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.41

THESIS

lllllllllllllllllllll

This is to certify that the

thesis entitled

THE ROLE OF NOREPINEPHRINE IN THE EXPRESSION
OF LORDOSIS IN FEMALE RATS

presented by
BECKY L. DAVIS

has been accepted towards fulfillment
of the requirements for

- M. 5. degree in ZOOLOGY

QMWW
/ Major professor
Date @2234? 7 L

07639 MSU is an Affirmative Action/Equal Opportunity Institution

 

 

LEBRARY
'Mi itigan 3%2‘

; University

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

DATE DUE DATE DUE DATE DUE

W 7’:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmative Action/Equal Opportunity Institution

c :\circ\datedue. omit-9.1

THE ROLE OF NOREPINEPHRINE IN THE EXPRESSION
OF LORDOSIS IN FEMALE RATS

BY

Becky L. Davis

A THESIS

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

MASTER OF SCIENCE

Department of Zoology

1992

ABSTRACT

THE ROLE OF NOREPINEPHRINE IN THE EXPRESSION
OF LORDOSIS IN FEMALE RATS

BY

Becky L. Davis

The importance of NE for lordosis in female rats and its
site of action in hormone-sensitive brain regions controlling
receptivity were examined. Ovariectomized, hormone-treated
female rats underwent surgical procedures and/or drug
treatments and were tested for sexual receptivity. It was
found that systemic injection of either an al- or B-antagonist
failed to block lordosis in receptive females and injection of
NE into the VMN of unreceptive females failed to facilitate
lordosis. Neurotoxic lesions of the ventral noradrenergic
bundle (VNAB) reduced NE concentrations in the VMN and MPN and
inhibited lordosis. Lordosis was restored with i.c.v.
administration of an al-receptor agonist. Neurotoxin
injection into either the VMN or MPN reduced NE concentrations
in these nuclei, but failed to alter lordosis. Furthermore,
injection of the al agonist into either the VMN or MPN of
VNAB-lesioned females failed to reinstate lordosis. These
results indicate that while NE is important for lordosis,
noradrenergic neurons terminating in either the VMN or MPN are
not essential for hormonal induction of sexual receptivity in

female rats.

 

1'0 my loving and thoughtful lather, Carolyn J. Burke

iii

ACKNOWLEDGEMENTS

I would first like to greatfully acknowledge my major
professor Dr. Lynwood Clemens for providing me with the
opportunity and financial support to complete this work. I
would also like to thank the members of my committee Drs. Fred
Dyer and Keith Lookingland for their time and patience. I
would especially like to extent appreciation to Dr. Keith
Lookingland for his invaluable assistance and guidance on a
major portion of this project. My appreciation must also go
to Dr. Jorge Manzanares for his extremely helpful technical
assistance. I also wish to sincerely thank my dear friend Dr.
Christine Wagner for her constant moral support and
companionship and Kevin Sinchak for his friendship and
helpfulness. Finally, I wish to acknowledge the guidance and
assistance of my best friend and husband Dr. Marc Bailie whose
patience and love has made the completion of this effort
possible.

iv

LIST OF TABLES . .
LIST OF FIGURES .
INTRODUCTION . .

EXPERIMENT 1:

METHODS .
RESULTS . .
DISCUSSION .
EXPERIMENT 2:
female rats . .
METHODS . .
RESULTS . .
DISCUSSION .
EXPERIMENT 3:
treated female rats
METHODS . .
RESULTS . .
DISCUSSION .
GENERAL DISCUSSION .

LIST OF REFERENCES .

NE in either
essential for lordosis in

the VMN or MPN is not
estrogen/progesterone-

TABLE OF CONTENTS

Injection of NE into the VMN
fails to facilitate lordosis in estrogen- -treated

Systemic injection of either a1-
or B-adrenergic antagonists fail to inhibit lordosis
in estrogen/progesterone-treated female rats

12

13

16

23

27

27

3O

30

34

35

41

49

58

66

LIST OF TABLES

Table 1 Effect of bilateral injections of 5-ADMP
into the ventral noradrenergic bundle (VNAB)
on amine concentrations in the ventromedial
nucleus (VMN) and medial preoptic nucleus (MPN)
of ovariectomized, estrogen/progesterone-treated
female rats . . . . . . . . . 42

vi

LIST OF FIGURES

Figure 1 Effects of propranolol on lordosis
in ovariectomized, estrogen/progesterone-
treated female rats of two different
strains . . . . . . . .

Figure 2 Effects of phenoxybenzamine
on lordosis in ovariectomized,
estrogen/progesterone-treated
female rats of two different strains .

Figure 3 Effects of propranolol on lordosis
in ovariectomized estrogen/progesterone-
treated female rats given various
treatments of estrogen . . . . .

Figure 4 Effects of phenoxybenzamine
on lordosis in ovariectomized
estrogen/progesterone-treated female
rats given various treatments of estrogen

Figure 5 Effect of bilateral injections
of NE into the ventromedial nucleus
(VMN) on lordosis in ovariectomized,
estrogen-treated female rats . . .

Figure 6 Effects of bilateral injections
of 5-ADMP into the ventral noradrenergic
bundle (VNAB) on lordosis in ovariectomized,
estrogen/progesterone-treated female rats

Figure 7 Effect of phenylephrine on
lordosis in VNAB-lesioned ovariectomized,
estrogen/progesterone—treated female rats

Figure 8 Effects of bilateral injections
of 5-ADMP into the ventromedial nucleus
(VMN) on NE concentrations in the VMN
and lordosis in ovariectomized,
estrogen/progesterone-treated female rats

18

20

23

25

32

44

46

48

Figure 9 Effects of bilateral injections
of 5-ADMP into the medial preoptic
nucleus (MPN) on NB concentrations
in the MPN and lordosis in ovariectomized,
estrogen/progesterone-treated female rats . . 51

Figure 10 Effects of administration of
phenylephrine into the ventromedial
nucleus (VMN) or medial preoptic
nucleus (MPN) on lordosis in VNAB-lesioned
ovariectomized, estrogen/progesterone-treated
female rats . . . . . . . . . 53

Figure 11 Effects of bilateral injections
of 5-ADMP into the medial amygdala
(mAMY) on NE concentrations in the
mAMY and lordosis in ovariectomized,
estrogen/progesterone-treated female rats . . 55

viii

INTRODUCTION

It has been well established that the interaction of
estrogen and progesterone are required for the full expression
of sexual behaviors in female rats (Beach, 1942; Young, 1961;
Edwards, et a1., 1968; Whalen, 1974). Proceptive behaviors
such as ear-wiggling and hopping/darting movements are used by
the female rat to attract the male (Beach, 1976). Receptivity
of the female is evaluated by observing the lordosis reflex
kBeach, 1976). This is an arched posture the receptive female
adopts when mounted by the male and maximizes the probability
that successful intromission will be achieved. Proceptive
behaviors and lordosis are thought to be controlled by
separate mechanisms. Whalen has demonstrated that the
probability of lordosis behavior occurring in ovariectomized
female rats is positively correlated with increases in both
estrogen and progesterone (Whalen, 1974). However, proceptive
behaviors are not greatly effected by increases in estrogen
but are strongly correlated with progesterone dose (Whalen,
.1974). These studies suggest that lordosis and ear-wiggling
and hopping/darting behaviors are separately effected by

estrogen and progesterone. Differences in control could occur

 

2
in separate brain regions due to differences in the action of
estrogen and.progesterone on various neurotransmitter systems.

