontal

9.0).

 

 

23

One wk after stereotaxic surgery all females were
injected with EB 72, 48, and 24 hr before behavioral
testing. The dose of EB administered varied with experi-
ment. In some experiments, 0.5 mg progesterone or 0.1 ml
sesame seed oil was injected intramuscularly 4-5 hr before
behavioral testing. Prior to intracerebral infusion, the
insert was removed and replaced by a 28 gauge infusion
insert which extended into the target site. The infusion
insert was connected by PE—20 polyethylene tubing to a
syringe mounted on a reciprocal microinfusion pump
(Harvard Apparatus). A 0.5 ul volume of solution was
delivered over 30 sec sequentially through each cannula
(1 ul/animal). Following infusion, the infusion insert
was replaced by the original insert. For intracerebral
infusions, cholinergic compounds were dissolved in an
artificial cerebrospinal fluid (CSF) containing 130 mM
NaCl, 25 mM Na H003, 5 mM NaZH P04, 30 mM KCL, 8 mM Mg 012,
and 13 mM CaCl2 (pH 6.8). The agents administered
included carbamylcholine chloride (carbachol, Sigma
Chemical 00.), hemicholinium—3 (HG-3, Aldrich Chemical
00.), and choline chloride (Sigma Chemical 00.). During
the test session each female was pretested for lordosis
and, immediately after, was bilaterally infused with the
appropriate drug or vehicle solution. The effects of
intracerebral treatment on lordosis were tested at various
times after infusion during 1—3 weekly sessions.

After the final behavioral test, all females were

 

 

 

24
anesthetized with ether or phenobarbital and perfused
intracardially with 0.9% saline followed by 10% buffered
formalin. Brains were removed and frozed coronal brain
sections (50 u) were taken. Implant locations were
verified following neutral red staining.

Data were analyzed with parametric analyses of
variance usually followed by Newman-Keuls range tests
(Winer, 1971) or with nonparametric Kruskall-Wallis
analyses of variance, Mann-Whitney U—tests, Wilcoxon
matched pairs T tests, or Fisher exact probability tests
(Siegel, 1956). Log transformations were made where

appropriate.

 

EXPERIMENT 1. Facilitation of Lordosis in Hormone—treated
Female Rats Following Intracerebral
Infusion of Carbachol

Intracerebral implants of cholinergic agonists in
crystalline form have been found to increase the inci-
dence of lordosis in female rats primed for 3 days with
1 ug EB (Clemens et al., 1980). The facilitation of
lordosis occurred 30-60 min after intracerebral treatment
and persisted up to 120 min. Unfortunately, the crys-
talline implant technique delivers a high, and often
inexact, dose of a drug (approximately 10 ug) to the brain
site, activating a behavioral response which may be sus-
tained for hours. Under these conditions, the effects of
repeated behavioral tests and adrenal steroid release may
contribute to the prolonged behavioral response often
observed. In Experiment 1, the ability of carbachol, a
cholinergic receptor agonist, to facilitate lordosis in
female rats was investigated using a microinfusion tech—
nique which provides precise control of drug dose and
limits the duration of drug stimulation. This procedure
should elicit a reliable, prompt, and transient facili-
tation of behavior.

The effect of carbachol on lordosis was evaluated
under a variety of hormone priming conditions. Systemic

administration of progesterone is known to activate

25

 

26
lordosis in female rats treated with EB (Boling and
Blandau, 1939). Since progesterone, as well as cho-
linergic agonists, facilitate lordosis in estrogen-primed
females, a common mechanism may underlie their actions.
In Experiment 1, the behavioral effects of progesterone,
administered by systemic injection, and carbachol, admin-
istered by intracerebral infusion, were compared. Female
rats were treated with either EB and progesterone, EB and
carbachol, or EB, progesterone, and carbachol. The

various doses of EB selected (0.13, 0.17, and 0.25 ug

 

for 3 days) activate only a low to moderate incidence of
lordosis, even when combined with progesterone treatment.
The doses of progesterone (0.5 mg) and carbachol (0.5
ug/cannula) activate a high incidence of lordosis when
combined with adequate amounts of estrogen. Utilizing

a series of low EB doses, in combination with optimum
doses of progesterone and/or carbachol, allowed comparison
of the behavioral effects of progesterone and carbachol, as

well as assessing the summation of their actions.

METHOD

Ovariectomized female rats were bilaterally implanted

 

with cannulae in the POA according to the procedure out-
lined in the General Method section (See Figure 1).

Implanted females received 3 weekly tests beginning 1 wk
after stereotaxic surgery. All females were primed with

0.13 ug EB at 72, 48, and 24 hr prior to the first weekly

arm 0...}... ...-5. . ,

 

Figure 1.

27

DOR

 

Distribution of cannula tips as verified
histologically in female rats from Experiment 1.
Only animals with implants in the areas indi—
cated were included in data analysis. The

plate represents a sagittal section takem 1.0 mm
lateral to the midline. Adapted from Albe—
Fessard et al. (1966). Abbreviations: ANT,
anterior; DOR, dorsal; ac, anterior commissure;
aha, anterior hypothalamic area; cc, corpus
callosum; poa, preoptic area; st, stria termin—
alis; v, ventricle; vmh, ventromedial hypo-
thalamus.

 

 

 

28
test. The priming dose of EB was increased to 0.17 ug
for the second weekly test and 0.25 ug for the third
weekly test. Approximately 4-5 hr before each test
females were injected with either 0.5 mg progesterone or
0.1 ml sesame oil. During a test session, each female
was pretested for lordosis and afterward bilaterally
infused with either carbachol (0.5 ug/cannula) or CSF
vehicle. The effects of intracerebral treatments on
lordosis were tested 15, 45, and 90 min after infusion.
Each female received the same combination of carbachol or

CSF with progesterone or oil over the 3 wks of testing.

RESULTS

Significant increases in L0 were observed following
preoptic infusion of carbachol (0.5 ug/cannula) under
all hormone regimens (Figure 2). At the 0.13 ug dose of
EB, analysis of variance revealed significant main effects
of intracerebral treatment (p<<.001) and time after infu-
sion (p <.001), as well as a significant treatment x time
interaction (p <.001). Females primed with EB and oil
displayed a higher incidence of lordosis 15 min (p< .05,
Newman—Keuls test) and 45 min (p <.05) after carbachol
infusion than after CSF infusion. Equivalent increases
in L0 were observed 15 min (p.<.05) and 45 min (p<:.05)
after carbachol infusion in females primed with EB and
progesterone. The incidence of lordosis in both groups

receiving carbachol returned to CSF levels by 90 min

 

Figue 2.

29

Mean lordosis quotients recorded from female
rats following bilateral infusion of carbachol
(0.5 ug/cannula) or artificial cerebrospinal
fluid (CSF) vehicle into the medial preoptic
area. Estradiol benzoate (EB) was injected
intramuscularly 72, 48, and 24 hr before
behavioral testing and the dose was increased
each wk for all females. Oil (0.1 ml, upper
panel) or progesterone (0.5 mg, lower panel)
was injected intramuscularly -5 hr before
testing. Each female received the same com—
bination of carbachol or CSF with progesterone
or oil over the 3 wk of testing. Intra-
cerebral treatment was administered immedi—
ately following the preinfusion test (PT).

The effect of intracerebral treatment on
lordosis was tested 15, 45, and 90 min after
infusion. For each treatment combination,

n = 10—14. *p <.05 (carbachol vs. CSF,
Newman—Keuls).

 

 

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31
after infusion.

Similar increases in L0 following carbachol treatment
were evident at the 0.17 ug dose of EB where significant
effects of treatment (p <.001), time (p‘<.001) and treat-
ment x time interaction (p <.001) were detected. Once
again females primed with EB, in COmbination with either
oil or progesterone, displayed a higher incidence of
lordosis 15 min (p <.05) and 45 min (p <.05) after carba-
chol infusion than after CSF infusion. There were no
significant treatment differences in behavior by 90 min
after infusion.

At the 0.25 ug dose of EB, analysis of variance indi-
cated significant main effects of treatment (p <.001) and
time (p <.001), and a significant treatment x time inter-
action (p <.001). In females primed with EB and oil, the
incidence of lordosis was significantly higher 15 min
(p <.05) and 45 min (p <.05) after carbachol infusion than
after CSF infusion. As at lower EB doses, LQ returned to
the CSF level by 90 min after carbachol infusion. In
females primed with 0.25 ug EB and progesterone, the inci-
dence of lordosis was higher after carbachol infusion than
after CSF infusion only at 15 min after intracerebral
treatment (p <.05).

Soliciting behaviors, such as ear wiggling and hopping
and darting, were not activated by any treatment

combination.

 

32

SUMMARY

In Experiment 1, female sexual behavior, indicated by
the incidence of lordosis, was significantly increased in
estrogen-treated female rats following bilateral infusion
of a cholinergic receptor agonist, carbachol (0.5 ug/
cannula), into the POA. Infusion of an artificial CSF
vehicle failed to facilitate lordosis. The incidence of
lordosis was normally highest 15 min after carbachol
infusion, began to wane by 45 min, and had returned to
control levels by 90 min. Centrally administered
carbachol activated lordosis at lower levels of estrogen

priming than did systemically administered progesterone.

 

EXPERIMENT 2. Brain Regions Implicated in Cholinergic
Mediation of Lordosis

A variety of evidence has implicated the POA, VMH, and
MRF in the control of hormone—dependent sexual behavior
in female rats. However, the ability of intracerebral
implants of estrogen to facilitate lordosis appears to
vary among these three brain regions. Although estrogen
implants in the POA or VMH have both been shown to acti-
vate lordosis, the VMH appears to be sensitive to a lower
level of estrogen stimulation (Barfield and Chen, 1977;
Rubin and Barfield, 1980). Furthermore, estrogen implants
in the MRF failed to facilitate lordosis, unless combined
with systemically administered progesterone (Yanase and
Gorski, 1976). Progesterone implants in the MRF also
activated lordosis in females treated systemically with
estrogen (Ross et al., 1971; Yanase and Gorski, 1976).
Both the POA and VMH concentrate estrogen from the circu-
lation more heavily than the MRF (Pfaff and Keiner, 1973).

In Experiment 2, the incidence of lordosis was
assessed in estrogen—primed female rats following micro—
infusion of the cholinergic receptor agonist, carbachol,
into several brain areas including the POA, VMH, and MRF.
Differences in the ability to concentrate and respond to

estrogen may determine the potential of carbachol to

33

 

34
facilitate lordosis following infusion into these brain

regions.

METHOD

Double—barrel cannulae were bilaterally implanted in
the POA, VMH, MRF, or frontal cortex of ovariectomized
female rats as described in the General Method section.
Following a 1 wk recuperative period, females were in-
jected intramuscularly with 0.13 ug EB 72, 48, and 24 hr
before behavioral testing. 0n the day of testing, females
were pretested for lordosis and afterward bilaterally
infused with either carbachol (0.5 ug/cannula) or CSF
vehicle. The effects of intracerebral treatments on
lordosis were tested 15, 45, and 90 min after infusion. On
the second wk of testing, 0.13 ug EB was again administered
intramuscularly to all females 72, 48, and 24 hr before
testing and intracerebral treatment (carbachol or CSF) was

reversed for each female.

RESULTS

Mean lordosis quotients : SEM are presented in Figure
3. For each site, nonparametric comparisons were made
between the change in lordosis frequency (postinfusion
minus preinfusion) following carbachol versus CSF with each
subject serving as its own control (Wilcoxon matched—pairs
T test). Significant increases in the incidence of lordo-
sis were observed 15 min (p <.005) and 45 min (p <.005)

after infusion of carbachol into the POA or VMH. The

 

Figure 3.

35

 

 

 

 

 

 

 

 

 

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sfifizzmfifi: gfizAi’T“?
I l I I I I
P IS 45 90 P 15 45 90

TIME AFTER INFUSION

Mean lordosis quotients recorded from female
rats following bilateral infusion of carbachol
(0.5 ug/cannula) or artificial cerebrospinal
fluid (CSF) vehicle into various brain areas.
All females were injected with 0.13 ug estradiol
benzoate 72, 48, and 24 hr before behavioral
testing. Carbachol or CSF was bilaterally
infused into the medial preoptic area (POA),
ventromedial hypothalamus (VMH), mesencephalic
reticular formation (MRF), or frontal cortex.
Intracerebral treatment was reversed for each
female over 2 wk of testing. Intracerebral
treatment was administered immediately
following the preinfusion test (P) and behav-
ioral effects were tested 15, 45, and 90 min
after infusion. For each site, n = 9-12.
*p<:.005 (carbachol vs. CSF, Wilcoxon T,
one-tailed).

 

36

frequency of lordosis was not significantly different from
CSF levels by 90 min after carbachol infusion in either
site. Proceptive behaviors, such as ear wiggling and
hopping and darting, were never observed during behavioral
tests. Infusion of carbachol into the MRF or cortex
failed to significantly facilitate lordosis on all tests.

Location of the insert cannula tips are presented in
Figure 4. Infusion sites which resulted in an L0 increase
of 40 or more were identified as positive. As indicated in
Table 1, Fisher exact probability tests revealed that carb-
achol induced lordosis in a significantly higher proportion
of females than CSF in both the POA (p <.005) and VMH

(p <-005)-

SUMMARY

In Experiment 2, the facilitation of lordosis
following intracerebral infusion of carbachol was shown to
be a site-specific phenomenon. Carbachol increased the
incidence of lordosis in estrogen-primed female rats
following bilateral infusion (0.5 ug/cannula) into either
the POA or the VMH. As in Experiment 1, the behavioral
response was highest 15 min after carbachol infusion and
returned to control levels within 90 min after infusion.
Carbachol failed to activate lordosis following infusion

into the MRF or frontal cortex.

 

Figurelh

37

Carbachol infusion sites in the medial preoptic
area (upper left), ventromedial hypothalamus
(upper right), frontal cortex (lower left), and
mesencephalic reticular formation (lower
right). An increase in lordosis quotient of 40
or more over the pretest recorded on at least 1
postinfusion test was considered a positive
effect (positive sites——circles; negative
sites--triangles). Histology was unavailable
for 1 ventromedial hypothalamic implant.
Adapted from Konig and Klippel (1974).

 

 

 

38

 

A 7470

A 7190

 

 

A 7020

6860

A

  

A 6670

A 1950

 

 

 

 

 

A 1610

 

 

 

 

A 10300

A 10050

0
2
8
9
A

 

A 9650

39

TABLE 1
Proportion of Female Rats ReSponding
Following Intracerebral Infusion of Carbachol

or Artificial CerebrOSpinal Fluid (CSF) Vehicle

 

 

 

 

 

PROPORTIONl
§22§ TREATMENT RESPONDING
POA Carbachol 9/12*

CSF vehicle 0/12
VMH Carbachol 10/12*
CSF vehicle 0/12
MRF Carbachol 1/9
CSF vehicle 1/9
Cortex Carbachol 4/11
CSF vehicle 1/11

 

1An increase in lordosis quotient of 40 or more
over the pretest recorded on at least one postinfusion
test was considered a positive response.

*p <.005 (carbachol vs. CSF, Fisher exact prob-
ability test, one-tailed).

EXPERIMENT 3. Inhibition of Lordosis in Hormone-treated
Female Rats Following Intracerebral
Infusion of Hemicholinium-3
EXPERIMENT 3a
Based on the ability of cholinergic agonists to
facilitate lordosis in female rats (Clemens et al., 1980,
Experiments 1 and 2), it was predicted that disruption of
central cholinergic activity in receptive females would
result in a reduction in the incidence of lordosis. In
Experiment 3a, the behavioral effects of a cholinergic
antagonist, HC-3, were examined in ovariectomized_female
rats brought into sexual receptivity by administration of
estrogen and progesterone. This anticholinergic agent has
been shown to retard the synthesis of acetylcholine by
competitively inhibiting transport of the precursor,
choline, across neuronal membranes (MacIntosh, Birks, and
Sastry, 1956; Gardiner, 1961; Guyenet, Lefresne, Rossier,
Beaujouan, and Glowinski, 1973). In Experiment 3a,
HC-3 was bilaterally infused near the dorsal-medial border
of the bed nucleus of the stria terminalis, an area shown
to heavily concentrate tritiated estradiol (Pfaff and

Keiner, 1973).