To examine the inhibitory' effects of a 'treatment on
proceptivity or lordosis, ovariectomized female rats are
treated with high doses of estrogen followed by progesterone.
This hormone treatment is thought to mimic the natural state
of estrous during which the female is receptive. If a given
treatment acts to block mechanisms of receptivity, the female
will not display some aspects of sexual behavior. Conversely,
in order to examine facilitation of a treatment on sexual
behavior, ovariectomized females are treated with low doses of
estrogen with or without subsequent progesterone
administration. This protocol exposes the animal to sub-
threshold levels of hormones and females do not exhibit
proceptive behaviors or lordosis when exposed to a: male.
Through the use of these models, compounds can be evaluated
for their role in mediating sexual behavior. By determining
the location and functional mechanisms of steroid responsive
neural circuits which control reproductive behaviors
researchers are able to gain incite into the action of
steroids on brain function.

Much work has been done on the brain circuitry involved
in sexual behaviors in the female rat. The location of
steroid-concentrating neurons in the central nervous system
suggests a pattern of circuitry functioning to control all

aspects of reproduction (Stumpf, et al., 1975; Pfaff and

3

Keiner, 1973). Through olfactory and visual cues, the estrous
female senses the males presence and will begin proceptive
behaviors to increase the males interest. As the aroused male
begins tactile contact and eventual mounting, the information
attained by the female through somatosensory stimulation is
transmitted via ascending pathways up the spinal cord to the
midbrain (for review see Pfaff, 1980). It is thought that
estrous-relevant information from the hypothalamus is
coordinated with motor output at the level of the midbrain,
resulting in lordosis.

Estrogen—concentrating neurons are found throughout the
spinal cord, medulla, and midbrain. These neurons could be
involved in relaying both ascending somatosensory input as
well as part of the descending motor output. High numbers of
estrogen-concentrating neurons are also located in the
hypothalamus, preoptic area, and limbic regions, areas known
to be involved in many of the physiologic as well as
behavioral aspects of reproduction. The distribution of
progestin receptors in the female rat brain also appears to be
widespread but high concentrations of estrogen-inducible
progestin receptors are found only in the preoptic region and
medial basal hypothalamus (primarily in the lateral part of
the ventromedial nucleus), and to a lesser extent in limbic
structures (Maclusky and McEwen, 1980; Parsons, et al., 1982).
A small percentage of the progestin receptor-containing

neurons in the preoptic and medial basal hypothalamus also

 

4

project to the midbrain (DonCarlos and Morrell, 1990). All of
these areas of the brain could be required to coordinate the
complex physical and behavioral requirements for successful
reproduction. These estrogen- and progestin-concentrating
regions correspond to sites in the brain where hormone
implants and lesions effect the sexual receptivity of
ovariectomized female rats.

Manipulations within the ventromedial nucleus (VMN) of
the hypothalamus appear to have dramatic effects on the sexual
behavior of ovariectomized female rats. Concentrated estrogen
implants into the VMN will induce lordosis without
progesterone treatment (Barfield, et al., 1983), while
cholesterol-diluted estrogen (a concentration more likely to
mimic physiological conditions) will produce a priming effect
and rats become receptive following systemic progesterone
injection (Rubin and Barfield, 1980; Davis, et al., 1982). In
support of the theory that estrogen acts in the VMN to
facilitate lordosis it was demonstrated that implants of an
antiestrogen into the VMN of female rats reduces lordosis in
females injected with estrogen and progesterone (Meisel, et
al., 1987). Progesterone also appears to have a major effect
on receptivity when implanted into the VMN. Female rats
primed with low doses of estrogen exhibit lordosis following
implants of progesterone into the VMN (Rubin and Barfield,
1983; Pleim and Barfield, 1988). Furthermore, antiprogestins

reduced progesterone—induced lordosis when implanted into the

 

5

VMN (Etgen and Barfield, 1986). In addition, electrolytic
destruction of the lateral aspect of the VMN abolished
estrogen- and estrogen/progesterone-induced lordosis (Mathews
and Edwards, 1977; Pfaff and Sakuma, 1979; Mathews, et al.,
1983), although some females did regain the ability to display
lordosis behavior (LaVaque and Rodgers, 1975; Okada, et al.,
1980). On the other hand, very large lesions that encompass
the entire VMN failed to interrupt hormone-induced receptivity
(Okada, et al., 1980). Evidence suggests that the VMN may be
a major site for the action of estrogen and progesterone to
mediate sexual behavior, but the contribution of other sites
in the circuit must also be considered.

Another brain region containing a high concentration of
steroid hormone receptors is the preoptic area (POA). Some
investigators have found that implantation of estrogen in the
POA is effective in inducing receptivity, although spread of
the concentrated estrogen to the VMN can not be ruled out
(Lisk, 1962; Yanase and Gorski, 1976). In contrast however,
Barfield and coworkers failed to observe an effect of dilute
estrogen implants in the POA of ovariectomized female rats
(Barfield, et al., 1983). Furthermore, implants of
antiestrogen into the POA failed to block
estrogen/progesterone-induced receptivity as it had in the VMN
(Meisel, et al., 1987). Also, unlike the VMN, progesterone
implants into the POA failed to have an effect on the

receptivity of female rats primed with low dose estrogen

 

6
(Rubin and Barfield, 1983; Pleim and Barfield, 1988) and
antiprogestins implanted into the POA failed to reduce
progesterone—induced lordosis (Etgen and Barfield, 1986).

As with the implant studies, lesions of the POA produced
different results than did VMN lesions. Powers and Valenstein
have shown that in estrogen/progesterone-treated
ovariectomized female rats, less estrogen is required to
induce lordosis in females with lesions in the medial preoptic
area (MPOA) (Powers and Valenstein, 1971). Bast demonstrated
that lesions in the medial preoptic nucleus (MPN) reduced the
receptivity induced by estrogen implants into the VMN (Bast,
et al., 1987). In addition, Leedy showed that the effect of
MPOA lesions on lordosis is dependant on location. Small
lesions in the ventral portion of the MPN were found to
facilitate receptivity in female rats, whereas lesions placed
more dorsally inhibited lordosis in ovariectomized
estrogen/progesterone-treated females (Leedy, 1984). These
results suggest that while neurons in the VMN are important in
the facilitation of lordosis, neurons of the MPOA may be
facilitatory and inhibitory' with regard to their role in
mediating hormone effects on receptive behaviors.

other regions of the brain which also concentrate steroid
hormones may also be important for the control of lordosis.
Lesions in estrogen-concentrating regions such as the amygdala
(Masco and Carrer, 1980), zona incerta (Dornan, et al., 1991),

and midbrain areas (Herndon, 1976; Edwards and Pfeifle, 1981;

 

7
Yamanouchi and Arai, 1982 and 1985) have been shown to affect
lordosis. Lesions in some areas of the midbrain have produced
dramatic deficits in lordosis behavior. It may' be that
pathways mediating sensory input or information coordinating
motor output important for the lordosis reflex are being
disrupted by lesions in these brain regions. Conversely,
implants of dilute estrogen into estrogen-concentrating brain
regions such as the diagonal band of Broca, lateral habenula,
amygdala, cortex (Barfield, et al., 1983), mesencephalic
reticular formation, and caudate-putamen (Yanase and Gorski,
1976) were ineffective in inducing receptivity. Others have,
however, facilitated lordosis in estrogen primed female rats
with progesterone implants in midbrain regions such as the
ventral tegmental area (VTA) (Luttge and Hughes, 1976;
Tennent, et al., 1982) and mesencephalic reticular formation
(Ross, et al., 1971; Yanase and Gorski, 1976), as well as the
habenula (Tennent, et al., 1982), hippocampus, and amygdala
(Franck and Ward, 1981). In contrast, other investigators
have failed to see an effect of progesterone implants into the
midbrain on lordosis (Rubin and Barfield, 1983; Pleim and
Barfield, 1988). Pleim, et al., have recently shown that
contralateral implants of progesterone in the VMN and VTA are
more effective at inducing lordosis in estrogen-primed females
than a unilateral implant in the VMN alone (Pleim, et al.,
1991). Although studies evaluating the effects of steroid

implants and lesions in regions of steroid-concentrating

 

8
neurons are somewhat ambiguous and inconsistent, these studies
do provide evidence that neural systems which control lordosis
are located throughout the brain. ‘

Steroid hormones act in many ways to effect neural
communication. One way in which estrogen and progesterone
produce their changes on neurons in the lordosis circuit is
through action on specific neurotransmitter systems. Most of
the known neurotransmitters and neuromodulatory peptide
systems have been examined for their effects on lordosis.
Some, such as the cholinergic neurons, appear to be directly
involved in the ability to lordose since cholinergic
antagonists can dramatically block receptivity in
estrogen/progesterone treated female rats (Clemens, et al.,
1980; Dohanich and Clemens, 1981). The involvement of
neuropeptide (ie. opiates, LHRH, substance P) and monoamine
(ie. NE, DA, 5HT) neurotransmitters in the lordosis circuit
are much less clear (for an overview see: Meyerson, et.al.
1985; De Vries, 1990).