METHOD
Ovariectomized female rats were bilaterally implanted

with cannulae in the stria terminalis according to the

40

 

 

41
procedure outlined in the General Method section (See
Figure 5). During the following 2 wk, all females were
injected intramuscularly with 2 ug EB 72, 48, and 24 hr
before behavioral testing. Progesterone (0.5 mg) was
injected intramuscularly 4-5 hr before testing. During the
test session, each female was pretested for lordosis and
afterward bilaterally infused with one of three solutions;
7.5 ug HC-3/cannula, 5 ug HC-3/cannula, or CSF Vehicle.
The effects of intracerebral treatments on lordosis were
tested 15 and 30 min after infusion. Each female received
the same hormonal and intracerebral treatment on 2

consecutive wk.

RESULTS

Intracerebral infusion of HC-3 (Figure 6) signifi-
cantly reduced the incidence of lordosis in females treated
with EB (2 ug) and progesterone (0.5 mg). The change in
L0 following HC-3 infusion (pretest minus test) was
compared to the change in L0 observed following CSF infu-
sion at correSponding times after intracerebral treatment
(Mann-Whitney U test). During the first wk of treatment,
significant reductions in L0 were recorded at 15 and 30 min
following infusion of 5 ug HC—3/cannula (at 15 min, p<:.05;
at 30 min, p <.01) or 7.5 ug HC-3/cannula (at 15 min,
p <.01; at 30 min, p <.01). During the second wk of
treatment, similar reductions in L0 were observed only
following infusion of the higher dose of HC-3 (7.5 ug/

cannula: at 15 min, p <.01; at 30 min, p <.01). Infusion

 

 

 

Figure.5.

42

Distribution of cannula tips as verified histo-
logically in female rats from Experiments 3.
Only animals with implants in the areas indi-
cated were included in data analysis. The
plate represents a sagittal section taken at
1.0 mm lateral to the midline. Adapted from
Albe-Fessard et al. (1966). Abbreviations:
ANT, anterior; DOR, dorsal; ac, anterior
commissure; aha, anterior hypothalamic area;
00, corpus callosum; poa, preoptic area; st,
stria terminalis; v, ventricle; vmh, ventro-
medial hypothalamus.

43

 

 

W STRIA TERMINALIS

N PREOPTIC AREA

MUNICORTEX

 

 

 

Figure 6.

44

 

 

 

 

 

P 15’ 30'
mm

80-

 

 

 

 

 

 

P 15’ 30’

CSF

(Sun) (7.5 Us)

Mean lordosis quotients (LQ) recorded from
female rats following bilateral infusion of
hemicholinium-3 (HO—3, 5 or 7.5 ug/cannula) or
artificial cerebrospinal fluid (CSF) vehicle
into the bed nucleus of the stria terminalis.
All females were injected with 2 ug estradiol
benzoate 72, 48, and 24 hr before behavioral
testing and 0.5 mg progesterone 4-5 hr before
testing. Each female received the same intra-
cerebral treatment over 2 wk of testing.
Intracerebral treatment was administered immedi-
ately following the preinfusion test (P) and
behavioral effects were tested 15 and 30 min
after infusion. *p <.05, **p <.01 (change in

L0 following HC—3 vs. change in L0 following

CSF at corresponding test times, Mann-Whitney U).

45
of 5 ug HC-3/cannula failed to alter lordotic behavior

during the second wk of treatment.

EXPERIMENT 3b

Significant reductions in lordosis were observed in
Experiment 3a following a single infusion of HC-3 into the
bed nucleus of the stria terminalis of receptive females.
However, behavioral inhibition was evident only at the
higher dose of HC-3 (7.5 ug/cannula) following a second
treatment on the subsequent wk. Since steroids are known
to reduce the effectiveness of various neurotropic agents
(Selye, 1971a, b), the behavioral tolerance to repeated
administration of HC-3 may be enhanced in animals treated
with estrogen and progesterone. In order to facilitate the
effects of HC-3 on lordosis and prevent the development of
tolerance, lower doses of EB were utilized in Experiment
3b. In addition, the possibility of a stimulatory action
of HC-3 was examined by testing females primed with a com-
bination of EB and progesterone which normally activates

only a low level of sexual receptivity.

METHOD

Ovariectomized female rats were bilaterally implanted
in the POA according to the procedure outlined in the
General Method section (See Figure 5). During the follow-
ing 2 wk, females were injected intramuscularly with 0.175
ug, 0.25 ug, or 0.5 ug EB 72, 48, and 24 hr before behav-

ioral testing. Progesterone (0.5 mg) was injected

 

 

 

46
intramuscularly 4-5 hr before testing. During the test
session, each female was pretested for lordosis and after-
ward bilaterally infused with either HC—3 (5 ug/cannula) or
CSF vehicle. The effects of intracerebral treatments on
lordosis were tested 30 min after infusion. Each animal
received the same priming dose of EB and intracerebral

treatment during both wk of testing.

RESULTS

Preoptic infusion of HC-3 (5 ug/cannula) caused
substantial reductions in lordotic behavior of females
primed with EB and progesterone (Figure 7). The change in
L0 following HC—3 infusion (pretest minus test) was compared
to the change in L0 observed following CSF infusion at
corresponding doses of EB (Mann-Whitney U test). During
the first wk of treatment, significant reductions in L0
were seen following HC-3 infusion in females receiving
0.5 ug EB plus progesterone (p <.01) or 0.25 ug EB plus
progesterone (p< .01). During the second wk of treatment,
a significant reduction in L0 following HC-3 infusion was
observed only in those females receiving 0.25 ug EB plus
progesterone (p <.01). A second exposure to HC—3 failed
to significantly reduce lordotic behavior in females
receiving 0.5 ug EB plus progesterone. The tolerance to
HC—3 displayed by females primed with this highest EB dose
was similar, though not as pronounced, as that seen in

Experiment 3a at a 2 ug priming dose of EB. Finally,

Figure 7.

47

[::J 9251551 POST csr 3232 POST HC-3

 

l00-

WEEKI

_L

80-

60-4

MEAN
L0

404

20-

 

 

 

 

 

 

 

 

 

 

   

**
LLEZL_.
P

30’ P 30’ P 30'

II"!

 

 

 

 

 

 

 

E8 DOSE

Mean lordosis quotients (LQ) recorded from
female rats following bilateral infusion of
hemicholinium-3 (HC—3, 5 ug/cannula) or
artificial cerebrospinal fluid (CSF) vehicle
into the medial preoptic area. Various doses
of estradiol benzoate (EB) were injected 72,
48, and 24 hr before behavioral testing and
0.5 mg progesterone was injected 4-5 hr before
testing. Each female received the same
hormone-intracerebral treatment combination
over 2 wk of testing. Intracerebral treatment
was administered immediately following the
preinfusion test (P) and behavioral effects
were tested 30 min after infusion. **p <.01
(change in L0 following HC-3 vs. change in

L0 following CSF at corresponding EB doses,
Mann-Whitney U).

48
no significant elevations in lordotic behavior occurred
in HC-3-treated females receiving 0.175 ug EB plus

progesterone.

EXPERIMENT 3c

Experiment 3c evaluated the specificity of the HC-3
effects reported in Experiments 3a and 3b. Choline,
possibly the rate limiting factor in acetylcholine syn-
thesis, is not produced centrally but rather is transported
to the neuron in a lipid-bound form (Ansell and Spanner,
1968). The quaternary ammonium compound, HC-3, is postu-
lated to competitively inhibit the transport of choline
across neuronal membranes to the site of acetylcholine syn-
thesis (MacIntosh et al., 1956; Gardiner, 1961; Guyenet
et al., 1973). Accordingly, the depletion in brain ace-
tylcholine content reported following intraventricular
infusion of HC-3 (Freeman, Choi, and Jenden, 1975) can be
prevented if choline chloride is infused along with HC-3
(Slater, 1968). In Experiment 3c, the ability of choline
chloride to prevent the inhibition of lordosis by HC—3 was
investigated. A dose of 120 ug/cannula of choline chlor-
ide was administered in order to reverse the effects of
5 ug/cannula of HC-3. Choline chloride doses 12-50 times
those of HC-3 are required to prevent significant reduction
in acetylcholine levels (Birks and MacIntosh, 1961; Slater
1968). A second control in Experiment 30 consisted of
infusion of an active dose of HC—3 into cortical brain

areas of females receiving estrogen and progesterone to

 

49
determine if HC-3 action was specific to particular brain
sites. No alteration in lordotic behavior was expected
from cortical infusion of HC-3 since this area does not

concentrate ovarian hormones (Pfaff and Keiner, 1973).

METHOD

Ovariectomized female rats were bilaterally implanted
in the POA according to the procedure outlined in the
General Method section (See Figure 5). During the follow-
ing wk, females were injected intramuscularly with 0.5 ug

EB 72, 48, and 24 hr before behavioral testing. Proges—

 

terone was injected intramuscularly 4-5 hr before testing.
During the test session, females were pretested for lordo-
sis and afterward bilaterally infused with a CSF solution
containing HC-3 (5 ug/cannula) or a CSF solution con-
taining HC-3 (5 ug/cannula) plus choline chloride (120
ug/cannula). The effects of intracerebral treatments on
lordosis were tested 30 and 60 min after infusion. A
single test session was conducted.

Bilateral implants were also placed in the frontal
cortex of ovariectomized females according to the procedure

outlined in the General Method section (See Figure 5).

 

During the following 2 wk, females were injected intra-
muscularly with 0.5 ug EB 72, 48, and 24 hr before behav-
ioral testing. Progesterone was injected intramuscularly
4—5 hr before testing. During the test session, all females
were pretested for lordosis and afterward bilaterally

infused with HC-3 (5 ug/cannula). The effects of

 

50
intracerebral treatment on lordosis were tested 30 and

60 min after infusion.

RESULTS

When HC—3 (5 ug/cannula) was infused in combination
with choline chloride (120 ug/cannula) the inhibitory
effect of HC-3 on lordosis was arrested (Figure 8). The
reductions in L0 observed at 30 and 60 min following HC—3
infusion were significantly greater than reductions seen
following infusion of HC—3 plus choline chloride (at 30 min,
p <.01; at 60 min, p <.05). The disruption of the inhibi—
tory effect of HC—3 achieved by choline chloride began to
wane by 60 min. There was no evidence of behavioral
inhibition following infusion of HC-3 (5 ug/cannula) into
the frontal cortex of females treated with EB and

progesterone (Table 2).

SUMMARY

A cholinergic antagonist was infused into forebrain
areas of ovariectomized female rats brought into sexual
receptivity by administration of estrogen and progesterone.
Bilateral infusion of HC—3 (5 or 7.5 ug/cannula), an ace—
tylcholine synthesis inhibitor believed to interfere with
choline uptake, decreased the incidence of lordotic behavior
displayed by females. This inhibition was prevented by
infusion of choline chloride (120 ug/cannula) along with
HC—3. HC-3 did not inhibit lordosis when infused into the
frontal cortex nor did HC—3 facilitate lordosis in unre-

ceptive females following infusion into the POA.

 

 

51

100]

80‘

60—

MEAN
LO

 

 

 

H C-3
4.

CHOLINE

Figure 8. Mean lordosis quotients (LQ) recorded from
female rats following bilateral infusion of
hemicholinium-3 (HC-3, 5 ug/cannula) or HC—3
(5 ug/cannula) plus choline chloride (120
ug/cannula) into the medial preoptic area.
All females were injected with 0.5 ug estra-
diol benzoate 72, 48, and 24 hr before be-
havioral testing and 0.5 mg progesterone 4—5
hr before testing. Intracerebral treatment
was administered immediately following the
preinfusion test (P) and behavioral effects
were tested 30 and 60 min after infusion.

*p <.05, **p.<.01 (change in L0 following HC—3
vs. change in L0 following HC-3 plus choline,
Mann—Whitney U).

 

 

52

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whomon as :m ens .m: .Nm opdoaCop Howemupmo m: m.o Spas oopoomcfl one; moadSow HH<*

 

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mussflcflaocofiEom

 

CHE or Gas om Pmopohm : CHE om SHE om Pmopopm c

 

N mmmg H MMMS *Ezmze<mme
Q<mmmmMD<mBzH

 

 

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mpmm mamsom 809m poonooom mpcoaposo mfimoohoq Coo:

N mqm<e

EXPERIMENT 4. Reversal of Hemicholinium—3 Inhibition
of Lordosis by Carbachol

In Experiments 1 and 2, stimulation of central cho-
linergic receptors by infusion of a receptor agonist,
carbachol, activated lordosis in hormone-primed female
rats. In Experiment 3, inhibition of acetylcholine syn-
thesis by infusion of a synthesis blocker, HC-3, reduced
the incidence of lordosis in hormone-primed female rats.
In Experiment 4, central cholinergic receptors were stimu-
lated by preoptic infusion of carbachol in an attempt to

reverse the inhibitory effects of HC-3 on lordosis.

METHOD

Ovariectomized female rats were bilaterally implanted
with cannulae in the POA according to the procedure outlined
in the General Method section (See Figure 9). Behavioral
tests were conducted 1 wk after stereotaxic surgery. Each
female was injected at 72, 48, and 24 hr prior to testing
with one of four EB doses: 0.13 ug, 0.17 ug, 0.25 ug, 0.5
ug. Progesterone (0.5 mg) was injected 4-5 hr before
testing. During the test session, each female was pre-
tested for lordosis and afterward bilaterally infused with
' a CSF solution containing HC-3 (1.25 ug/cannula) or a CSF
solution containing HC-3 (1.25 ug/cannula) plus carbachol

(0.5 ug/oannula). The effects of intracerebral treatments

53

Figure 9.

54

00!!

A

(I __-—-"'-'

    

Distribution of cannula tips as verified
histologically in female rats from Experiment
4. Only animals with implants in the areas
indicated were included in data analysis. The
plate represents a sagittal section taken 1.0
mm lateral to the midline. Adapted from Albe-
Fessard et al. (1966). Abbreviations: ANT,
anterior; DOR, dorsal; ac, anterior commissure;
aha, anterior hypothalamic area; cc, corpus
callosum; poa, preoptic area; st, stria termin—
alis; v, ventricle; vmh, ventromedial hypo—
thalamus.

 

 

55
on lordosis were tested 15, 45, and 90 min after infusion.

A single behavioral test session was conducted.

RESULTS

Carbachol (0.5 ug/cannula) blocked the inhibitory
effect of HC-3 (1.25 ug/cannula) on lordosis in females
primed with various doses of EB (0.13, 0.17, 0.25, 0.5 ug)
in combination with progesterone (0.5 mg). Reduction in
the incidence of lordosis was most pronounced 45 min after
infusion of HC-3 at all doses of EB treatment (Figure 10,
upper panel). In order to compare the effects of HC—3, and
HC—3 plus carbachol, over all four doses of EB, linear
regression lines of L0 on log transformed EB doses were
generated. The lines illustrated in the lower panel of
Figure 10 represent regression functions of pretest and 45
min test LQ recorded at the four doses of EB priming.
Analysis of variance indicated that regression functions
were statistically equivalent for pretests before HC—3,
pretests before HC—3 plus carbachol, and tests 45 min
after HC—3 plus carbachol. The regression line for tests
45 min after HC-3 was significantly different from the
counterpart line for tests 45 min after HC—3 plus carbachol
(p <.05). The regression line for tests 45 min after HC-3
was also significantly different from a regression line
formed by pooling the regression functions of the three
other lines (p< .01). In addition, at the 0.13 ug dose of

EB, a substantial and transient increase in the incidence

 

 

Figure 10.

56

Upper panel. Mean lordosis quotients recorded
from female rats following bilateral infusion

of hemicholinium—3 (HO-3, 1,25 ug/cannula) or
HC-3 (1.25 ug/cannula) plus carbachol (0.5 ug/
cannula) into the medial preoptic area. Var-
ious doses of estradiol benzoate (EB) were
injected 72, 48, and 24 hr before behavioral
testing and 0.5 mg progesterone was injected

4—5 hr before testing. Each female received
only one behavioral test session. Intracerebral
treatment was administered immediately following
the preinfusion test (PT). The effect of intra-
cerebral treatment on lordosis was tested 15, 45,
and 90 min after infusion. For each treatment
combination, n = 9—10. Lower panel. Linear re-
gression lines of lordosis quotient on loglo
transformed EB doses for pretests and 45 min
tests.