One of the first neurotransmitters to be examined for its
role in receptivity was norepinephrine. Several lines of
evidence suggest that hypothalamic noradrenergic neurons
mediate the stimulatory effects of gonadal steroids on sexual
receptivity in female rats. Some investigators have shown
that microinjection of norepinephrine or adrenergic receptor
agonists directly into the VMN (Foreman and Moss, 1978;

Fernandez-Guasti, et al., 1985) or MPN (Foreman and Moss,

 

9

1978) induce lordosis in ovariectomized, estrogen—treated
female rats. Neurotoxin-induced lesions of hypothalamic
noradrenergic neurons impair lordosis in ovariectomized,
estrogen/progesterone-treated female rats (Hansen, et al.,
1981). In addition, NE release in the VMN is elevated in
ovariectomized, estrogen/progesterone—treated rats displaying
high levels of lordosis behavior (Vathy and Etgen, 1989).

Noradrenergic innervation of the hypothalamus originates
from perikara located in subcoeruleus nuclei of the pons-
medulla (i.e. A1, A2, A5, A7; Dahlstrom and Fuxe, 1964;
Lindvall and Bjérklund, 1983). Axons of these neurons ascend
to the diencephalon via the ventral noradrenergic bundle
(VNAB) and terminate in virtually all regions of the
hypothalamus including the VMN and MPN (Olsen and Fuxe, 1972;
Jacobowitz and Palkovits, 1974; Moore and Bloom, 1979;
Palkovits, 1981). These noradrenergic neurons also
concentrate estrogen and project to regions containing a high
number of estrogen—concentrating neurons (Heritage, et al.,
1977).

Ovarian hormones have been shown to affect noradrenergic
transmission both pre- and post-synaptically. Evidence
suggests that estrogen and progesterone may influence the
uptake and release of norepinephrine from synaptosomes
(Janowsky and Davis, 1970). In addition, estrogen and
progesterone influence the levels of KCl-stimulated.NE release

in the VMN of anesthetized female rats (Vathy and Etgen,

 

10

1988). Priming doses of estrogen have also been shown to
influence norepinephrine turnover in related areas such as the
diagonal band of Broca, periventricular nucleus and lateral
septum (Renner, et al., 1986). Further studies show that NE
levels are elevated in the VMN of receptive females when
exposed to a male (Vathy and Etgen, 1989). Lastly, estrogen
has been shown to increase the firing rate of A1 noradrenergic
neurons which project to the hypothalamus (Kaba, et al.,
1983). These findings suggest that steroid hormones may
influence NE transmission by their direct presynaptic action
on the noradrenergic neuron.

In addition to the direct actions on noradrenergic
neurons, ovarian hormones can also affect noradrenergic
transmission by modulating postsynaptic adrenergic receptors
on hormone-concentrating target neurons. Estrogen and
progesterone have been shown to differentially alter the
binding characteristics of selective radioligands to al-, a2-,
and B-adrenergic receptors in the amygdala and hypothalamus of
ovariectomized female rats (Agnati, et al., 1980). Estrogen
effects the density of al-adrenergic receptors in the MPN,
median eminence and VMN and alters the diurnal rhythm of a1-
receptor density in the MPN and median eminence (Weiland and
Wise, 1987; Sortino, et al., 1989). These studies provide
evidence that estrogen and progesterone, at least in part, act

at the level of the adrenergic receptor to produce changes in

 

ll
noradrenergic transmission which modulates behavioral
responses.

It is conceivable that the noradrenergic system could be
one of the components of the lordosis circuit and might
function to transmit incoming information from the spinal cord
and brain stem to other brain regions such as the
hypothalamus. The responsiveness of this pathway could be
acted upon by estrogen and/or progesterone to coordinate
information important for the performance of receptive
behavior. The overall purpose of the following experiments
was to evaluate the importance of NE for hormone dependant
receptivity and to determine the location of NE influence

within the brain circuity controlling reproductive behaviors.

 

EXPERIMENT 1: SYSTEMIC INJECTION OF EITHER 021- OR B-
ADRENERGIC ANTAGONISTS FAIL TO INHIBIT LORDOSIS IN
ESTROGEN/PROGESTERONE TREATED FEMALE RATS

Although NE is believed to be important for the control
of reproductive behaviors in the female rat, the specific
adrenergic receptor subtype(s) involved and the role of these
receptors in mediating lordosis is unclear. Through the use
of pharmacologic manipulations, noradrenergic receptors have
been traditionally classified into a1-, a2- and B-adrenergic
receptors. It is generally accepted that al- and B-receptors
are located on the postsynaptic neuron and produce their
effects through second messenger systems utilizing
phosphoinositide hydrolysis and adenylate cyclase,
respectively. Alpha-2 receptors are presumably located on the
presynaptic terminal of the noradrenergic neuron and function
to inhibit the release of NE. However, there are some 02 type
receptors located postsynaptically in the brain as well
(Janowsky and Sulser, 1987).

To establish the importance of either al- or B-adrenergic
receptors for the transmission of information resulting in
sexual behavior, the pharmacological agents phenoxybenzamine
or propranolol were used to block these receptors in
estrogen/progesterone-treated female rats. Phenoxybenzamine
is an a-receptor antagonist and is moderately selective for
the al-adrenergic receptor. Propranolol is a nonselective, B-
adrenergic receptor antagonist (Weiner, 1985). Both compounds

penetrate into the brain and produce their effects by binding

12

 

13
to their specific receptors thereby blocking the binding and
action of endogenous norepinephrine.

The purpose of the present study was to determine if
estrogen/progesterone-induced lordosis is mediated by the
stimulation of either the al— or B-adrenergic receptor, since
steroids have been shown to affect the binding characteristics
of these receptor types (Agnati, et al., 1980). This study
demonstrates that systemic injections of either the al-
receptor antagonist phenoxybenzamine or the B-receptor
antagonist propranolol is ineffective at inhibiting

estrogen/progesterone-induced lordosis in female rats.

METHODS

Animals

Sherman strain female rats weighing 200-250 g were
obtained from Camm Research Co. (Wayne, NJ). Long Evans
strain female rats weighing 200-250 g were obtained from
Charles Rivers Laboratories (Wilmington, MA). All animals
were maintained in a temperature- (21 1 1° C) and light-
(lights on between 2100 and 1100 h) controlled environment,
and provided food (Wayne Lablox) and tap water ad libitum.
For all experiments, rats were bilaterally ovariectomized
under sodium pentobarbital anesthesia (30 mg/kg; i.p.), and
one week later treated with estradiol benzoate (0.5 ug/0.1
ml/rat; i.m.) 72, 48, and 24 hours, and progesterone (500

ug/O.1 ml/rat; i.m.) 5 hours before a behavioral pre-test for

 

14
sexual receptivity. Only female rats attaining a lordosis
quotient of 70 or greater in the behavioral pre-test, thereby
demonstrating a response to exogenous hormone treatment, were
used in the present studies.
REESE

Estradiol benzoate (Sigma Chemical Co., St Louis, MO) and
progesterone (Sigma) were dissolved in sesame oil.
Propranolol hydrochloride (Sigma) was dissolved in 0.9%
saline. Phenoxybenzamine (a gift from Smithkline Beecham,
Swedeland, PA) was dissolved in 50% propylene glycol (Sigma).
Treatment

For each of the present studies, twenty female rats were
randomly assigned to one of the following treatment groups and
then randomly reassigned to receive a second treatment the
following week. For all experiments, females were injected
with estradiol benzoate (see appropriate figure legend for
dose and time of treatment) and progesterone (500 ug/0.1
ul/rat; i.m.) 4 hours before behavioral testing.