 

 

 

MEAN LORDOSIS QUOTIENT

 

57

 

  

10°—
._. HC-3 °~,
\
0---o HC-3+carbachol ‘ ‘o--..°.——‘°
o
1' ‘x‘
80- I ~.‘
I' \
I \
A I \ A
A\\ X
60- ° A
‘1"
:\\ A
l \
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I" ‘0 A ’ °‘\ A
" \\ °\ \‘
1 ‘\ ‘ ‘\
20- 1' ‘\ ‘°
i “o
I
'1 A
°\A\‘_ A/
PT IS’ 45’ 90' PT 15’ 45’ 90’ PT IS’ 45’ 90’ PT ‘5’ 45’ 90’
0.13 ug 0.17 ug 0.25 ug 0.5 ug
EB PRIMING DOSE
(+0.5 Mg PROGESTERONE)
HC-3+CARB (PT) HC-3 (PT)
I
100 j” HC-3+CARB (45’)
’I ”l
80
.—
z
E
5 HC-3 (45')
I
D 60
O
2
m
0 40
O
n:
0
d
20

 

0.2 5 0.5

log 23 oosr:

 

 

58
of lordosis was observed after infusion of HC-3 plus carb-
achol (Figure 10, upper panel), similar to that seen in
Experiment 1 after infusion of carbachol alone.

Results indicate that the incidence of lordosis on
pretests varied as a function of the dose of EB which was
administered in conjunction with progesterone. This
relationship between L0 and EB dose was maintained 45 min
after infusion of HC—3 plus carbachol. In contrast, LQ was
dependent on EB dose 45 min after infusion of HC—3 alone,
but was reduced at all doses of EB priming.

Infusion of HC—3 consistently inhibited the display of
soliciting behaviors, as well as lordosis. However, carb—
achol failed to maintain soliciting behaviors in females

treated with HC-3 plus carbachol.

SUMMARY

In Experiment 4, the inhibition of lordosis by HC-3
(1.25 ug/cannula), an acetylcholine synthesis blocker, was
prevented when carbachol (0.5 ug/cannula), a cholinergic

receptor agonist, was infused along with HC—3.

 

 

EXPERIMENT 5. Alterations in Cholinergic Binding Sites
within Specific Brain Areas Following
Estrogen Treatment
The results of Experiments 1-4 indicate that pharma-
cological manipulations of the cholinergic system influence
the sexual behavior displayed by female rats. In particu-
lar, stimulation of cholinergic receptors in certain brain

regions increased the probability of lordosis, but only

 

following pretreatment with low doses of estrogen (Experi-
ments 1 and 2). This estrogen—dependent facilitation of
lordosis by a cholinergic receptor agonist suggests that
estrogen may alter specific cholinergic processes within
the brain. Recent evidence has, in fact, confirmed an
increase in cholinergic muscarinic binding in certain
brain regions following estrogen treatment of female rats
(Rainbow, DeGroff, Luine, and McEwen, 1980). Two days of
systemic treatment with 10 ug EB increased in 33332 mus-
carinic binding in whole hypothalami, as well as in more
discrete preparations of VMH and anterior hypothalami.

In Experiment 5, muscarinic binding was analyzed in
various brain areas from female rats which had been treated
with several doses of estrogen (0.125, 1, or 10 ug EB
for 3 days). Muscarinic agonists have been shown to facili-
tate lordosis following implantation or infusion into the
POA or VMH of females primed with 0.13 ug EB (Experiments

59

 

60
1 and 2) or 1 ug EB (Clemens et al., 1980). It was
suspected that these low priming doses of EB might increase
the sensitivity of muscarinic receptors in the POA and VMH,
thereby promoting the action of cholinergic receptor
agonists. An increase in receptor sensitivity could result
from alterations in receptor number or receptor affinity
(Scatchard, 1949). Changes in muscarinic binding were
assessed in zitrg using 3H quinuclidinyl benzilate (QNB),

a high affinity muscarinic antagonist.

METHOD

Two wk after ovariectomy, female rats were injected
intramuscularly with EB (0.125, 1, or 10 ug) or 0.1 cc
sesame seed oil 72, 48, and 24 hr before sacrifice°
Following decapitation, the brain was rapidly removed and
various areas were dissected in ice—cold saline according
to the method of Luine, Khylchevskaya, and McEwen (1974).
Brain areas assayed included the POA, medial basal hypo—
thalamus (MBH), and portions of the caudate nucleus and
parietal cortex. Fresh tissues were homogenized in 1 ml
of ice-cold 320 mM sucrose° Homogenates were centrifuged
at 1000 g for 10 min at 4° C. Twenty-five ul aliquots of
low—speed supernatant (5—30 ug protein) were incubated for
1 hr at 370 C in 207 ul of potassium phosphate buffer con-
taining 320 mM sucrose, 10 mM potassium phosphate, 1 mM
EDTA, and 0.5 mM ascorbate (pH 7.4), with 1 nM 3H l—QNB
(specific activity 40 Ci/mmole, New England Nuclear). The

reaction was terminated by filtration through Whatman GF/B

 

 

61
filters under vacuum. Filters were transferred to glass
scintillation vials and allowed to dry. Ten ml of Econo-
fluor counting solution (New England Nuclear) was added
and samples were counted 24 hr later at 45% efficiency.
In order to determine the amount of nonspecific binding,
parallel incubations were run in a potassium phosphate
buffer containing 1 nM 3H QNB and 1 uM atropine sulfate
(Sigma Chemical 00.). Specific binding was defined as
.binding in the absence of atropine sulfate minus binding
in the presence of atropine sulfate. Protein content of
each sample was determined by the method of Bradford (1976)
with bovine-serum albumin as standards. In this assay
system, 3H QNB binding increased linearly over a protein
concentration range of 5—60 ugo Further, the cholinergic
receptor agonist, carbachol, inhibited 3H QNB binding
(IC50= 60 uM) as reported previously (Yamamura and Synder,
1974; Jones and Sumikawa, 1981). In order to differentiate
changes in binding site number from changes in binding site
affinity, isotherms were generated by incubating 25 ul
aliquots of supernatant with several concentrations of

3H QNB (0.025—1 nM).

RESULTS

The effects of EB treatment on 3H QNB binding are
presented in Figure 11 and Table 3. Systemic injection of
EB for 3 days increased specific binding of 3H QNB in the
MBH of female rats at 1 ug (p< .05, vs. oil, two-tailed

Mann—Whitney U-test following Kruskall—Wallis analysis of

 

 

Figure 11.

62

5
o
0

Q
0
O

8
o

  
 

QNB BOUND
(fmoI/mg protein)
8
o

N
O
O

ESTRADIOL BENZOATE DOSE
Wewaw

Specific binding of 3H quinuclidinyl benzi-
late (QNB) in synaptosomal preparations of
tissue from the medial basal hypothalamus
(MBH; left) and medial preoptic area (POA;
right) of female rats. Ovariectomized females
were injected intramuscularly with estradiol
benzoate (EB, 0.125, 1, or 10 ug) or 0.1 ml
sesame seed oil 72, 48, and 24 hr before sacri-
fice (n = 5—11 per treatment). Specific
binding was defined as total binding in the
presence of 1 nM 3H QNB minus nonspecific
binding in the presence of 1 nM 3H QNB and 1
uM atropine sulfate. (*}><.05, ** p< .01;

EB VS. oil, two-tailed Mann-Whitney U).