To determine the effect of adrenergic antagonists on
receptivity, estrogen (0.5 ug/rat; i.m. at 72, 48, and 24
hours before testing) and progesterone (500 ug/rat; i.m. at 4
hours before testing)-treated female rats of Sherman and Long
Evans strains were injected with either propranolol (1, 4 or
20 mg/kg; i.p.) or its vehicle (0.9% saline; 1 ml/kg; i.p.);
or with either phenoxybenzamine (1, 4 or 20 mg/kg; s.c.) or

its vehicle (50% propylene glycol; 1 ml/kg; s.c.) 2 hours

 

15
prior to behavioral testing. Two different strains of rats
were tested since results from preliminary studies were not in
agreement with a previous report.

To determine if the effect of adrenergic antagonists on
lordosis is dependant on estrogen treatment, Sherman strain
female rats were injected with estrogen (either 0.125, 0.25,
or 0.5 ug/rat; i.m. at 72, 48, and 24 hours before testing; or
4 or 10 ug/rat; i.m. at 48 hours before testing) and
progesterone (500 ug/rat; i.m. at.44 hours before testing)
followed by injections of either propranolol (4 mg/kg) or its
vehicle (0.9% saline; 1 ml/kg; i.p.) 2 hours before behavioral
testing, or in a separate experiment, phenoxybenzamine (4
mg/kg) or its vehicle (50% propylene glycol; 1 ml/kg; s.c.) 2
hours before behavioral testing.

Behavioral Testing

In all studies, female rats were tested for sexual
receptivity by placing them with a sexually experienced male
Long Evans rat which had been adapted to the testing arena
(45x50x58 cm Plexiglas cage). Lordosis behavior was measured
as a lordosis quotient (LQ) which is defined as the frequency
of lordosis postures to ten mounts divided by ten and
multiplied by 100 (LQ = number of lordosis responses/10 x
100). A mount was counted when the male palpated the female's
flank with his forepaws and exhibited pelvic thrusting. Each

test session was limited to 10 mounts.

 

16
Statistics
Lordosis quotients were analyzed.with.Kruskal-Wallis one—
way analysis of variance by ranks followed by the Mann-Whitney
U test for comparisons between two groups (Siegle, 1956).
Differences were considered significant if the probability of

error was less than 5%.

RESULTS

As shown in Figure 1, injection of 1, 4, or 20 mg/kg of
the B-antagonist propranolol given 2 hours after progesterone
treatment and 2 hours prior to behavioral testing failed to
inhibit receptivity in either the Sherman or Long Evans strain
of ovariectomized estrogen/progesterone-treated female rats.
Likewise, in a similar experiment, injection of the al-
antagonist, phenoxybenzamine (1, 4 or 20 mg/kg) failed to
inhibit receptivity in either of these two strains (Figure 2).
These results suggest that neither 3— nor al-receptor blockade
is sufficient to inhibit the high level of receptivity induced
by estrogen and progesterone in these two strains of female
rats.

To determine if the inability of either of the adrenergic
receptor antagonists to block estrogen/progesterone induced
receptivity was dependant on concentration or duration of
exposure to estrogen, ovariectomized females were primed with

various concentrations of estrogen that were administered

 

 

17

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21
over various time schedules. As shown in Figure 3,
estrogen/progesterone-treated female rats given 0.125 pg of
estrogen 72, 48 and 24 hours before testing were less
receptive than females treated with the higher doses of
estrogen, and propranolol failed to inhibit lordosis as
compared to the vehicle-treated females for any of the
estrogen-treatment groups. Likewise as shown in Figure 4,
estrogen/progesterone-treated females were less receptive when
primed with lower doses of estrogen, and phenoxybenzamine
failed to inhibit lordosis in any of the estrogen-treatment
groups. These results indicate that systemic administration
of the adrenergic antagonists propranolol and.phenoxybenzamine
does not block estrogen/progesterone induced lordosis

regardless of estrogen-treatment and level of receptivity.

DISCUSSION

The role of adrenergic receptor subtypes in mediating
hormone induced receptivity in female rats has remained
unclear. In contrast to the results of the present study,
Fernandez-Guasti and coworkers found that systemic injection
of the al-antagonists phenoxybenzamine or prazosin, or the B-
antagonist propranolol inhibited lordosis in
estrogen/progesterone-treated females when given 2 hours after
progesterone injection (Fernandez-Guasti, at al., 1985a).
These results suggest that either al- or B-receptor blockade

is sufficient to inhibit lordosis and that activation of both

 

 

22

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23

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26
receptor types are required for receptivity. In the present
experiment, however, neither injection of the al-antagonist
phenoxybenzamine or the B—antagonist propranolol into
estrogen/progesterone-treated female rats inhibited lordosis,
regardless of the female strain used. The failure to decrease
the occurrence of lordosis was not dependant on estrogen dose
or the level of receptivity in females. These results are in
agreement with Davis and Kohl, who also failed to see an
effect of phenoxybenzamine on lordosis (Davis and Kohl, 1977).
Interestingly, these investigators found that systemic
injections of the az-agonist, clonidine, inhibited lordosis in
estrogen/progesterone-treated females (possibly asziresult of
inhibiting presynaptic NE release). Further, they found that
this inhibition was blocked by pretreatment with the (:2-
antagonist yohimbine. Variations in the results from various
laboratories might possibly be due to an inconsistency of
systemic injections to provide the required concentration of
antagonist at the sites of its action. It is unlikely that
the conflicting results are due to female rat strain

differences.

 

EXPERIMENT 2: INJECTION OF NE INTO THE VMN FAILS TO
FACILITATE LORDOSIS IN ESTROGEN-TREATED FEMALE RATS

Various lines of evidence lend strong support to the
notion that the VMN is an important part of the brain
circuitry controlling hormone-mediated sexual behavior. There
are also data which indicate that NE is in some way involved
in these pathways. Conceivably, NE could be acting in the VMN
to mediate the effects of estrogen and progesterone on
lordosis since NE is found to increase in this region during
sexually receptive behavior in hormone-treated female rats
(Vathy and Etgen, 1989).

In the following study, unreceptive estrogen-primed
female rats were used to determine if increases in NE in the
VMN could facilitate lordosis. The females used for this
study were treated with low doses of estrogen (without
progesterone treatment) and would not display receptivity when
exposed to a male. The results indicate that injection of NE
into the area of the VMN only, is not sufficient to induce
receptivity in female rats primed only with low doses of

estrogen.