 

 

 

63

 

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PSMOHMHQmHm oz .Pxop mnp CH anfipommv ohm hdmmd map Mo mHHumQ .EMm H sflopohg we

9mm UCZoQ mHHmOHMHoQO mPMHHNCwQ HhQHUHH05QHSd mm HoEw mm Ummmmhgxo opd mosaw>m
.APCmEmehP
9mm Halo n Qv moaMHHowm whomwn p: :m 6cm .m: .Nm UomemHCHEUw mm; HHo ho mm

 

 

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HmH H mmmH mmH H moom wmm H mowH mom H wmmH sweepzm
mpdbsmO

HNH H mmsH 90H H HHoH NNH H momH Nomm H osmH waHoo
HapmHHmm

m: 0H m: H ms mmH.o HHHo <mm<

 

 

PsosPMoHB Ammv mpdoasmm Hoawmgpmm wQHSOHHom swampsm mpmusmv USN
xmppoo Hapwflamm map CH wsfidsflm oHCHHmomSS

m MHm<E

64
variance) and 10 ug doses (p <.01). No significant
alteration in 3H QNB binding in the MBH was detected
following treatment with 0.125 ug EB for 3 days. In
contrast, administration of EB decreased specific binding
of 3H QNB binding in the POA at 1 and 10 ug doses (p <.01),
but failed to alter binding at the 0.125 ug dose. EB
treatment did not significantly affect 3H QNB binding at
any dose level in the parietal cortex or caudate putamen
as indicated by Kruskall—Wallis analysis of variance
(Table 3).

Analyses of the binding kinetics for MBH and POA
tissue are presented in Figure 12. An estimate of binding
site affinity is represented by the apparent dissociation
constant or KD (negative reciprocal of the slope). An
estimate of binding site number per mg of protein is
provided by the Bmax (intercept of the x axis). EB (10 ug)
increased the number of muscarinic binding sites per mg
protein in the MBH, indicated by an increase in Bmax’ and
decreased the number of muscarinic binding sites per mg
protein in the POA, indicated by a decrease in Bmax‘ EB
did not alter binding site affinity (KD) in either the
MBH or POA.

SUMMARY

The results of Experiment 5 confirm the increase in
muscarinic binding previously reported (Rainbow et al.,
1980) in the MBH of female rats treated with 10 ug EB.

A similar increase in binding was observed at 1 ug EB.

 

 

 

Figue 12.

65

Scatchard plot of specific 3H quinuclidinyl
benzilate (QNB) binding in the medial basal
hypothalamus (MBH; upper) and medial preoptic
area (POA; lower) of female rats treated with
estradiol benzoate (EB, 10 ug) or 0.1 ml sesame
seed oil 72, 48, and 24 hr before sacrifice.
Binding isotherms were generated by incubating
synaptosomal preparations with several concen—
trations of 3H QNB (0.025—1 nM). The free con-
centration of H QNB present in the incubation
golume was defined as total H QNB added minus
H QNB specifically bound. KD represents an
estimate of binding site affinity. Bmax
represents an estimate of binding site number
per mg of protein. Each point is the mean of
2—3 determinations.

 

KD (nM) Bmax (fmol/mg)
OIL EB OIL EB
MBH .044 .043 647 819

POA .024 .023 1001 599

 

 

BOUND/FREE (fmoI/mg protein—nM) x10'4

66

4‘ MBH

 

 

 

260 600 Iobo

     

POA

 

 

260 660 Iobo

BOUND (fmol/mg protein)

 

67
However, muscarinic binding was not enhanced in the MBH
following treatment with 0.125 ug EB, a priming dose found
to be effective in behavioral Experiments 1 and 2.
Furthermore, EB treatment (1 or 10 ug) actually reduced
muscarinic binding in the POA. Scatchard analysis indicated
that the alterations in muscarinic binding induced by
estrogen were due to changes in the number of binding sites

rather than affinity of binding sites.

 

 

 

DISCUSSION

Cholinergic activity in specific areas of the female
rat brain may play a significant role in the regulation of
sexual behavior. The potential of ovarian hormones to
control biological functions by altering protein synthesis,
and specifically neurotransmitter activity, forms the basis
of this hypothesis. In the present series of experiments,
female sexual behavior, indicated by the incidence of
lordosis, was significantly increased in estrogen—primed
female rats following bilateral infusion of a cholinergic
receptor agonist, carbachol (0.5 ug/cannula), into the POA
or VMH. The behavioral response was prompt and transient,
occurring within 15 min after infusion and persisting for
less than 90 min. At low levels of estrogen priming, intra-
cerebral carbachol activated lordosis more effectively than
systemic progesterone. Carbachol failed to facilitate
lordosis following infusion into the MRF or frontal cortex,
suggesting site—specificity. Furthermore, forebrain in-
fusion of an agent which retards acetylcholine synthesis,
HC-3 (1.25, 5, or 7.5 ug/cannula), reduced the incidence
of lordosis in estrogen-progesterone-treated females. This
action was specific to the cholinergic system since carb-
achol (0.5 ug/cannula) or choline chloride (120 ug/cannula)
prevented the inhibition of lordosis by HC-3. Finally,

68

 

 

 

 

 

69
estrogen treatment (1 or 10 ug EB for 3 days) was found to
increase cholinergic muscarinic binding in the MBH and
decrease muscarinic binding in the POA. However, an
expected increase in muscarinic binding within these areas
was not observed at the estrogen-priming dose (0.125 ug)
utilized in behavioral experiments.

These experiments confirm and extend recent findings
which indicate that central cholinergic activity may in-
fluence hormonally—activated sexual behavior. An increase
in lordotic behavior has been observed following systemic
administration of nicotine to estrogen-primed females (Fuxe
et al., 1977). This facilitative action of nicotine was
prevented by prior treatment with a nicotinic receptor
blocker but was unaltered by pretreatment with dopaminergic
antagonists. More recently, muscarinic agonists in
crystalline form were found to increase lordosis in
estrogen-primed females when implanted into either the POA
or the MRF (Clemens et al., 1980). The facilitation of
lordosis by central stimulation of muscarinic receptors
was blocked by pretreatment with atropine sulfate, a cho-
linergic muscarinic receptor blocker.

Earlier reports by Lindstrom described both facili—
tative and inhibitory roles of cholinergic stimulants in
the mediation of lordosis. Cholinergic agonists pilo-
carpine, oxotremorine, and arecoline were initially found
to reduce lordotic behavior in females treated with estro-

gen and progesterone (Lindstrom and Meyerson, 1967). A

 

 

7O
serotonergic mechanism appeared to be implicated, however,
since pretreatment with parachlorophenylalanine blocked the
inhibitory effect of pilocarpine (Lindstrom, 1971). Pilo-
carpine and oxotremorine were later shown to increase lor-
dotic behavior in estrogen-primed females 4 hr after sys—
temic injection (Lindstrom, 1973). The facilitation was
blocked by atropine sulfate, reinforcing a cholinergic
interpretation. However, the enhancement of lordosis was
attributed to a drug-induced release of adrenal progestins

since adrenalectomy abolished the increase. The discrep-

 

ancy between the effects observed by Lindstrom following
systemic administration of cholinergic agents and data
recorded in our laboratory may be related to the mode of
drug delivery. Intracerebral administration of cholinergic
agonists in specific brain areas has been found to activate
a prompt lordotic response which persisted in the absence

of the adrenal glands (Clemens et al., 1980). A facili—

 

tation of lordosis by manipulations of the cholinergic
system appears to be predicated on the discrete chemical
stimulation of certain areas of the rat brain.

In Experiment 1, bilateral infusion of carbachol into
the POA facilitated lordosis in ovariectomized female rats
under several estrogen and estrogen-progesterone regimens.
Estrogen and progesterone were administered systemically;
carbachol was administered intracerebrally. As shown in
Figure 2, the various doses of estrogen (0.13, 0.17, and

0.25 ug EB) activated only low levels of lordosis even

71
when combined with progesterone (0.5 mg). However, when
carbachol was infused into the POA (0.5 ug/cannula) of
females receiving estrogen or estrogen and progesterone, a
significant increase in the incidence of lordosis was
observed. Carbachol facilitated lordosis at lower doses of
estrogen priming than did progesterone. Furthermore, the
combination of carbachol and progesterone did not increase
the incidence of lordosis in estrogen—primed females beyond
that achieved by carbachol alone. Although the possibility
exists that the doses of carbachol and progesterone were
not behaviorally comparable, even higher doses of proges-
terone (0.75 or 1 mg) fail to increase lordosis signifi—
cantly at these low levels of estrogen priming (Clemens,
Huang, and Caldwell, unpublished data). These doses of
estrogen probably do not induce sufficient progestin re—
ceptors for progesterone to achieve a behavioral effect
(Moguilewsky and Raynaud, 1977; Blaustein and Wade, 1978;
MacLusky and McEwen, 1978). The disparity observed between
the behavioral effects of carbachol and progesterone may
indicate that carbachol and progesterone achieve their
effects by separate mechanisms.