METHODS
Animals
Sherman strain female rats weighing 200-250 g were
obtained from Camm Research Co. (Wayne, NJ). All animals were
maintained in a temperature- (21 1 1° C) and light- (lights on
between 2100 and 1100 h) controlled environment, and provided

27

 

28

food (Wayne Lablox) and tap water ad libitum. Females were
bilaterally ovariectomized under sodium pentobarbital
anesthesia (30 mg/kg; i.p.), and one week later treated with
estradiol benzoate (0.5 ug/0.1 ml/rat; i.m.) 72, 48, and 24
hours, and progesterone (500 ug/0.1 ml/rat; i.m.) 5 hours
before a behavioral pre-test for sexual receptivity. Only
females attaining a lordosis quotient of 70 or greater in the
behavioral pre-test, thereby demonstrating a response to
exogenous hormone treatment, were used in the present studies.
REESE

Estradiol benzoate (Sigma Chemical Co., St Louis, MO) and
progesterone (Sigma) were dissolved in sesame oil.
Norepinephrine hydrochloride (Sigma) was dissolved in
artificial CSF immediately before use.
Intracerebral Injections into the VMN

Ten animals receiving VMN injections of norepinephrine or
its vehicle were implanted with stainless steel guide cannula
7 days prior to the experiment. Rats were anesthetized with
sodium pentobarbital (30 mg/kg; i.p.) and positioned in a
stereotaxic frame (David Kopf Instruments, Tujunga, CA) with
the incisor bar set at the horizontal plane (Kbnig and
Klippel, 1963). Bilateral 23-gauge stainless-steel guide
cannulae were implanted 2.3 mm posterior to bregma, 10.8 mm
from the midline and 7.5 mm below dura, and anchored to the
skull with stainless-steel screws and dental cement. On the

day of the experiment, fifteen minutes prior to behavioral

 

29

testing, norepinephrine (200 ng/side) or its artificial CSF
vehicle (0.5 ul/side) were injected bilaterally using a 10 ul
Hamilton microsyringe connected to a 30 gauge stainless-steel
injector which protruded 1 mm beyond the tip of the cannula
guide and into the VMN. Following completion of the study,
animals were anesthetized with sodiunlpentobarbital (60 mg/kg;
i.p.) and perfused with 0.9% saline followed by a 10% formalin
solution. The brains were removed and frontal sections (50
um) through the VMN were prepared using a microtome with a C02
freezing stage. Mounted brain sections were stained with
cresyl violet and examined for cannulae placement in the VMN.
only data from females with cannulae placed in the VMN were
used for statistical analyses.
Behavioral Testing

In the present study, ten VMN-cannulated female rats were
randomly assigned to one of two treatment groups (NE or its
vehicle) and, one week later, reassigned to the second
treatment group. This design allowed each female to be tested
twice, receiving both treatments. All females were injected
with a low dose of estradiol benzoate (0.175 ug/0.1 ml/rat;
i.m.) 72, 48, and 24 hours before behavioral testing. Female
rats were tested for sexual receptivity by placing them with
a sexually experienced male Long Evans rat which had been
adapted to the testing arena (45x50x58 cm Plexiglas cage).
Lordosis behavior was measured as a lordosis quotient (LQ)

which is defined as the frequency of lordosis postures to ten

 

30

mounts divided by ten and multiplied by 100 (LQ = number of
lordosis responses/10 x 100). A mount was counted when the
male palpated the female's flank with his forepaws and
exhibited pelvic thrusting. Each test session was limited to
10 mounts.
Statistics

Lordosis quotients were analyzed using the Mann—Whitney
U test for-comparisons between two groups (Siegle, 1956).
Differences were considered significant if the probability of

error was less than 5%.

RESULTS

As shown in Figure 5, bilateral injections of NE (400
ng/rat; 200 ng/side) into the VMN failed to facilitate
receptivity in ovariectomized females rats treated with low
doses of estrogen as compared to the non-receptive, vehicle-
treated controls. These results indicate that injections of
NE in the area of the VMN are not sufficient to facilitate
receptivity in ovariectomized females treated with low doses

of estrogen.

DISCUSSION
Several lines of evidence suggest that NE transmission in
the VMN is important for the display of lordosis. This may,
however, only be a minor portion of the whole circuitry

responsible for expression of sexual behavior. NE has been

 

 

31

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32

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33

shown to increase in the area of the VMN of
estrogen/progesterone-treated receptive female rats, although
it is not known if NE is increasing in other brain regions
important for lordosis as well (Vathy and Etgen, 1989). In
contrast to the findings of the present study, NE injections
into the VMN have been shown to facilitate lordosis in
estrogen only-treated female rats (Foreman and Moss, 1978;
Fernandez-Guasti, et al., 1985b). This effect was blocked by
systemic injection of either an al- or B-antagonist
(Fernandez-Guasti, et al., 1985b), suggesting that NE action
may be required at both al- and B-receptors in the area of the
VMN.

In the present study, injections of NE into the VMN
failed to facilitate lordosis in estrogen-treated female rats.
The reason for differences between the present study and other
investigators findings is unclear but might be due to several
factors. There could be variations in the sites of injection
between experiments or differences in NE concentration
available at the sites, even though doses of NE were
comparable. In other studies, leakage of NE from the VMN to
other important brain areas cannot be ruled out. Although NE
may act at different adrenergic receptor subtypes to inhibit
or facilitate lordosis, these results suggest that exogenous
increases of NE in the VMN alone is insufficient to induce
lordosis in ovariectomized female rats treated with low doses

of estrogen.

 

EXPERIMENT 3: NE IN EITHER THE VMN OR MPN IS NOT ESSENTIAL
FOR LORDOSIS IN ESTROGEN/PROGESTERONE-TREATED FEMALE RATS

Further evidence to support a role of NE in the control
of lordosis is demonstrated by various lesion studies.
Electrolytic— and neurotoxin-induced lesions of the ventral
noradrenergic bundle (VNAB) deplete NE concentrations in the
basal forebrain and disrupt lordosis in ovariectomized
estrogen/progesterone-treated female rats (Hansen, et al.,
1981). It is not known, however, if loss of NE in the VMN,
MPN, or other regions following these lesions is responsible
for the reduction in lordosis.

The following experiments were performed in an attempt to
determine the site of NE action in the control of lordosis.
The effects of selective neurotoxin (5-ADMP)-induced lesions
of noradrenergic neurons terminating in the ‘VMN, MPN, or
medial amygdala (mAMY) on lordosis were examined in
ovariectomized, estrogen/progesterone-treated female rats.
The results reveal that while neurotoxin-induced depletion of
NE 1J1 the basal forebrain inhibits lordosis, selective NE
depletion in either the VMN, MPN, or mAMY is not accompanied
by a loss of lordosis. These data suggest that noradrenergic
neurons terminating in any one of these nuclei are not
essential, by themselves, for the induction of sexual

receptivity in ovariectomized female rats by gonadal steroids.

34

3 5
METHODS

Animals

Sherman strain female rats weighing 200—250 g were
obtained from Camm Research Co. (Wayne, NJ). All animals were
maintained in a temperature- (21 t 1° C) and light- (lights on
between 2100 and 1100 h) controlled environment, and provided
food (Wayne Lablox) and tap water ad libitum. For all
experiments, rats were bilaterally ovariectomized under sodium
pentobarbital anesthesia (30 mg/kg; i.p.), and one week later
treated with estradiol benzoate (0.5 ug/0.1 ml/rat; i.m.) 72,
48, and 24 hours, and progesterone (500 ug/0.1 ml/rat; i.m.)
5 hours before a behavioral pre-test for sexual receptivity.
Only female rats attaining' a lordosis quotient of 70 or
greater in the behavioral pre-test, thereby demonstrating a
response to exogenous hormone treatment, were used in the
present studies.
REESE

Estradiol benzoate (Sigma Chemical Co., St Louis, MO) and
progesterone (Sigma) were dissolved in sesame oil. Fluoxetine
hydrochloride (Eli Lilly and Co., Indianapolis, IN) was
dissolved in 0.9% saline. 5—Amino-2,4,dihydroxy-a-
methylphenylethylamine dihydrobromide (5-ADMP; synthesized by
Dr. John R. Palmer of The Upjohn Co., Kalamazoo, MI) was
dissolved in 0.3% saline containing 0.1% ascorbic acid.
Phenylephrine hydrochloride (Sigma) was dissolved in

artificial CSF. Drugs were administered as indicated in the

 

36

legends of the appropriate table and figures; doses of
fluoxetine, 5-ADMP and phenylephrine were calculated as free
base.
Neurochemical Lesions of the VNAB