Lindstrom (1973) suggested that the ability of system—
ically administered cholinergic agonists, pilocarpine and
oxotremorine, to facilitate lordosis was dependent upon a
drug-induced release of adrenal steroids. In contrast, it
has been reported that the lordotic response elicited by

intracerebral treatment with muscarinic agonists persisted

 

 

72

in the absence of the adrenal glands (Clemens et al., 1980).
Data from Experiment 1 confirm this result; systemic pro-
gesterone failed to facilitate lordosis at low levels of
estrogen priming, whereas, intracerebral carbachol was
effective at these same estrogen doses. It is doubtful
that progesterone released from the adrenals would exert
an action at low estrogen levels when an optimum dose of
exogenous progesterone was ineffective.

The transient nature of the behavioral response to

stimulation by carbachol also disputes the importance of

 

adrenal steroid action. Maximal levels of lordosis were
observed 15 min after intracerebral infusion of carbachol
(Figure 2). The response began to wane at 45 min and had
returned to control levels by 90 min. Lordosis activated
by adrenal steroids would likely persist at high levels
beyond 90 min after release. For example, the incidence

of lordosis observed following ether—induced release of

 

adrenal steroids in female rats has been found to increase
linearly from the time of exposure to ether before peaking
at 120—180 min (Franck, 1977). The prompt behavioral
response to intracerebral treatment followed by a gradual
decline to control levels suggests a discrete stimulation
of central cholinergic receptors by carbachol.

In Experiment 2, the facilitation of sexual behavior
by intracerebral infusion of carbachol was found to be a
site—specific phenomenon. Specifically, bilateral infusion

of carbachol (0.5 ug/cannula) into either the POA or the

 

73
VMH increased the incidence of lordosis in estrogen—primed
female rats. However, carbachol failed to activate lor-
dosis following infusion into the MRF or frontal cortex.
The potential of carbachol to facilitate lordosis following
infusion into a particular brain region may be related to
the ability of that region to concentrate and respond to
estrogen.

Intracerebral implants of estrogen have been shown to
be effective in activating sexual behavior when located in
the VMH (Barfield and Chen, 1977; Davis, McEwen, and Pfaff,
1979; Rubin and Barfield, 1980). It was suspected that
carbachol might induce an even greater behavioral response
following infusion into the VMH compared to the POA.
However, the degree of facilitation of lordosis was similar
following cholinergic stimulation of the VMH and POA at
this dose of estrogen priming. Since other behavioral
results suggest that the VMH is uniquely sensitive to
estrogen, regional differences in the response of the POA
and VMH to cholinergic stimulation may be evident at lower
estrogen priming doses or lower carbachol doses.

Neither the MRF nor cortex concentrates estradiol as
efficiently as the POA and VMH (Pfaff and Keiner, 1973).
Furthermore, estrogen implants in the MRF facilitated lor—
dosis only when combined with systemic progesterone (Ross
et al., 1971; Yanase and Gorski, 1976). Since these areas
appear to be less sensitive to estrogen it is not sur—

prising that estrogen-dependent cholinergic effects were

 

 

74
not observed at the low dose of estrogen priming (0.13 ug
EB for 3 days) found to be effective in the POA and VMH.
Although microinfusion of carbachol in the MRF has proven
to be ineffective, it has been reported previously that
crystalline carbachol activated lordosis when implanted in
the MRF of female rats primed with 1 ug EB for 3 days
(Clemens et al., 1980). The effectiveness of crystalline
carbachol in the MRF may be related to the high dose of
carbachol delivered to brain tissue (approximately 10 ug/
cannula) and a potentially wider locus of stimulation.

A variety of evidence suggests that the POA and VMH
exert different actions in mediating female sexual behavior.
For example, although lesions of the VMH inhibited lordosis
in estrogen-treated female rats (Mathews and Edwards, 1977),
lesions of the POA increased the potential of estrogen—
treated females to display lordosis (Powers and Valenstein,
1972). Furthermore, electrical stimulation of the VMH has
been reported to facilitate lordosis (Pfaff and Sakuma,
1979) while stimulation of the POA inhibited lordosis
(Malsbury and Pfaff, 1973). These results seem to suggest
that the VMH exerts excitatory control over estrogen-
induced lordosis and the POA exerts tonic inhibitory
control. Appropriately, estrogen has been shown to
increase the number of cells in the VMH diSplaying detect-
able spontaneous activity while decreasing their number in
the POA (Bueno and Pfaff, 1976).

However, cholinergic stimulation of either the VMH or

 

 

 

 

 

75
POA under identical conditions facilitated lordosis in
estrogen-treated female rats in Experiment 2. This finding
may indicate that intracerebral stimulation with the cho-
linergic agonist carbachol activates an excitatory circuit
in the VMH but suppresses an inhibitory circuit in the POA.
Alternatively, both inhibitory and excitatory components of
a neural circuit controlling lordosis could exist in the
POA along with an excitatory component in the VMH. This
latter suggestion is supported by the heterogeneous
response of POA neurons observed during somatosensory
stimulation of the perineum (Bueno and Pfaff, 1976).

The possibility that the facilitation of lordosis by
chemical stimulation of the VMH or POA occurs after the
cholinergic agent has diffused away from the site of
infusion cannot be entirely dismissed at present. However,
a behavioral response that results from the diffusion of
carbachol across the brain substance which separates the
POA and VMH seems unlikely. Available evidence indicates
that, in general, drug diffusion is largely limited to a
1 mm radius around the infusion site when infusion volumes
of less than 1 ul are employed (Rech, 1968; Myers, Tytell,
Kawa, and Rudy, 1971).

In Experiment 3, a significant inhibition of sexual be—
havior was observed following intracerebral treatment with
an agent known to disrupt cholinergic function. HC—3, a
compound which retards acetylcholine synthesis (MacIntosh et

al., 1956; Gardiner, 1961; Guyenet et al., 1973), reduced the

 

 

 

76
incidence of lordosis in estrogen-progesterone-treated
females following bilateral infusion into the POA or stria
terminalis. Similar reductions in the incidence of
lordosis have been observed following systemic treatment
with high doses (10 mg) of the cholinergic receptor
antagonist, atropine sulfate (Singer, 1968). The ability
of cholinergic antagonists such as HC—3 and atropine
sulfate to inhibit lordosis in sexually receptive female
rats indicates that cholinergic activity in Specific areas

of the brain may play an important role in the mediation

 

of this hormone-dependent behavior.

The effect of HC-3 reported in Experiment 3 was both
localized and specific. The inhibition of lordosis was
observed only after HC-3 infusion into sites known to con-
centrate estrogen (Pfaff and Keiner, 1973). Furthermore,
HC-3 failed to significantly reduce the incidence of lor-
dosis when choline chloride was added to the infusate. The
decline in acetylcholine levels following intraventricular
infusion of HC—3 can be counteracted by simultaneous
infusion of choline chloride (Slater, 1968). Evidence
from.in 31352 preparations indicates that HC-3 inhibits the
high affinity uptake of choline necessary for the intra-
neuronal synthesis of acetylcholine(MacIntosh et al., 1956;
Gardiner, 1961; Guyenet et al., 1973). However, the
mechanism by which HC-3 alters in vixg acetylcholine syn-
thesis is not completely understood. Although intraven-

tricular infusions of low doses of HC—3 (0.3—10 ug)

77
effectively deplete acetylcholine levels, concomitant

1LPG-choline uptake has not been

inhibition of central
demonstrated (Robinson, 1970; Freeman et al., 1975).
Choline chloride does prevent both depletion of acetyl-
choline levels (Slater, 1968) and high affinity choline
uptake (Robinson, 1970) when infused with higher intraven—
tricular doses of HC-3 (10—300 ug). A recent report
(Freeman, Macri, Choi, and Jenden, 1979) suggests, however,
that lower in £112 doses of HC-3 probably reduce endogenous
acetylcholine levels by inhibiting high affinity uptake of
choline. In Experiment 30, the inhibitory effects of HC-3
(5 ug/cannula) on lordosis were counteracted by simultaneous
administration of choline chloride (120 ug/cannula). This
dose of HC-3 may have been effective in arresting choline
uptake within a localized area around the cannula tip.
Presumably the functional integrity of cholinergic processes
within this region is vital to the full display of lordosis.
Behavioral inhibition was observed within 15 min after
HC—3 infusion in Experiment 3a and within 30 min after HC-3
infusion in Experiments 3b and 30. Similarly, whole brain
acetylcholine levels have been reported to be significantly
depleted within 10 min after intraventricular infusion of
HC-3 (Slater, 1968), while lowest acetylcholine levels were
detected after several hours (Freeman et al., 1975, 1979).
In Experiment 30, the inhibition of lordosis by HC-3 was
completely prevented 30 min after infusion of HC-3 with

choline chloride. However, by 60 min after infusion, the

78
behavioral inhibition by HC-3 was only partially arrested.
This waning effect may have been the result of a rapid
transport of excess choline from the brain (Ansell and
Spanner, 1968).

A tolerance to the inhibitory effects of HC-3 was
observed in some groups during the second wk of behavioral
testing. This tolerance to HC-3 was prevented when the dose
of HC-3 was increased (Experiment 3a) or when the priming
dose of EB was decreased (Experiment 3b). Consequently, it
seems likely that repeated steroid treatment induced a
behavioral tolerance to HC-3 by the second wk of testing.
This explanation seems most tenable since the occurrence of
tolerance to HC-3 was clearly dependent upon the relative
amount of EB administered and the period of exposure.

Selye (1971 a, b) has demonstrated the protection afforded
by steroids against toxic levels of neurotropic agents.
Estrogen may heighten the activity of various enzyme
systems which degrade and inactivate the neurotropic drug.
Alternatively, repeated steroid exposure may have accel—
erated acetylcholine synthesis such that the lower dose of
HC—3 (5 ug/cannula) was less effective on the second wk.

In Experiment 4, bilateral infusion of HC—3 (1.25 ug/
cannula) into the POA also reduced the incidence of lordosis
below pretest levels in females receiving various doses of
EB (0.13, 0.17, 0.25, and 0.5 ug) and progesterone (0.5 mg).
At each EB dose, the greatest degree of behavioral sup-

pression was recorded 45 min after infusion of HC—3.

 

 

79
Endogenous acetylcholine levels are reported to be signifi-
cantly depleted at 1 hr after intraventricular infusion of
comparable quantities of HC-3 (Robinson, 1970). Lordosis
was maintained at pretest levels when carbachol (0.5 ug/
cannula) was infused along with HC-3 in females receiving
0.17, 0.25, and 0.5 ug EB with progesterone. Since both
carbachol (Experiment 4) and choline chloride (Experiment
3c) were found to counteract the inhibitory effect of HC-3
on lordosis, a specific involvement of the cholinergic
system is clearly indicated.

The ability of cholinergic manipulations to influence
sexual behavior suggests tnrtovarian hormones alter cho-
linergic processes within the brain. Neuronal exposure to
estrogen might promote acetylcholine synthesis, alter cho-
linergic receptor binding, or both. Recently, the activity
of choline acetyltransferase was reported to be elevated
significantly in the POA of ovariectomized female rats
following EB treatment, suggesting increased acetylcholine
synthesis (Luine, Khylchevskaya, and McEwen, 1975; Luine,
Park, Tong, Reis, and McEwen, 1980). In Experiment 4, the
contribution of acetylcholine synthesis was minimized by
treating the POA with an agent, HC-3, which reduced intra-
neuronal synthesis of acetylcholine. Nevertheless, carb-
achol infusion maintained lordosis at pretest levels
previously recorded for each dose of EB. Even in the
absence of significant acetylcholine synthesis, carbachol

facilitated lordosis in direct proportion to the priming

 

 

 

80
dose of EB. This observation may suggest that estrogen
acts to enhance cholinergic processes other than synthesis.
It is possible that estrogen increased the number or
affinity of cholinergic receptors in the POA. Higher doses
of EB (0.25 or 0.5 ug) would promote greater enhancement of
cholinergic receptor sensitivity than lower doses of EB
(0.13 or 0.17 ug), thereby accomodating greater behavioral
effectiveness of a receptor mimetic such as carbachol.
However, we cannot exclude the possibility that HC-3
reduced acetylcholine synthesis in varying degrees as a
function of the dose of EB administered. Consequently,
acetylcholine synthesis may be an important factor at
certain EB doses, even in the presence of HC-3.

In order to determine directly whether estrogen alters
the sensitivity of cholinergic muscarinic receptors in the
brain, specific binding of a high-affinity radiolabelled
muscarinic antagonist, 3H QNB, was analyzed in discrete
areas of the female rat brain in Experiment 5. Previous
evidence indicated that high doses of estrogen (10 ug EB
for 2 days) increased the number of muscarinic binding
sites in whole hypothalamus, and specifically in the VMH
and anterior hypothalamus (Rainbow et al., 1980). It was
hypothesized that lower estrogen priming doses (0.125 or 1
ug EB for 3 days) used in behavioral experiments (Experi-
ments 1 and 2, Clemens et al., 1980) would also increase
muscarinic binding in the POA and MBH, and consequently

facilitate the action of cholinergic receptor agonists.

 

81
However, in Experiment 5, the estrogen priming dose (0.125
ug EB) found to be effective in Experiments 1 and 2 failed
to increase muscarinic binding in either the POA or MBH.
Higher estrogen doses (1 or 10 ug EB) did increase binding
in the MBH, confirming earlier findings (Rainbow et al.,
1980). Scatchard analysis revealed that this alteration
was the result of an increase in the number of muscarinic
binding sites (Bmax) rather than an increase in binding site
affinity (KD). Surprisingly, higher doses of estrogen (1
or 10 ug EB) decreased muscarinic binding in the POA,
specifically by reducing the number of binding sites.
There was no effect of estrogen treatment on muscarinic
binding in two control areas, the parietal cortex and
caudate putamen.

The failure to detect increases in muscarinic binding
in the POA and MBH at the lowest estrogen dose (0.125 ug EB)
may be due to the insensitivity of the assay system. Alter—
natively, alterations in the number of binding sites for a
cholinergic agonist such as carbachol might not be evident
when receptors are labelled with a cholinergic antagonist
such as 3H QNB. Evidence now indicates that muscarinic
binding sites exist in three distinct forms with different
affinities for agonists but similar affinities for antag-
onists (Birdsall, Burgen, Hiley, and Hulme, 1976). The
distribution of the three agonist binding sites varies
throughout the rat brain and high affinity sites have been

suggested to represent degraded forms of the low affinity

 

 

 

82
site (Wamsley, Zarbin, Nigel, Birdsall, and Kuhar, 1980).
Consequently, the effect of estrogen on the level of the
biologically active low affinity receptor may be concealed
in an assay system using a muscarinic antagonist such as
3H QNB.

Although infusion of carbachol into the POA con-
sistently facilitated lordosis in estrogen—primed females
(0.125 ug EB), a decrease in muscarinic binding sites was
observed in the POA following estrogen treatment at higher

doses (1 or 10 ug EB). This result was quite unexpected

 

and indicates that the cholinergic mechanism controlling
lordosis may be complex. For example, evidence suggests
that estrogen may not alter the same cholinergic parameters
in the POA as in the MBH. In particular, the activity of
CAT, the primary enzyme regulating acetylcholine synthesis,
has been reported (Luine et al., 1975) to increase in the
POA of female rats treated with estrogen (5 ug EB for 3

days). Since this hormonal regimen failed to alter CAT

 

activity in the MBH, it appears that estrogen may modify
cholinergic activity in different brain areas by different
mechanisms. Thus, estrogen might promote cholinergic
function in the POA by elevating CAT activity and conse—
quently acetylcholine synthesis, and in the MBH by increas—
ing the number of cholinergic binding sites. Together,
present evidence suggests that cholinergic activity in the
POA and VMH exerts an important influence of female sexual

behavior and that estrogen promotes cholinergic function

83

in these areas, but by different mechanisms.

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