Rats were anesthetized with Equithesin (3 ml/kg; i.p.)
and positioned in a stereotaxic frame (David Kopf Instruments,
Tujunga, CA, U.S.A.) with the incisor bar set 3 mm below the
horizontal plane. The needle of a 5 pl Hamilton syringe was
inserted into the VNAB at coordinates A 0.0 mm, L 11.3 mm, V -
6.8 mm from dura (15), and bilateral injections of either 5-
ADMP (8 ug/side) or its vehicle (0.3% saline containing 0.1%
ascorbic acid; 0.3 ul/side) were made over a 1 minute period.
The needle remained in the brain for an additional 10 minutes
after injection to reduce the reflux of the neurotoxin back up
the needle track. One hour prior to administration of S-ADMP
rats were injected with fluoxetine (10 mg/kg; s.c.) to prevent
the uptake of neurotoxin into 5-hydroxytryptaminergic neurons.
Rats were allowed to recover for 7 days following surgery
before behavioral testing was performed.
Neurochemical Lesions of the VMN. MPN and mAMY

Rats were anesthetized with Equithesin (3 ml/kg; i.p.)
and positioned in a stereotaxic frame (David Kopf Instruments,
Tujunga, CA) with the incisor bar set 2.4 mm below the
horizontal plane (Kénig and Klippel, 1963). For the VMN, the
needle of a 5 ul Hamilton syringe was inserted at coordinates

A 4.4 mm, L 10.7 mm, V -8.8 mm from dura, and bilateral

37

injections of either 5-ADMP (2 pg/side) or its vehicle (0.3%
saline containing 0.1% ascorbic acid; 0.3 pl/side) were made
over a 1 minute period. For the MPN, the needle of a 5 p1
Hamilton syringe was inserted at coordinates A 6.8 mm, L i0.7
mm, V —7.7 mm from dura, and two bilateral injections of
either 5-ADMP (2 pg/site/side) or its vehicle (0.3% saline
containing 0.1% ascorbic acid; 0.3 pl/site/side) were made
over a 2 minute period by injecting for the first minute at
the most ventral position (7.7 mm below dura) and then raising
the needle to inject more dorsally (7.0 mm below dura) for the
final minute. For the mAMY, the needle of a 5 pl Hamilton
syringe was inserted at coordinates A 2.1 mm, L 13.2 mm, V -
8.0 mm from dura, and bilateral injections of either 5—ADMP (3
pg/side) or its vehicle (0.3% saline containing 0.1% ascorbic
acid; 0.3 pl/side) were made over a 1 minute period. The
needle remained in the brain for an additional 10 minutes
after the final injection to reduce the reflux of the
neurotoxin back up the needle track. One hour prior to
injection of 5-ADMP rats were injected with fluoxetine (10
mg/kg; s.c.) to prevent uptake of neurotoxin into 5-
hydroxytryptamine (5HT) neurons. Rats were allowed to recover
for 7 days following surgery before behavioral testing was
performed.
Lateral Ventricular Cannulation

Animals receiving intracerebroventricular (i.c.v.)

injections of phenylephrine or its vehicle were implanted with

38

a stainless steel guide cannula into a lateral cerebral
ventricle 7 days prior to the experiment. Rats were
anesthetized with Equithesin (3 ml/kg; i.p.) and positioned in
a stereotaxic frame (David Kopf Instruments, Tujunga, CA) with
the incisor bar set 2.4 mm below the horizontal plane (K6nig
and Klippel, 1963). A 23-gauge stainless-steel guide cannula
was implanted such that the tip was 1.4 mm lateral to bregma
and 3.2 mm below dura, and anchored to the skull with
stainless-steel screws and dental cement. On the day of the
experiment, phenylephrine or its artificial CSF vehicle were
injected in a volume of 5 p1 with a 10 pl Hamilton
microsyringe connected to a 30 gauge stainless-steel injector
which protruded 1 mm beyond the tip of the cannula guide and
into the lateral ventricle.
Intracerebral Injections into Hypothalamic Nuclei

Animals receiving intracerebral (i.c.) injections of
phenylephrine or its vehicle into the VMN or MPN were
implanted with stainless steel guide cannula 7 days prior to
the experiment. Rats were anesthetized with Equithesin (3
ml/kg; i.p.) and positioned in a stereotaxic frame (David Kopf
Instruments, Tujunga, CA) with the incisor bar set 2.4 mm
below the horizontal plane (Kbnig and Klippel, 1963). For the
VMN, bilateral 23—gauge stainless steel cannula were implanted
$0.7 mm from midline, 2.6 mm posterior to bregma and 8.8 mm
below dura. For the MPN, bilateral 23-gauge stainless steel

cannula were implanted $0.7 mm from midline, 2.4 mm posterior

”4.

 

39

to bregma and 7.4 mm below dura. Phenylephrine (1 pg/side) or
its artificial CSF vehicle (0.3 pl/side) were injected
bilaterally into the VMN or MPN with a 10 pl Hamilton
microsyringe connected to a 30 gauge stainless-steel injector
30 minutes prior to behavioral testing.
Behavioral Testing

For all experiments, females were injected with estradiol
benzoate (0.5 pg/0.1 ml/rat; i.m.) 72, 48, and 24 hours, and
progesterone (500 pg/0.1 ml/rat; i.m.) 5 hours before
behavioral testing. Female rats were tested for sexual
receptivity by placing them with a sexually experienced male
Long Evans rat which had been adapted to the testing arena
(45x50x58 cm Plexiglas cage). Lordosis behavior was measured
as a lordosis quotient (LQ) which is defined as the frequency
of lordosis postures to ten mounts divided by ten and
multiplied by 100 (LQ = number of lordosis responses/10 x
100). A mount was counted when the male palpated the female's
flank with his forepaws and exhibited pelvic thrusting. Each
test session was limited to 10 mounts.
Tissue Dissection and Neurochemical Analyses

Within one hour following behavioral testing, animals
were decapitated and brains were removed from the skull and
frozen on aluminum foil placed directly over dry ice. Frontal
brain sections (600 pm) beginning approximately at 9220 pm
(Kbnig and Klippel, 1963) were prepared in a cryostat (-9°C),

and the VMN and MPN were dissected from these sections

 

40
according to a modification (Lookingland and Moore, 1985) of
the method of Palkovits (Palkovits, 1973). Tissues samples
were placed in 60 p1 of 0.1 M phosphate-citrate buffer (pH
2.5) containing 15% methanol and stored at -20°C until
assayed.

On the day of the assay tissue samples were thawed,
sonicated for 3 s (Sonicator Cell Disruptor, Heat Systems-
Ultrasonic, Plainview, NY), and centrifuged for 30 s in a
Beckman 152 Microfuge. NE, dopamine (DA) and 5HT
concentrations in supernatants were measured by high
performance liquid chromatography with electrochemical
detection as described previously (Chapin, et al., 1986).
Tissue pellets were dissolved in 1.0 N NaOH and assayed for
protein (Lowry, et al., 1951).
statistics

Statistical analyses of monoamine concentrations were
performed using the Student's t test to compare differences
between two groups, and one-way analysis of variance followed
by the least significant difference test for the comparison of
multiple groups (Steel and Torrie, 1960). Lordosis quotients
were analyzed with Kruskal-Wallis one-way analysis of variance
by ranks followed by the Mann-Whitney U test for comparisons
between two groups (Siegle, 1956). Differences were
considered significant if the probability of error was less

than 5%.

4 1
RESULTS

As shown in Table 1, NE concentrations were
significantly reduced to 35% of control in the VMN and to 30%
of control in the MPN 7 days following bilateral injections of
5-ADMP into the VNAB. In contrast, S—ADMP had no effect on
the concentrations of DA or 5HT in these brain regions. 5-
ADMP injections into the VNAB significantly reduced lordosis
quotients in ovariectomized, estrogen/progesterone-treated
female rats (Figure 6), and this effect was reversed by i.c.v.
administration of the al-adrenergic receptor agonist
"phenylephrine (Figure 7). Taken together these results
suggest that neurotoxin-induced disruption of noradrenergic
neurons is associated with a deficit in sexual receptivity in
female rats.

To determine if the reduction in sexual receptivity
following 5-ADMP—induced lesions of the VNAB resulted from
loss of noradrenergic neuronal projections to the VMN or MPN,
lordosis quotients were determined in ovariectomized,
estrogen/progesterone-treated rats in which noradrenergic
terminals in these hypothalamic nuclei were selectively
lesioned. As shown in Figure 8, injection of 5-ADMP directly
into the VMN reduced NE concentrations in the VMN to 17% of
control, but failed to alter lordosis quotients as compared
with vehicle—treated controls. Similarly, direct injection of
5-ADMP into the MPN reduced NE concentrations in the MPN to

17% of control, but failed to alter lordosis quotients as

 

42

Table 1 Effect of bilateral injections of 5—ADMP into the
ventral noradrenergic bundle (VNAB) on amine concentrations in
the ventromedial nucleus (VMN) and medial preoptic nucleus
(MPN) of ovariectomized, estrogen/progesterone-treated female
rats.

Amine Concentration (ng/mg protein)

 

 

 

NE DA 5HT
VMN vehicle 14.5 i 1.l§ 1.2 i 0.1 6.1 i 0.3'
5—ADMP# 5.1 i 0.7* 1.1 i 0.1 6.4 i 0.4
MPN vehicle 24.3 i 1.5 6.7 t 2.7 10.4 i 0.5
5-ADMP 7.2 i 0.7* 5.5 i 2.2 10.3 i 0.5
S values represent the means i 1 S.E.M. of 9-11

determinations.

# rats were injected with 5—ADMP or its vehicle into the VNAB
and killed by decapitation 7 days later.

* values for 5-ADMP-treated rats that are significantly
different from vehicle—treated controls (p<0.05).

 

43

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49
compared with vehicle-treated controls (Figure 9). In
addition, injection of phenylephrine into either the VMN or
MPN of VNAB-lesioned rats failed to reinstate lordosis
quotients to the levels determined in the sham-lesioned
controls (Figure 10). As was the case for the VMN and MPN,
direct injection of 5-ADMP into the mAMY reduced NE
concentrations in the mAMY to 19% of control, but failed to
alter lordosis quotients as compared with vehicle-treated
controls (Figure 11). Taken together these results indicate
that noradrenergic neurons terminating in either the VMN, MPN,
or mAMY are not by themselves necessary for sexual receptivity

in ovariectomized, estrogen/progesterone-treated female rats.

DISCUSSION

Neurons in the VMN and MPN are believed to play an
important role in the mediation of hormone-induced sexually
receptive behaviors in female rats. However, the involvement
of NE in these and other lordosis relevant regions are
unclear. In the past it has been thought that NE (under the
influence of steroid hormones) acts on neurons of a specific
brain region within the neural circuit responsible for sexual
behavior.

In the present study, disruption of subcoeruleus
noradrenergic neurons in the VNAB following i.c.
administration of the selective noradrenergic neurotoxin 5-

ADMP (Jarry, et al., 1986) resulted in the loss of lordosis

 

 

50

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51

 

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55

 

 

 

 

 

 

 

 

 

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5—ADMP

VEHICLE

56

behavior in ovariectomized, estrogen/progesterone-treated
female rats. These results are consistent with previous
reports of an inhibitory effect of electrolytic- and
neurotoxin-induced lesion of the VNAB on lordosis behavior
(Hansen, et al., 1981), and indicate that subcoeruleus
noradrenergic neurons are important for the control of sexual
receptivity. The results of the present study also indicate
that the facilitative effects of NE on female sexual
receptivity are mediated by al-adrenergic receptors since
i.c.v. injection of the al-adrenergic receptor agonist
phenylephrine restores lordosis in VNAB-lesioned female rats,
although the involvement of B—adrenergic receptors can not be
ruled out.

A number of reports have implicated either the VMN or MPN
as possible sites of action of NE on lordosis in female rats.
In the present study, however, depletion of NE in either the
VMN or MPN did not reduce lordosis in ovariectomized,
estrogen/progesterone-treated female rats. In addition,
injection of the al-adrenergic receptor agonist phenylephrine
into either the VMN or MPN of hormone-treated females with
VNAB-lesions failed to restore lordosis. These results
suggest that noradrenergic neurons in the VMN or MPN alone are
not sufficient to induce lordosis in ovariectomized,
estrogen/progesterone-treated female rats.

Another region of the brain reported to be important for

sexual receptivity in female rats is the corticomedial

 

57

amygdala, and disruption of noradrenergic innervation to this
brain region occurs following VNAB lesions (Fallon, et al.,
1978). The amygdala contains neurons that concentrate labeled
estrogen (Pfaff and Keiner, 1973; Stumpf, et al., 1975), and
electrolytic lesions in the anterior portion of the
corticomedial amygdala disrupts lordosis, while
electrochemical stimulation of this region facilitates
lordosis in ovariectomized, steroid-treated female rats (Masco
and Carrier, 1980). However, in the present study, depletion
of NE in the region of the mAMY, as in either the VMN or MPN,
failed to prevent hormone induced sexual receptivity.
Alternatively, NE may play a neuromodulatory role in
facilitating lordosis, and noradrenergic innervation to
multiple brain nuclei (possibly including the VMN, MPN, or
mAMY) may be required for full expression of sexual
receptivity in female rats.

In conclusion, although previous reports indicate that
the VMN and MPN are important hypothalamic regions for the
control of lordosis, the results of the present study indicate
that noradrenergic neurons terminating in either one of these
regions are, in and of themselves, not essential for gonadal
steroid induction of sexual receptivity in ovariectomized

female rats.

GENERAL DI SCUSS ION

Sexually receptive behavior in female rats requires
estrogen and progesterone for full expression (Whalen, 1974).
Although multiple sites of action for estrogen and
progesterone probably exist, evidence suggests that these
hormones exert a major facilitatory effect on female sexual
receptivity through action on neurons located in the VMN and
MPN of the hypothalamus. Neurons located in these regions
concentrate labeled estrogen (Pfaff and Keiner, 1973; Stumpf,
et al., 1975) and progesterone (MacLusky and McEwen, 1980;
Parsons, et al., 1982; DonCarlos and. Morrell, 1990), and
ovariectomized female rats become receptive when given VMN
implants of estrogen and systemic injections of progesterone
(Rubin and Barfield, 1980). Conversely, implants of
antiestrogens into the VMN block the stimulatory effects of
estrogen/progesterone treatment on receptivity in
ovariectomized female rats (Meisel, et al., 1987). In
addition, electrolytic lesions of the VMN abolish lordosis
(Pfaff and Sakuma, 1979; Mathews, et al., 1983). Electrolytic
lesions of the dorsal region of the MPN reduce lordosis in
ovariectomized female rats given systemic injections of
estrogen and progesterone (Leedy, 1984), and in ovariectomized
female rats implanted with estrogen in the VMN and injected
systemically with progesterone (Bast, et al., 1987). Thus,
while neurons in the VMN and MPN are implicated in mediating

gonadal steroid-induced sexually receptive behaviors, the

58

 

59
identity of the neurotransmitters involved in this action are
less well defined.

There are several lines of evidence to suggest that NE is
involved in the brain circuitry controlling hormone-mediated
sexual receptivity. Ovarian hormones have been shown to
influence the transmission of noradrenergic neurons by
effecting uptake and release (Janowsky and Davis, 1970; Vathy
and Etgen 1988), firing rates (Kaba, et al., 1983), and NE
turnover (Renner, et al., 1986). In addition, estrogen-
concentrating noradrenergic neurons located in the pons-
medulla project to basal forebrain areas which also contain
estrogen-concentrating neurons. Results from Experiment 3
demonstrate that selective neurotoxic lesions of the
subcoeruleus noradrenergic neurons which ascend in the VNAB,
deplete NE in basal forebrain regions and also result in the
loss of lordosis behavior in ovariectomized,
estrogen/progesterone-treated female rats. These results are
consistent with previous findings that electrolytic- and
neurotoxin-induced lesions of the VNAB inhibit lordosis
behavior (Hansen, et al., 1981). Together, the results of
these studies give strong evidence that subcoeruleus
noradrenergic neurons are important for the control of sexual
receptivity.

The results from Experiment. 3 also suggest that the
facilitative effects of NE on female sexual receptivity are

mediated by al-adrenergic receptors, since lordosis behavior

 

 

60
was restored in VNAB-lesioned female rats following i.c.v.
injection of the al receptor agonist phenylephrine.
Unfortunately, most studies performed in an attempt to
determine the adrenergic receptor subtype involved in the
control of lordosis have yielded inconsistent information.
The results from Experiment 1 indicate that blocking a1
receptors in estrogen/progesterone—treated female rats with
systemic injections of the al-antagonist phenoxybenzamine,
does not inhibit receptivity. The failure of
phenoxybenzamine, as well as the B-receptor antagonist
propranolol, to prevent.hormone-mediated sexual receptivity in
Experiment 1, is in disagreement with. the findings of a
previous report (Fernandez-Guasti, et al., 1985a). In
contrast to the present study, Fernandez-Guasti and coworkers
found that systemic injections of either al- or B-adrenergic
receptor antagonists inhibit lordosis in
estrogen/progesterone-treated female rats. Their results
suggest that both of these adrenergic receptor subtypes are
required for the facilitative action of NE on lordosis. In
support of the results from Experiment 1, Davis and Kohl were
also unable to block lordosis with systemic injections of
phenoxybenzamine, however they did inhibit receptivity when
estrogen/progesterone—treated female rats were injected with
the a2 receptor agonist clonidine (Davis and Kohl, 1977).
This effect was blocked by the 02 receptor antagonist

yohimbine, suggesting that either the presynaptic a2 receptor

 

61

was functioning to inhibit the release of NE or perhaps a
postsynaptic a2 receptor was involved. It is unclear why
systemic studies are in disagreement with respect to the
adrenergic receptors involved and their function. Considering
the vast complexity of the systems and ‘variabilities in
procedures it may not be a direct enough approach to the
question. A better method may be to deplete the source of NE
and replace it with a selective agonist as was done in
Experiment 3. Results from the study using this model gives
strong support to the hypothesis that al-adrenergic receptor
stimulation is necessary for lordosis. It is certainly likely
that other receptor subtypes are involved as well.

Although NE does appear to be involved in the control of
hormone-mediated lordosis, the location of NE's action within
the neural circuit is unclear. To address this question, a
number of investigators have performed experiments applying
adrenergic agonists and antagonists, as well as NE, directly
into areas of the brain thought to be important for the
control of lordosis. As with the systemic studies, the direct
application of adrenergic compounds into specific brain
regions have produced conflicting results. One of the first
studies in which selective adrenergic receptor ligands were
injected into lordosis relevant brain regions suggested that
blockade of (11 receptors or stimulation of B-receptors in the
VMN or MPN facilitated lordosis in estrogen-treated female

rats (Foreman and Moss, 1978). In addition, stimulation of al

 

62
receptors or blockade of B-receptors in either of these
regions reduced receptivity in estrogen-treated females.
These results indicate that al stimulation in the VMN or MPN
inhibits lordosis while B-receptor stimulation in either of
these regions facilitates lordosis in female rats. In this
same study, injections of NE into the area of the VMN or MPN
was found to facilitate lordosis. Others have shown however,
that NE injection into the area of the MPN inhibited lordosis
in female rats primed with estrogen and progesterone (Caldwell
and Clemens, 1986). In yet another study, a combined
injection of a2- and B-receptor agonists into the VMN of
estrogen—treated female rats facilitated lordosis, while
separately they did not (Fernandez-Guasti, et al., 1985b).
Also in this study, NE injection into the VMN facilitated
lordosis in estrogen-treated rats. Etgen has recently shown
that implants of the (11 antagonist prazosin into the VMN of
estrogen/progesterone-treated female rats prior to
progesterone treatment, prevents the females from becoming
receptive (Etgen, 1990). This would indicate that al-
receptors in the VMN are necessary for lordosis. In
Experiment 2 of the present study, NE injections into the VMN
of estrogen-treated female rats failed to facilitate
receptivity. It is thought that NE functions selectively in
brain regions such as the VMN or MPN to effect lordosis.
However, as seen with systemic studies, results from site

specific studies are conflicting and inconsistent and do not

 

63
agree with regard to the role NE plays in the display of
lordosis, specific to these brain regions.

Although NE is required for lordosis, its action may not
be at a particular site but throughout various regions within
the neural circuits controlling receptivity. Convincing
evidence that NE is not required in a specific region
important for sexual behavior is given by the results of
Experiment 3. Depletion of NE in either the VMN or MPN did
not reduce lordosis in ovariectomized, estrogen/progesterone-
treated female rats. Furthermore, although i.c.v. injection
of the al-adrenergic receptor agonist phenylephrine restored
lordosis in VNAB-lesioned females, selective injection of
phenylephrine into either the VMN or MPN of VNAB-lesioned
females failed to reinstate receptivity. These results
strongly suggest that noradrenergic neurons in the VMN or MPN
alone are not necessary for lordosis in ovariectomized,
estrogen/progesterone-treated female rats. Although NE
concentrations have been shown to increase in the VMN when
estrogen/progesterone-treated female rats are receptive (Vathy
and Etgen, 1989), it is quite possible that NE concentrations
are elevated in other areas as well.

NE transmission may be required in various brain regions
either simultaneously or in a coordinated manner to effect the
general excitation of neurons within the hormone-sensitive

circuit which controls receptivity. Without the influence of

 

64
NE, neurons may be unable to reach the necessary level of
excitation.

In conclusion, although previous reports indicate that
regions such as the VMN and MPN are important for the control
of lordosis and NE is in some way required for this behavior,
the results of the present study indicate that noradrenergic
neurons terminating in either the VMN or MPN are not in and of
themselves essential for gonadal steroid induction of sexual
receptivity in ovariectomized female rats. Although a1-
receptors stimulation most likely functions to increase the
transmission resulting in lordosis it is probable that this is
not specific to one brain region but is a generalized effect.
Other adrenergic receptor subtypes are probably involved in
the hormone-sensitive neural pathway for lordosis as well, but
their specific involvement has not been addressed using the
NE—depleted female rat model.

Further studies utilizing NE-depleted female rats could
possibly clear up some of the discrepancies associated with
the function of NE in the control of lordosis. By this method
the female's endogenous source of NE is drastically reduced,
and adrenergic stimulation replacement can be controlled as to
locale and receptor subtype. It would be interesting to
determine if a B-adrenergic agonist could restore receptivity
in VNAB—lesioned females as did the al-adrenergic agonist.
Neuropeptides thought to be involved in the control of

lordosis could also be administered to VNAB-lesioned females

65

to investigate interactions between these neuromodulators and
the noradrenergic system. I

It is important to keep in mind the type of hormone—
treatment models that are being used when comparing the
results from different studies. The varying effects of
steroid hormones and time frames of action are not yet
understood and may greatly influence the effects of NE on the
neurons controlling sexual receptivity. It would be
interesting" to investigate ‘the ‘temporal effects of NE on
estrogen/progesterone induced receptivity. The reduced
concentrations of NE at the time of hormone treatment could

play a significant role in the modulation of lordosis.

 

 

 

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Bast, J. D.; Hunts, C.; Renner, K. J.; Morris, R. K.;
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